museum national d`histoire naturelle

MUSEUM NATIONAL
D’HISTOIRE NATURELLE
Ecole Doctorale Sciences de la Nature et de l’Homme – ED 227
Année 2008
N° attribué par la bibliothèque
|_|_|_|_|_|_|_|_|_|_|_|_|
THESE
Pour obtenir le grade de
DOCTEUR DU MUSEUM NATIONAL D’HISTOIRE NATURELLE
Discipline : Recherche clinique en pathologie aviaire
Présentée et soutenue publiquement par
Yannick ROMAN
Le 22 septembre 2008
Contribution à la validation de l’électrophorèse des
protéines plasmatiques comme outil diagnostique en
médecine aviaire appliquée à la conservation
Sous la direction de : Madame le Professeur BOMSEL-DEMONTOY Marie-Claude
Sous la co-direction de : Monsieur SAINT JALME Michel, Maître de conférences
Composition du jury :
Mme Jeanne BRUGERE-PICOUX,
Professeur , ENVA,
Président
Mme Marie-Claude BOMSEL-DEMONTOY,
Professeur, MNHN, DJBZ,
Directeur de Thèse
M. Michel SAINT JALME,
Maître de conférences, MNHN, UMR 5173,
Co-Directeur
M. Jacques DUCOS DE LAHITTE,
Professeur, ENVT,
Rapporteur
M. Karim ADJOU,
Maître de conférences, ENVA,
Rapporteur
M. Jean-Yves JOUGLAR,
Maître de conférences, ENVT,
Examinateur
on.
REMERCIEMENTS
Je remercie tout d’abord ma collègue et amie Marie-Claude BOMSEL-DEMONTOY pour
avoir accepté d’occuper la charge de Directeur de cette Thèse. Merci de m’avoir défendu
jusqu’au bout avec ton dynamisme habituel.
Je tiens également à remercier mon collègue et ami Michel SAINT JALME, pour ses conseils,
la rigueur de ses corrections et tous les bons moments que nous avons partagés ensemble. Je
pense pouvoir dire que c’est lui qui m’a appris à regarder le monde avec un regard de
« chercheur ».
Je tiens à remercier tout particulièrement Mme Jeanne BRUGERE-PICOUX, M. Jacques
DUCOS DE LAHITTE, M. Karim ADJOU et M. Jean Yves JOUGLAR, d’avoir accepté de
faire partie de mon jury de thèse.
Je remercie très chaleureusement mon collègue et ami Daniel CHASTE-DUVERNOY,
biologiste en laboratoire de diagnostic médical à Torcy, qui a pris en charge la totalité des
dosages des protéines totales et des bilans lipidiques de cette thèse, ainsi qu’un bon nombre
d’électrophorèses. Sans lui, rien ne se serait fait, car c’est lui qui m’a appris tout ce que je sais
en diagnostic de laboratoire, et c’est lui qui, le premier, a attiré mon attention sur ce
formidable examen complémentaire qu’est l’électrophorèse des protéines plasmatiques chez
les oiseaux. Merci encore pour toutes ces heures passées à répondre à mes questions retorses.
Je suis également extrêmement reconnaissant de l’aide fournie gracieusement par le
laboratoire SEBIA, et en particulier par Geneviève HENNACHE pour la réalisation des
électrophorèses capillaires sur le Capillarys2©. Ma reconnaissance envers Patrick
TROLLIET, n’a pas de bornes. Il a en effet toujours été là pour me donner au cours de
milliers de coups de téléphone les bons conseils techniques.
Je remercie chaleureusement Bertrand BED’HOM de la station INRA de Jouy en Josas pour
son aide inestimable dans identification de l’apolipoprotéine A-I et dans l’investigation de la
ponte, Alain GUILLOT de la station INRA de Jouy en Josas pour la réalisation pratique de la
spectrométrie de masse et David GOURICHON de la station INRA de Tours-Nouzilly pour
les prélèvements des poules pondeuses.
Ma gratitude va également au Muséum national d’Histoire naturelle, ainsi qu’au Conseil
Général Seine maritime pour leur support financier en termes de réactifs d’analyse et
d’investissement matériel. Je tiens en particulier à remercier Geneviève BERAUDBRIDENNE, Directrice du Département des jardins botaniques et zoologiques du MNHN,
ainsi que Isabelle MARAVAL, Directeur de la culture et de la jeunesse au CG76 et Benoit
PROUST, directeur adjoint de la culture et de la jeunesse au CG 76, pour avoir accepté que je
réalise cette thèse en parallèle à mon travail de Dr. vétérinaire au Parc de Clères.
Toute ma gratitude va également à la Direction du Parc zoologique de Clères, et en particulier
à Alain HENNACHE et à Paul ASTOLFI. Merci pour toutes ces années que nous avons passé
ensemble, et pour tout ce que j’ai pu apprendre à votre contact. Je suis également très
reconnaissant envers l’ensemble de l’équipe des soigneurs animaliers du Parc de Clères et en
particulier Didier CATTEVILLE et Cyrille DUMAIS, à l’équipe du laboratoire Bio-VSM et
en particulier à Karine NAUDIN qui a pris en charge les dosages de protéines totales et les
lipidogrammes, à Jean-Louis LIEGEOIS de l’académie de fauconnerie du Puy du fou qui m’a
permis de réaliser des prélèvements sur de nombreuses espèces de rapaces, à Dorothée
ORDONNEAU du zoo de Lille pour sa collaboration à la première partie de la thèse et pour
les prélèvements des amazones, à Norin CHAI et Charly PIGNON pour nos expériences
communes et pour les prélèvements de pigeons.
Je tiens, bien sûr à remercier mes amis Yan, coco, Manue, Pilouch, Laurent, Arnaud, Jean-Mi,
Tapu, Norin, Charly, Ced pour leur indéfectible amitié.
Une personne a été la clé de voute de cette thèse : Julie, mon amie dont le soutien moral, ainsi
que l’aide concrète durant cette thèse auront été essentiels.
Je remercie enfin toute ma famille et en particulier ma Mère, ma Grand-mère, mon Grandpère, ma tante Jacky, mon oncle Alain, mon cousin Florian et Delphine pour leur soutien.
TABLE DES MATIERES
INTRODUCTION GENERALE................................................................................................ 1
LES OISEAUX EN ELEVAGE CONSERVATOIRE ........................................ 1
Contexte général................................................................................................................. 1
Contexte sanitaire des élevages conservatoires d’oiseaux ................................................. 2
L’ELECTROPHORESE DES PROTEINES SANGUINES .............................. 3
Rappels ............................................................................................................................... 3
Historique ................................................................................................................... 3
Principes généraux de l’électrophorèse des protéines sanguines ............................... 5
Techniques d’électrophorèse utilisées en laboratoire de diagnostic .......................... 8
L’électrophorèse des protéines plasmatiques comme moyen diagnostique en médecine
aviaire ............................................................................................................................... 13
Utilisation de l’électrophorèse des protéines sanguines dans le contexte de la
médecine humaine et vétérinaire...................................................................................... 13
Contexte : une grande diversité d’espèces aviaires et de techniques
électrophorétiques ............................................................................................................ 14
Définition des fractions protéiques et du rapport albumine / globuline ................... 16
Interprétation des électrophorégrammes .................................................................. 18
OBJECTIFS DE RECHERCHE .............................................................................. 25
CHAPITRE I : variations interspécifiques des électrophorégrammes aviaires ....................... 27
Article 1: Description et identification d’un pic en alpha de grande amplitude composé
d’apolipoprotéine A-I, sur les profils d’électrophorèse des protéines plasmatiques
réalisés sur gel d’agarose, chez le pigeon domestique (Columba livia), le milan noir
(Milvus migrans) et l’amazone aourou (Amazona amazonica). ...................................... 27
Cet article a été soumis à la revue Veterinary Clinical Pathology. ............................................... 27
Article 2: Localisation du fibrinogène et de l’albumine sur les profils d’électrophorèse des
protéines plasmatiques de cinq espèces d’oiseaux taxonomiquement distinctes. ............ 47
CHAPITRE II : variations physiologiques des électrophorégrammes aviaires ....................... 59
Article 3: Influence de la mue sur les profils d’électrophorèse des protéines plasmatiques de
l’oie à tête barrée (Anser indicus). ................................................................................... 59
Article 4 : Influence de la ponte sur les profils d’électrophorèse des protéines plasmatiques
de la poule pondeuse (Gallus gallus)................................................................................ 79
CHAPITRE III : variations des électrophorégrammes aviaires liées à des interférences
analytiques................................................................................................................................ 97
Article 5: Effets de l’hémolyse sur la concentration en protéines totales et sur les profils
d’électrophorèse des protéines plasmatiques chez les oiseaux. ....................................... 97
Article 6: Effets de la lipémie sur la concentration en protéines totales et sur les profils
d’électrophorèse des protéines plasmatiques chez le milan noir (Milvus migrans)....... 113
CHAPITRE IV : Comparaison de techniques électrophorétiques ........................................ 127
Article 7: L’électrophorèse des protéines plasmatiques chez les oiseaux: comparaison d’une
méthode semi-automatique d’électrophorèse en gel d’agarose, Hydrasys©, avec une
méthode automatisée d’électrophorèse capillaire de zone, Capillarys2©...................... 127
DISCUSSION GENERALE : apport de nos travaux à la compréhension globale des
électrophorégrammes aviaires................................................................................................ 149
DIVERSITE TAXONOMIQUE DES OISEAUX ............................................ 149
APPORTS A LA COMPREHENSION DES EFFETS DE LA PONTE ET
DE LA MUE ....................................................................................................................... 151
APPORTS A LA COMPREHENSION DES EFFETS DE L’HEMOLYSE
ET DE LA LIPEMIE ........................................................................................................ 153
COMPARAISON DE L’UTILISATION, DES DEUX TECHNIQUES LES
PLUS UTILISEES ACTUELLEMENT EN LABORATOIRE DE
DIAGNOSTIC MEDICAL : L’ELECTROPHORESE EN GEL D’AGAROSE
ET L’ELECTROPHORESE CAPILLAIRE DE ZONE ......................................... 155
V
CONCLUSION ...................................................................................................................... 157
REFERENCES BIBLIOGRAPHIQUES ............................................................................... 159
LISTE DES ABRÉVIATIONS UTILISÉES ......................................................................... 174
ILLUSTRATION DES ESPECES D’OISEAUX ETUDIEES.............................................. 175
INTRODUCTION GENERALE
I.
LES OISEAUX EN ELEVAGE CONSERVATOIRE
A. Contexte général
La perte de diversité biologique menace de nos jours le fondement même des processus permettant
l’équilibre de la vie sur Terre. La biosphère est actuellement confrontée à un taux d’extinction 10 à 100 fois
supérieur au taux naturel d’extinction (UICN 1, 2008). Ayant démarré approximativement il y a 100000 ans,
et coïncidant étroitement avec la croissance démographique et la répartition des être humains sur terre,
l’extinction des espèces a ainsi progressé à un rythme sans précédent depuis la dernière grande extinction du
Crétacé (Leakey & Lewin, 1996). Ce phénomène est connu sous le nom d’extinction de l’Holocène et
constitue la sixième extinction massive. Certains biologistes estiment que plus de la moitié des espèces
vivantes aujourd’hui pourraient s’éteindre d’ici 2100 (Leakey & Lewin, 1996 ; Wilson, 2002). En 2007,
l'UICN considérait que 16306 espèces de plantes et d’animaux sur une liste de 41415 espèces évaluées
étaient menacées et confrontées à un sérieux risque d’extinction dans un proche avenir, ce qui représentait
approximativement une espèce de mammifère sur quatre, une espèce d'oiseau sur huit, et un tiers des
amphibiens (UICN, 2008).
Les menaces pesant sur la diversité biologique in situ s’accroissent sans cesse et les espèces doivent survivre
dans des environnements de plus en plus anthropisés (Hannah & Bowles, 1995). Elles incluent les
changements climatiques (Migley et al., 2002 ; Thomas et al., 2004), l’utilisation non durable des ressources,
l’introduction d’espèces allochtones à caractère invasif (Williamson, 1996), l’émergence de maladies (Berger
et al., 2004), la chasse intensive (Benett et al., 2002). Cependant, la plus importante est la destruction des
habitats entraînant la fragmentation, la simplification et la disparition des biotopes (Sauders et al., 1991 ;
Debinski & Holt, 1999 ; Koh et al., 2004). La perte et la dégradation des habitats touchent 89% des oiseaux,
83% des mammifères et 91% des plantes, inscrits dans la liste rouge de l’UICN (UICN 2008). Le taux
d’extinction est si important, que certaines espèces disparaitront avant même d’être découvertes (UICN,
2008). D’ici 2050, suivant les scénarii, les changements climatiques pourraient en outre entrainer une
disparition de 15 à 37% des espèces vivantes (Thomas et al., 2004).
La mise en danger de la biodiversité est difficile à contrôler à court ou à moyen terme, si bien qu’un
nombre croissant d’espèces pourraient être condamnées à disparaitre, malgré les mesures de conservation in
situ. C’est de ces constatations qu’est né le concept de conservation ex situ reposant sur le principe de
l’«Arche de Noé ». La convention sur la diversité biologique, adopté lors du Sommet de la Terre à Rio de
1
UICN : International Union for Conservation of Nature
1
Janeiro en 1992 par 168 pays la définit comme la « préservation d’une composante de la diversité biologique
en dehors de son habitat naturel ».
Les parcs zoologiques à travers le monde constituent une des principales réserves de populations
captives dont le but est la conservation ex-situ. Ils sont organisés en réseaux dans le cadre d’associations
internationales comme l’EAZA 2 en Europe et gèrent ainsi des populations captives à visée d’élevages
conservatoires qui pourront, à moyen ou long terme, servir de support à des programmes de réintroduction
(Olney, 2005). Ils sont désormais considérés comme des maillons de la fragile chaine qui s’est constituée
pour tenter d’endiguer l’extinction de nombreuses espèces.
Les parcs zoologiques peuvent de plus être de véritables laboratoires de recherche pour des espèces
conservées ex situ, tant dans le domaine de l’éthologie que de la zoologie ou de la génétique… mais aussi de
la pathologie qui peut être plus facilement étudiée, évaluée et souvent jugulée. Ce sont ainsi des modèles
expérimentaux que l’on peut pour partie transposer dans la nature. La médecine vétérinaire y a sa place et
son intérêt à tout niveau de la conservation et de la réintroduction d’espèces menacées.
B.
Contexte sanitaire des élevages conservatoires d’oiseaux
L’établissement de populations captives à visée conservatoire implique inévitablement des
modifications importantes de conditions de vie, comme par exemple pour les oiseaux, une sédentarisation.
D’autre part, la rationalisation des élevages conjuguée à la problématique de présentation des collections
animales en parc zoologique conduit souvent à des densités importantes d’animaux. Ces deux facteurs,
facilitent la transmission des agents pathogènes (Fowler, 1996).
D’autres facteurs tels le stress favorisent l’apparition de maladies inhérentes à la captivité et aux
manipulations. Le stress peut être responsables de l’élévation des taux de corticostéroïdes sanguins, et ainsi
entrainer une immunodépression (Fowler, 1996). Malgré les précautions prises dans le cadre des
programmes d’élevage, les taux de consanguinité sont souvent plus élevés que dans la nature, ce qui peut
concourir à affaiblir les défenses naturelles des animaux. Enfin, le mélange d’individus de sensibilités
différentes aux maladies (animaux d’espèce ou d’âge différent) est souvent néfaste puisqu’il permet la mise
en contact d’animaux plus ou moins naïfs d’un point de vue immunitaire avec des réservoirs potentiels de
germes pathogènes (Fowler, 1996).
Les conditions inhérentes aux élevages conservatoires sont donc grandement propices à l’apparition
et à la transmission de maladies pouvant s’avérer dévastatrices pour les populations captives. La prévention
de ces maladies est une préoccupation quotidienne du personnel scientifique travaillant dans ces institutions
(Miller, 1999). Il est indispensable que les animaux conservés ex situ jouissent d’une bonne santé. En effet,
la problématique de réintroduction en milieu naturel ne peut être menée à bien sans un contrôle des
2
EAZA : European Association of Zoo and Aquaria
2
problèmes sanitaires. Il faut impérativement éviter de répandre dans des populations dont les effectifs sont
déjà affaiblis des agents pathogènes dont les impacts seraient catastrophiques (Woodford, 201).
La mise en place de mesures de prophylaxie sanitaire, destinées à prévenir l’apparition et la
propagation de maladies repose sur un certain nombre de moyens diagnostiques (Wolff, 1996), surtout chez
les oiseaux qui n’expriment, en général, que très tardivement des symptômes. Or il apparait qu’un grand
nombre d’examens complémentaires (Radio-immuno assay, ELISA) sont caractéristiques d’espèces. Dans le
cadre des élevages d’oiseaux en parc zoologique, la variété des taxons détenus, ainsi que leur éloignement
phylogénétique par rapport aux espèces à intérêt économique majeur (poulet, faisan, canard) en limite
souvent l’usage. Le cout des analyses, ainsi que la possibilité de les réaliser en grand nombre dans le cadre
de protocoles de dépistage ont également leur importance. C’est dans ce contexte que l’électrophorèse de
protéines plasmatiques est utilisée en routine au Parc zoologique de Clères depuis maintenant sept ans. Cet
examen complémentaire s’est révélé extrêmement utile dans le cadre de mesures de prophylaxie offensive et
défensive contre la tuberculose aviaire et nous a permis de faire baisser la prévalence de la maladie.
L’électrophorèse des protéines plasmatiques présente de nombreux avantages : possibilité de réaliser de
grandes séries d’analyses, faible cout, etc.
II. L’ELECTROPHORESE DES PROTEINES SANGUINES
A. Rappels
1.
Historique
L'invention de l'électrophorèse est issue de la convergence de multiples travaux en physique, en
chimie et en biologie. Historiquement, les premières lois de l’électrostatique et de l’électricité apparaissent
dès le début du XVIIIe siècle avec les travaux de Charles de Coulomb (1736-1806) qui définit les premières
lois de l'électrostatique, Alessandro Volta (1745-1827) qui invente la première pile électrique, André Marie
Ampère (1775-1836) qui définit les notions de tension et d'intensité d'un courant électrique et établit en 1827
les lois de l'électrodynamique. En 1859, l’allemand Georg Hermann Quincke (1834-1924) découvre qu’il est
possible de déplacer des particules colloïdales sous l’action d’un champ électrique : ce phénomène est appelé
la cataphorèse. Par la suite, Hermann Von Helmholtz (1821-1894) met en évidence un phénomène similaire
en milieu liquide, l'électro-osmose : dans un champ électrique, il observe que des particules chargées se
déplacent vers le pôle de signe opposé à leur charge. En 1892, S.E Linder et H. Picton imaginent exploiter
cette observation pour la séparation de particules chargées : des molécules portant plus de charges doivent
migrer plus vite que celles en portant moins. En 1937, le biologiste suédois Arne Wilhelm Kaurin Tisélius
(1902-1971) concrétise cette idée en séparant les protéines contenues dans des liquides biologiques
complexes comme du sérum sanguin et du lait, ce qui lui vaut le prix Nobel en 1948 (Tiselius, 1937; Kyle &
Shampo, 2005). Au départ, la séparation des protéines se faisait donc en milieu liquide dans un tube en verre
3
et le repérage des substances séparées était réalisé par des moyens optiques complexes et coûteux (Tiselius,
1937; Mahenc & Sanchez, 1980). Dès le début des années 50, la technique est améliorée stabilisant la phase
liquide sur un support solide. C’est l’électrophorèse sur support. Le premier support utilisé est tout d’abord
un support de papier (Kunkel & Tiselius, 1951), vite remplacé par J. Kohn en 1957 par l’acétate de cellulose
(Kohn, 1957; Rocco, 2005). Cette amélioration permet l’utilisation de voltages plus élevés, ce qui permet
une séparation électrophorétique plus rapide. La détection des protéines, par coloration est plus simple et
moins couteuse que dans l’appareil de Tiselius (Mahenc & Sanchez, 1980).
Dès 1955, O. Smithies propose l’utilisation d’un gel d’amidon pour stabiliser la phase aqueuse (Smithies,
1955). L’intérêt d’un gel réside dans une plus grande porosité limitant le tamisage moléculaire. Ce type de
support combine les avantages des techniques précédentes et permet d’obtenir une résolution accrue. Les
améliorations suivantes ont été apportées par l’utilisation de gels de polyacrylamide (Raymond & Wang,
1960) et d’agarose (Elevitch, 1966). Ces supports sont plus faciles à préparer et permettent encore une
amélioration de la résolution (Mahenc & Sanchez, 1980; Bienvenu et al., 1998). En 1967, Hjerten est le
premier à décrire l’utilisation de l’électrophorèse capillaire de zone, qui est en fait une variante améliorée du
système de Tiselius (Hjerten, 1967). La séparation des protéines est effectuée dans des tubes capillaires, ce
qui permet l’utilisation de très hauts voltages, sans échauffement de l’échantillon, puisque la chaleur produite
se dissipe aisément. La quantification des fractions protéiques est réalisée directement par mesure de
l’absorbance à travers la paroi du tube (Feuilloley et al., 1999; Bossuyt, 2003). Depuis ses débuts en 1937
l’électrophorèse n’a ainsi eu de cesse de s’améliorer et est devenue un outil indispensable dans de nombreux
laboratoires de recherche et dans l’industrie. L’électrophorèse est actuellement couramment utilisée en
médecine humaine et en médecine vétérinaire des mammifères, où on l’utilise en particulier pour séparer les
protéines présentes dans les fluides biologiques. La technique la plus utilisée actuellement en laboratoire de
diagnostic est l’électrophorèse en gel d’agarose (Daunizeau, 2003). Elle tend à être supplantée dans les
laboratoires ayant des débits d’analyse importants, par l’électrophorèse capillaire. Notre étude a donc été
basée exclusivement sur l’utilisation de l’électrophorèse en gel d’agarose et de l’électrophorèse capillaire en
médecine aviaire, cette dernière n’ayant a ce jour encore jamais été étudiée chez l’oiseau. Ces deux
méthodes, d’utilisation fréquente en milieu médical sont ainsi facilement accessibles à tout praticien
vétérinaire.
4
2.
Principes généraux de l’électrophorèse des protéines sanguines
Du latin scientifique electricitas, dérivé du grec êlektron (ambre jaune ayant la propriété d’attirer les
corps légers lorsqu’on l’a frotté), relatif à l’électricité et du grec pherein porter, l'électrophorèse est une
méthode de séparation de particules chargées électriquement par migration différentielle sous l'action d'un
champ électrique (Dauzat et al, 1988). Des particules chargées placées dans un champ électrique créé par
une tension continue se déplacent vers le pôle de signe opposé à leur charge à une vitesse proportionnelle à
cette charge. Cette opération, s'appliquant à un ensemble hétérogène de particules chargées, entraîne la
séparation de ces particules : c'est la séparation électrophorétique ou électrophorèse.
a.
Charge électrostatique d’une protéine
Les protéines sont des molécules amphotères. Elles possèdent à la fois des fonctions acides
(groupement carboxylique : R - COOH) et basiques (groupement amine : R – NH2). Selon le pH du milieu
dans lequel elles se trouvent, les protéines vont se comporter plutôt comme des acides ( R – COOH Ù R –
COO- + H+) ou comme des bases ( R – NH2 + H+ Ù R – NH3+), acquérant ainsi une charge nette plutôt
négative ou positive en fonction du pH (Lassus, 2001). Pour chacune de ces molécules il existe un pH
caractéristique de la molécule, où la charge nette est nulle puisque les charges négatives et positives des
fonctions acides et basiques s’annulent. C’est le pH isoélectrique ou pHi. La charge q d'une protéine est donc
fonction de son pH isoélectrique et du pH de la solution tampon (Lafont, 2005). Une molécule en solution à
un pH supérieur à son pHi, se comporte comme un acide et cède des protons. Il y a donc prédominance des
groupements COO-. Elle acquiert une charge apparente négative et se comporte comme un anion. La
différence pH - pHi détermine l'intensité de la charge q d'une particule : plus cette différence est grande en
valeur absolue, plus la charge est importante (Lafont, 2005). Dans le cadre de l’électrophorèse des protéines
sanguines, les pH utilisés sont basiques. Les protéines, chargées négativement, migrent donc vers l’anode.
b.
La force coulombienne et la force de friction, principaux acteurs de
l’électrophorèse
La force coulombienne F subie par une particule de charge électrique q soumise à un champ
électrostatique E se définit comme suit: F = q . E
avec F en Newtons, q en Coulombs et E en V.m-1.
Soumise à cette force électrostatique, cette particule va se déplacer à une vitesse v constante de telle sorte
que: F = qE = γv où γ est un cœfficient de friction. La mobilité électrophorétique µ de la particule est définie
comme étant telle que v = µ E (Mahenc & Sanchez, 1980).
5
Très rapidement, lors de son déplacement, la particule va se déplacer à vitesse constante, la force
coulombienne s’équilibrant avec des forces de frottements. Ces forces de frottement visqueux sont
proportionnelles au diamètre de la particule et à la viscosité du milieu.
La mobilité électrophorétique d'une particule est donc proportionnelle à sa charge et inversement
proportionnelle à la viscosité du milieu et à sa taille.
c.
Autres phénomènes complexes intervenant lors de l’électrophorèse:
Le courant d'électro-endosmose est un phénomène intervenant dans l’électrophorèse sur support et
dans l’électrophorèse capillaire. Dans les conditions expérimentales de pH imposées par l’utilisation de la
solution tampon, le support ou le tube capillaire se charge dans la plupart des cas négativement. Cette charge
négative attire les cations du tampon et crée ainsi une double couche électrique dite double couche de Stern.
Sous l’effet du champ électrique, les cations excédentaires de la double couche se mettent en mouvement
vers la cathode en entraînant l’apparition d’un courant liquidien dirigé du pôle positif vers le pôle négatif. Ce
courant liquidien est lié à la migration des cations entraînant le solvant par effet d’osmose. Il accélère ou
ralentit la migration des molécules, suivant qu'elles migrent vers la cathode ou vers l'anode (Feuilloley et al.,
1999; Daunizeau, 2003; Lafont, 2005; Perrin, 2006). Il peut même dans le cas de l’électrophorèse capillaire,
être plus puissant que les forces électriques, ce qui fait que des protéines chargées négativement peuvent
globalement migrer vers la cathode (Feuilloley et al., 1999 ; Bossuyt, 2003).
Le passage du courant dans tout matériau conducteur s'accompagne de son échauffement par effet
Joule. Ce dégagement de chaleur entraîne la formation de courants liquidiens de convection. Ces courants
élargissent les zones de séparation et diminuent ainsi la résolution de la séparation électrophorétique
(Mahenc & Sanchez, 1980 ; Perrin, 2006). Pour limiter l’impact des courants de convection, le support est en
général réfrigéré. Dans le cas de l’électrophorèse capillaire, les dégagements de chaleur sont facilités par la
surface de contact importante du tube capillaire avec l’air environnant (Feuilloley et al., 1999; Bossuyt,
2003).
Dans l’électrophorèse sur support, l’utilisation d’un support destiné à stabiliser la phase liquide du
système s’accompagne également de l’apparition de quelques phénomènes pouvant modifier la mobilité
électrophorétique des protéines à séparer :
•
Le phénomène de tamisage moléculaire : lors de l’électrophorèse, les molécules qui se déplacent ont
tendance à heurter les mailles du support. Plus le substrat est poreux, moins le phénomène est important.
Dans le cas d’un support peu poreux, les pores du support sont de petite taille et les molécules ont tendance à
être ralenties, voire même arrêtées à l’endroit de leur dépôt pour les plus grosses d’entre elles. Ce phénomène
est négligeable lors d’utilisation de gels d’agarose pour les électrophorèses de protéines (Daunizeau, 2003).
•
Les phénomènes d’adsorption : les charges apparues sur le support, de même que des interactions
hydrophobes peuvent parfois attirer, voire fixer des molécules sur le support (Mahenc & Sanchez, 1980;
6
Daunizeau, 2003). Ces phénomènes ont tendance à diminuer la résolution de la séparation électrophorétique.
Ils sont limités dans le cas de l’utilisation de certains supports comme les gels d’agarose.
•
Les courants d'évaporation : le passage du courant dans tout matériau conducteur s'accompagne de son
échauffement par effet Joule. Dans le cas d'une électrophorèse sur support, cet effet peut entraîner un
échauffement du support de migration conduisant à une évaporation de l'eau de la solution tampon. Cet effet
est maximal au milieu de la bande. Il s'établit ainsi un courant liquidien depuis chaque extrémité vers le
centre de la bande (Daunizeau, 2003 ; Lafont, 2005). A l’extrême, il peut se produire au centre du support
une hyper concentration saline et un resserrement des mailles lorsque le support est un gel. Pour limiter ce
phénomène, on utilise en général des cuves réfrigérées.
d.
Paramètres influant le résultat d’une électrophorèse
De toutes les notions précédentes, il est possible de déduire que la migration électrophorétique d'une
molécule dépend de très nombreux facteurs parmi lesquels on peut citer :
•
Les caractéristiques intrinsèques des molécules de l’échantillon comme leur charge électrique et leur
encombrement stérique.
•
La quantité de l’échantillon qui doit être de quelques microgrammes dans le cadre de l’électrophorèse de
zone à proprement parler. Dans le cas contraire, il existe des risques que le champ électrique ne soit plus
uniforme après un temps de migration donné, comme c’est le cas dans l’isotachophorèse (Mahenc &
Sanchez, 1980)
•
L’intensité du champ électrique doit être adaptée à la nature des molécules à séparer. La séparation de
grosses molécules requiert plutôt des champs électriques de faibles ampleur (quelques V/cm) alors que celle
de petites molécules requiert des champs plus forts (jusqu’à 100 V/cm) (Mahenc & Sanchez, 1980). Le
champ électrique doit être suffisamment puissant pour que le temps de séparation soit court. Il est cependant
important de garder à l’esprit que l’utilisation de champs électriques forts entraîne des dégagements de
chaleur par effet Joule (Mahenc & Sanchez, 1980).
•
La température doit être maintenue la plus basse possible. Elle a en effet tendance à augmenter du fait de
l’effet Joule (Daunizeau, 2003). Une température élevée augmente la diffusion des molécules par
mouvement brownien et par les courants de convection et diminue ainsi la résolution d’une électrophorèse.
En électrophorèse des protéines, il est important que la température n’atteigne pas la limite fatidique de 60°C
où ces dernières sont irréversiblement dénaturées (Mahenc & Sanchez, 1980).
•
Les caractéristiques physico-chimiques du solvant :
Le pH conditionne la charge des protéines en fonction de leur pHi. Il est donc important qu’il soit
constant pour que le déplacement des molécules soit reproductible. On utilise donc une solution tampon dont
le pH est fixé.
La viscosité du solvant conditionne l’importance des forces de frottement. Comme nous l’avons vu
précédemment, la mobilité électrophorétique d’une molécule est inversement proportionnelle au cœfficient
7
de viscosité du solvant. Plus une molécule est volumineuse, plus l’effet de la viscosité du solvant est
important. Plus la viscosité est importante et plus les phénomènes de convection et de diffusion sont limités
(Mahenc & Sanchez, 1980).
La force ionique du tampon dépend de la concentration des différents ions et de leur valence (Daunizeau,
2003). Une concentration en électrolytes élevée dans la solution tampon par rapport à la concentration en
électrolytes de l’échantillon permet de maintenir un pH constant et un gradient de potentiel uniforme dans le
système (Viera-Nunes, 1999). Lorsque la force ionique du tampon augmente, la mobilité électrophorétique
des molécules à séparer diminue (Mahenc & Sanchez, 1980).
Il est important que le tampon soit homogène et que sa composition reste identique pendant toute la
durée de la migration. Pour cela, il suffit que les compartiments anodiques et cathodiques soient de volumes
suffisamment élevés pour que l’on puisse négliger l’évolution de la composition de l’électrolyte par
phénomène d’électrolyse au cours de l’électrophorèse (Mahenc & Sanchez, 1980).
Certains ions minéraux peuvent se fixer sur les protéines, diminuer leur charge nette et entraîner leur
floculation. Le choix d’une solution tampon est primordial pour une séparation donnée (Mahenc & Sanchez,
1980).
•
Les caractéristiques physico-chimiques du support vont conditionner l’intensité des phénomènes de
tamisage moléculaire, d’adsorption et de courants liquidiens. Un bon support d’électrophorèse doit présenter
les caractéristiques suivantes (Daunizeau, 2003) : bonne résistance mécanique, porosité adaptée aux
molécules à séparer, inertie chimique et insolubilité, phénomènes d’électro-endosmose et d’adsorption
mineurs, structure et composition adapté au mode de révélation. Seule la maîtrise parfaite de l'ensemble de
ces paramètres permet l'obtention de résultats répétables, et donc utilisables.
3.
Techniques d’électrophorèse utilisées en laboratoire de diagnostic
a.
L’électrophorèse de zone en gel d’agarose
Une installation d’électrophorèse de base compte 4 unités principales (Daunizeau, 2003) :
-
Un générateur de courant continu.
-
Une cuve contenant deux bacs de tampon dans lesquels plongent les électrodes reliées au générateur,
ainsi qu’un système permettant d’installer le gel de telle sorte qu’il baigne dans les bacs de tampon à
chacune de ses extrémités.
-
Des bacs permettant la coloration et la décoloration des gels une fois la migration terminée.
-
Un système de lecture, le densitomètre intégrateur.
Les cuves commerciales disponibles sont de deux grands types : verticales ou horizontales. Dans tous
les cas, le système est rempli avec un électrolyte doué de propriétés tampon suffisantes pour maintenir un pH
constant. De l’électrolyte en large excédent est contenu dans les compartiments anodiques et cathodiques
dans lesquels plongent l’anode et la cathode permettant d’établir un champ électrique aux bornes du support
8
dont les extrémités sont immergées dans la solution. La concentration de l’analyte est en général très faible
par rapport à la quantité d’électrolyte, ce qui garantit un champ électrique constant pendant la durée de la
migration (Mahenc & Sanchez, 1980; Daunizeau, 2003).
La migration est réalisée dans la phase liquide qui imprègne le milieu gélifié d’agarose, la matrice,
dont le rôle est de stabiliser la phase liquide (Mahenc & Sanchez, 1980; Daunizeau, 2003; Lafont, 2005;
Guyot-Ferreol, 2006). Celle-ci permet de limiter les courants de convection, de réduire l’influence des
vibrations et de faciliter la manipulation (Daunizeau, 2003). L'agarose est constitué de longues chaînes où
alternent deux monomères (le β D galactose lié en 1-3 et le 3-6 anhydro αL galactose lié en 1-4). Il se gélifie
à basse température par formation d'une multitude de ponts hydrogènes entre les longues molécules linéaires
de monomères qui le composent (pas de liaison covalente). ll est très hydrophile et peut, à très basse
concentration (de l'ordre de 1%) former des gels solides et très poreux dont la taille des pores dépend de la
concentration en agarose (Lassus 2001; Gautier 2006). Sa grande porosité le rend très utile pour séparer les
molécules ou les complexes moléculaires de grande taille. A la concentration de 1 %, ce système est
d’ailleurs proche de la veine liquide, l’effet de tamisage moléculaire étant quasi-inexistant.
Les tensions appliquées sont de l’ordre de 200 à 300 V pendant une vingtaine de minute. On utilise
en général un tampon TRIS-Véronal à pH d’environ 8,6. Les points isoélectriques des protéines sont
généralement compris entre 2,7 et 7,3 (Lassus, 2001). Dans du tampon TRIS-Véronal, les protéines se
comportent comme des anions et migrent vers l’anode (Kaneko, 1997; Daunizeau, 2003).
Après migration, les protéines présentes sur le gel sont précipitées à l’aide d’acide acétique. Les
précipités protéiques se trouvent imbriqués dans les mailles du support, ce qui a pour intérêt de les fixer à
l’endroit qu’elles occupaient au moment de l’arrêt du générateur (Daunizeau, 2003).
Les protéines fixées sur le support sont ensuite colorées au rouge ponceau ou à l’amidoschwarz, puis le
support est décoloré et séché. La lecture des gels est effectuée à l’aide d’un densitomètre intégrateur. La
courbe obtenue par lecture densitométrique du support permet la détermination des différents pourcentages
de fractions protéiques. Après mesure de la concentration en protéines totales par la réaction du biuret, il est
donc possible de calculer les valeurs absolues de chaque fraction (Daunizeau, 2003).
L’électrophorèse en gel d’agarose est la technique la plus fréquemment utilisée en biologie médicale
pour la séparation des protéines sériques ou plasmatiques. Elle n’a cependant été que relativement rarement
abordée dans les publications sur les oiseaux par rapport à des techniques aujourd’hui abandonnées en
diagnostic, comme l’électrophorèse sur acétate de cellulose.
Les méthodes totalement manuelles d’électrophorèses, manquant de répétabilité et de reproductibilité
sont de plus progressivement abandonnées au profit de méthodes semi-automatiques. Notre étude de
l’électrophorèse des protéines plasmatiques chez les oiseaux a donc été basée sur les toutes dernières
techniques semi-automatiques qui ont permis à cette méthode de diagnostic de se simplifier et de gagner en
fiabilité.
9
b.
Utilisation d’un système semi-automatisé d’électrophorèse sur gel d’agarose
Sebia Hydrasys © dans le cas de notre étude
L’Hydrasys est un système semi-automatique créé et commercialisé en 1997 par la société SEBIA,
leader sur le marché de l’électrophorèse en laboratoire médical. L’intérêt majeur de l’utilisation de ce
système repose sur la grande répétabilité et reproductibilité des résultats obtenus grâce à l’automatisation de
la plupart des étapes de l’électrophorèse : application de l’échantillon sur le gel, migration, séchage du gel,
puis coloration (Bossuyt, 1998a). Cette grande répétabilité, ainsi que le fait que ce système permette la
gestion semi-automatique de grandes séries d’échantillons le rendent tout à fait adapté comme outil de
recherche dans notre étude.
Cet automate est composé de deux compartiments : un compartiment de migration dans lequel a lieu
le dépôt des échantillons sur le gel, la migration, puis le séchage du gel, et un compartiment de coloration
dans lequel le gel est coloré au noir amidon, décoloré, puis séché. Un fois coloré, le gel est lu par un scanner
haute résolution piloté par un logiciel qui permet l’acquisition des courbes d’électrophorèse (Phoresis ©
version 5.50). Dans notre étude, les courbes ont été redécoupées manuellement.
Figure 1 : Automate Sebia Hydrasys©. Le fonctionnement de ce système est semi-automatique. Il comporte
un compartiment de migration et de séchage du gel (A) et un compartiment de coloration (B).
10
Figure 2 : Exemple de gel 15/30 Hydragel protein © après migration et coloration au noir amidon. De 1 à
10 : coq (Gallus gallus) ; de 11 à 18 : pigeon domestique (Columba livia) ; de 19 à 26 : Milan noir (Milvus
migrans).
Figure 3 : Découpage d’une courbe de colombiforme à l’aide du logiciel Phoresis ©.
11
Par rapport au système traditionnel d’électrophorèse décrit précédemment, le système Hydrasys
présente quelques variantes :
•
L’application de l’échantillon est réalisée par l’automate à l’aide d’applicateurs microporeux.
•
Les cuves sont remplacées par des mèches en éponge imprégnées d’une solution tampon. Ces mèches
sont elles-même au contact des électrodes. Elles sont remplacées à chaque analyse.
•
Les gels sont fabriqués industriellement, et non de manière extemporanée comme dans les systèmes
traditionnels. Un contrôle qualité garantit que les caractéristiques physico-chimiques des gels sont
rigoureusement identiques.
•
Le maintien des gels à température constante durant la migration et le séchage sont assurés par un
système à effet Peltier associé à un échangeur thermique. Ceci permet d’obtenir des variations importantes
de température en des temps extrêmement courts.
•
La fixation des protéines dans le gel est obtenue par séchage rapide.
•
La coloration est prise en charge automatiquement dans le compartiment de coloration. Il s’agit d’une
coloration au noir amidon.
Les interventions du technicien se résument donc à charger les applicateurs avec 10µL de plasma ou de
sérum, à placer le gel et l’applicateur dans le compartiment de migration, à choisir et à démarrer le
programme de migration, à transférer le gel séché dans le compartiment de coloration, à choisir et à démarrer
le programme de coloration et à réaliser l’acquisition des courbes dans le scanner et leur découpage à l’aide
du logiciel Phoresis ©.
Les kits utilisés dans notre étude pour l’électrophorèse des protéines sanguines des oiseaux étaient
des kits Hydragel 15/30 proteine ©. Ces kits comprenaient les gels, les râteaux d’application, les mèches
tamponnées, ainsi que le colorant nécessaire. Ces consommables étaient donc remplacés systématiquement
entre chaque analyse. La séparation électrophorétique était réalisée sur des gels d’agarose à 8g/L dans un
tampon Tris-barbital à pH 9,2, à puissance constante de 22 W jusqu’à accumulation de 33V.h. Pendant la
migration, la température était maintenue à 20°C.
c.
L’électrophorèse capillaire de zone
L’électrophorèse capillaire de zone est réalisée dans des tubes de silice de très faible diamètre (25 à
100 µm), d’où une surface de contact proportionnellement très importante avec l’échantillon à analyser et
avec la solution tampon (Viera-Nunes, 1999). Le faible diamètre des capillaires permet une dissipation très
efficace de la chaleur générée par l’effet Joule et autorise l’utilisation de champs électriques très élevés
(Bossuyt, 2003; Perrin, 2006). En présence d’un tampon aqueux de pH très alcalin, les groupements silanol
de la paroi du capillaire sont chargés négativement et induisent un courant d’électro-endosmose important :
le flux électro-osmotique. Celui-ci assure un « pompage » des molécules de l’anode vers la cathode sous la
forme d’un front plat, d’où une efficacité séparative très élevée (Feuilloley et al., 1999; Bossuyt, 2003). Dans
ce courant liquidien, les molécules cationiques voient leur mobilité propre s’ajouter au flux électro-
12
osmotique et gagnent rapidement la cathode. C’est le contraire pour les molécules anioniques qui sont
ralenties par le flux électro-osmotique mais finissent tout de même par rejoindre l’extrémité cathodique du
tube, le flux électro-osmotique surpassant leur vitesse de déplacement dans le tampon (Feuilloley et al.,
1999; Bossuyt, 2003). La détection est réalisée tout le long de la migration, à l’extrémité cathodique du tube,
par mesure de l’absorbance ou par fluorescence induite par un laser (Feuilloley et al., 1999; Bossuyt, 2003).
L’électrophorèse capillaire est utilisée dans le domaine médical depuis 1994 (Bossuyt, 2003). Cette
technique, complètement automatisée, surpasse l’électrophorèse de zone classique par sa rapidité d’exécution
et sa répétabilité (Feuilloley et al., 1999; Bossuyt, 2003). Nous avons été les premiers, au cours de cette
étude, a utiliser cette technique chez l’oiseau.
B.
L’électrophorèse des protéines plasmatiques comme moyen
diagnostique en médecine aviaire
1.
Utilisation de l’électrophorèse des protéines sanguines dans le contexte de la
médecine humaine et vétérinaire
Utilisée à des fins médicales chez l’homme depuis plus de 50 ans, l’électrophorèse des protéines
sériques a bénéficié de toutes les améliorations acquises au fil des années. D’abord réalisées sur support de
papier, puis sur acétate de cellulose, les électrophorèses sont maintenant couramment réalisées sur gel
d’agarose, et en électrophorèse capillaire (Dimpopullus, 1961; Bossuyt, 1998a; Le carrer, 1998; Daunizeau,
2003). Chez l’homme, l’électrophorèse des protéines sériques peut aider au diagnostic de syndromes
néphrotiques, de syndromes inflammatoires ou de cirrhoses hépatiques. Toutefois, son utilisation la plus
importante demeure le diagnostic des gammapathies monoclonales accompagnant certains types
d’hémopathies malignes (Dimpopullus, 1961; Bossuyt, 1998a; Le carrer, 1998; Daunizeau, 2003). Le fait
d’utiliser du sérum au lieu de plasma permet d’éviter que le fibrinogène ne masque d’éventuels pics
monoclonaux à la jonction beta-gamma (Daunizeau, 2003).
Ce n’est qu’une dizaine d’années après le début de son utilisation en médecine humaine que cette
technique est apparue dans le domaine de la médecine vétérinaire, principalement, des carnivores
domestiques et des chevaux (Amog et al., 1977; Groulade, 1978, 1985 ; Trumel et al., 1996; ). L’utilisation
de l’électrophorèse des protéines sériques des mammifères domestiques diffère peu de son utilisation chez
l’homme. Cependant, si l’utilisation de l’électrophorèse est devenue complètement courante dans les
laboratoires de médecine humaine, son usage dans les laboratoires vétérinaires demeure encore assez peu
développé.
Chez les oiseaux, l’électrophorèse des protéines sanguines est utilisée en tant que moyen diagnostic
depuis une quinzaine d’années. Son utilisation en médecine aviaire est donc beaucoup plus récente que chez
les mammifères (Cray & Tatum, 1998; Werner & Reavill, 1999; Cray et al., 2007). L’électrophorèse des
13
protéines sanguines est principalement utilisée dans le diagnostic de phénomènes inflammatoires liés à des
affections bactériennes, virales ou parasitaires chez les oiseaux. Elle est donc le plus souvent réalisée sur
plasma en médecine aviaire. Les plasmas sont en effet moins sujets à l’hémolyse que les sérums, et ils
contiennent le fibrinogène, protéine caractéristique de la phase aiguë de l’inflammation (Hawkey & Hart,
1988; Hochleitner et al., 1994; Cray & Tatum, 1998 ; Fudge, 2000). L’électrophorèse des protéines permet
chez l’oiseau, de combler l’absence totale de techniques de dosages de protéines spécifiques de
l’inflammation tels que nous les connaissons chez les mammifères (dosage de la C Reactive Protein par
exemple). Utilisée conjointement au dosage des protéines totales par la réaction du Biuret, l’électrophorèse
des protéines est de plus reconnue en médecine aviaire comme étant la méthode la plus adaptée à la
détermination de la concentration en albumine et en globulines d’un plasma (Spano et al., 1980 ; Lumeij et
al., 1990 ; Lumeij et al., 1996 ; Harr, 2002). Le faible volume de plasma nécessaire pour réaliser une analyse
rend cette technique utilisable, même chez des espèces de petite taille.
Les indications de cet examen complémentaire sont donc, chez l’oiseau, proches de celles d’une
numération / formule sanguine. En revanche, sa mise en œuvre est beaucoup plus simple et beaucoup plus
rapide que celle de la réalisation d’un hémogramme, qui implique obligatoirement une numération manuelle
par technique indirecte (Lucas & Jamroz, 1961 ; Campbell, 1995).
Nombreux sont les auteurs qui considèrent cet examen complémentaire comme un outil diagnostic
fiable en médecine aviaire (Cray & Tatum, 1998 ; Werner & Reavill, 1999 ; Cray et al. 2007). Certains
auteurs attirent néamoins l’attention (sans donner de précision sur les paramètres utilisés) sur des différences
de résultats entre laboratoires, en particulier pour des fractions de faible amplitude (Rosenthal et al. 2005).
Malgré ses possibles indications, l’utilisation de l’électrophorèse des protéines plasmatiques en
médecine aviaire est encore très marginale. Le praticien est en effet confronté à un manque cruel de données
validées scientifiquement dans ce domaine.
2.
Contexte : une grande diversité d’espèces aviaires et de techniques
électrophorétiques
Les oiseaux constituent une des classes les plus diversifiées du règne animal, avec selon Sibley and
Monroe (1990) 9672 espèces réparties en 2057 genres, 144 familles et 23 ordres. Cette diversité
taxonomique se reflète par une diversité des profils obtenus lors d’électrophorèse des protéines plasmatiques
(Sibley & Hendrickson, 1970; Cray & Tatum 1998; Zaias et al., 2000; Blanco & Hofle, 2003). Certaines
études montrent que des protéines de même nature peuvent migrer sur des distances différentes en fonction
des espèces.
Ainsi, bien que de poids moléculaires identiques, l’albumine de la perruche callopsite
(Nymphicus hollandicus) migre au niveau des alpha-globulines du poulet (Gallus gallus) (Archer & Battison,
1997). De telles différences de mobilités électrophorétiques s’expliqueraient par des distributions de charge
de surface différentes. Une telle variabilité est une source de difficulté dans l’interprétation de ces
électrophorégrammes et est probablement responsable d’une sous-utilisation de cet examen complémentaire
14
chez les oiseaux. En absence de repères normatifs, l’utilisateur est en effet tenté de se détourner de
l’électrophorèse pour des examens complémentaires d’apparence plus simple à interpréter. Il est donc
important de pouvoir dégager les grandes lignes communes aux électrophorégrammes des oiseaux. Les
résultats de cette thèse permettront ainsi de faciliter l’établissement d’un diagnostic pathologique et parfois
systématique pour les oiseaux et en particulier ceux inscrits dans les programmes de conservation.
Des valeurs de références ont d’ores et déjà été établies, pour de nombreux taxons, comme les
Psittaciformes (Clubb et al., 1991; Margolin, 1995; Cray et al., 2007), les Falconiformes (Ivins et al., 1986;
Ferrer et al., 1987; Lumeij et al., 1998; Tatum et al., 2000; Del Pilar Lanzarot et al., 2001; Blanco & Hofle,
2003 ; Spagnolo et al., 2006), les Columbiformes (Balasch et al., 1974; Lumeij & De Bruijne, 1985;
Gayathri & Hegde, 2006), les Galliformes (Torres-Medina et al., 1971; Balash et al. 1973; Filipovic et al.,
2007), les Anseriformes (Balash et al. 1974; Driver et al. 1981), les Ciconiiformes (Del Pilar Lanzarot et al.
2005), les Pelicaniformes (Balash et al., 1974 ; Zaias et al. 2000). Cependant, il apparait que de nombreuses
publications sont contradictoires en ce qui concerne le nombre de fractions séparées, les valeurs de
concentration de ces fractions, voire même l’aspect général des courbes d’électrophorèse. Chez les pigeons
et les rapaces, par exemple des publications récentes font état de manière plus ou moins explicite d’une
fraction alpha d’intensité importante (Tatum et al., 2000 ; Blanco & Hofle, 2003 ; Gayathri & Hegde, 2006).
Cette fraction n’est pas mentionnée dans la plupart des référence antérieures. De telles différences semblent
liées à l’utilisation de paramètres électrophorétiques, voire de techniques différentes. Ceci est parfaitement
illustré par l’exemple de la poule chez qui cinq bandes ont été séparées par électrophorèse à frontière mobile
(Moore, 1948), cinq à six par électrophorèse sur support de papier (Common & Mc Kinley, 1953 ; McKinley
et al, 1953; Torres-Medina et al., 1971), six sur gel d’agarose (Filipovic et al., 2007), six à huit sur support
d’acétate de cellulose (Longenecker et al., 1967 ; Beg & Clarkson, 1970 ; Torres-Medina et al., 1971; Balash
et al. 1973), 12 à 15 avec des gels d’amidon (Amin, 1961 ; Ogden et al., 1962) et jusqu’à de 10 à 19 à l’aide
de gels de polyacrylamide (Glick, 1968 ; Harris & Sweeney, 1969 ; Torres-Medina et al., 1971).
Notre étude sera donc basée sur l’utilisation de l’électrophorèse en gel d’agarose, technique
actuellement la plus répandue dans les laboratoires de diagnostic médical. L’utilisation d’un système semiautomatisée également largement répandu en laboratoire médical, tel que l’Hydrasys © permettra à tout
praticien de s’y référer directement. Nous aborderons également l’utilisation de l’électrophorèse capillaire,
technique émergente en médecine humaine, dont l’utilisation n’a jamais été étudiée chez l’oiseau.
15
3.
Définition des fractions protéiques et du rapport albumine / globuline
Les publications les plus nombreuses concernent principalement les Psittaciformes et les
Falconiformes. Les électrophorégrammes de ces espèces d’oiseaux sont le plus souvent découpés en une
fraction pré-albumine, une fraction albumine, une ou deux fractions alpha, une fraction beta et une fraction
gamma (Werner & Reavill, 1999 ; Cray, 2000 ; Harr, 2002). Les fractions alpha, beta et gamma sont
qualifiées de globulines (Kaneko, 1997 ; Cray, 2000). Chez le poulet 84 protéines ont été identifiées dans le
plasma (Corzo et al., 2004). Hormis la fraction albumine présentant un pic d’allure monoclonale, les
fractions séparées par électrophorèse sont donc composées, de plusieurs protéines.
Figure 4: Exemple d’électrophorégramme chez l’oie à tête barrée (Anser indicus).
L’albumine constitue la fraction anodique la plus intense. La finesse de ce pic est un bon indicateur
de la qualité de l’électrophorèse (Kaneko, 1997). L’albumine représente approximativement 30 à 70 % des
protéines totales chez les Psittacidés et les rapaces diurnes (Tatum et al., 2000 ; Cray et al.,
2007).
Synthétisée par le foie, elle joue un rôle important de réservoir protéique et de transport des acides aminés
entrant dans sa composition (Kaneko, 1997). En raison de son abondance et de sa petite taille, cette protéine
joue de plus un rôle fondamental dans le maintien de la pression oncotique sanguine. Elle a enfin un rôle
majeur en tant que protéine de transport. L’albumine permet de solubiliser dans le plasma des molécules qui
seraient peu ou pas solubles en milieu aqueux et d’éviter leur élimination par le rein (Kaneko, 1997). Chez
16
l’oiseau, comme chez l’homme, l’albumine est considérée comme une protéine négative de la phase aiguë de
l’inflammation, c'est-à-dire que sa concentration diminue en cas de processus inflammatoire (Wicher et al.,
1991; Monnet et al., 2002 ; Upragarin, 2005).
La fraction pré-albumine se situe en position anodique par rapport au pic d’albumine. Cette fraction
représente de 0 à 4 % des protéines totales chez les rapaces (Tatum et al., 2000). Il s’agit le plus souvent
d’une fraction de faible intensité pouvant cependant ponctuellement atteindre des valeurs avoisinant 20 à 30
% des protéines totales dans certaines espèces de psittacidés comme les inséparables (Agapornis sp.), les
loris (Lorius sp.), les eclectus (Eclectus sp.) et les cacatoès (Cacatua sp.) (Cray, 2000 ; Cray et al., 2007). La
fraction pré-albumine est en particulier composée de transthyrétine, une protéine de transport des hormones
thyroïdiennes et du retinol (Cookson et al., 1998 ; Harr, 2002). La transthyrétine est, comme l’albumine
considérée comme une protéine négative de la phase aiguë de l’inflammation (Cray et al., 2007).
Les alpha-globulines constituent la fraction la « plus rapide » des globulines. Elles sont pour la
plupart synthétisées par le foie (Kaneko, 1997). Elles représentent 8 à 10 % des protéines totales chez les
Psittacidés (Cray et al., 2007), et de 10 à 25% chez les rapaces (Tatum et al., 2000). La fraction alpha est une
fraction de protéines très hétérogène présentant des rôles biologiques variables (Alpha lipoprotéines, alpha-1
antitrypsine, alpha-1 glycoprotéine acide, alpha-2 macroglobuline, haptoglobine) (Cray, 1997 ; Cray &
Tatum, 1998 ; Chamanza et al., 1999). Hormis l’alpha-lipoprotéine qui correspond aux High Density
Lipoproteins (HDL) responsables principalement du transport du cholestérol et des phopholipides, ces
protéines sont des protéines positives de la phase aiguë de l’inflammation (Chamanza et al., 1999). Leur
concentration augmente en cas de phénomène inflammatoire.
Les beta-globulines sont elles aussi principalement synthétisées par le foie (Kaneko, 1997). Cette
fraction représente 10 à 25 % des protéines totales chez les psittacidés et les rapaces (Tatum et al., 2000 ;
Cray et al., 2007). La fraction beta est, comme la fraction alpha, une fraction très hétérogène constituée de
protéines présentant des rôles biologiques différents (fibrinogène, transferrine, beta-lipoproteine,
complément) (Cray & Tatum, 1998). Là aussi, hormis la beta-lipoproteine qui correspond aux Very Low
Density Lipoproteins (VLDL) responsables du transport des lipides, ces protéines sont pour la plupart des
protéines positives de la phase aiguë de l’inflammation. La transferrine, protéine négative de l’inflammation
chez les mammifères est une protéine positive de l’inflammation chez les oiseaux (Chamanza, 1999).
Le fibrinogène est un très bon indicateur de l’inflammation (Hawkey & Hart, 1988). C’est pourquoi
la plupart des auteurs choisissent d’utiliser du plasma plutôt que du sérum pour réaliser les électrophorèses
des protéines sanguines chez les oiseaux (Lumeij, 1987 ; Hochleitner et al., 1994; Cray & Tatum, 1998)
La fraction gamma représente environ cinq à 15 % des protéines totales (Tatum et al., 2000 ; Cray et
al., 2007). Elle est constituée d’immunoglobulines mais elle peut, chez certaines espèces, contenir des
protéines migrant normalement dans la fraction beta (beta-lipoprotéines…) (Cray & Tatum, 1998 ; Werner &
Reavill, 1999). Il s’agit principalement chez les oiseaux des IgM, des IgA et des Ig Y (correspondant aux
IgG des mammifères) synthétisées par les lymphocytes B et les plasmocytes (Glick, 2000). Leur élévation est
la preuve d’une stimulation antigénique Kaneko, 1997).
17
Par convention, chez les oiseaux, le rapport albumine / globuline (A/G) est calculé en divisant la
somme des fractions albumine et pré-albumine par la somme des globulines (Lumeij, 1987; Cray & Tatum,
1998 ; Werner & Reavill, 1999). Un tel rapport est en général supérieur à 1 (Tatum et al., 2000 ; Cray et al.,
2007). Dans les heures qui suivent le début d’un processus inflammatoire, le foie recrute des hépatocytes
destinés à la synthèse des protéines positives de l’inflammation. Ces protéines, principalement présentes dans
les fractions alpha et beta permettent la mise en place de la première ligne de défense non spécifique de
l’organisme. En même temps, la synthèse des protéines négatives de l’inflammation, comme l’albumine ou
la transthyrétine à tendance à être régulée à la baisse pour faciliter la synthèse des protéines positives de
l’inflammation par le foie (Kushner & Feldman, 1976; Wicher et al., 1991). De plus, au bout de quelques
jours, la présence d’une stimulation antigénique entraine la synthèse d’immunoglobulines et donc
l’augmentation des gamma-globulines (Ivins et al. 1986). Le rapport A/G est donc le paramètre le plus fiable
à interpréter d’un point de vue clinique (Lumeij, 1987), puisque tout processus inflammatoire entraine très
rapidement une inversion caractéristique de ce rapport.
4.
Interprétation des électrophorégrammes
De nombreux phénomènes peuvent induire des modifications des électrophorégrammes plasmatiques
des oiseaux. Or il est capital, pour pouvoir poser un diagnostic de distinguer ce qui est réellement
pathologique, de ce qui est physiologique voire artéfactuel.
a.
Modifications pathologiques des électrophorégrammes aviaires :
Les modifications pathologiques des électrophorégrammes aviaires sont surtout connues à travers
des publications reposant sur des cas cliniques. Comme le soulignent Cray et Tatum (1998), l’électrophorèse
des protéines plasmatiques semble permettre de poser un diagnostic précoce, puisque dans le cas de la
chlamydophilose, par exemple, des changements importants au niveau des électrophorégrammes des oiseaux
malades apparaissent avant que les sérologies ne se révèlent positives.
La lecture d’un électrophorégramme débute tout d’abord par le calcul et l’interprétation du rapport
A/G et de la concentration en protéines totales. En général, une diminution du rapport A/G signe un
processus inflammatoire en cours (Lumeij, 1987; Cray & Tatum, 1998 ; Werner & Reavill, 1999). Cette
baisse peut être liée à une hypo-albuminémie et/ou à une hyperglobulinémie. Une hypo-albuminémie peut
être observée dans les premiers stades d’une hypoprotéinémie (Cray et al., 1995 ; Kaneko, 1997 ; Werner &
Reavill, 1999). Dans le cas d’un rapport A/G normal, l’observation d’une hyperprotéinémie signe le plus
souvent un état de déshydratation de l’animal (Cray et al., 1995; Kaneko, 1997). La mise en évidence d’une
hypoprotéinémie peut être liée à un défaut de synthèse protéique (malnutrition, malabsorption, insuffisance
18
hépatique), à des fuites protéiques (diarrhée, insuffisance rénale, hémorragie ou exsudation massive), ou à
une hyperhydratation (Lumeij, 1987 ; Cray et al, 1995 ; Kaneko, 1997).
En l’absence de normes établies, l’interprétation plus fine, fraction par fraction est plus délicate chez
l’oiseau. Le praticien doit donc se référer à un électrophorégramme normal de l’espèce concernée, ou mieux
à des valeurs de références personnelles. La fraction pré-albumine est une fraction de très faible intensité
dont les variations sont peu interprétables. L’albumine est une protéine négative de l’inflammation, dont la
concentration diminue en cas de processus inflammatoire. La diminution de la fraction albumine caractérise
en général une affection chronique. Des hypo-albuminémies peuvent également être constatées lors
d’hypoprotéinémie (Kaneko, 1997 ; Werner & Reavill, 1999). Une augmentation de la fraction alpha est le
plus souvent liée à un état inflammatoire de l’oiseau, voire à une insuffisance rénale (Cray et al, 1995 ;
Kaneko, 1997 ; Werner & Reavill, 1999). L’augmentation de la fraction beta est, comme pour la fraction
alpha, le plus souvent lié à une inflammation systémique (Hawkey & Hart, 1987 ; Cray et al., 1995 ; Kaneko,
1997 ; Werner & Reavill, 1999). Elle s’observe particulièrement en cas d’hépatite (Werner & Reavill, 1999).
Des diminutions des fractions alpha et beta peuvent être observées en cas d’hypoprotéinémie. La fraction
gamma étant principalement constituée d’immunoglobuline, une augmentation de cette fraction signe en
général la présence d’une infection accompagnée d’une stimulation antigénique du système immunitaire
(Cray et al., 1995 ; Kaneko, 1997 ; Werner & Reavill, 1999). L’installation d’une réaction immunitaire
signe donc la présence d’un phénomène inflammatoire qui dure au moins depuis plusieurs jours (Ivins et al.,
1986). Plus rarement chez l’oiseau, un pic monoclonal dans la région gamma peut être le signe d’une
hémopathie maligne (Werner & Reavill, 1997). Une diminution de la fraction gamma peut être constatée en
cas d’immunodépression, ou dans le cadre plus général d’une hypoprotéinémie (Jacobson et al., 1986 ;
Kaneko, 1997 ; Werner & Reavill, 1999).
Les modifications des électrophorégrammes observées dépendent du stade d’évolution de la
maladie. Lors d’aspergillose respiratoire chez l’oiseau, on constate ainsi en début d’évolution de la maladie
une diminution du rapport A/G liée principalement à une diminution de l’albuminémie et à une hyper-beta
globulinémie (Cray et al., 1995 ; Reidarson & McBain, 1995 ; Werner & Reavill, 1999 ; Ivey , 2000). Le
passage à la chronicité se traduit par une hypo-albuminémie aggravée et par une augmentation de la fraction
gamma (Cray et al., 1995 ; Werner & Reavill, 1999). Une fois les défenses de l’hôte submergées, on observe
une diminution de toutes ces fractions (Reidarson & McBain, 1995). Le rapport A/G, et notamment
l’intensité de l’hypoalbuminémie sont un bon paramètre pour estimer le pronostic de la maladie (Reidarson
& McBain, 1995).
Des affections bactériennes chroniques comme la tuberculose aviaire à Mycobacterium avium sont
également décrites comme entrainant une hypo-albuminémie (Blanco & Hofle, 2003), ainsi qu’une
augmentation importante des fractions alpha (Tatum et al., 2000 ; Blanco & Hofle, 2003), beta (Hoefer,
1996 ; Cray et al., 1995, Werner & Reavill, 1999 ; Tatum et al., 2000 ; Blanco & Hofle, 2003) et gamma
(Cray et al., 1995, Werner & Reavill, 1999 ; Tatum et al., 2000 ; Blanco & Hofle, 2003). Lors de
chlamydophilose aviaire aiguë à Chlamydophila psittaci, on constate chez les rapaces, comme chez les
19
psittacidés, une hypo-albuminémie associée à une augmentation marquée de la fraction beta et à une
augmentation modérée de la fraction gamma (Cray et al., 1997 ; Werner & Reavill, 1999 ; Blanco & Hofle,
2003). Le passage à la chronicité s’accompagne d’un retour du profil électrophorétique vers la normalité, les
fractions beta et gamma demeurant plus élevées que la normale (Cray et al., 1997 ; Werner & Reavill, 1999).
Lors d’hépatite chronique, on observe une diminution des fractions albumines et alpha-2 et une augmentation
globale de la fraction beta (Cray et al., 1995). Des maladies virales entrainant une immunodépression de
l’oiseau, comme la PBFD 3 chez les psittacidés sont connues pour entrainer une hypoprotéinémie liée entre
autres à une hypo-gamma globulinémie (Jacobson et al., 1986). Des affections parasitaires comme la
sarcosporidiose à Sarcocystis falcatula, coccidie dont le cycle passe par une forme enkystée au niveau des
muscles striés chez les perroquets de l’ancien monde ont été décrites comme étant responsables d’une
augmentation polyclonale modérée de la fraction beta et d’une hyper-gamma globulinémie d’intensité
moyenne (Cray et al., 1996). Enfin en cas de maldigestion, chez des jeunes cacatoès noirs (Probosciger
aterrimus), Romagnano et al. (1996) ont décrit une hypoprotéinémie liée à une hypo-albuminémie et à une
hypo-gamma globulinémie.
b.
Modifications physiologiques des électrophorégrammes chez les oiseaux
Le cycle de vie de tout oiseau est marqué par des événements comme la croissance, la mue ou la
ponte. De tels phénomènes ont des répercussions métaboliques importantes et sont donc susceptibles d’être
responsables de modifications substantielles des électrophorégrammes aviaires. La prise en compte de telles
modifications est capitale pour éviter toute confusion avec des modifications liées à des processus
pathologiques.
Le phénomène de mue implique une augmentation importante du métabolisme basal, en raison du
cout énergétique lié au renouvellement des plumes, de la diminution de l’isolation thermique du plumage et
de modifications comportementales de l’oiseau qui recherche une alimentation plus riche en acides aminés
soufrés destinés à la synthèse de la kératine (King, 1981 ; Walsberg, 1983; Klaassen, 1995). Les plumes sont
composées de 95% de protéines, et représentent 25% de la matière sèche d’un oiseau (King, 1981; Murphy
& King, 1992). Leur renouvellement s’accompagne d’une augmentation significative des synthèses
protéiques de l’organisme, et d’une dépression passagère de l’immunité à médiation humorale (Mrosovsky &
Sherry, 1980; Murphy & Todd, 1995; Kuenzel, 2003). Les effets de la mue sur l’électrophorèse des protéines
plasmatiques n’ont jusqu’à maintenant été abordés que dans une étude réalisée sur le canard colvert mâle
(Driver, 1981). Cet auteur observe que la mue s’accompagne d’une diminution des protéines totales. Les
taux plasmatiques d’albumine semblent 10 à 20 % plus bas durant la mue qu’après, tandis que les valeurs de
la fraction alpha 2 semblent plus faibles au moment de la mue qu’avant. Cependant, cette publication est la
seule existant sur ce sujet, et les techniques ont évolué depuis 25 ans.
3
PBFD : Psittacine Beak and Feather Disease
20
La ponte constitue une perturbation métabolique majeure au cours de la vie de l’oiseau. Au cours de
la ponte, l’oiseau exporte ainsi des quantités importantes de lipides et de protéines destinées à la fabrication
des œufs. Les protéines présentes dans le blanc d’œuf sont synthétisées directement au niveau des différentes
portions de l’oviducte. En revanche, aucun des lipides et des protéines du jaune d’œuf ne sont synthétisées
dans l’ovaire. Tous sont apportés par le sang et proviennent en majorité du foie dont l’activité de synthèse
protéique est multipliée par 3 et la lipogenèse multipliée par 10 lors de la maturité sexuelle (Sauveur & De
Revier, 1988a). De nombreuses protéines plasmatiques, comme les Very Low Density Lipoproteins (VLDL)
ou les vitellogénines, ainsi que des protéines précurseur du blanc d’œuf sont présentes en quantité importante
dans le sang durant la période de ponte (Hermier et al., 1989). De tels phénomènes ont été étudiés dès les
années 50 dans un but de recherche en utilisant différentes techniques d’électrophorèse des protéines
sanguines. Pour certains auteurs, la ponte ne s’accompagne d’aucune modification de la concentration en
protéines totales du plasma (Sturkie & Newman, 1951). D’autres auteurs, en revanche ont noté une élévation
des protéines totales une semaine avant la ponte, suivi d’un retour à la normale dès la ponte du premier oeuf
(Vanstone et al., 1955 ; Gayathri & Edge, 2006). Les modifications les plus régulièrement observées sur les
électrophorégrammes d’oiseaux en ponte sont une augmentation de la fraction pré-albumine (Brand et al.,
1951; Kristjanson et al., 1963; Lush, 1963; Kuryl & Gasparska, 76,85). Au niveau de la fraction alpha,
Vanstone et al. (1955) et Polat et al. (2004) décrivent que la fraction alpha-1 diminue, pour devenir quasiinexistante chez les oiseaux en ponte, ce qui est en contradiction avec Kuryl & Gasparska (1976) qui
décrivent une augmentation de la fraction post-albumine durant la même période. Certains auteurs décrivent
l’apparition d’un pic monoclonal au niveau de la fraction beta durant la ponte (Cray & Tatum, 1997; Werner
& Reavill, 1999). Ceci n’est pas en accord avec les affirmations de Kuryl & Gasparska (1985) qui ont décrit
la diminution des fractions pré et post transferrine lors de la ponte, ou de Lush (1963) qui a décrit la présence
de deux fractions post-transferrine évoluant en sens inverse durant la même période. Enfin, plusieurs auteurs
ont décrit une augmentation de la fraction gamma au cours de la ponte (Vanstone et al., 1955; Elliott &
Bennet, 1971; Kuryl 1985). La ponte semble de plus s’accompagner d’une diminution du rapport A/G
(Sturkie & Newman, 1951; Kaneko, 1989). Les études citées ci-dessus présentent ainsi de nombreuses
contradictions, ce qui est très probablement lié au fait que ces études sont basées sur des techniques
électrophorétiques différentes. La plupart des études sont très anciennes et les techniques ont bien évolué
dans les 15 dernières années. Il serait donc intéressant de réactualiser ces données avec des techniques telles
que l’électrophorèse en gel d’agarose, dont l’usage est
actuellement le plus répandu comme outil
diagnostique.
La croissance d’un organisme implique une utilisation importante d’acides aminés transportés sous
la forme de protéines dans le sang. En raison des besoins accrus lors de la croissance, le foie accélère la
synthèse de protéines plasmatiques (Filipovic et al., 2007). Enfin, la maturation progressive du système
immunitaire avec l’âge permet la production accrue d’immunoglobulines (Filipovic et al., 2007). Tous les
auteurs s’accordent à dire que la concentration en protéines totales augmente de manière constante durant la
croissance d’un oiseau (Vanstone et al., 1955 ; Cuenca, et al., 1995 ; Muller, 1995 ; Work, 1996 ; Filipovic
21
et al., 2007). La concentration en albumine semble suivre étroitement celle des protéines totales (Clubb,
1991 ; Cuenca et al, 1995 ; Work, 1996 ; Filipovic et al., 2007), bien que pour Muller (1995) il n’y ait pas de
différence significative entre des oiseaux jeunes et adultes chez le faisan de Colchide (Phasianus colchicus).
Chez le fuligule à dos blanc (Aythya valisineria ), Kocan & Pitt (1976) ont mis en évidence que la fraction
pré-albumine était plus faible chez les jeunes oiseaux que chez les adultes. D’autres auteurs n’ont enregistré
aucune différence significative chez le grand tétras (Tetrao urogallus) (Cuenca et al, 1995).
D’une manière générale, les globulines semblent également augmenter lors de la croissance d’un
oiseau (Medway & Kare, 1958 ; Muller et al., 1995 Work, 1996 ; Filipovic et al., 2007). Ceci semble être le
résultat de l’augmentation des fractions alpha (Kocan & Pitt, 1976 ; Clubb, 1991 ; Muller et al., 1995 ;
Filipovic et al., 2007), beta (Kocan & Pitt, 1976 ; Muller et al., 1995 ; Filipovic et al., 2007) et gamma
(Clubb, 1991 ; Muller et al., 1995 ; Filipovic et al., 2007). Cependant, chez le grand tétras, Cuenca et al.
(1995) n’ont mis en évidence aucune différence significative entre les jeunes et les adultes pour les fractions
alpha et beta. Certains auteurs rapportent que le rapport A/G tend à augmenter au cours de la croissance chez
les psittacidés (Clubb, 1991) alors que d’autres décrivent une diminution de ce rapport durant la même
période chez le poulet (Medway & Kare, 1958 ; Muller et al., 1995 ; Filipovic et al., 2007). Pour d’autres
auteurs, il n’existe aucune différence significative entre des oiseaux jeunes et adultes (Cuenca et al, 1995).
Les taux de protéines contenues dans la ration alimentaire d’un oiseau sont connus pour avoir un
impact sur la concentration en protéine totale et sur les différentes fractions protéiques (Leveille &
Sauberlich, 1961 ; Polat et al., 2004 ; Bunchasak et al., 2005). Une ration protéique concentrée
s’accompagne en effet d’une augmentation de la concentration plasmatique en protéines totales (Leveille &
Sauberlich, 1961 ; Polat et al., 2004 ; Bunchasak et al., 2005). Cependant, les effets de la richesse en
protéines de la ration sur les différentes fractions ne font pas l’unanimité. Pour Leveille & Sauberlich (1961),
chez le poulet, une augmentation du pourcentage de protéines dans la ration s’accompagne d’une
augmentation de la concentration en albumine sans augmentation des globulines. Pour Bunchasak et al.
(2005), elle s’accompagne d’une augmentation de toutes les fractions protéiques à l’exception de la fraction
alpha qui diminue, et d’une diminution du rapport A/G. Enfin, pour Polat et al. (2004), chez l’autruche, elle
s’accompagne uniquement d’une augmentation des fractions alpha et d’une diminution de la fraction gamma.
c.
Effet d’interférences analytiques sur les électrophorégrammes des oiseaux
On définit une interférence analytique comme l’effet d’une substance présente dans l’échantillon à
analyser, qui entraine une erreur de mesure de la concentration ou de l’activité d’un analyte (Kroll & Elin,
1994). Chez les oiseaux, deux sources d’interférences sont très fréquentes : l’hémolyse et la lipémie.
L’hémolyse se définit comme la rupture des cellules sanguines et la libération de leur contenu
intracellulaire dans le plasma. La libération d’hémoglobine dans le plasma lui donne une coloration
caractéristique rosée à rouge. L’hémolyse est le plus courant des artéfacts rencontrés en électrophorèse des
protéines (Werner & Reavill, 1999). Chez les oiseaux, environ 4,5% des prélèvements réalisés sont
22
hémolysés (Fudge, 2000). Dans cette classe, les hématies sont plus volumineuses que celles des mammifères
(Lucas & Jamroz, 1961) et souffrent probablement plus du passage dans l’aiguille durant la prise de sang.
Les cas d’hémolyse intra-vasculaires sont rares chez les oiseaux (0,3% des prélèvements), et l’hémolyse est
donc le plus souvent lié à des mauvaises manipulations du prélèvement par l’opérateur durant la phase préanalytique ou analytique : passage du sang sous pression dans le fût de l’aiguille lors de la prise de sang,
délai trop important entre la collection de l’échantillon et sa centrifugation, homogénéisation brutale du
prélèvement sanguin, congélation du sang total (Guder, 1986 ; Fudge, 2000). L’hémolyse est connue pour
entrainer des erreurs de dosage des protéines totales. Elle est le plus souvent responsable de valeurs
artéfactuellement trop hautes (Andreasen et al., 1996, 1997). Chez les oiseaux, les effets de l’hémolyse sur
l’électrophorèse des protéines sont relativement peu documentés. Ils sont décrits par Werner & Reavill
(1999) dans un article de synthèse, et ont été étudiés dans une publication plus générale sur l’utilisation de
l’électrophorèse chez les psittacidés (Cray et al. 2007). La présence d’hémoglobine libre dans un plasma
semble entrainer une augmentation de la fraction gamma. Cependant, ces deux études étaient respectivement
basées sur la description de cas isolés et sur un effectif expérimental faible.
Chez l’oiseau, les principales causes de lipémie sont la lipémie postprandiale et la lipémie de ponte.
Dans les deux cas, la présence de lipoprotéines dans le plasma diffracte la lumière et lui donne une
apparence trouble, voire franchement lactescente. La lipémie postprandiale est un phénomène courant chez
les oiseaux que l’on met rarement à jeun avant une prise de sang (Meinkoth & Allison, 2007). Les
prélèvements lipémiques représentent 2,87% des prélèvements réalisés chez les oiseaux (Fudge, 2000). La
lipémie est connue chez l’homme pour entrainer des erreurs de dosage des protéines totales par la méthode
du biuret. Elle est décrite comme étant responsable de valeurs artéfactuellement trop hautes ou trop basses en
fonction du matériel d’analyse utilisé (Meinkoth & Allison, 2007). Cependant, peu de références décrivent
les effets de la lipémie sur l’électrophorèse des protéines plasmatiques chez les oiseaux. Comme pour les
effets de l’hémolyse, ils sont décrits succinctement par Werner & Reavill (1999) dans leur article de
synthèse, et ont été étudiés dans une publication plus générale sur l’utilisation de l’électrophorèse chez les
psittacidés (Cray et al. 2007). La lipémie serait responsable de l’apparition d’un pic au niveau de la fraction
beta. Mais, comme pour la description de l’hémolyse, ces études s’appuient sur des effectifs expérimentaux
faibles. De plus, ils ne donnent aucune indication quand à l’origine de la lipémie étudiée. Or il est très
probable que les effets de la lipémie de ponte, liés principalement à la présence de VLDL (Hermier et al.,
1989), soient fort différents de ceux de la lipémie postprandiale, liée principalement à la présence de
portomicrons chez les oiseaux (Klasing, 2000).
23
24
III. OBJECTIFS DE RECHERCHE
Les recherches menées dans le cadre de cette thèse visent à renforcer les connaissances sur
l’électrophorèse des protéines pour permettre l’utilisation de cette technique en tant qu’outil diagnostique
dans les élevages conservatoires d’oiseaux où l’approche sanitaire est primordiale pour une bonne gestion de
ces espèces en captivité et une possible réintroduction dans leur milieu naturel. Ce travail s’articule autour de
sept articles réunis en quatre chapitres qui ont pour objectif de combler point par point les carences que nous
avons soulignées lors de notre analyse bibliographique.
De notre étude bibliographique, il est ressorti que la classe des oiseaux est très diversifiée. En
l’absence d’approche globale et de repères normatifs, il est difficile pour un utilisateur éventuel de mettre en
pratique, chez l’oiseau, un examen complémentaire tel que l’électrophorèse des protéines plasmatiques.
Notre premier chapitre est donc consacré à l’étude des différences interspécifiques des électrophorégrammes
des oiseaux. Ce chapitre s’articule lui-même autour de deux articles. Le premier article s’est attaché à
identifier la composition d’un pic monoclonal de grande amplitude situé au niveau des alpha-globulines chez
les colombiformes, les falconiformes et les psittaciformes. L’identification de la protéine responsable d’un
tel pic permet une meilleure interprétation des electrophorégrammes dans ces taxa. Le deuxième article a
visé à localiser la bande correspondant au fibrinogène sur des électrophorégrammes de cinq espèces
d’oiseaux phylogénétiquement éloignées. Nous avons proposé que cette protéine facile à localiser serve de
point de comparaison des espèces les unes par rapport aux autres.
Le deuxième chapitre a été consacré aux modifications des électrophorégrammes aviaires en relation
avec différents état physiologiques. Le cycle annuel de vie d’un oiseau passe par diverses phases, comme
la ponte et la mue. Ces étapes modifient profondément le métabolisme des individus, avec un impact peu
connu sur les électrophorégrammes. Or il est capital de pouvoir distinguer les modifications liées à de tels
phénomènes, de celles liées à un véritable processus pathologique. Ce chapitre s’articule autour de deux
articles. Le premier a visé à décrire les modifications des électrophorégrammes au cours de la mue chez l’oie
à tête barrée (Anser indicus), tandis que le deuxième a abordé les modifications liées à la ponte chez la poule
domestique (Gallus gallus).
Le troisième chapitre traite de l’influence des interférences analytiques sur les résultats de
l’électrophorèse en gel d’agarose chez les oiseaux. En effet, à l’heure actuelle, peu de données
bibliographiques traitent des interférences analytiques liées aux phénomènes d’hémolyse ou de lipémie. Or,
ces phénomènes sont fréquents lors d’un prélèvement sanguin chez l’oiseau. Ce chapitre s’appuie sur deux
articles. Dans le premier, nous avons comparé les effets de l’hémolyse sur les électrophorégrammes de deux
espèces phylogénétiquement éloignées, comme le milan noir (Milvus migrans) et l’oie à tête barrée (Anser
indicus). Le deuxième article a été consacré à l’étude de l’interférence de la lipémie postprandiale sur les
électrophorégrammes du milan noir (Milvus migrans).
Notre synthèse bibliographique a enfin mis en évidence que la grande majorité des publications
traitant de l’électrophorèse des protéines chez les oiseaux sont basées sur des techniques qui ne sont plus
25
utilisées en laboratoire médical de nos jours. Or les résultats obtenus en utilisant des techniques différentes
sont difficilement comparables. Dans la quatrième et dernière partie nous nous sommes donc employés à
remettre à jour les connaissances concernant l’utilisation, chez l’oiseau, des techniques les plus modernes
utilisées actuellement en diagnostic médical : électrophorèse en gel d’agarose et électrophorèse capillaire.
Cette dernière partie repose sur un article dont le but était de comparer les résultats obtenus par
électrophorèse en gel d’agarose à l’aide d’un système semi-automatisé avec ceux obtenus par électrophorèse
capillaire de zone à l’aide d’un système complètement automatisé.
La double approche, à la fois appliquée et fondamentale de ce travail s’inscrit parfaitement dans la
problématique de gestion de collections du Département des Jardins Botaniques et Zoologiques du Muséum
national d’Histoire naturelle. Il correspond également aux attentes réglementaires en terme de recherche
appliquée à la conservation, telles qu’énoncées dans l’arrêté du 25 Mars 2004 réglementant le
fonctionnement des parcs zoologiques. Enfin, il apporte les informations nécessaires à une utilisation
raisonnée de l’électrophorèse des protéines plasmatiques dans le diagnostic et le screening des maladies en
élevage conservatoire.
Les articles de cette thèse sont présentés dans la mise en forme correspondant à la revue dans
laquelle ils ont été soumis.
26
CHAPITRE I : variations interspécifiques des électrophorégrammes
aviaires
Article 1: Description et identification d’un pic en alpha de grande amplitude composé
d’apolipoprotéine A-I, sur les profils d’électrophorèse des protéines plasmatiques réalisés sur
gel d’agarose, chez le pigeon domestique (Columba livia), le milan noir (Milvus migrans) et
l’amazone aourou (Amazona amazonica).
Cet article a été soumis à la revue Veterinary Clinical Pathology.
Résumé
Notre utilisation courante de l’électrophorèse des protéines plasmatiques en tant qu’outil diagnostique nous a
permis d’observer la présence d’un pic monoclonal dans la région alpha des électrophorégrammes de
nombreuses espèces d’oiseaux, ce qui n’est qu’exceptionnellement évoqué dans la littérature. Le but de cette
étude était d’étudier l’intensité et la position d’un tel pic et de déterminer sa composition chez l’amazone
aourou (Amazona amazonica), le milan noir (Milvus migrans) et le pigeon domestique (Columba livia).
Cette étude a été conduite sur 12 oiseaux de chaque espèce. Les profils obtenus en électrophorèse sur gel
d’agarose ont été utilisés pour quantifier la fraction alpha. L’électrophorèse en gel d’agarose haute résolution
a été utilisée pour isoler et exciser la bande alpha à étudier. Cette bande a été analysée par spectrométrie de
masse afin de déterminer sa composition. Les profils électrophorétiques des trois espèces étudiées étaient
tous caractérisés par la présence d’un pic fin et intense au niveau de la région alpha. Cette fraction
représentait approximativement 20% des protéines totales chez l’amazone aourou et jusqu’à 36% chez le
milan noir et le pigeon. L’analyse spectrométrique de ce pic nous a permis d’identifier sans aucune
ambigüité l’apolipoprotéine A-I dans ces trois espèces. La protéine identifiée correspondait à la forme
mature circulante de cette protéine. Cette apolipoprotéine joue un rôle clé dans le métabolisme du cholestérol
via les High Density Lipoproteins. L’importance des taux plasmatiques d’apo A-I observés dans cette étude
pourrait être liée au fait que chez les oiseaux, contrairement aux mammifères, la synthèse de cette protéine a
lieu dans la plupart des tissus. Ceci pourrait constituer une adaptation à leur métabolisme particulier des
lipides. L’apo A-I est connue comme étant une protéine négative de la phase aiguë de l’inflammation, c'està-dire que son taux plasmatique diminue lors d’un phénomène inflammatoire. L’interprétation des
électrophorégrammes et le calcul du rapport A/G chez ces espèces doivent par conséquent tenir compte de
telles particularités.
27
28
Description and identification of a high amplitude alpha globulin peak of Apolipoprotein A-I
in agarose gel plasma electrophoresis of rock pigeons (Columba livia), black kites (Milvus
migrans) and orange-winged parrots (Amazona amazonica).
RH: an apolipoprotein A-I peak in avian plasma proteinograms
Yannick ROMAN, Bertrand BED’HOM, Alain GUILLOT, Julie LEVRIER, Daniel CHASTEDUVERNOY, Marie-Claude BOMSEL-DEMONTOY and Michel SAINT JALME.
From the Museum national d’Histoire naturelle (MNHN), DJBZ, Le Parc de Clères, Clères, France (Roman,
Levrier) ; the INRA, AgroParisTech, UMR1236 GDA, Domaine de Vilvert, 78352 Jouy en Josas, France
(Bed’Hom), the INRA, PAPSS, Domaine de Vilvert, 78352 Jouy en Josas, France (Guillot), the Laboratoire
Bio-VSM, Torcy, France (Chaste-Duvernoy), the Museum national d’Histoire naturelle (MNHN), DJBZ,
Ménagerie du Jardin des Plantes, Paris, France (Bomsel-Demontoy) and the Museum national d’Histoire
naturelle (MNHN), EGB, UMR 5173 - CERSB - MNHN, CNRS, Paris IV, Paris, France (Saint Jalme).
Corresponding author: Yannick Roman (yannick.roman@cg76.fr).
29
Background: The common use of plasma protein electrophoresis as a diagnostic tool in our institute has
allowed us to notice the presence of a strong monoclonal alpha peak in the proteinograms of many bird
species, which is seldom found in the literature. Objective: Our aim was to analyse the intensity and position
of such a peak and to identify its composition in orange-winged parrots (Amazona amazonica), black kites
(Milvus migrans) and rock pigeons (Columba livia). Methods: This study was conducted on 12 birds of each
species. Electrophoresis patterns were used to quantify the alpha fraction. High resolution agarose gels were
used to isolate and sample the protein band of interest and to identify it by de novo sequencing approach
using an ion trap mass spectrometer. Results: Electrophoresis patterns of the three species displayed a strong
and thin peak in the alpha region. This fraction represented about 20% of the total proteins in parrots, and up
to 36% in kites and pigeons. Analysis of the alpha peak unequivocally identified apolipoprotein A-I in all
three species. This protein corresponded to the mature circulating form. Conclusions: Apo A-I plays a
pivotal role in cholesterol homeostasis. High levels of apo A-I could be related to the fact that in birds,
contrary to mammals, apo A-I synthesis occurs in most tissues. This could be the result of an adaptation to
their peculiar fat metabolism. Apolipoprotein A-I is a negative acute phase protein. Interpretation of
electrophoregrams and A/G ratios in these species should therefore take this information into account.
Keywords: Plasma protein electrophoresis, agarose gel electrophoresis, apolipoprotein A-I, alpha globulin,
bird.
30
Within the last 15 years, the application of protein electrophoresis to clinical avian medicine has
received the attention of several publications, and it is now commonly recognized to be a reliable diagnostic
tool for the evaluation, diagnosis, and monitoring of a variety of diseases and conditions.1-4 Plasma proteins
are traditionally identified according to their electrophoretic mobility and immuno-reactivity as either
albumin or globulins.4 Albumin is the most abundant plasma protein. Globulins are traditionally separated
into fractions called alpha, beta and gamma. The alpha and beta fractions contain acute phase proteins whose
concentrations increase in the presence of an inflammatory condition. The gamma fraction contains
immunoglobulins.2,4 Some authors have demonstrated high intertaxonomic variations of plasma
electrophoresis patterns.3,5-7 In order to improve interpretation of avian electrophoregrams, several authors
established reference ranges in bird taxa such as Psittaciformes,8-10 Falconiformes,7, 11-16 Columbiformes,17-19
Galliformes,20-22 Anseriformes,17,23 Ciconiiformes,24 and Pelicaniformes.6,17
Our regular use of plasma protein electrophoresis as a diagnostic tool led to the observation of a significant
peak in the vicinity of alpha globulins, in various taxa such as Columbiformes, Falconiformes, Strigiformes,
Psittaciformes, Phoenicopteriformes, Charadriiformes and Ciconiiformes (Yannick Roman, unpublished
data). Although the amplitude of this peak seems to be a specific feature of each species, such particularities
are not even mentioned in most publications relevant to this topic. The presence of a very significant peak in
some species could thus lead to electrophoregram interpretation errors. Nevertheless, three recent
publications have more or less explicitly mentioned the existence of this peak. In Falconiformes and
Strigiformes, Tatum et al. described a high-amplitude monoclonal alpha-1 globulin fraction that was
positioned just at the edge of the albumin peak.14 In the case of Falconiformes, Blanco & Hofle did not
clearly point out the presence of such a peak,7 although the reference values they published confirm the
significance of the alpha fraction, which alone could have represented between 22% and 31% of the total
protein content. More recently, Gayathri & Hegde found that pigeons have relatively high alpha globulin
content, in comparison with ducks and turkeys.19 However, none of these publications sheds any light on the
molecular origin of this peak.
The purpose of the present study was thus to verify the presence, and determine the composition of a
strong peak in the vicinity of the alpha globulins, in the agarose gel electrophoregram of three
phylogenetically distant bird species: rock pigeons (Columba livia), black kites (Milvus migrans) and orangewinged parrots (Amazona amazonica).
31
Materials and methods
Experimental animals
The study was conducted on 12 rock pigeons, Columba livia (5.7) held at the zoological park of Clères
(France), 12 black kites, Milvus migrans (7.5) held at the Académie de fauconnerie du Puy du fou (France)
and 12 orange-winged parrots (Amazona amazonica) of unknown sex. All birds were clinically examined,
and determined to be healthy.
Samples
Blood samples were taken outside the breeding season, from September to December depending on the
species. Blood samples were taken from the right jugular vein of the black kites and orange-winged parrots,
and from the brachial vein of the rock pigeons, using 23 G needles and 2 ml syringes (respectively: Terumo
Neolus 23 G, and Terumo Syringe 2 ml, Terumo Europe N.V., Leuven, 3001, Belgium). Two millilitres of
blood were drawn from each bird, collected on lithium heparin (Venosafe vacutainers, Terumo, Leuven,
Belgium), and centrifuged at 3000 g for 5 minutes. Analyses were carried out on the resulting plasma, since
it is commonly accepted that this medium is preferable to serum, for protein electrophoresis in birds. Plasma
is indeed less prone to haemolysis than serum, and contains fibrinogen, a protein characteristic of the acute
phase of inflammatory conditions.3,25 Two aliquots of each plasma sample were then stored in cryotubes (20°C) until they were analysed (Micronic systems, Lelystad, Holland). They were then thawed and
rehomogenised by gentle mixing 1 hour before analysis.
Total plasma protein concentration measurements
Total protein concentration was determined by the Biuret reaction, using a Roche Integra 400 wet chemistry
analyser (Roche Integra 400©, Roche diagnostics, Meylan, 38242, France). Readings were made at a
wavelength of 552 nm.
Agarose gel electrophoresis
Agarose gel electrophoresis of plasma proteins was carried out using a Hydrasys© semi-automated system
(Sebia, Evry, France), using the Hydragel protein 15/30© set (Sebia, Evry, France) which is the most
commonly used kit for blood protein electrophoresis in medical laboratories. The system was operated
according to the manufacturer’s instructions, using version 7.00 F0.1 of the system software. Ten microlitres
of plasma samples were distributed manually onto the sample template applicator and were allowed to
diffuse for a period of 5 minutes in a wet chamber. Application of the samples to the gel (30s),
electrophoresis (~ 7 min) and drying of the gel (65°C for 10 min) were all performed automatically in the
migration compartment of the instrument. The temperature was maintained at 20°C using a Peltier device
during the complete migration process, and drying was obtained by heating the gels to 65°C. Electrophoretic
separation was obtained on 8 g/l agarose gels in a Tris-barbital buffer at pH 9.2 and a constant power level of
20W, until 33 Vh had been accumulated. Once dried, the gels were manually transferred to the staining
32
compartment of the instrument in which staining (4 min with 4 g/l amidoblack in an acidic solution),
destaining (3 times, for 3 min, 2 min and 1 min respectively, with 0.5 g/l of citric acid solution) and drying
(75°C for 8 min) were carried out automatically. Once these operations had been completed, the gels were
scanned with a high resolution Epson perfect V700 photo scanner (Epson France, Nanterre, France).
Electrophoretic curves and dosages of the different fractions were acquired using Phoresis© software,
version 5.50 (Sebia, Evry, France).
If present, pre-albumin was included to the albumin fraction. Albumin was identified as the strongest anodal
peak. The beta peak was defined as the fibrinogen peak.26 Globulins were divided into 5 fractions. Alfa
fractions were located between the albumin and beta peaks, and gamma fractions were located beyond the
beta peak. As previously described in other studies relevant to avian protein electrophoresis, the A/G ratio
was calculated by dividing the sum of the prealbumin and albumin fractions by the sum of the globulin
fractions.1,3
Sampling of alpha bands from the gels
For each species, one plasma sample was chosen randomly. The electrophoresis was performed on Hydragel
15HR© high resolution gels (Sebia, Evry, France) in order to separate the fractions from each other as well
as possible, and to avoid sampling errors. Accurate positioning of the alpha fraction to be extracted from a
non-dried gel was achieved by superimposing it with a coloured gel. The Sebia hydrasys© system was
indeed shown to have excellent reproducibility, thanks to the automation of most steps of the analytical
procedure, including sample application, migration and staining.27
With the gel used for sampling of the alpha fraction, the semi-automatic process was interrupted before
drying. The gel used to locate the band corresponding to the alpha fraction was dried and stained according to
the normal procedure.
Gels strips containing the protein band of interest were sampled, placed in Eppendorf tubes (Eppendorf GA,
Hambourg, Germany), and sent under refrigerated conditions (3°C) to the the PAPSS proteomic platform at
INRA in Jouy en Josas, for peptide de novo sequencing and protein identification by homology search.
In-gel digestion and sample preparation
Each gel slice was washed twice with 200 µl 50mM ammonium carbonate in a 50% acetonitrile solution,
dried at room temperature, and digested overnight at 37°C with 100 ng of sequencing grade modified trypsin
(Promega, Madison, WI, USA) in 2 µl of 50 mM NH4HCO3. The resulting peptides were extracted as
follows: the supernatant from trypsin hydrolysis was first transferred into a new tube, and the gel slices were
extracted once with 25 µl of buffer extract-B (50 mM ammonium carbonate), and twice with 25 µl of buffer
extract-C (formic acid 0.1% acetonitrile 50%). For each extraction, the gel slices were incubated for 15 min
at room temperature under gentle shaking conditions. The three extracts were pooled with the original
trypsin digest supernatant, and dried for 1 h in a Speed-Vacuum concentrator (Savan, Thermo Fisher,
33
Illkirch, France). The peptides were then re-suspended in 25 μl of precolumn loading buffer (0.08%
trifluoroacetic acid and 2% acetonitrile in water), prior to LC-MS/MS analysis.
Mass spectometry analysis
LC-MS/MS analysis (Liquid Chromatography with tandem Mass Spectrometry) was performed with an
Ultimate 3000 LC system (Dionex, Voisins le Bretonneux, France) connected by means of a
nanoelectrospray interface to a linear ion trap mass spectrometer (LTQ, Thermo Fisher, USA), thus enabling
peptide separation, ionisation and fragmentation to be carried out.
Four microliters of tryptic peptide mixtures were loaded at a flow rate of 20 μl/min onto a precolumn
(Pepmap C18; 0.3 X 5 mm, 100Å, 5 µm; Dionex). After 4 minutes, the precolumn was connected to the
separating Pepmap C18 nanocolumn (0.075 X 15 cm, 100Å, 3 μm, Dionex) and the gradient was set to 300
nl/min. All peptides were separated in the nanocolumn, using modified buffer elut-B with a linear gradient of
acetonitrile ranging between 2% and 36 %, for a period of 18 minutes. The eluting buffers were: buffer elutA: 0.1% formic acid, 2% acetonitrile and buffer elut-B: 0.1% formic acid, 80% acetonitrile. The total
runlength was 50 min, including the regeneration step. Ionization was performed at the liquid junction, with
a spray voltage of 1.3 kV applied to a non-coated capillary probe (PicoTip EMITER 10 μm ID; New
Objective, USA). The peptide ions were analysed using the Nth-dependent method as follows: (i) full Ms
scan (m/z 300-2000), (ii) ZoomScan (scan of the 3 major ions), (iii) MS/MS on these 3 ions using classical
peptide fragmentation parameters: Qz = 0.25, activation time = 30 ms, collision energy = 40%.
Database search
Protein identification was carried out using local Peaks Studio 4.2 software (Bioinformatic Solution,
Waterloo,
Ontario,
Canada),
together
with
the
FASTS
tools
available
at
the
website:
http:www.ebi.ac.uk/fasta33/.
The raw data was first loaded into Peaks Studio, and filtered in order to eliminate the noisy spectra. The
filtered MS/MS spectra were rapidly translated into amino acid sequences, with several de novo sequencing
parameters: parent and fragment-mass error tolerances of 0.5 Da, trypsin as the protease, with a maximum of
one missed cleavage allowed, partial oxidation of methionine. All of the sequences produced by Peaks
Studio were then filtered, so as to retain only those with peaks scoring higher than 50 % on doubly charged
ions. These short sequences were analysed with FASTS against the Uniref100 protein database, with
MDM20 as the selected matrix. The identified proteins were classified by homology scoring, according to
their E value.
Statistical analysis
Descriptive statistical analysis was carried out using Microsoft Excel 2003©. Systat 7.0 software (Systat 7.0,
Systat Software Inc., London, United Kingdom) was used for the investigation of intersexual differences. In
34
view of the small size of the studied population, the Kruskal and Wallis one way variance analysis test was
used.
Results
Plasma protein electrophoresis
The electrophoretic patterns of the three studied species were found to be relatively similar (Figures
1-3). Indeed, they were all characterised by a significant alpha fraction peak, whose intensity
represented approximately 20% of the total proteins for the orange-winged parrots, and
approximately 36% of the total proteins for both the black kites and the rock pigeons (Table 1).
These peaks were intense and narrow, similar to those of albumin, thus giving the impression that
only one protein was overwhelmingly present. The electrophoregrams of the orange-winged parrots
and rock pigeons contained a prealbumin fraction of very weak amplitude which was included in
the albumin fraction.
No significant difference was found between males and females, in the case of black kites. In the
case of pigeons, the females had significantly lower total protein concentration and gamma-1
fraction values than the males (p < 0.05).
Figure 1: Example of plasma electrophoresis patterns for an orange-winged parrot (Amazona amazonica).
The alpha band of interest is indicated with an arrow in both the electrophoresis pattern and the gel.
35
Figure 2: Example of plasma electrophoresis patterns for a black kite (Milvus migrans). The alpha band of
interest is indicated with an arrow in both the electrophoresis pattern and the gel.
Figure 3: Example of plasma electrophoresis patterns for a rock pigeon (Columba livia). The alpha band of
interest is indicated with an arrow in both the electrophoresis pattern and the gel.
36
Table 1: Plasma protein electrophoresis results in orange-winged parrots, black kites and rock pigeons.
Results are expressed in g/l, in the form of their mean ± SD.
Species
Total protein
Albumin
Alpha-1
Rock pigeon
27.08 ± 1.70
10.57 ± 1.27
10.56 ± 0.71
Black kite
33.51 ± 3.50
14.54 ± 2.48
11.96 ± 0.82
43.49 ± 3.35
25.85 ± 2.36
8.55 ± 2.57
Orangewinged parrot
Alpha-2
Beta
Gamma-1
Gamma-2
3.01 ± 0.46
0.70 ± 0.41
1.23 ± 0.38
0.61 ± 0.08
3.45 ± 0.80
0.97 ± 0.37
2.00 ± 0.67
1.25 ± 0.43
4.51 ± 1.66
3.33 ± 0.86
Identification of alpha-1 peaks by LC-MS/MS analysis
There was no difficulty with any of the species in sampling the band corresponding to the alpha peak, since
this band was clearly distinguishable from the others (Figures 1-3).
LC-MS/MS analysis of peptides resulting from trypsin digestion of proteins located in the alpha region is
presented in figure 4. The search of homology with the sequences translated by peaks studio software on
FASTS against the Uniprot database allowed us to unequivocally identify the apolipoprotein A-I as the only
protein in the alpha peak for the three studied bird species. The sequences obtained in this work showed the
highest similarity with pro-apo-lipoproteins from duck, chicken and quail. The alignment of our sequences
with that of chicken, duck and quail revealed that we did not detect any peptide containing the 24 N-terminal
amino acids.
37
Figure 4: Sequence alignment of pro-apoliprotein A-I from Human (Homo sapiens, UniProt P02647),
Chicken (Gallus gallus, UniProt P08250), Quail (Coturnix japonica, UniProt P32918) and Duck (Anas
platyrhynchos, UniProt O42296), with peptides resulting from trypsin digestion of the protein contained in
the alpha band in the orange-winged parrot (Amazona amazonica), the black kite (Milvus migrans) and the
rock pigeon (Columba livia). The peptide sequences displayed here correspond to the peptide sequences
determined by LC-MS/MS and de novo sequencing approach.
38
Discussion
The striking result of this study is that, for the first time, a high amplitude monoclonal peak
identified as the apolipoprotein A-I has been pointed out in the alpha globulin region of three different
species, belonging to three distant bird taxa (Psittaciformes, Falconiformes and Columbiformes).
Of all the publications written in the past 30 years on the subject of Falconiformes and
Columbiformes, only three have more or less explicitly mentioned the existence of a significant alpha
fraction, in agreement with the findings described in the present paper.7,14,19 It is interesting to note that these
three papers are also the only ones to have used agarose gel electrophoresis with Falconiformes and
Columbiformes, since the analysis of all other references was based on cellulose acetate electrophoresis. The
poorer resolution of the latter technique could be the reason for which the alpha peak was not observed. As
far as the Psittaciformes are concerned, no previous publication has described the presence of a peak in the
vicinity of the alpha globulins. In their recent paper on the topic of plasma protein electrophoresis in
Psittacidae, although they made use of agarose gel electrophoresis, Cray et al. did not publish any reference
values for which the alpha fractions exceeded 7% of the total protein content.10 This could be due to the fact
that their electrophoretic parameters were different to those used in our study, thus highlighting the variability
of results, according to the chosen electrophoretic technique.
The comparison of peptide sequences obtained in the present work with protein sequences present in
Uniprot database allowed apolipoprotein A-I to be unequivocally identified as the only protein contained in
the alpha peak of the three studied species. The absence of identification of the expected 24 N-terminal
amino acids, based on the alignment with the chicken sequence, could mean that the apolipoprotein found in
the three species we worked on were present in a mature circulating form. This lacking sequence corresponds
to a 18 amino-acid prepeptide, which may act as a signal peptide, and to a 6 amino-acid propeptide.28-30
Indeed, the conversion of proapo A-I to mature apo A-I requires the proteolytic cleavage of these 24 aminoacids.29 Some sequences were not identified. Indeed, trypsin systematically cuts the peptides at the Cterminal of lysine or arginine and, if they are too small, some peptides can not be identified by mass
spectrometry.
Apolipoprotein A-I is known to be the most common apolipoprotein, of the high density lipoproteins
(HDL) found in both birds and mammals.29,31,32 With hydrophilic regions interacting with polar surface
lipids, as well as many hydrophobic regions interacting with core lipids, such an amphipathic protein
contributes to the lipoprotein’s structure and stability, and facilitates the solubilisation of hydrophobic lipids
in the aqueous environment of the blood.33 High density lipoproteins are composed mainly of phospholipids,
cholesteryl esters and cholesterol.34-37 In birds, they are responsible for the redistribution of cholesterol,
cholesterol esters and other non polar lipids to the peripheral tissues, and for returning them to the liver for
reuse or excretion via the bile.33,38 Apo A-I therefore plays a pivotal role in cholesterol homeostasis, through
reverse cholesterol transport.33,39
39
In the present study, apo A-I appeared to be very abundant, since alpha peaks were shown to represent up to
1/3 of the total protein content in pigeons and black kites. This is consistent with other publications which
have observed that HDL and cholesterol levels in pigeons are higher than those in most other animal species,
with total plasma cholesterol levels averaging 300 mg/dl, and 75-80% of this cholesterol being present in the
form of HDL.34,35 However, until now similar results have never been published for the case of
Psittaciformes and Falconiformes.
When compared to mammals, the large quantities of observed apo A-I could be attributed to the fact
that the expression patterns of apo A-I in birds are significantly different to those in mammals. Indeed, it has
been demonstrated that in chickens, apo A-I synthesis occurs in most tissues (notably the liver, intestine,
kidney, ovary, heart, muscles, brain and skin), whereas in mammals, its synthesis is limited to the liver and
the intestine.39-42 Apolipoprotein A-I has furthermore been demonstrated to be present in the Very Low
Density Lipoproteins of chickens,32 and the Low Density Lipoproteins of geese,37 whereas this is not the
case in mammals. High levels of apo A-I could be the result of an adaptation to the birds’ peculiar fat
metabolism. Indeed, in birds, the fat metabolism plays a key role, since fatty acids are known to be their
primary source of fuel during long distance flights.43,44 This could therefore explain the lower peak observed
in orange-winged parrots (a sedentary species), when compared to black kites and pigeons which are migrant
birds. Cholesterol is furthermore known to be a major constituent of cell membranes.45 Such a high apo A-I
level could also be the result of an adaptation to potentially high cell membrane turnover, in animals which
are known to have a significantly higher metabolism than mammals of the same weight.46-48 Further studies
should be carried out in order to investigate apo A-I peaks in other bird species, and should be made over a
range of physiological conditions requiring strong metabolic changes, such as egg laying, moulting, premigratory hyperphagia and migration. Such investigations should lead to a more thorough understanding of
the role of apo A-I in birds.
The fact that apo A-I is found in such large quantities in the three studies species, and that it is
responsible for a peak in the alpha region, must be taken into account when interpreting their
electrophoregrams. Such a peak could in fact be incorrectly interpreted as resulting from an inflammatory
phenomenon, since numerous positive acute phase proteins (positive APP) such as alpha-1 antitrypsin or
alpha-2 macroglobulin have been reported to migrate into the alpha region.3 In addition, it seems that apo AI, like albumin, is considered to be a negative acute phase protein (negative APP) in chickens,49-51 showing
that, contrary to positive APP, its plasmatic concentration decreases in the presence of an inflammatory
phenomenon. Indeed, acute inflammation involves alterations to the metabolism and changes in gene
regulation in the liver, which recruits increasing numbers of hepatocytes for the synthesis of acute phase
proteins within the first hours of the inflammatory condition. At the same time, negative APP synthesis tends
to be down-regulated, thereby increasing the liver’s capacity to synthesize positive APP.50,52 In addition, it
has been demonstrated that during the course of an inflammatory reaction in humans, the Amyloid A Serum
displaces the apo A-I from the HDL. This release of apo A-I into the bloodstream is accompanied by an
acceleration of its catabolism.53
40
By convention in birds, the A/G ratio is defined as the sum of the prealbumin and albumin fractions divided
by the sum of the globulin fractions.1,3 Clinically speaking, this parameter is very relevant since both
prealbumin and albumin correspond to negative APP in birds,10,49,50 whereas globulins contain mainly
positive APP.3 Any inflammatory condition therefore leads to a rapid and strong inversion of the A/G ratio.
In the three species studied here, the strong apo A-I peak corresponds to a negative APP. For these species a
new ratio, defined by the sum of the prealbumin, albumin and apo A-I fractions, divided by the sum of the
other globulins, may therefore be more clinically relevant than the classical A/G ratio.
Acknowledgements
We wish to thank the « Plateau d’Analyse Protéomique par Séquençage et Spectrométrie de masse» (PAPSS,
INRA, Jouy-en-Josas) which performed the mass spectrometry experiments, J. L. LIEGEOIS from the
Académie de fauconnerie du Puy du fou (France) for the black kite blood samples, P. TROLLIET from Sebia
for his invaluable technical support, the Museum national d’Histoire naturelle and the Conseil Général de
Seine Maritime (France) for their financial support, and Glenn Lund from Techtrans Consulting for the
english proofreading.
41
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46
Article 2: Localisation du fibrinogène et de l’albumine sur les profils d’électrophorèse des
protéines plasmatiques de cinq espèces d’oiseaux taxonomiquement distinctes.
Cet article a fait l’objet d’une conférence lors du 9° congrès de la European Association of Avian
Veterinarians (EAAV), Zurich, Suisse, 2007. Il a été soumis à la Revue de Médecine Vétérinaire.
47
48
Location of the fibrinogen and albumin fractions in plasma protein electrophoresis agarose gels of five
taxonomically distinct bird species
Localisation du fibrinogène et de l’albumine sur les profils d’électrophorèse des protéines plasmatiques de
cinq espèces d’oiseaux taxonomiquement distinctes
Y. ROMAN1*, J. LEVRIER1, D. ORDONNEAU2, D. CHASTE-DUVERNOY3, M.C. BOMSELDEMONTOY4, M. SAINT JALME5.
1
Muséum national d’Histoire naturelle (MNHN), DJBZ, Le Parc de Clères, 32 avenue du Parc, 76690
Clères, France
2
Zoo de Lille, avenue Mathias Delobel, 59800 Lille, France
3
Laboratoire Bio-VSM, 3 bis rue Pierre Mendès France, 77200 Torcy, France
4
Muséum national d’Histoire naturelle (MNHN), DJBZ, Ménagerie du Jardin des Plantes, 57 rue Cuvier,
75005 Paris, France
5
Muséum national d’Histoire naturelle (MNHN), EGB, UMR 5173 - CERSB - MNHN, CNRS, Paris IV, 57
rue Cuvier, 75005 Paris, France
*Corresponding author: Yannick Roman (yannick.roman@cg76.fr).
Summary
Although plasma protein electrophoresis is an invaluable diagnostic tool in bird medicine, but high intertaxonomic variations sometimes make their interpretation difficult for practitioners. The purpose of this
study was to improve global understanding of avian plasma electrophoresis patterns by locating the
fibrinogen and albumin, and by comparing migration distances of the latter in different bird species. This
study was conducted on 80 birds from five different species: 15 peafowls (Pavo cristatus), 18 bar-headed
geese (Anser indicus), 12 rock doves (Columba livia), 21 black kites (Milvus migrans) and 14 orange-winged
parrots (Amazona amazonica). Protein electrophoresis was performed on both plasma and serum. For each
bird, significant differences between fraction concentrations were taken into account to determine the
position of the fibrinogen peak on both electrophoresis curves and agarose gels in each species. Once the
fibrinogen was located, measurements were performed on the gels with a calliper square in order to
determine the migration distances of both fibrinogen and albumin. Albumin was observed to migrate to
different locations in different species. On the other hand, comparison of the fibrinogen migration distance
between species allowed us to split the five studied species into two groups, corresponding to the avian
phylogenetic classification. In the Galloanserae infraclass including, in the case of the present study, the
peafowls and bar headed geese, the fibrinogen migrated over a significantly greater distance than in the case
of the Neoaves infraclass, which includes the pigeons, black kites and orange-winged parrots. Fibrinogen
may thus be a useful feature for the conventional naming of different protein fractions in avian species.
49
Keywords
Protein electrophoresis, Fibrinogen, Albumin, Serum, Plasma
Running title
Fibrinogen and albumin in avian plasma protein electrophoresis
Résumé
L’électrophorèse des protéines plasmatiques est un outil diagnostique très utile chez l’oiseau, bien que
d’importantes variations inter-taxonomiques rendent parfois l’interprétation des profils quelque peu difficile
pour le praticien. Le but de cette étude était d’améliorer la compréhension globale des profils
électrophorétiques chez les oiseaux par la localisation du fibrinogène et de l’albumine et par la mesure des
distances de migration de ces protéines sur gel d’agarose. Cette étude a été menée sur 80 oiseaux appartenant
à cinq espèces différentes : 15 paons bleus (Pavo cristatus), 18 oies à tête barrée (Anser indicus), 12 pigeons
domestiques (Columba livia), 21 milans noirs (Milvus migrans) et 14 amazones aourou (Amazona
amazonica). Les différences des valeurs brutes des différentes fractions mesurées sur plasma et sur sérum ont
permis de localiser la position du fibrinogène sur les profils électrophorétiques et les gels d’agarose. Une fois
le fibrinogène localisé, les distances de migration du fibrinogène et de l’albumine ont été mesurées
directement sur le gel à l’aide d’un pied à coulisse. L’albumine a migré sur des distances différentes pour les
différentes espèces. La comparaison des distances de migration du fibrinogène nous a en revanche permis de
scinder les cinq espèces étudiées en deux groupes distincts correspondant à la classification phylogénétique
des oiseaux proposée par Lecointre et Le Guyader (2001). Dans l’infraclasse des Galloanserae comprenant
dans notre cas l’oie à tête barrée et le paon bleu, le fibrinogène a migré significativement plus loin que dans
l’infraclasse des Neoaves comprenant ici le pigeon, le milan noir et l’amazone aourou. Le fibrinogène
pourrait être un bon point de repère pour nommer les fractions de manière conventionnelle dans les
différentes espèces d’oiseaux.
Mots clés
Electrophorèse des protéines, Fibrinogène, Albumine, Serum, Plasma
Titre courant
Fibrinogène et Albumine sur les electrophorégrammes des protéines chez l’oiseau
50
INTRODUCTION
Plasma protein electrophoresis is an invaluable diagnostic tool in avian medicine [1, 15]. However, high
intertaxonomic variations have been observed in avian electrophoresis patterns [14, 16], which make their
interpretation difficult for practitioners.
A previous study of avian albumin has shown that the same proteins can migrate over different distances,
depending on species. For albumin, these differences have been attributed to variations in conformation and
surface charge distribution [1].
Fibrinogen is one of the most abundant acute phase proteins in birds. In the case of an inflammatory
condition, its increase is fast and massive [6]. Plasma is obtained by immediate centrifugation of a blood
sample mixed with an anticoagulant, whereas serum is prepared by allowing the blood sample to clot before
centrifugation. Serum and plasma are thus quite similar, except for the fact that plasma contains clotting
factors, fibrinogen and an anticoagulant, which are not present in the serum [7]. Total protein values have
been demonstrated to be about 1,5g/L higher in plasma than in serum, due to the presence of fibrinogen [10].
This significant difference can be helpful in locating fibrinogen, by comparing plasma versus serum
electrophoresis patterns in the same species.
The aim of this study was thus to locate fibrinogen in the electrophoresis patterns of five different species,
and to compare migration distances of this protein on electrophoresis gel. The location of this protein may
then improve our understanding of protein electrophoresis in birds. To this end, agarose gel protein
electrophoresis was carried out on the sera and plasmas of 15 peafowls (Pavo cristatus), 18 bar-headed geese
(Anser indicus), 12 rock doves (Columba livia), 21 black kites (Milvus migrans) and 14 orange-winged
parrots (Amazona amazonica).
MATERIAL AND METHODS
The study was conducted on 80 healthy birds from five different species: 15 peafowls (Pavo
cristatus) and 18 bar-headed geese (Anser indicus) held at the Zoological Park of Clères (France), 12 rock
doves (Columba livia) held at the Menagerie du Jardin des plantes (France), 21 black kites (Milvus migrans)
held at the Académie de fauconnerie du Puy du fou (France), and 14 orange-winged parrots (Amazona
amazonica) held at the Zoological Park of Lille (France). Protein electrophoresis was carried out on both
plasma and serum. Four millilitres of blood were drawn from each bird, using 23 G needles and five
millilitre syringes (respectively: Terumo Neolus 23 G, and Terumo 5 ml Syringe, from Terumo Europe N.V.,
Leuven, Belgium). For the plasma, two millilitres of blood were collected in lithium heparin tubes (Venosafe
vacutainers, Terumo, Leuven, Belgium) and immediately centrifuged at 3000 g for 5 minutes. Lithium
heparin was used as the anticoagulant of choice for plasma chemistry determinations, because of its low
biological activity and lack of interference with analyte detection (HRUBEC et al, 2002). For the sera, two
millilitres of blood were collected in dry tubes which were coated with a clotting activator (Venosafe
vacutainers, Terumo, Leuven, Belgium) and were then incubated at 30°C for 1h30, before being centrifuged
51
at 2000 g for 10 minutes. The plasmas and sera were then immediately stored in cryotubes for several weeks
at -20°C (-4°F) in order to guarantee that all measurements be carried out under the same conditions. The
samples were then thawed and rehomogenised, by gentle mixing one hour before analysis.
Total protein concentration was determined using the Biuret method with the Roche Integra 400 wet
chemistry analyser (Roche diagnostics GmbH, Mannheim, Germany). Readings were made at a wavelength
of 552 nm.
Agarose gel electrophoresis was carried out using a Hydrasys© semi-automated system (Sebia, Evry,
France), using the Hydragel protein 15/30© set (Sebia, Evry, France) which is the most commonly used kit
for protein electrophoresis in medical laboratories. Plasma aliquots were loaded onto the gel and
electrophoretic separation was obtained on 8 g/l agarose gels in a Tris-barbital buffer (pH 9.2), at 20°C and a
constant power level of 20W, until 33 V-h had been accumulated. Once dried and coloured with amidoblack
in the same system, the gels were scanned with a high resolution Epson expression 1680 pro flat scanner.
The electrophoretic curves and quantification of the different fractions were carried out with version
7.00 F0.1 of Sebia’s Phoresis software. Albumin was identified as the biggest and most anodal peak, and it
was not decided to assess the pre-albumin fraction separately because of its negligible contribution. The
globulins were divided into several fractions, depending on species, in order to study each peak separately.
Since their composition was unknown, these fractions were given arbitrary denominations (G1 to G6).
Migration distances were measured directly on the gels. These measurements were made from the edge of
the gels with a 6 inch (15 cm) dial calliper square (General, Montreal, Canada).
Systat 7.0 software (SPSS inc© 1997) was used for all analyses. Because of the small size of the
studied population, we used Wilcoxon’s signed rank test for the comparison of serum and plasma
electrophoresis patterns in the same species, and Mann and Whitney’s U test for the comparison of
fibrinogen and albumin migration distances between the studied species.
52
RESULTS
a. Fibrinogen localisation: intraspecific differences between plasma and serum protein electrophoresis.
Due to inter-taxonomic differences, the globulins were scattered into four fractions for the Amazon parrots,
bar-headed geese and rock doves, five fractions for the black kites, and six fractions for the peafowls. These
fractions were arbitrarily named G1 to G6 (Figure 1).
For each species, significant differences between plasma and serum protein electrophoresis fractions were
investigated. The values of the G2 fraction in Rock doves, the G3 fraction in bar headed geese, black kites
and orange winged parrots, and the G4 fraction in peafowls appeared to be significantly higher in the plasma
than in the serum (respectively: Z= -3.059, p ≤ 0.01; Z = -3.724, p ≤ 0.01; Z = -4.015, p ≤ 0.01; Z = -3.296, p
≤ 0.001; Z= -3.408, p ≤0.01). For the G3 fraction in rock doves, the albumin, G2 and G4 fractions in bar
headed geese, the G5 fraction in black kites, the albumin and G1 fractions in orange winged parrots, and the
albumin, G1, G2 and G3 fractions in peafowls, the measured plasma values appeared to be significantly
lower than those measured in serum (p ≤ 0.01).
b. Comparison of albumin and fibrinogen migration distances between bird species.
Fibrinogen was located in each species by comparing the results from plasma and serum protein
electrophoresis (Figures 2). No interspecific difference was found in the fibrinogen migration distances
between peafowls and bar-headed geese, on the one hand, and between black kites, orange-winged parrots
and rock doves, on the other hand. We found significant differences in fibrinogen migration distance
between peafowls and black kites (p ≤ 0.01, U=0,), between peafowls and orange-winged parrots (p ≤ 0.01,
U = 2), between peafowls and rock doves (p ≤ 0.01, U = 180), between bar-headed geese and black kites (p ≤
0.01, U = 0), between bar-headed geese and orange-winged parrots (p ≤ 0.01, U = 2.5), and between barheaded geese and rock doves (p ≤ 0.01, U = 216).
No interspecific difference was found in albumin migration distance between bar-headed geese and black
kites. We found significant differences in albumin migration distance between peafowls and black kites (p ≤
0.01, U = 296), between peafowls and orange-winged parrots (p ≤ 0.01, U=0), between peafowls and rock
doves (p ≤ 0.01, U=180), between peafowls and bar-headed geese (p ≤ 0.01, U = 268), between bar-headed
geese and orange-winged parrots (p ≤ 0.01, U=0), between bar-headed geese and rock doves (p ≤ 0.01,
U=216), between black kites and pigeons (p ≤ 0.01, U=240), between orange-winged parrots and pigeons (p
≤ 0.01, U=12.5), and between orange-winged parrots and black kites (p ≤ 0.01, U = 14).
53
Figure 1: examples of plasma and serum protein electrophoresis patterns of five bird species. Plasma (Grey
curve) and serum (Black curve) patterns are superimposed. Asterisks represent significant differences
between plasma and serum (Wilcoxon's test: p ≤ 0.01), due to fibrinogen loss during the clotting process.
54
Figure 2: plasma (P) and serum (S) protein electrophoresis agarose gels of five avian species. The location of
the fibrinogen peaks is indicated by the two arrows. The albumin location is indicated by the letter "A".
DISCUSSION
This study demonstrates that in all of the studied species, only one fraction appears to have plasma
values higher than those found in serum. This difference is due to fibrinogen, which is present in plasma, but
not in serum, because of the clotting process [7]. In the present study, this difference allowed us to locate
fibrinogen in electrophoresis patterns and gels. For practitioners dealing with poorly known avian species,
the fibrinogen can thus be easily located, by comparing plasma and serum patterns. The location of
fibrinogen, which is a major acute phase protein in birds [6], will enable practitioners to improve their
interpretation of observed electrophoresis patterns.
Depending on species, other differences were found between the plasma and serum values of some of the
protein fractions. However, concerning the latter, the serum values were only slightly higher than those
found in the plasma. These small differences may have been induced by artefactual changes, related to
haemolysis and protein leakage from blood cells in the case of sera. Indeed, due to the longer period required
for clotting, the fluid components were in contact with blood cells for a longer period of time for sera than
for plasmas [5, 7].
The comparison of fibrinogen migration distances between the studied species supports the
conclusion that fibrinogen migrates to the same locations, for peafowls and bar-headed geese, on the one
hand and for black kites, orange-winged parrots and rock doves, on the other hand. By considering the
fibrinogen migration distances, we can therefore split the five species into two groups. By comparison with
the avian phylogenetic tree shown in Figure 3 [9], these two groups appear to be well matched with two
distinct monophyletic groups: galliformes and anseriformes, corresponding to the Galloanserae infraclass on
the one hand, and psittaciformes, columbiformes and falconiformes, corresponding to the Neoaves infraclass
55
on the other hand. This distinction could be related to differences in the structure or surface charge
distribution of fibrinogen between the two infraclasses. As far as the authors are aware, such a result has
never been documented in previous studies. This phenomenon thus merits further investigation, in order to
examine a greater variety of bird taxa than in the present study.
The comparison of albumin migration distances reveals that this protein seems to migrate to different
locations, depending on species, except for the case of the black kite and the bar-headed goose. Furthermore,
the migration distance standard deviations were very high for albumin, by comparison with those of
fibrinogen, which were very low. Contrary to albumin, fibrinogen thus seems to migrate to almost the same
location in agarose gels. This could be related to the fact that albumin is a small and fast migrating molecule,
whereas fibrinogen is a large and slow migrating one [8]. This makes it easier to read albumin variations than
those of fibrinogen.
All authors familiar with this topic agree with the observation that fibrinogen migrates to a beta location on
bird electrophoregrams [2, 3, 5, 8, 13, 15]. In order to establish a standardised method of defining protein
fractions in avian plasma protein electrophoresis patterns, this protein, which is easy to identify by
comparing plasma and serum electrophoregram patterns, could be used as a reference for the naming of the
different fractions. In this way, fibrinogen would correspond to the "Beta" peak, the fractions located
between the albumin and fibrinogen peaks would therefore be named "Alfa" peaks, and the fractions beyond
the fibrinogen peak would be referred to as "Gamma" peaks.
Figure 3: avian phylogenetic tree (LECOINTRE and LE GUYADER, 2001).
56
AKNOWLEDGMENTS
We wish to thank the Académie de fauconnerie du Puy du fou, in particular J.L. LIEGEOIS, for supplying
the kites' blood samples, the Parc zoologique de Lilles, in particular D. ORDONNEAU for supplying the
amazon parrots' blood samples, the Ménagerie du jardin des plantes (MNHN), in particular N. CHAI and
C.P. PIGNON for supplying the rock doves' blood samples. We are also grateful to P. TROLLIET from
Sebia for his invaluable technical support, to the Museum national d’Histoire naturelle and to the Conseil
Général de Seine Maritime (France) for their financial support, and to Techtrans Consulting for the English
proofreading.
REFERENCES
1. ARCHER F.J. BATTISON AL. : Differences in electrophoresis patterns between plasma albumins of the
cockatiel (Nymphicus hollandicus) and the chicken (Gallus gallus domesticus), Avian Path., 1997, 26, 865870.
2. CRAY C., BOSSART G., HARRIS D.: Plasma protein electrophoresis: principles and diagnosis of
infectious diseases. In Proceedings of the 17th AAV conference, Lake Worth, 1995, 55-59.
3. CRAY C., TATUM L.: Application of protein electrophoresis in avian diagnostic testing, J. Avian Med.
Surg., 1998, 12, 4 -10.
4. FUDGE A.M.: Avian laboratory medicine. In: A.M. FUDGE (éd) : Laboratory medicine; avian and exotic
pets, W.B. Saunders company, Philadelphia, 2000, 1-184.
5. FUDGE A.M., SPEER B.: Selected controversial topics in avian diagnostic testing, Seminars Avian Exot.,
Pet Med., 2001, 10, 96-101.
6. HAWKEY C., HART M.G.: An analysis of the incidence of hyperfibrinogenemia in birds with bacterial
infections, Avian Path., 1988, 17, 427-432.
7. HRUBEC T.C., WHICHARD J.M., LARSEN C.T., PIERSON F.W.: Plasma versus serum: specific
differences in biochemical analyte values, J. Avian Med. Surg., 2002, 16, 101-105.
8. KANEKO J.J.: Serum proteins and the dysproteinemias. In: J.J. KANEKO (éd): Clinical biochemistry of
domestic animals, Academic press, New York, 1989, 142-164.
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9. LECOINTRE G., LE GUYADER H. : Oiseaux; annexe 6. In G. Lecointre and H. Le Guyader (éds):
Classification phylogénétique du vivant 2° edition, Belin, Paris, 2001, 515.
10. LUMEIJ J.Y., MC LEAN B.: Total protein determination in pigeon plasma and serum: comparison of
refractometric methods with the Biuret method, J. Avian Med. Surg, 1996, 10, 150-152.
11. ORDONNEAU D., ROMAN Y., CHASTE-DUVERNOY D., BOMSEL M.C.: Plasma electrophoresis
reference ranges in various bird species. In Proceedings of the 8th EAAV conference, Arles 2005: 283-289.
12. ROMAN Y., ORDONNEAU D., CHASTE-DUVERNOY D., SAINT JALME M., BOMSEL M.C.,
HINGRAT Y.: Early detection of an acute inflammatory condition by plasma protein electrophoresis an
haematology in peafowls. In Proceedings of the 8th EAAV conference, Arles, 2005, 290-297.
13. ROSENTHAL K.L.: Avian protein disorders. In A.M. FUDGE (ÉD): Laboratory medicine; avian and
exotic pets, W.B. Saunders company, Philadelphia, 2000, 171-173.
14. SIBLEY C.G., HENDRICKSON H.T.: A comparative Electrophoretic study of avian plasma proteins,
The Condor, 1970, 72, 43-49.
15. WERNER L.L., REAVILL D.R.: The diagnostic utility of serum protein electrophoresis, Vet clin. North
Am. Exot. Anim. Prac., 1999, 2, 651-662.
16. ZAIAS J., FOX W.P., CRAY C., ALTMAN N.H.: Hematologic, plasma protein, and biochemical
profiles of brown pelicans (Pelecanus occidentalis), Am. J. Vet. Res., 2000, 61, 771-774.
58
CHAPITRE II : variations physiologiques des électrophorégrammes
aviaires
Article 3: Influence de la mue sur les profils d’électrophorèse des protéines plasmatiques de
l’oie à tête barrée (Anser indicus).
Cet article a été soumis au Journal of Wildlife diseases.
Résumé
L’électrophorèse des protéines plasmatiques est actuellement reconnue comme étant un examen
complémentaire fiable en médecine aviaire. Cependant, l’influence de phénomènes physiologiques
circannuels tels que la mue, sur les électrophorégrammes aviaires, sont peu connus. Pourtant, la mue
représente une période de changements hormonal et métabolique drastiques. L’objectif de cette étude était
d’observer de manière détaillée chez l’oiseau les effets de la mue sur la concentration en protéines totales et
sur les profils électrophorétiques. A cet effet, des prélèvements sanguins ont été réalisés tous les 15 jours, sur
19 oies à tête barrée (Anser indicus), de la mi-mai à la mi-août. Ces prélèvements ont été utilisés pour
déterminer les concentrations en protéines totales et réaliser des électrophorèses des protéines plasmatiques
sur gel d’agarose. Les stades de mue des oiseaux ont été évalués à l’occasion des prélèvements. L’oie à tête
barrée a été choisie comme modèle dans cette étude, car ces oiseaux renouvellent simultanément la totalité
de leurs rémiges sur une période très brève. La concentration en protéines totales et les valeurs absolues des
fractions albumine, alpha-2, beta et gamma ont été observées comme étant à leur minimum durant la mue,
tandis que celles des fractions pré-albumine et alpha-1 étaient à leur maximum durant la même période. Cette
étude constitue, une publication de référence des effets de la mue sur les profils électrophorétiques des
oiseaux. L’augmentation des fractions pré-albumine et alpha-1 est vraisemblablement liée à une
augmentation des taux plasmatiques des hormones thyroïdiennes durant la période de mue. La diminution
des fractions albumine, alpha-2, beta et gamma est probablement liée à une mobilisation des réserves
protéiques et énergétiques en faveur du renouvellement des plumes, ainsi qu’à une expansion du système
circulatoire au niveau des follicules en croissance. D’un point de vue clinique, les changements des profils
électrophorétiques associés à la mue se sont révélés moins significatifs que prévus et ne semblent pas être
assez importants pour induire en erreur le praticien vétérinaire.
59
60
Roman, Bomsel-Demontoy,,Levrier, Chaste-Duvernoy and Saint Jalme
Molt and plasma protein electrophoresis in bar headed geese
Influence of molt on plasma protein electrophoresis in bar-headed geese (Anser indicus)
Yannick ROMAN1,6, Marie-Claude BOMSEL-DEMONTOY2, Julie LEVRIER1 , Dorothée ORDONNEAU3,
Daniel CHASTE-DUVERNOY4 and Michel SAINT JALME5
1
Muséum national d’Histoire naturelle (MNHN), DJBZ, Le Parc de Clères, 32 avenue du Parc, 76690
Clères, France
2
Muséum national d’Histoire naturelle (MNHN), DJBZ, Ménagerie du Jardin des Plantes, 57 rue Cuvier,
75005 Paris, France
3
Zoo de Lille, avenue Mathias Delobel 59800 Lille
4
Laboratoire Bio-VSM, 3 bis rue Pierre Mendès-France, 77200 Torcy, France
5
Muséum national d’Histoire naturelle (MNHN), EGB, UMR 5173 - CERSB - MNHN, CNRS, Paris IV, 57
rue Cuvier, 75005 Paris, France
6
Corresponding author (tel: +33(2)35332308, fax: +33(2)35331166, e-mail: yannick.roman@cg76.fr)
61
ABSTRACT
Plasma protein electrophoresis is now commonly recognized to be a very reliable diagnostic tool in avian
medicine. However, the influence of circannual phenomena such as molt on protein electrophoregrams is
poorly documented. Yet, the molt is a period of heavy hormonal and metabolic change in birds. The purpose
of this study was to investigate the effects of molt on total protein concentration and electrophoresis patterns
in birds. Nineteen bar headed geese (Anser indicus) were blood sampled from mid May to mid August, at 15
day intervals. At the same time, the birds’ molting stage was checked. Total protein concentrations were
measured and plasma agarose gel electrophoresis were performed on these samples. The geese were chosen
as a model, because these birds molt over a very short period. The total protein concentration, albumin,
alpha-2, beta and gamma fractions were at their minimum values during molt, whereas the prealbumin and
alpha-1 fractions rose to their maximum levels. This study provides baseline information relevant to changes
occurring in avian proteinograms throughout the molt. The increase in the prealbumin and alpha-1 fractions
may be related to an increase in plasma thyroid hormones during molt. The decrease observed in albumin,
alpha-2, beta and gamma fractions may be related to a protein and energy shift, directed towards feather
growth, as well as to an expansion of the circulatory system located around the feather follicles. From a
clinical point of view, the observed changes associated with molting were less significant than initially
expected, and are not likely to be strong enough to mislead the practitioner.
KEYWORDS
Molt, Plasma, Protein, Electrophoresis, Bird
62
INTRODUCTION
Serum protein electrophoresis is commonly used in human laboratory medicine. Over the last 15
years, its use has been extrapolated to avian medicine and it is now commonly recognized as a very reliable
diagnostic tool in this field (Cray and Tatum, 1998; Werner and Reavill, 1999). However, some circannual
physiological events, such as molting, may be liable to induce changes that could potentially mislead the
practitioner. Feathers are indeed composed of 95% protein and represent nearly 25 % of a bird’s dry mass
(Murphy and King, 1992). The loss and regeneration of feathers during the molt occurs over a period of a
few weeks to a few months, and may therefore be associated with heavy protein transport and synthesis. The
molt is furthermore a period of intensive energy demand (Klaassen, 1995). Oxygen consumption has been
shown to be nine to 46 % greater in molting birds than in non molting birds (Walsberg, 1983). This energy
expenditure arises from several components: the energy content of feathers, the cost of biosynthesis of
feather material, body temperature regulation due to decreased insulation and changes in activity, and the
energy intake required to supply the sulphur-containing amino acids necessary for feather synthesis
(Klaassen, 1995). In addition to this metabolic aspect, studies have shown that a significant increase in total
body protein synthesis, osteoporosis, loss of body fat, and depletion of the immune system occur during this
event (Mrosovsky and Sherry, 1980; Murphy and Todd, 1995; Kuenzel, 2003). Finally, at this period of time,
heavy hormonal changes occur. Thyroid hormones increase concomitantly with a decrease in sexual steroids
(Sauveur and De Reviers, 1988). In ducks and geese, the post-nuptial molt coincides with high
triiodothyronin (T3) and tetraiodothyronin (T4) plasma levels, and low levels of circulating luteinising
hormone (LH) and testosterone (Assenmasher and Jallageas, 1978; John et al., 1983). In Canada geese, the
growth hormone (GH) was observed to be at its highest level during molt (John et al. 1983). Corticosterone
levels were shown to be either stable (Rehder et al. 1986; Otsuka et al. 1998; Piersma and Ramenofsky,
1998), or to decrease, according to which species was studied (Romero and Remage-Healey, 2000; Rich and
Romero, 2001).
A great deal has been written about molting within the last 40 years, with particular attention having
been paid to addressing the specific types of molt of various birds species(Delacour, 1964; Oring, 1968;
Saint Jalme et al. 1995; Todd, 1996), the comprehension of the physiological mechanisms of molting
(Assenmasher and Jallageas, 1978; John et al., 1983; Walsberg, 1983; Rehder et al. 1986; Klaassen, 1995;
Piersma and Ramenofsky, 1998; Johnson, 2000; Romero and Remage-Healey, 2000; Rich and Romero,
2001; Otsuka et al. 2004), and the evaluation of methods used to induce molting in domestic birds (Brake,
1993, Berry, 2003; Park et al. 2004). However, our review of the literature identified only one reference
dealing with plasma electrophoretic changes occurring during molt, from the point of view of the veterinary
practitioner. This study, conducted on mallard ducks, reported changes in the albumin and α2 globulin
fractions determined using cellulose acetate electrophoresis (Driver, 1981). This is however the only study to
have dealt with this topic, and electrophoretic techniques have improved considerably over the last 20 years.
63
The purpose of the present study was therefore to more thoroughly investigate changes in the
electrophoresis patterns in birds during molt, through the example of the bar headed goose (Anser indicus).
Understanding these changes could help the practitioner with the interpretation of avian electrophoregrams,
and improve understanding of avian molt physiology. To this end, geese were chosen as a model, because
these birds molt only once a year, at the end of the reproductive season (Delacour, 1964). This prebasic (or
postnuptial) molt leads to the growth of both flight and body feathers. All of the wing feathers are shed and
replaced simultaneously, to such an extent that, in general, geese completely loose their flight capacity for
five to six weeks (Delacour, 1964; Todd, 1996). In barnacle and Canada geese, wing feather elongation was
showed to last 35 to 40 days, with growth speeds averaging 7.5 mm daily (Owen and Ogilvie, 1979; Todd,
1996). One could therefore expect such an intense phenomenon to have a significant impact on plasma
protein electrophoresis patterns.
This study was conducted in 2004, on a group of 19 one year old bar headed geese, which
were blood sampled at 15-day intervals, from mid May to mid August. These samples were used to
investigate the influence of molting on agarose gel plasma protein electrophoresis.
MATERIAL AND METHODS
Experimental animals
The present study was conducted in 2004, on 19 bar headed geese (Anser indicus) held at the Clères
zoological park (France). The group of geese was composed of 7 males and 12 females, which had been
hand reared one year before the study, in order to limit the effects of handling stress resulting from blood
sampling. All these birds were furthermore sexually immature at the time this study was performed, which
allowed us to get rid of potential changes related to the reproduction status. The geese were housed outdoors
in a two hectare meadow, and were dewormed with ivermectin (200µg/kg), twice a year in spring and
autumn. In mid June, one male goose fell ill and was excluded from the study.
Samples
Bar headed geese were blood sampled at intervals of 15 days, from mid May to mid August. Two milliliters
of blood were drawn from each bird at the brachial vein and collected on lithium heparin (Venosafe
vacutainers, Terumo Europe, Leuven, Belgium). Since hemoglobin is known to interfere with protein
concentration determination and plasma electrophoresis, care was taken to avoid hemolysis by taking blood
samples with 21 G needles and 2 ml syringes (Terumo Europe, Leuven, Belgium). Heparinised blood
samples were centrifuged at 3000 g for 5 minutes and the plasma samples were stored in cryotubes (Micronic
systems, Lelystad, Holland) at a temperature of -20°C (-4°F) until they were analysed. The samples were
then thawed and rehomogenised by gentle mixing 1 hour before analysis. Analyses were carried out on
plasma, since for protein electrophoresis in birds this medium is less prone to hemolysis than serum, and
64
because it contains fibrinogen, which is a characteristic acute phase protein (Cray and Tatum, 1998;
Hochleithner, 1994).
Molt investigation
Members of the Anserinae subfamily are known to molt only once a year, at which time both body and flight
feathers are replaced (Delacour, 1964). Samples were sorted into four chronological stages, which were
devised for the purposes of our study. These stages were based on the molt chronology described by
Delacour (1964): A. Basic plumage I; B. Molt of the body feathers; C. Primary and secondary remige
development (blood feathers measuring 1/3 to 2/3 of the final feather length); D. Basic plumage II. Heinroth
(cited by ROMERO et al. (2005)) described three phases of feather growth: “firstly, a slow initial phase
confined to the feather germ at the base of the follicle, secondly a long phase of daily elongation during the
major part of which growth is more or less linear, thirdly a progressive slowing down of growth, a
withdrawal of pulp from the calamus and the cornification of the feather base”. Stage “B and C” therefore
respectively corresponded to body and wing feather elongation.
Molt was assessed every 15 days at the times when the birds were captured for blood sampling. Molt score
was not investigated more precisely in order to limit the handling of the birds and therefore to limit the
potential stress artifacts on protein electrophoresis (Grieninger, 1978; Chamanza et al. 1999). Geese were all
synchronous in molting. During the sample collection in mid June and at the beginning of July all of them
were respectively at the growth phase of body feathers (B) and flight feathers (C). The other samples
corresponded to birds with basic plumage (A or D).
Total protein concentration
Total protein concentration was determined by the Biuret reaction, using a Roche Integra 400 wet chemistry
analyser (Roche diagnostics GmbH, Mannheim, Germany). Readings were made at a wavelength of 552 nm.
Plasma protein agarose gel electrophoresis
Agarose gel electrophoresis of plasma proteins was carried out using a Hydrasys© semi-automated system
(Sebia, Evry, France), using the Hydragel protein 15/30© set (Sebia, Evry, France). It was operated
according to the manufacturer’s instructions, using version 7.00 F0.1 of the system software. Ten microlitres
of plasma samples were manually distributed onto the sample template, and were allowed to diffuse for a
period of 5 minutes in a wet chamber. Application of the samples to the gel, electrophoresis, and drying of
the gel were all performed automatically in the migration compartment of the instrument. The temperature
was maintained at 20°C (68°F) using a Peltier device during the complete migration process, and drying was
obtained by heating the gels to 65°C. Electrophoretic separation was obtained on 8 g/l agarose gels in a Trisbarbital buffer (pH 9.2), at a constant power level of 20W, until 33 Vh had been accumulated. Once dried,
the gels were manually transferred to the staining compartment of the instrument where amido-black
staining, destaining and drying were performed automatically. Once these operations had been completed,
65
the gels were scanned with a high resolution Epson perfect V700 photo scanner (Epson France, Nanterre,
France). Electrophoretic curves and dosages of the different fractions were acquired using Phoresis©
software, version 5.50 (Sebia, Evry, France). Albumin was identified as having the strongest anodal peak.
The beta peak was defined as the fibrinogen peak (Roman et al., 2007). Globulins were divided into 5
fractions. The alfa fractions were located between the albumin and beta peaks, and the gamma fraction was
located beyond the beta peak. As previously described in other studies of avian protein electrophoresis, the
A/G ratio was calculated by dividing the sum of the prealbumin and albumin fractions by the sum of the
globulin fractions (Lumeij, 1987; Cray and Tatum, 1998).
Statistical analysis
Systat 7.0 software (SPSS inc. © 1997) was used for all analyses. Because of the small size of the
studied population, we used Friedman’s two-way variance analysis and Wilcoxon’s signed rank
test.
66
RESULTS
The bar headed geese electrophoresis patterns were divided into six fractions (Figure 1): one prealbumin fraction, one albumin fraction, two alpha fractions, one beta fraction and one gamma fraction.
Figure 1: example of plasma electrophoresis patterns of a Bar headed goose (Anser indicus).
In both sexes, total protein concentration appeared to vary significantly as a function of time
(Friedman’s test: p = 0). In both males and females, total protein concentration significantly decreased from
mid June to early July (respectively: Z = -2.366, p < 0.02; Z = -2.667, p < 0.01) (Figure 2). Thereafter, total
protein concentration remained at a low value until the end of the study.
67
Figure 2: variation of total plasma protein concentration during the molt in males (
females (
)
and
) bar headed geese (Anser indicus). This graph makes use of measurements based on
bi-monthly blood samples, taken between mid May and mid August. Molt stages are indicated as: A.
Basic plumage I; B. Molt of the body feathers; C. primary and secondary flight feathers development; D.
Basic plumage II. The data points are expressed as: mean ± SEM.
All fractions appeared to vary significantly as a function of time, from the beginning to the end of the
study, in both sexes (Friedman’s test: p < 0.01). Several fractions were observed to increase during molt, in
both males and females. The pre-albumin fraction increased from mid June to early July (Figure 3a,). This
increase was significant in females (Z = 2.981, p < 0.05) and only marginally significant in males (Z = 1.859,
p = 0.063). Thereafter, it significantly decreased in both sexes, from early July to mid July (Z = -2.201, p <
0.05 and Z = -2.824, p < 0.01, for males and females respectively). The alpha-1 fraction tended to be higher,
around the molt period (Figure 3c). However, it reached its maximum value earlier in females than in males.
Alpha-1 fraction increased significantly from early June to mid June in females (Z = 2.824, p < 0.05),
reaching its maximum value during the molt of the body feathers. Thereafter, it decreased significantly from
early July to mid August (Z = -1.961, p < 0.05). In males, the alpha-1 fraction tended to increase from mid
May to early July, reaching a maximum during molt of the flight feathers. This increase was only marginally
significant (Z = 1.859, p = 0.063). The alpha-1 fraction then remained stable from early June to early August,
and significantly decreased until the end of the study (Z = -2.201, p < 0.05).
Some other fractions decreased at the time of molting. The albumin concentration decreased from mid June
to early July, in both males and females (respectively: Z = -2.366, p < 0.02; Z = -3.059, p < 0.01) (Figure
3b). The albumin fraction then increased significantly for males (Z = 2.201, p < 0.05), from early July to mid
68
July. The alpha-2 fraction decreased significantly in males and females from early July to mid July
(respectively: Z = -2.366, p < 0.02; Z = -2.903, p < 0.01), reached its minimum value during molt, and then
increased significantly from early July to mid July (respectively: Z = 2.201, p < 0.05; Z = 3.059, p <0.01)
(Figure 3d). The gamma fraction decreased significantly in both males and females from early June to early
July (respectively: Z = -2.366, p < 0.02; Z = -2.118, p < 0.05), and again increased significantly from early
July to early August (respectively: Z = 1.992, p < 0.05; Z = 2.040, p < 0.05) (Figure 3f). The beta fraction
significantly decreased in both males and females, from mid June to early July (respectively: Z = -2.366, p <
0.02; Z = -2.824, p < 0.05) and then remained stable until the end of the study (Figure3e).
In both sexes, the A/G ratio did not appear to fluctuate in relation to the molt period.
Except for the alpha-1 and gamma fractions, no significant difference was found between males and
females for any of the measured parameters. In general, the alpha-1 fraction was observed to be significantly
higher in females than in males (p < 0.02), except from early June to early August when no significant
difference was found. The gamma fraction was observed to be significantly higher in females than in males
(p < 0.05).
69
Figure 3: variation of (a) pre-albumin, (b) albumin, (c) alpha-1, (d) alpha-2, (d) beta and (e) gamma fractions
concentration during the molt in males (
)
and females (
) bar headed geese (Anser
indicus). This graph makes use of measurements based on bi-monthly blood samples, taken between mid
May and mid August. Molt stages are indicated as: A. Basic plumage I; B. Molt of the body feathers; C.
primary and secondary flight feathers development; D. Basic plumage II. The data points are expressed as:
mean ± SEM.
70
DISCUSSION
The present study provides baseline information relevant to the changes which occur in avian
proteinograms throughout the molt. It clearly stresses that the molt coincides with an indisputable decrease in
total protein concentration, and albumin, alpha-2, beta and gamma fractions, followed by an increase in total
protein, and albumin, alpha-2 and gamma fractions once the molt is finished. In addition, our study reveals
an increase in the prealbumin during the molt. In the same manner, alpha-1 fraction was higher around the
molt, than after or before. This study is the first to have investigated the effects of molt in plasma proteins of
both male and female birds.
Our results match those of previous publications, which also showed a decrease in albumin and total
protein concentrations during the molt of chickens (Gildersleeve et al., 1983), passerine birds (De Graw and
Kern, 1985) and tropical seabirds (Work, 1996). For the case of mallard ducks, Driver (1981) demonstrated
that the total protein concentration decreased significantly, reaching a minimum during molt, and then
increased. The albumin concentration was described to be 10 to 20 % lower during, than after molt, whereas
the alpha-2 fraction was shown to be lower during molt than beforehand. However, no change was found in
the other fractions. This could be due to the fact that this particular study was based on a smaller sample size,
and made use of a less accurate electrophoresis technique (cellulose acetate electrophoresis), than the work
presented here. Our results therefore provide additional data concerning changes observed in the prealbumin,
alpha-1, beta, and gamma fractions.
In the study reported here, total protein concentration decreased mainly as the result of
reduced albumin, alpha 2 and gamma fractions during the molt. Albumin is the most abundant plasma
protein. Besides other important roles as a carrier protein, or in the control of osmotic pressure of the blood,
albumin also plays the role of a mobile source of amino acids in a nutritional emergency (Butler, 1971;
Kaneko, 1989). The observed decline in albumin concentration thus suggests that this amino acid reserve
was used for feather production. In molting passerine birds, Murphy and Todd (1995) demonstrated an
acceleration of the whole body protein turnover, which could enable an increase in the animal’s metabolic
plasticity and allow such adaptations. The fact that the albumin values increased again after the molt in male
birds, before decreasing again, could be explained by a “rebound” effect related to the competition between
the homeostatic phenomenon aiming at restoring albumin to its original concentration, and the consumption
of this latter as an amino-acid reserve used for feather production.
Our study highlights the fact that the gamma fraction, which is known to contain immunoglobulins (Kaneko,
1989; Cray and Tatum, 1998; Werner and Reavill, 1999), tended to decrease during the molt, which is a
period of intense energy expenditure (Klaassen, 1995). Energetically expensive conditions which are not
directly related to molting, such as immune responses (OTS et al., 2001; Martin et al., 2003), may be
temporarily reduced (Raberg et al., 1998). Depletion of the immune system during molt was first
demonstrated in the Smyth line (SL) chicken, which expresses the gene of an auto-immune disease similar to
vitiligo, resulting in the loss of melanocytes from the feathers. During molt, some of the new emerging
feathers are pigmented, suggesting that the autoimmune disease is not completely expressed during this
71
period, because the immune system is transiently depleted (Boyle and Smyth, 1984). This is in agreement
with a recent publication which showed that triiodothyronine (T3) has an immunosuppressive effect on
humoral immunity in Eider ducks (Somateria mollissima) (Bourgeon and Raclot, 2007). A dramatic increase
in the concentration of this hormone, which is well known to occur throughout the molt (Assenmacher and
Jallageas, 1978; John et al., 1983), is probably responsible for a transfer of energy towards feather
replacement, resulting (in the case of our study) in a decrease in the gamma fraction, followed by an increase
once the molt was over.
A decrease in some protein fractions may also be related to hemodilution effects, resulting from expansion of
the circulatory system located around the feather follicles. Some studies have indeed showed that in
passerine birds, molting causes an increase in the plasma volume (Chilgren and De Graw, 1977; De Graw
and Kern, 1985). The changes observed in the alpha-2 or beta fractions may be related to one or more of the
phenomena described above.
Our study also shows that prealbumin and alpha-1 fractions tend to increase during the molt. Increases in the
plasma levels of thyroid hormones, which appear to function by promoting feather replacement and
regulating the impaired homeothermy of the defeathered bird (Johnson, 2000), may have contributed to the
increase in these fractions. Indeed, it has been demonstrated that in the chicken, both triiodothyronine (T3)
and thyroxine (T4) selectively stimulate the synthesis of four major plasma proteins in hepatocyte cultures,
including fibrinogen and α1-globulin M (hemopexin) both of which are acute phase proteins, lipoproteins,
and prealbumin B (Hertzberg et al. 1981; Grieninger et al., 1986). A short period of exposure, of the order of
30 minutes, is sufficient to trigger a nearly full thyroid hormone effect on plasma protein synthesis
(Hertzberg et al. 1981). On the other hand, in 1987, another study demonstrated for the first time the
existence of an association between an increase in the concentration of plasma transthyretin and molting in
birds (Cookson et al. 1988). This carrier protein, also called thyroxine binding pre-albumin, may be
responsible for the increase in the prealbumin fraction during remige molt observed in this study. The fact
that we observed the beta fraction, which is known to contain fibrinogen, to decrease during the molt remains
unexplained.
Except for the alpha-1 fraction, main changes observed herein in the plasma protein fractions closely
coincided with the molt of the flight feathers and may correspond to maximum changes in the geese’s protein
metabolism. The alpha-1 fraction reached its maximum value 15 days earlier in females than in males. This
could be related to a slightly earlier molt in females than in males that our relatively rough molt investigation
method was not able to point out. However, a thorough investigation of feather molt would not have been
feasible in this kind of study, since the potential stress induced by frequent handlings of the birds would have
resulted in changes in the proteinograms (Grieninger, 1978; Chamanza et al. 1999).
The main differences observed between males and females occurred in the alpha-1, beta and gamma
fractions. The values of these fractions were in fact shown to be greater in females than in males. Such
differences between males and females have seldom been investigated in birds, outside the breeding
season. In black storks (Ciconia nigra) higher gamma values were observed in females than in males, but
72
this finding remains unexplained (Lanzarot et al., 2005). In the study reported here, such differences
might have been caused by the presence, unbeknown to the authors, of underlying inflammatory
conditions in some of the birds in the female group. Further studies should be carried out in order to
investigate differences between male and female electrophoregrams, outside the breeding season.
In any case, from the clinical point of view, changes related to the molt were less significant than
initially expected, and may not be strong enough to mislead the practitioner, since the variations recorded
during the course of the molt were weak in comparison with the observed inter-individual variations.
ACKNOWLEDGEMENTS
We wish to thank P. Trolliet from Sebia for his invaluable technical support, the team from bio VSM, and
more especially Mrs. K. Naudin from bio VSM, who carried out the total protein measurements. We wish to
thank the team from the Parc Zoologique de Clères, and in particular D. Catteville and C. Dumais who
provided us with invaluable help in handling and rearing the birds. We are grateful to the Muséum National
d’Histoire Naturelle and the Conseil Général de Seine Maritime (France) for their financial support. We also
appreciate the proofreading assistance provided by Techtrans Consulting.
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77
78
Article 4 : Influence de la ponte sur les profils d’électrophorèse des protéines plasmatiques de
la poule pondeuse (Gallus gallus)
Cet article a été soumis à la revue Veterinary Clinical Pathology.
Résumé
L’électrophorèse des protéines plasmatiques est de plus en plus communément admise comme étant un
examen complémentaire fiable en médecine des oiseaux. Cependant, l’influence de phénomènes circannuels
tels que la ponte, sur les électrophorégrammes aviaires sont méconnus. Pourtant, la période de ponte est une
période d’intense bouleversement métabolique et hormonal pour l’oiseau. L’objectif de cette étude était donc
d’aborder les effets de la ponte sur les électrophorégrammes aviaires. La poule pondeuse a été prise pour
modèle dans notre étude car cette dernière est capable de pondre en moyenne un œuf toutes les 25 heures
pendant plusieurs mois. Vingt poules (Gallus gallus) ont été prélevées mensuellement de la mi-octobre à la
mi-mars. Ces poules ont commencé à pondre début janvier. Les œufs ont été ramassés et comptabilisés
chaque jour. Les concentrations plasmatiques en triglycérides, en cholestérol et les valeurs absolues des
fractions alpha-1, beta-1 et beta-2 ont augmenté de manière importante dès l’entrée en ponte. Pendant la
même période, la fraction alpha-3, ainsi que la concentration en cholestérol HDL ont fortement diminué. Les
concentrations en albumine et en protéines totales ont augmenté régulièrement du début à la fin de l’étude.
Cette étude constitue une publication de référence des effets de la ponte sur les profils électrophorétiques des
oiseaux. L’augmentation des fractions beta-1 et beta-2 est liée à la mise en circulation de VLDLy à
destination des follicules chez la poule pondeuse. La diminution de la fraction alpha-3 correspond à une
diminution de la concentration plasmatique en HDL durant la même période. D’un point de vue clinique, les
changements observés sont susceptibles d’induire des erreurs d’interprétation des électrophorégrammes
aviaires en simulant la présence d’un processus inflammatoire aigu conduisant à une augmentation de la
fraction gamma.
79
Effects of egg-laying on plasma protein electrophoresis in hens (Gallus gallus)
RH: egg-laying and plasma protein electrophoresis in hens
Yannick Roman, Bertrand Bed’Hom, David Gourichon, Julie Levrier, Daniel Chaste-Duvernoy and
Marie-Claude Bomsel-Demontoy, Michel Saint Jalme.
From the Muséum national d’Histoire naturelle (MNHN), DJBZ, Le Parc de Clères, Clères, France (Roman,
Levrier), the INRA, AgroParisTech, UMR1236 GDA, Domaine de Vilvert, 78352 Jouy en Josas, France
(Bed’Hom), the INRA, Pôle Expérimental Avicole de Tours, UE1295, centre de Tours-Nouzilly, 37380
Nouzilly (Gourichon), the Laboratoire Bio-VSM, Torcy, France (Chaste-Duvernoy), the Muséum national
d’Histoire naturelle (MNHN), DJBZ, Ménagerie du Jardin des Plantes, Paris, France (Bomsel-Demontoy),
and the Muséum national d’Histoire naturelle (MNHN), EGB, UMR 5173 - CERSB - MNHN, CNRS, Paris
IV, Paris, France (Saint Jalme). Corresponding author: Yannick Roman (yannick.roman@cg76.fr).
80
Background: plasma protein electrophoresis is now commonly recognized to be a very reliable diagnostic
tool in avian medicine. However, the influence of circannual phenomena such as egg-laying on protein
electrophoregrams is poorly documented. Yet, the laying period is a period of heavy hormonal and metabolic
change in birds. Objective: The purpose of this study was to investigate the effects of egg-laying on total
protein concentration and electrophoresis patterns in birds. Methods: 20 hens (Gallus gallus) were blood
sampled monthly from mid October to Mid March. Hens started to lay eggs in early January. Eggs were
collected each day. Total protein, triglyceride, total cholesterol and HDL-cholesterol concentrations and
sample turbidity were measured and plasma agarose gel electrophoresis were performed on these samples.
The hens were chosen as a model, because these birds are able to lay one egg every 25 hour for several
months. Results: Triglyceride, cholesterol, alpha-1, beta-1 and beta-2 fraction concentrations started to
increase as soon as hens began to lay eggs. During the same period, the alpha-3 fraction and HDLCholesterol concentration were observed to decrease. Total protein and albumin concentrations appeared to
increase throughout this study. Conclusions: This study provides baseline information relevant to changes
occurring in avian proteinograms throughout the egg-laying. Increases in the beta-1 and beta-2 fractions
were related to the release in the bloodstream of VLDLy targeted to the vitellus in laying hens. The decrease
in the alpha-3 fraction corresponds to a decrease in the HDL plasma concentration during the same period.
From a clinical point of view, the observed changes associated with egg-laying could have completely
misled the practitioner by simulating an acute inflammatory condition leading to increases in the beta
fraction.
Keywords: Egg-laying, Plasma, Protein, Electrophoresis, bird
81
Plasma protein electrophoresis has proven to be very useful in avian medicine, as a routine screening test and
diagnostic tool. Used in conjunction with total protein measurements, with the Biuret reaction, it permits the
evaluation, diagnosis, and monitoring of a variety of diseases and conditions in birds.1-4 However, some
circannual physiological events such as egg-laying may induce heavy changes in plasma proteinograms,
which could potentially mislead the practitioner. Indeed, egg-laying is an event which, during the life of a
bird, produces a major metabolic perturbation. Egg-laying has been demonstrated to be responsible for a
22% increase in the basal metabolic rate in European starlings (Sturnus vulgaris),5 and for an increase of up
to 38-51% in laying hens.6 This energy expenditure is probably due to the synthesis of fatty acids, yolk
precursors and egg white proteins, the transport of these precursors to the egg, the laying down of the shell,
transfer of the egg through the oviduct, and maintenance of the reproductive organs.6 Heavy hormonal
changes occur within this period of time. The release of a gonadotropin-releasing hormone (GnRH) by the
hypothalamus induces the release of a follicle-stimulating hormone (FSH) and a luteinising hormone (LH)
by the pituitary gland. The latter stimulates the production of oestrogen by the ovary, which then triggers the
production of egg-yolk precursors.7-9 During egg-laying, the bird thus exports significant quantities of lipids
and proteins, which are needed for the production of eggs. In laying hens, eggs are indeed composed of an
average of 12% proteins and 12% lipids.10 While egg-white proteins are directly synthesized by the oviduct
wall, the lipids and proteins contained in the vitellus are synthesized by the liver and transported to the
vitellus by the bloodstream. When hens reach sexual maturity, liver protein synthesis increases threefold, and
lipid synthesis increases by a factor of 10.7 In domestic fowls, ducks and pigeons, the follicles undergo a
rapid growth phase 6 to 11 days prior to ovulation (up to 16 days in some penguin species).9,11 During this
period in hens, 2 g of protein per day are deposited in the yolk, which increases in volume by a factor of
3500 to 8000.9 Multiple serum proteins, including Very Low Density Lipoproteins (VLDL) and
vitellogenins, are therefore present in large quantities in the bloodstream of chickens during the laying
period.12,13
Such a phenomenon has been investigated by plasma and serum protein electrophoresis for research
purposes. For some authors, egg-laying does not correspond to an increase in total protein concentration.14
On the other hand, some other authors have noticed an increase in total protein quantity, one week before
egg-laying, followed by a return to normal levels at the inception of egg-laying.15,16 The most commonly
observed modification in the electrophoregrams of egg-laying birds is an increase in the pre-albumin
fraction.17-20 Concerning the alpha fraction, McKinley et al. (1953) and Polat et al. (2004) described a
decrease in alpha-1 fraction, until it becomes virtually inexistent, in egg-laying birds.21,22 This is
contradictory to the findings of Kuryl and Gasparska (1976), who described an increase in post-albumin
fraction during this same period.20 Some authors have documented a monoclonal increase in beta fraction
associated with egg-laying.3,4 This is contradictory to the statements of Kuryl and Gasparska (1985), who
described a decrease in pre- and post-transferrin fractions during egg-laying,20 and to the findings of Lush
(1963) who described two post-transferrin fractions which varied with opposite tendencies during the same
period.19 Finally, some authors have observed an increase in the gamma fraction during egg-laying.15,20,23
82
Egg-laying also appears to be associated with a decrease in the A/G ratio.14,24 The above-mentioned studies
thus present numerous contradictions, which can probably be accounted for by significant differences in the
electrophoretic techniques used. Most of these studies were made many years ago, and the techniques used
for the analysis of serum and plasma proteins have significantly progressed over the last 15 years. The only
study carried out using agarose gel produced results which differ from those of other studies, since it found
that in ostriches (Struthio camelus) the only changes which occurred during egg-laying were a reduction in
the alpha-1 fraction.22
The purpose of the present study was therefore to use agarose gel electrophoresis to implement a
more thorough investigation of the changes which occur in avian plasma proteins during egg-laying, through
the example of the chicken (Gallus gallus domesticus). A detailed understanding of these changes may help
the practitioner in future interpretations of avian electrophoregrams, and improve our understanding of egglaying physiology in birds. To this end, chickens were chosen as a model. In this particular species, the
domestication process has led to birds whose egg-laying is spread over a long cycle (40 to 50 weeks, with up
to 300 eggs per year), and for which brooding is eliminated through daily collection of the eggs.7 One could
therefore expect such an intense phenomenon to have a significant impact on plasma protein electrophoresis
patterns.
Our study was conducted on 20 hens which were blood sampled monthly, from October 2007 to
March 2008. The hens were 45 days old at the beginning of the study, and started to lay eggs in January. The
effects of egg-laying were studied by using agarose gel plasma protein electrophoresis to analyse the
collected samples.
Materials and methods
Experimental animals
The study was conducted on 20 White Leghorn laying hens, (Gallus gallus) held by the UE997 INRA,
Génétique Factorielle Avicole, Tours-Nouzilly (France), who were all born on September the 6th 2007. At
the beginning of the study, they were housed in collective cages (13 to 14 birds per cage), with simulated 10hour days. Egg-laying was triggered on January 6th by transferring the hens into individual cages, with
simulated 16 hour days. The hens started to lay eggs 10 days after their transfer into the laying cages and the
eggs were collected manually every day at 10 am in real time.
The hens were fed on commercial chick starter at first, and then on broiler pellets until they reached
maturity. They were then given laying hen pellets, as soon as they had been transferred to the laying battery.
In all cases, feeding was provided ad libitum.
The chicks were vaccinated with attenuated vaccines, by intramuscular injection for Marek’s disease
(Nobilis Rismavac©, Nobilis, Beaucouze, France) on September 6th 2007, and orally for infectious bronchitis
(Nobilis H120©, Nobilis, Beaucouze, France) on the same day. They were vaccinated for infectious
bronchitis (Nobilis IB 4-91©, Nobilis, Beaucouze, France) on September 16th, for infectious bursal disease
(Nobilis Gumboro D78©, Nobilis, Beaucouze, France) on September 23rd, for the Newcastle disease (Pestos
83
HB1©, Merial, Villeurbanne, France) on October 1st, for infectious bursal disease (Bursine 2©, Fort Dodge,
Tours, France) on October 14th, for the Newcastle disease and infectious bronchitis (Respectively: Nobilis
ND clone 30 © and Nobilis IB 4-91©, Nobilis, Beaucouze, France) on October 28th, for swollen head
syndrome (Aviffa RTI©, Merial, Villeurbanne, France) on November 11th, and for encephalomyelitis
(Encephalo nobilis ©, Nobilis IB 4-91©, Nobilis, Beaucouze, France) on December 9th.
At the time of their transfer to the laying cages, the hens were given intramuscularly booster doses of
inactivated vaccine against the Newcastle disease, infectious bronchitis, egg drop syndrome, swollen head
syndrome (Gallimune 407 ND+IB+EDS+ART ©, Merial, Villeurbanne, France) and infectious bursal
disease (Gumboriffa ©, Merial, Villeurbanne, France).
Samples
Blood samples were taken monthly in 2007, on October 23rd, November 20th, December 17th, and in 2008, on
January 21st, February 18th and March 10th, each time at 9 am. The blood samples were taken from the
brachial vein, using 22 G needles and 2 ml syringes (respectively: Terumo Neolus 22 G, Leuven, Belgium
and BD Plastipack, Octeville, France). Two millilitres of blood were drawn from each bird, collected in
lithium heparin tubes (Venosafe vacutainers, Terumo, Leuven, Belgium) and centrifuged at 3000 g for 10
minutes. Analyses were carried out on the resulting plasma. The plasma samples were split into two aliquots,
of which one was immediately sent by refrigerated parcel post (4°C) to the Bio-VSM laboratory for plasma
chemistry analysis and turbidity measurements. The analyses were thus carried out the day after the samples
were taken. The other aliquots were stored in cryotubes (-20°C) until they were needed for protein and
lipoprotein analysis (Micronic systems, Lelystad, Holland). All samples were thawed and re-homogenised by
gentle mixing on March 20th, one hour before analysis.
Total protein, triglyceride, total cholesterol and HDL-cholesterol (HDL-C) concentrations, and
turbimetry measurements
Total protein concentration was determined with the Biuret reaction, using a Roche Integra 400 wet
chemistry analyser (Roche Integra 400©, Roche diagnostics, Meylan, 38242, France). Readings were made
at a wavelength of 552 nm. Triglyceride concentrations were determined with the same analyser, at a
wavelength of 512 nm, using Trinder’s reaction (GOP/PAP). Total cholesterol concentration was determined
by an enzymatic colorimetric method: cholesterol esters were cleaved by the action of cholesterol esterase in
order to yield free cholesterol and fatty acids. Cholesterol oxidase then catalyzed the oxidation of cholesterol,
producing cholest-4-en-3-one and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide
reacted with phenol and 4-aminoantipyrine to form a red quinone-imine dye, whose colour intensity was
directly proportional to the cholesterol concentration. The readings were taken at 512 nm. The same principle
was used to measure HDL-C concentrations, except for the fact that in this case, enzymes coupled with
polyethylene glycol (PEG) to the amino groups were used. In the presence of magnesium ions and dextran
sulfate, water-soluble complexes are formed with LDL, VLDL, and chylomicrons, which become resistant to
84
PEG-modified enzymes, contrary to HDL. Turbidity measurements were carried out with the same analyser
at 659 nm, since other forms of interference (such as hemolysis) are known to be low at this wavelength.25
Light of a known intensity “I0” was passed through the sample, on the other side of which the transmitted
intensity “I” (“I0” less the absorbed light) was measured. The turbidity τ = ln (I0/I) was recorded in
nephelometric turbidity units (NTU).26,27
Plasma protein and lipoprotein agarose gel electrophoresis
Agarose gel electrophoresis were carried out using a Hydrasys© semi-automated system (Sebia, Evry,
France), using the Hydragel protein 15/30© set for plasma protein electrophoresis, and the Hydragel 30
lipoprotein © set for plasma lipoprotein electrophoresis (Sebia, Evry, France). It was operated according to
the manufacturer’s instructions, using version 7.00 F0.1 of the system software. Ten microlitres of plasma
samples were manually distributed onto the sample template applicator, and were allowed to diffuse for a
period of 5 minutes in a wet chamber. Application of the samples to the gel, electrophoresis, and drying of
the gel were all performed automatically in the migration compartment of the instrument. The temperature
was maintained at 20°C using a Peltier device during the complete migration process, and drying was
obtained by heating the gels to 65°C. Electrophoretic separation was obtained on 8 g/L agarose gels, in a
Tris-barbital buffer, at pH 9.2 and a constant power level of 20W, until 33 Vh had been accumulated, for
protein electrophoresis, and at pH 8.5 and a constant voltage of 160V for lipoprotein electrophoresis. For
plasma protein electrophoresis, once they had been dried, the gels were manually transferred to the staining
compartment of the instrument where amido-black staining, destaining and drying were performed
automatically. For plasma protein lipoprotein electrophoresis, the gels were manually coloured with Sudan
black stain provided in the Hydragel 30 lipoprotein © set (Sebia, Evry, France). Once these operations had
been completed, the gels were scanned with a high resolution Epson perfect V700 photo scanner (Epson
France, Nanterre, France). Electrophoretic curves and quantification of the different fractions were acquired
using version 5.50 of the Phoresis© software (Sebia, Evry, France).
Statistical analysis
Descriptive statistical analysis was carried out using Microsoft Excel 2003©. Systat 7.0 software (Systat 7.0,
Systat Software Inc., London, United Kingdom) was used for all aother analysis. In view of the small size of
the studied population, Friedman’s two way analysis of variance and Wilcoxon’s signed rank test were used
to investigate parameters of the same birds at different times. Kruskal and Wallis’ one way variance analysis
test was used to investigate the differences between hens, which laid eggs on the day the sample was drawn,
and the other hens, and between the hens which laid eggs on the day following that of the sample, and the
other hens. Correlations were investigated using Spearman’s rank correlation test.
85
Results
The Egg-laying curve is displayed in Figure 1. The hens began laying eggs on January 16th.
Figure 1: egg laying curve of the hens. This graph is represents the mean number of eggs layed per hen and
per day from January 15th to March 31st.
The hens’ plasma protein electrophoresis patterns were divided into eight fractions, which were then
classified into one prealbumin fraction, one albumin fraction, three alpha fractions, two beta fractions and
one gamma fraction (Figure 2).
Figure 2: examples of plasma electrophoresis patterns of a hen (Gallus gallus). Patterns from the same bird
before the laying period (Grey curve) and during the laying period (Black curve) were superimposed. The
time samples when were drawn is indicated with arrows.
86
Figure 3: variation of (a) albumin, (b) alpha-1, (c) alpha-3, (d) beta-1, (d) beta-2 and (e) gamma fraction
concentrations around the beginning of egg-laying in hens (Gallus gallus). This graph is based on
measurements of monthly blood samples, taken between mid October 2007 and mid March 2008. The data
points are expressed as: mean ± SEM. Asterisks represent significant differences between two successive
measures (Wilcoxon’s test: p<0.05).
87
Figure 4: example of protein and lipoprotein electrophoresis agarose gels of hens before and during the
laying period.
Figure 5: variation of (a) total plasma protein, (b) triglyceride, (c) total cholesterol and (d) HDLcholesterol concentrations around the beginning of egg-laying in hens (Gallus gallus). This graph is based
on measurements of monthly blood samples, taken between mid October 2007 and mid March 2008. The
data points are expressed as: mean ± SEM. Asterisks represent significant differences between two
successive measures (Wilcoxon’s test: p<0.05).
88
Some of the measured parameters appeared to show dramatic changes, corresponding to the periods
(in January) during which the hens began to lay eggs. The beginning of the laying period was thus associated
with significant increases in alpha-1 (3-4 fold), beta-1 (1.3-1.5 fold) and beta-2 (1.5-2 fold) fractions, and
with a significant decrease in alpha-3 fraction (1.2-1.3 fold) (Figure 2 and 3). The gamma fraction appeared
to be significantly higher in March than in December (p < 0.05; Z = 2.539), indicating an increase between
these two dates (Figure 3). Increases in beta-1 and beta-2 fractions were due to the appearance, as soon as
egg-laying had begun, of a new band located in the beta fraction (Figure 4). This band was characterised by
its variable position, which ranged from that of the beta-1 peak to that of the beta-2 peak, and by its irregular
anodal front. It resulted in a monoclonal peak, migrating between, and partially obscuring, the beta-1 and
beta-2 peaks (Figure 2).
On the other hand, some fractions, such as pre-albumin and alpha-2, did not appear to be subject to variations
related to the reproductive status of the birds, since no significant change was recorded at the time when the
hens began laying eggs. The total protein and albumin concentrations were generally observed to increase as
a function of time, throughout this study (Figure 5).
The A/G ratio decreased significantly from October to November, following which it then increased
significantly until December, and then decreased again when the hens began to lay, as a result of the
presence of increased alpha-1, beta-1 and beta-2 fractions.
The hens’ lipidograms were divided into two fractions: fast migrating lipoproteins and slow migrating
lipoproteins (Figure 4). The fast migrating lipoproteins may correspond to the High Density Lipoproteins
(HDL). However, in the case of the slow migrating lipoproteins, it was difficult or even impossible, in laying
hens, to distinguish between the Very Low Density Liprotein (VLDL), and the Low density Lipoprotein
(LDL) fractions.
The beginning of the laying period also corresponded to major changes in lipid measurements values and
lipidograms. This is the case for triglyceride and total cholesterol, whose concentrations increased
significantly at the beginning of egg-laying (Figure 5). During this period, the triglyceride concentration
increased 20-30 fold, and the total cholesterol concentration increased 1.7 fold. At the same time, the HDL-C
concentration decreased significantly, by 3 to 8 fold. Such changes were also observed in the hens’
lipidograms (Figure 4). Slow migrating lipoproteins, which represented only 31.4 ± 7.7 % of the blood’s
lipoproteins before egg-laying, represented 86 ± 9% of the latter after egg-laying had begun. In the
meantime, the fast migrating lipoproteins dramatically decreased in the same proportions.
Plasma samples drawn during the laying period usually had a milky visual appearance. Their turbidity tended
to increase, from the beginning of the study in October until the month of February. However, changes
recorded after the beginning of egg-laying in January did not appear to be significant, due to wide interindividual variations.
In October, November and December, before egg-laying had begun, the total cholesterol and HDL-C
concentrations were strongly correlated (Respectively: p < 0.01, n = 20, Rs = 0.875; p < 0.01, n = 20, Rs =
0.837; p < 0.02, n = 20, Rs = 0.527). Once egg-laying began, these correlations disappeared. On the other
89
hand, the triglyceride and total cholesterol concentrations appeared to be strongly related in January,
February and March (Respectively: p < 0.01, n = 20, Rs = 0.576; p < 0.01, n = 20, Rs = 0.750; p < 0.01, n =
20, Rs = 0.750), which was not the case before egg-laying. The correlation between HDL-C concentrations
and the alpha-3 fraction was highly significant throughout this study, indicating that HDL could migrate to
the alpha-3 fraction (respectively: p < 0.01, n = 20, Rs = 0.654; p < 0.01, n = 20, Rs = 0.794; p < 0.02, n =
20, Rs = 0.533; p < 0.01, n = 20, Rs = 0.823; p < 0.02, n = 20, Rs = 0.558; p < 0.02, n = 20, Rs = 0.530). On
the other hand, the triglyceride concentration appeared to be strongly correlated with the sum of the beta-1
and beta-2 fractions, from January to March (Respectively: p < 0.01, n = 20, Rs = 0.720; p < 0.02, n = 20, Rs
= 0.548; p < 0.01, n = 20, Rs = 0.750), although this was not the case from October to December.
No significant difference was found, for any of the parameters, between those hens, which laid eggs
on the day the sample was drawn, and the other hens, or between the hens which laid eggs on the day
following that of the sample, and the other hens.
The HLD-C, Albumin, and beta-2 fraction concentrations appeared to be correlated to the “laying intensity”,
which was expressed by the average number of eggs laid per hen, per day, for the period ranging from 5 days
before until 5 days after the drawing of samples. (Respectively: p < 0.02, n = 52, Rs = -0.334; p < 0.01, n =
52, Rs = 0.369; p < 0.05, n = 52, Rs = 0.305).
Discussion
The present study provides baseline information relevant to the changes which occur in avian
proteinograms throughout the egg-laying period. It clearly stresses that egg-laying coincides with an
indisputable increase in alpha-1, beta-1 and beta-2 fractions. In addition, our study reveals a decrease in the
alpha-3 fraction during the same period. Total protein and albumin concentrations increased throughout this
study. Some of these recorded changes are likely to completely mislead the practitioner. Indeed, increases in
the beta fraction and subsequent decreases in the A/G ratio are usually characteristic of an ongoing
inflammatory condition.3,4,24 It is therefore essential to take into account a bird’s physiological status, at the
time samples are drawn, in order to correctly interpret its plasma protein electrophoregrams.
The changes observed in the alpha-1, alpha-3, beta-1 and beta-2 fractions, as well as in the
triglyceride, total cholesterol and HDL-C concentrations, appeared suddenly at the beginning of the birds’
egg-laying process. In addition, it was found that the variations in HDL-C, albumin, and beta-2 fraction
concentrations are correlated with egg-laying intensity.
Throughout our study, increases in total protein concentration occurred monotonically, from the beginning to
the end of the study period, and may be related mainly to the growth of the chickens. Indeed, in growing
chickens and pheasants, total protein concentration has already been described to significantly increase with
growth, due to increases in most of the protein fractions.15,28,29 This result matches that reported by Sturkie
and Newman (1951), who did not observe any increase in total protein concentration directly related to the
egg-laying condition in hens.14 Contrary to other numerous publications,17-20 we did not observe any change
in the pre-albumin fraction. Nevertheless, the increase in alpha-1 fraction observed in the present study could
90
correspond to the increases in pre-albumin fraction reported in the other studies. Changes in electrophoretic
mobility related to the use of agarose gel electrophoresis, instead of other techniques such as moving
boundary, starch gel and polyacrylamide electrophoresis, could have been responsible for such a
phenomenon. Indeed, the presence or absence of the pre-albumin fraction has been described to be partially
determined by the nature of the buffer solution used.15 The decrease in alpha-3 fraction found in the present
study may correspond to the decrease in pre-transferrin fraction described by Kuryl and Gasparka (1985),
using polyacrylamide gel electrophoresis.20 Concerning the changes found in the beta fraction, our results
closely match the suggestions of Cray and Tatum (1997) and Werner and Reavill (1999).3,4 Changes in the
beta fraction during egg-laying occur in the form of a monoclonal peak which merges with the beta-1 and
beta-2 fractions. The slight increase in gamma fraction observed in the present study, during the egg-laying
period, is also consistent with the findings of several previous studies.15,20,23 The decreases observed in the
A/G ratio during egg-laying have already been documented by other authors,24,24 and may be related to the
dramatic increase in globulin fractions, in particular the alpha-1, beta-1 and beta-2 fractions, in the case of
our study. The observed decrease in A/G ratio, between the sample taken in October and that taken in
November, could possibly be attributed to the vaccinations carried out on the 1st and 14th of October.
As far as we know, our study is only the second to have investigated the effects of egg-laying in
birds, using plasma protein electrophoresis performed on agarose gel. In a previous study carried out on
ostriches (Struthio camelus), Polat et al. (2004) found no changes in any of the protein fractions, except for
the alpha-1 fraction.22 In addition, these authors described a significant decrease in this latter fraction, as
opposed to the increase found in the present study. The small sample size used by Polat et al. could explain
the fact that no changes were recorded in fractions other than alpha-1. However, such significant differences
may also be due to inter-species differences. Changes in plasma protein levels which occur during egg-laying
may depend on the balance between the rate of serum protein production by the liver and the rate at which
these proteins are used for egg production.15
The hen model used in our study had the advantage of being unaffected by the potential influences
associated with hatching of the eggs, since the eggs were collected as they were laid. However, laying hens
are able to lay up to 300 eggs per year, at mean intervals of 25 hours.7 It would thus be of considerable
interest, in future studies, to study the effects of egg-laying under natural conditions, in wild species whose
broods are less prolific than in the case of the laying hen (e.g. an average of 10 eggs per clutch for
anseriformes, wild galliformes, or struthionidae).30-33
In the present study, we have revealed dramatic changes in avian lipidograms. Triglyceride
concentrations increased strongly as soon as egg-laying began. This increase in triglyceride concentration is
a result of a dramatic estrogen-induced increase in VLDL levels, which become the main lipoprotein class
involved in the transport of triglycerides in laying birds.13,34,35 Indeed, in laying hens, Hermier et al. (1989)
have reported that the VLDL concentration increases from 2 to 300-900 mg/dl.13 During egg-laying, the liver
packages and secretes triglycerides and phospholipids in special yolk-targeted VLDL called VLDLy.36,37 In
our study, such VLDLy may have migrated between the beta-1 and beta-2 fractions, as suggested by the
91
excellent correlation between the triglyceride concentration and the beta fraction value during the laying
period. The VLDLy have half the size of normal VLDL, and are more uniform in diameter,35 which may
explain the thin monoclonal band reported in the beta fraction of laying hens. The appearance of this thin
band closely corresponds to the description of the beta-lipoprotein band in human electrophoregrams: its
anodal front is irregular, and its position varies, depending on its plasma concentration.38
Herein, the decrease of HDL-C concentration during the laying period is consistent with the changes
reported in the lipidograms, and with other publications which describe a strong decrease in HDL plasma
concentration and apolipoprotein A-I synthesis by the liver.13,39,40 The very high correlation between the
HDL-C concentration and the alpha-3 fraction value throughout this study may indicate that HDL migrate to
the alpha-3 fraction in hens.
The excellent correlation between total cholesterol and HDL-C concentrations is a reflection of the fact that
this class of lipoprotein has been shown to contain up to 75-80% of the circulating cholesterol in some bird
species.34,41 After egg-laying has begun, the excellent correlation between total cholesterol and triglyceride
concentrations may indicate that most of the cholesterol is transported by VLDLy during egg-laying.
Vitellogenins, whose concentrations is also known to increase during egg-laying, and whose density is lower
than that of HDL,12,34 could be responsible for the increase in alpha-1 fraction observed in this study.
However, further studies would be needed before this hypothesis can be confirmed.
The fact that, in the current study, no difference was found between hens which had laid an egg on the day of
the blood sample, and the other hens, appears to be normal since other authors have already observed
changes related to egg-laying in hens, as early as two weeks before laying of the first egg.15
The laying hens used in this study seem to have provided a good model for the investigation of
changes related to egg-laying. However, we cannot exclude that some of the changes observed in our study
might have resulted from the vaccination protocol, or from the stress induced by the transfer of the hens into
individual cages. Both of these phenomena could have contributed to an increase in the alpha or beta
fractions, and the vaccinations could have been at least partly responsible for the observed increase in
gamma fraction.42-44 However, the changes in lipid measurements values and lipidograms recorded in our
study closely match those described in the literature relevant to egg-laying.13,34,35,39,40 This may also be the
case for the strong changes recorded in the proteinograms, which are furthermore very well correlated with
the latter measurements.
Acknowledgements
We wish to thank P. TROLLIET from Sebia, for his invaluable technical support, as well as the team from
bio VSM, in particular Mrs. K. Naudin, who carried out the total protein measurements. We thank the
Museum national d’Histoire naturelle and the Conseil Général de Seine Maritime (France) for their financial
support. We also appreciate the assistance provided by Techtrans Consulting, in the proofreading of this
document.
92
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CHAPITRE III : variations des électrophorégrammes aviaires liées à
des interférences analytiques
Article 5: Effets de l’hémolyse sur la concentration en protéines totales et sur les profils
d’électrophorèse des protéines plasmatiques chez les oiseaux.
Cet article a été soumis au Journal of wildlife diseases.
Résumé
Durant les 15 dernières années, l’utilisation de l’électrophorèse des protéines plasmatiques en médecine
aviaire a fait l’objet de quelques publications scientifiques et cet examen complémentaire est actuellement
reconnu comme étant un outil fiable dans le diagnostic de nombreuses maladies. Malheureusement, cette
technique est toujours sous-utilisée en médicine aviaire en raison de nombreux facteurs pouvant interférer
avec les profils électrophorétiques, dont l’hémolyse. L’hémolyse se définit comme la libération du contenu
cellulaire des érythrocytes et d’autres cellules sanguines dans le liquide extracellulaire. Ce phénomène est
commun, particulièrement en médecine aviaire et trouve principalement son origine dans des fautes de
manipulation des prélèvements sanguins. En médecine humaine l’hémolyse est connue pour constituer une
des principales interférences analytiques pouvant conduire à l’obtention d’un résultat erroné. L’influence
d’une telle interférence sur les électrophorégrammes aviaire n’est que succinctement décrite dans un article
de synthèse bibliographique et n’a fait que brièvement l’objet d’une publication traitant plus largement de
l’utilisation de l’électrophorèse des protéines plasmatiques chez les psittacidés. L’objectif de cette étude était
donc d’étudier de manière plus approfondie l’effet de cette interférence sur les électrophorégrammes des
oiseaux et d’analyser d’éventuelles différences interspécifiques. A cet effet, des prélèvements sanguins ont
été réalisés sur 28 milans noirs (Milvus migrans) et 19 oies à tête barrée (Anser indicus) et séparés en deux
aliquotes. L’un des deux a été plongé dans de l’azote liquide durant cinq secondes pour entrainer une
hémolyse par congélation-décongélation avant centrifugation. Les plasmas hémolysés et non hémolysés ont
été utilisés pour mesurer les concentrations en protéines totales et en hémoglobine plasmatique et déterminer
les profils d’électrophorèse des protéines. Dans les deux espèces étudiées, l’hémolyse a entrainé une
surestimation de la quantification des protéines totales. Chez l’oie à tête barrée, l’hémolyse s’est soldée par
une augmentation de la fraction gamma. Chez le milan noir, cette augmentation a été observée, non
seulement dans la fraction gamma, mais également dans la fraction beta, ce qui met en évidence des
différences interspécifiques avec l’oie à tête barrée. Dans les deux espèces, les changements observés lors de
l’hémolyse des prélèvements évoquent fortement ceux qui sont décrits lors de processus inflammatoires
chroniques. Ils seraient donc susceptibles d’induire en erreur le praticien dans l’établissement d’un
diagnostic.
97
98
Roman, Bomsel-Demontoy, Levrier, Chaste-Duvernoy and, Saint Jalme
Hemolysis effects on avian plasma protein concentration and electrophoresis
Effect of hemolysis on plasma protein levels and plasma electrophoresis in birds
Yannick ROMAN1,5, Marie-Claude BOMSEL-DEMONTOY2, Julie LEVRIER1 , Daniel CHASTEDUVERNOY3 and Michel SAINT JALME4
1
Muséum national d’Histoire naturelle (MNHN), DJBZ, Le Parc de Clères, 32 avenue du Parc, 76690
Clères, France
2
Muséum national d’Histoire naturelle (MNHN), DJBZ, Ménagerie du Jardin des Plantes, 57 rue Cuvier,
75005 Paris, France
3
Laboratoire Bio-VSM, 3 bis rue Pierre Mendès-France, 77200 Torcy, France
4
Muséum national d’Histoire naturelle (MNHN), EGB, UMR 5173 - CERSB - MNHN, CNRS, Paris IV, 57
rue Cuvier, 75005 Paris, France
5
Corresponding author (tel: +33(2)35332308, fax: +33(2)35331166, e-mail: yannick.roman@cg76.fr)
99
ABSTRACT
Within the last 15 years, application of protein electrophoresis in clinical avian medicine has been the focus
of several publications and it is now commonly recognized to be a reliable diagnostic tool for many
pathologic conditions in birds, even though it is seldom pathognomonic for a specific disease. Unfortunately,
this technique is still underused in avian medicine because many factors may interfere with electrophoresis
patterns. Hemolysis is one of these factors. Hemolysis can be defined as the release of intracellular
components from erythrocytes and other blood cells into the extracellular fluid. This phenomenon is
common, especially in birds, and is mainly related to improper specimen handling. In human laboratory
medicine, it is known to be an interference factor that can lead to erroneous results. The influence of
hemolysis on protein electrophoresis in birds has only been quoted in a review article and investigated in a
single study in psittacidae. The aim of this study was therefore to investigate more thoroughly this effect and
to analyse potential interspecific differences. Blood samples were drawn from 28 black kites (Milvus
migrans) and 19 bar headed geese (Anser indicus) and separated into two aliquots. One of them was dipped
into liquid nitrogen for five seconds in order to cause freeze-thawing hemolysis before centrifugation. Total
plasma protein concentration, plasma hemoglobin concentration and plasma protein electrophoresis patterns
were determined for both hemolysed and non hemolysed samples. In both species, hemolysis resulted in
falsely high total plasma protein concentration. In bar headed geese, hemolysis caused a rise in the gamma
fraction. In black kites, this rise involved not only gamma fraction but also beta fraction stressing
interspecific differences. In both species, these changes could have mimicked a chronic inflammatory
condition with its resulting antigenic stimulation, and could have therefore completely misled a practitioner.
KEYWORDS
Hemolysis, Interference, Protein Electrophoresis, Bird.
100
INTRODUCTION
Serum protein electrophoresis has been extensively used for decades with human specimens for diagnostic
purposes (Dimpopullus, 1961; Daunizeau, 2003). It consists in a separation of serum proteins into different
protein fractions by an electric field. The acquisition of the electrophoregrams using a densitometer allows
technicians to obtain electrophoretic curves. The concentrations of the different fractions are then calculated
by multiplying the percentage of the area under a given curve times the total protein concentration
determined by the Biuret reaction (Daunizeau, 2003).
In human laboratory medicine, this technique is commonly used to evaluate, diagnose, and monitor a variety
of diseases and conditions. Indeed levels of different serum proteins rise or fall in response to such disorders
as cancer, intestinal or kidney protein-wasting syndromes, disorders of the immune system, liver
dysfunction, impaired nutrition, chronic fluid-retaining conditions and inflammatory conditions (Le Carrer,
1998; Daunizau, 2003).
The use of protein electrophoresis in veterinary medicine and especially in avian medicine is far more recent
than in human medicine. Within the last 15 years, application of protein electrophoresis in clinical avian
medicine has been the focus of several publications and some avian veterinarians have recognized plasma
protein electrophoresis to be a reliable diagnostic tool for many pathologic conditions (Lumeij, 1987; Cray
and Tatum, 1998; Werner and Reavill, 1999). Unfortunately, this technique is still underused in avian
medicine because many factors may interfere with electrophoresis patterns. Hemolysis is one of these factors
needing to be investigated more thoroughly.
In human laboratory medicine, hemolysis is considered as one of the main interference factors, which is
defined by Kroll and Elin (1994) as “the effect of a substance present in the sample that alters the correct
value of the result usually expressed as concentration or activity for an analyte”. It mainly consists of the
release into the extracellular fluid of hemoglobin and other intracellular components from erythrocytes,
following damage or disruption of the cell membranes. Release of hemoglobin usually gives a characteristic
pink to red tinge to the plasma or the serum (Kirschbaumweg, 2001; Lippi et al., 2006). Hemolysis is quite a
common phenomenon in both human and veterinary medicine (Andreasen et al. 1996, 1997; Benlakehal et
al., 2000; Thomas, 2001). In avian medicine, 4.55% of the samples were demonstrated to be hemolysed
(Fudge, 2000). In birds, in vivo hemolysis is scarce (0.3% of the samples) (Fudge, 2000). It can be a feature
during acute lead toxicosis, and has been described in shore birds, after ingestion of crude oil (Fry and
Addiego, 1987; Fudge, 2000). In vitro hemolysis is more common in birds, since it has been described to
affect up to 4.25 % of the specimens (Fudge, 2000). Hemolysis is related to improper specimen handling
during all steps of the pre-analytical phase, like forcing blood through small needles during sampling, long
storage of the blood before centrifugation or excessive agitation when mixing (Guder, 1986).
Constituents released in the plasma or in the serum from broken blood cells can interfere with laboratory
analysis in several ways which are already heavily documented in human medicine (Benlakehal et al., 2000;
Pontet, 2000; Vermeer et al., 2007) and have been investigated in birds in only two studies (Andreasen et al.
101
1996, 1997). Dealing with plasma or serum total protein concentration measurements, hemolysis is known to
lead to artefactual high values (Andreasen et al., 1996, 1997; Pontet, 2000; Vermer et al., 2007). Fewer
studies report to the influence of hemolysis in agarose gel protein electrophoresis. In mammals, and
especially in humans, hemolysis is known to generate artefacts in serum electrophoresis: hemoglobinhaptoglobin complexes move to the alpha 2 fraction and free hemoglobin migrates to the beta fraction
(Bossuyt et al., 1998; Benlakehal et al., 2000; Thomas, 2001). In some circumstances, this mistake can
completely mislead the practitioner (Alanio-Brechot et al., 2006). In birds this phenomenon is poorly
documented. It has only been described by Werner and Reavill (1999) in a review article and recently
investigated in psittacine birds by Cray et al. (2007). It is described to cause an increase in the gamma
fraction. However, these studies were respectively based on isolated cases and were only investigated in
psittacine birds.
The purpose of the study reported here was therefore to investigate more thoroughly the effects of hemolysis
in plasma protein concentration measurements and electrophoresis patterns and to investigate potential
interspecific differences basing on two distant taxa.
MATERIALS AND METHODS
This study was conducted with 19 bar headed geese (Anser indicus) held at the Clères zoological park
(France) and 28 black kites (Milvus migrans) held at the Académie de fauconnerie du Puy du fou (France).
The blood samples were performed on the occasion of a veterinary screening protocol conducted in October
and November 2007 respectively. All the birds looked clinically healthy. All analyses were carried out on
plasma. Blood samples were taken from the brachial vein for bar headed geese and from the right jugular
vein for black kites, using 21 G needles and 5 ml syringes (respectively: Terumo Neolus 23 G, and Terumo
Syringe 5 ml, Terumo Europe N.V., Leuven, 3001, Belgium). Four millilitres of blood were drawn from each
bird and collected in lithium heparin tubes (Venosafe vacutainers, Terumo, Leuven, Belgium). Heparinised
blood samples were immediately split into two dry tubes (Venosafe vacutainers, Terumo, Leuven, Belgium).
Once all blood samples were performed, one of them was centrifuged at 3000 g for 5 minutes while the other
one was first dipped into liquid nitrogen for 5 seconds in order to cause freeze-thawing hemolysis (Thomas,
2001; Lippi et al., 2006). Plasmas were then stored in cryotubes (Micronic systems, Lelystad, Holland) until
analysis, at -20°C (-4°F), for one month in black kites and two month in bar headed geese.
Samples were thawed and rehomogenised by gentle mixing 1 hour before analysis.
Total plasma protein concentration was determined by the Biuret reaction using a Roche Integra 400 wet
chemistry analyser (Roche Integra 400©, Roche diagnostics, Meylan, 38242, France). Readings were made
at a wavelength of 552 nm.
Agarose gel electrophoresis was carried out using a Hydrasys© semi-automated system (Sebia, Evry,
France), using the Hydragel protein 15/30© set (Sebia, Evry, France) which is the most commonly used kit
for protein electrophoresis in medical laboratories. Plasma aliquots were loaded onto the gel and
102
electrophoretic separation was obtained on 8 g/l agarose gels in a Tris-barbital buffer (pH 9.2), at 20°C at
constant power level of 20W, until 33 Vh had been accumulated. Once dried and coloured with amidoblack
in the same system, gels were read with a densitometer Preference (Sebia, Evry, France). This densitometer
enabled us to obtain electrophoresis patterns, to define protein fractions and to measure Area Under the
Curve (AUC) for each fraction. Albumin was identified as the biggest and most anodal peak and was decided
to include pre-albumin fraction. The beta peak was defined as corresponding to the fibrinogen peak.
Globulins were divided into 4 to 5 fractions depending on the species. Alpha fractions were located between
albumin and beta peaks, and gamma peaks were located beyond the beta peak. As previously described in
other studies about avian protein electrophoresis, the A/G ratio was calculated by dividing the sum of
prealbumin and albumin by the sum of the globulin fractions (Lumeij, 1987; Cray and Tatum 1998). Effect
of hemolysis in electrophoresis patterns was investigated by comparing Areas Under the Curves (AUC) for
each fraction rather than protein concentration values, since in hemolysed samples total protein concentration
was supposed to be erroneous (Andreasen et al. 1996, 1997).
Hemoglobin concentration was estimated by the oxy-hemoglobin method, as described by Howlett (2000),
except we used 40 µl of plasma instead of 20 µl of blood. Plasma were diluted in 5 ml of a 0.04 % ammonia
solution and placed on a roller mixer for 3 minutes. Samples were then transferred in spectrophotometric
cuvettes for immediate reading. Absorbances were read at 540 nm with a Helios Delta spectrophotometer
(Thermo electron corporation, Courtaboeuf, France). Before processing the samples, the spectrophotometer
was set to zero using the dilution solution alone as a blank. Machine readings were then directly converted
into g/l referring to a calibration graph established thanks to a commercially available hemoglobin standard
(Coulter 5C control, Beckman Coulter inc., Roissy, France).
Migration distances were measured directly on the gels. These measures were made from the edge of the gels
with a 6’’ dial calliper square (General, Montreal, Canada).
Systat 7.0 software (SPSS inc. © 1997) was used for all analyses. Considering the small size of the
population studied, Wilcoxon’s signed rank test, Mann and Whitney’s U test and Spearman rank correlation
were used.
RESULTS
In both species tested, freeze-thawing hemolysis resulted in a significant increase in plasma
hemoglobin (Tables 1 and 2). After hemolysis, mean hemoglobin concentration values raised from 0.07 to
1.13 g/l in bar headed geese and from 0.08 to 0.5 g/l in black kites.
In both species, freeze-thawing hemolysis resulted in a significant increase in total protein concentration and
total Area Under the Curve (AUC). Total protein concentration furthermore appeared to be very well
correlated to plasma hemoglobin concentration (Spearman’s test: n = 19, Rs = 0.859, p < 0.01; n = 28, Rs =
0.486, p < 0.01).
103
Bar headed geese electrophoresis patterns were divided into five fractions (Fig. 1) and black kite
electrophoresis patterns into six (Fig. 2). Black kites’ patterns showed a high alpha 1 peak and two gamma
peaks.
In both species tested, freeze-thawing hemolysis resulted in a significant increase in total Area Under the
Curve (AUC) (Tables 1 and 2). Main changes related to hemolysis occurred in the albumin and gamma
fractions.
Table 1: effects of hemolysis on total protein concentration, hemoglobin concentration and electrophoresis
patterns of bar headed geese (Anser indicus).
Parameter
Total protein
concentration (g/l)
Hemoglobin
concentration (g/l)
Total AUCb
Albumin AUCb
Alpha 1 AUCb
Alpha 2 AUCb
Beta AUCb
Gamma AUCb
A/G
a
Mean ± SD
b
Non-hemolysed
samplea
Hemolysed
samplea
56.4 ± 3.6
67 ± 8.1
Wilcoxon
Wilcoxon
matched pairs matched pairs
p value
Z value
3.783
< 0.01*
0.07 ± 0.03
1.13 ± 0.58
3.823
< 0.01*
1707.6 ± 288.1
950.9 ± 169.2
92.9 ± 17.3
243.5 ± 63.2
331.5± 60.8
88.8 ± 22.9
1.26 ± 0.14
1897.6 ± 328.9
805.8 ± 129.3
91 ± 15.5
236.3 ± 58
344.4 ± 72.6
420.1 ± 199.6
0.8 ± 0.25
2.575
-3.501
-0.805
-1.730
0.926
3.823
-3.823
0.01*
< 0.01*
0.42
0.08
0.36
< 0.01*
< 0.01*
Area Under the Curve
* Significant differences between groups; Wilcoxon’s test: p ≤ 0.05.
Table 2: effects of hemolysis on total protein concentration, hemoglobin concentration and electrophoresis
patterns of black kites (Milvus migrans).
Parameter
Total protein
concentration (g/l)
Hemoglobin
concentration (g/l)
Total AUCb
Albumin AUCb
Alpha 1 AUCb
Alpha 2 AUCb
Beta AUCb
Gamma 1 AUCb
Gamma 2 AUCb
A/G
a
Mean ± SD
b
Non-hemolysed
samplea
Hemolysed
samplea
Wilcoxon
matched pairs
Z value
Wilcoxon
matched pairs
p value
35.5 ± 3.5
37.7 ± 3.7
3.496
< 0.01*
0.08 ± 0.05
0.5 ± 0.42
4.623
< 0.01*
1352.5 ± 84.9
604.3 ± 33.6
450 ± 52
27.4 ± 5.5
171 ± 40
26.7 ± 8.7
69.8 ± 19
0.81 ± 0.09
1419.7 ± 99.1
557.2 ± 37.3
423.2 ± 43.7
30 ± 5.3
222.2 ± 37.4
89.9 ± 51.1
93.5 ± 19.4
0.65 ± 0.09
3.142
-3.803
-4.076
1.526
4.463
4.623
4.623
-4.623
0.02*
< 0.01*
< 0.01*
0.13
< 0.01*
< 0.01*
< 0.01*
< 0.01
Area Under the Curve
* Significant differences between groups; Wilcoxon’s test: p ≤ 0.05.
104
Figure 1: examples of plasma electrophoresis patterns of a bar headed goose (Anser indicus). Patterns from
the non hemolysed sample (Grey curve) and the hemolysed sample of the same bird
(Black curve) were
superimposed. Asterisks represent significant differences between hemolysed and non hemolysed samples
(Wilcoxon’s test: p ≤ 0.05).
Figure 2: examples of plasma electrophoresis patterns of a Black kite (Milvus migrans). Patterns from the
non hemolysed sample (Grey curve) and the hemolysed sample of the same bird (Black curve) were
superimposed. Asterisks represent significant differences between hemolysed and non hemolysed samples
(Wilcoxon’s test: p ≤ 0.05).
105
The hemolysed samples showed a significant decrease in the albumin fraction AUC in both species. Changes
in these fractions represented a 17 % average decrease in bar headed geese and a 8 % average decrease in
black kites.
In both species, hemolysed samples showed a significant increase in the gamma fraction AUC. Changes in
this fraction represented a 441 % average increase of the gamma fraction in bar headed geese and a 235 %
average increase in the gamma 1 fraction in black kites. In addition, these values appeared to be very well
correlated to hemoglobin concentration (Spearman’s test: n = 19, Rs = 0.900, p < 0.01; n = 28, Rs = 0.561, p
< 0.01).
Hemolysis resulted in a significant decrease in the A/G ratio in the both species tested.
While In bar headed geese, hemolysis didn’t show any significant impact in fractions other than albumin or
gamma, in black kites hemolysed samples also showed a significant decrease in the alpha 1 fraction AUC
and a significant increase in the beta and the gamma 2 fraction AUC. Changes in these fractions represented
a 8 % average decrease in the alpha1 fraction, a 30 % increase in the beta fraction and a 32 % average
increase in the gamma 2 fraction. In addition, alpha 1 fraction was shown to be negatively correlated to
hemoglobin concentration (Spearman’s test: n = 28, Rs = -0.588, p < 0.01) while beta fraction appeared to be
positively correlated to hemoglobin concentration (Spearman’s test: n = 28, Rs = 0.412, p < 0.05).
Measures on the gels showed that hemoglobin migrated over a significantly longer distance in black
kite than in bar headed goose (Mann and Whitney’ test: U = 474, p = 0). In addition, the band corresponding
to the beta peak appeared to migrate over a significantly shorter distance in black kite than in bar headed
goose (Mann and Whitney’s test: U = 0, p = 0). This resulted in a significantly shorter distance between beta
peak and hemoglobin peak in black kite than in geese (Mann and Whitney’s test: U = 0, p = 0).
DISCUSSION
This study clearly stresses that hemolysis is an important interference factor in both total plasma protein
concentration measurement and plasma protein electrophoresis in birds. Hemolysed samples show higher
total protein concentration. In bar headed geese, hemoglobin migrates to the gamma fraction. In black kites,
hemolysis results in an increase in both beta, gamma1 and gamma 2 fractions, denoting interspecific
differences. This increase appears to primarily concern the gamma 1 fraction, as suggested by its high
average increase. In black kites, hemoglobin may therefore mainly migrate to the gamma1 fraction, the more
immediate vicinity between hemoglobin and beta peaks being responsible for the significant increase in beta
fraction. In both species, these changes may mimic a chronic inflammatory condition with a resulting
antigenic stimulation (Kaneko, 1989; Werner and Reavill 1999). Hemolysis interference may therefore
mislead the practitioner. Further studies should investigate this phenomenon in other species and try to
define a threshold value for hemoglobin concentration in birds beyond which samples should be preferably
rejected. In this study, measuring PCV before and after hemolysis could have helped us to assess the
proportion of lysed cells following the freezing of the samples.
106
In a hemolysed sample, constituents released in the plasma or serum from broken blood cells can interfere
with laboratory results in three ways:
1. Addition interference: this interference applies to analytes which intracellular concentration is far more
important than plasma concentration. Leakage of these intracellular components lead to increased
concentration in plasmas or sera (Kirschbaumweg, 2001; Lippi et al., 2006).
2. Chemical interference: constituents released from blood cells can interfere with chemical reactions used
to analyse for plasma or serum components (Kirschbaumweg, 2001; Lippi et al., 2006).
3. Optical interference: hemoglobin strongly absorbs light at 540 nm. Hemolysis therefore increases
absorption in this wavelength range and causes an apparent increase in the concentration of analytes
measured in this range. In wet reagent analysers, endpoint assays read between 505 and 570 nm have
been demonstrated to be subjected to interference by hemoglobin (Dorner et al., 1983; Kirschbaumweg,
2001; Lippi et al., 2006).
Total protein concentration measurement may therefore be prone to both addition and optical interferences,
since on the one hand hemoglobin itself is a protein (Pontet, 2000), and on the other hand the Biuret reaction
used to carry on this measurement was based on an endpoint assay read at 553 nm. This phenomenon has
been quite well documented in birds (Andreasen et al., 1996, 1997). Hemolysis is described to induce
artefactually higher total protein values (Lippi et al., 2006), even if this analysis is not extremely sensitive to
interference by hemoglobin (Andreasen et al., 1996, 1997). However, effects of this interference can vary
from one chemical analyser to another (Andreasen et al., 1996, 1997; Meinkoth and Allison, 2007). In our
study, total protein concentration was measured with a Biuret blanked method which was said not to be
significantly affected by sample hemolysis as long as hemoglobin concentration was under 5 g/l (Cobas
integra 400 users manual 2006-10 V1 FR). Nevertheless, in both species, plasmas coming from frozenthawed blood samples showed increased amounts of total proteins. This may be mainly related to addition
interference, since optical interference may have been reduced by the blanking procedure. This may explain
the good correlation between hemoglobin and total protein concentrations.
As demonstrated in this study, electrophoresis patterns of hemolysed bird’s sample mainly show a rise in
the gamma fraction. These changes differ from those observed in mammals in which hemoglobinhaptoglobin complexes move to the alpha 2 fraction whereas free hemoglobin migrates to the beta 1 fraction
(Bossuyt et al., 1998; Benlakehal et al., 2000; Thomas, 2001). In mammals, moderate hemolysis levels first
lead to increases in the alpha 2 fraction while beta fraction starts to increase through heavier hemolysis as
soon as haptoglobin binding capacity is overtaken (Benlakehal et al., 2000). Haptoglobin is a protein that
binds hemoglobin with high affinity in order to inhibit its strong oxidative activity (Gutteridge, 1987) and to
avoid it to pass through the glomeruli which may lead to renal failure (Lim et al., 2000). However, a recent
publication shows that haptoglobin does not exist in chickens and may be replaced by another hemoglobin
binding protein called PIT54 in the neognathae subclass. PIT 54 has been identified to be a soluble member
of the family of scavenger receptor cystein-rich proteins and seems to exist only in birds, basing on the
currently available genomic data (Wicher and Fries, 2006). This protein is completely different from
107
haptoglobin, which may partly explain that no rise in alpha fraction was observed in electrophoresis of
hemolysed samples in this study.
The significant decreases observed in albumin fraction in bar headed geese and in albumin and alpha 1
fractions in black kites may be related to a dilution phenomenon. Indeed, blood cell lysis leads to
intracellular fluid release in the plasma (Lippi et al., 2006).However, these variations may not be clinically
relevant, since they are lower than inter-individual variability in both species (see table 1 and 2).
As for any biochemical parameter, good quality of the sample warrants reliability of the results and
care must be taken during both sample collection and processing.
In human medicine, in vitro hemolysis has been documented to occur with mechanical destruction, freezing,
hyperosmotic shock, detergents, exhaustion of glucose in the sample or increased fragility due to inherited
diseases (Guder, 1986). The practitioner should therefore avoid rapidly forcing blood through small needles,
long storage of the blood before centrifugation or excessive agitation when mixing. Furthermore, as
suggested by other authors, plasma should be used preferably to serum, for protein electrophoresis in birds
(Lumeij, 1987; Cray and Tatum, 1998; Werner and Reavill; Hochleithner, 1994). Indeed, the inevitable
hemolysis occurring during the processing of a serum can be a significant problem, since the fluid
component of the blood is in contact for a longer period of time than for plasma (two hours according to the
National Committee for Clinical Laboratory Standards) (Fudge, 2000; Hrubec et al., 2002). The presence of
fibrinogen in the plasma is an advantage rather than an inconvenient in birds, since the fibrinogen has been
demonstrated to be to be a very good indicator of inflammation in birds (Hawkey and Hart, 1988). In the
field of avian medicine, electrophoresis is mainly used in the diagnosis of inflammatory condition. The
presence of a fibrinogen peak in avian electrophoregrams, may therefore not be considered to be an issue, as
it is the case in the human medicine field where it is currently used for the diagnosis of monoclonal
gammapathies (Le carrer, 1998; Daunizeau, 2003).
In case of sample hemolysis, another blood sample should preferably be performed
(Kirschbaumweg, 2001; Martinez-Subiela, 2002; Meinkoth and Allison, 2007).
As shown in this study, effects of hemolysis not only depend on the analyser and the method used, but also
on the species. It is therefore difficult to suggest hard and fast guidelines about sample management.
Samples should always be collected in transparent containers so that the practitioner could estimate sample
hemolysis after centrifugation. Hemolysed samples may preferably be discarded since it is visible to the
naked eye as a pink to red hue of the plasma or the serum as soon as extracellular concentration of
hemoglobin is above 0.6 g/l (9.3 mol/l) (Lippi et al., 2006) and since at this concentration it was
demonstrated in this study to induce significant changes. In case it is impossible to get another sample,
results may be given back with a warning about hemolysis artefacts addressed to the clinician: “warning,
hemolysed sample: total protein concentration and gamma fraction may be overestimated”.
108
ACKNOWLEDGEMENTS
We wish to thank J. L. LIEGEOIS from the Académie de fauconnerie du Puy du fou (France) for the black
kite blood samples, P. TROLLIET from Sebia for his invaluable technical support, the team from bio VSM,
and more especially K. Naudin from bio VSM, who carried out the total protein measurements, the team
from the Parc Zoologique de Clères, the Museum national d’Histoire naturelle and the Conseil Général de
Seine Maritime (France) for their financial support, and Glenn Lund from Techtrans Consulting for the
English proofreading.
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HRUBEC, T. C., WHICHARD, J. M., LARSEN, C. T., and F. W. PIERSON. 2002. Plasma versus serum:
specific differences in biochemical analyte values. Journal of Avian Medicine and Surgery 16: 101-105.
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Animals, 4th edition, J. J. KANEKO (ed.). Academic Press Inc., San Diego, California, pp. 142-165.
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KIRSCHBAUMWEG, L. T. 2001. Haemolysis as an influence and interference factor. The Journal Of The
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LIM, Y. K., JENNER, A., ALI, A. B., WANG, Y., HSU, S. I., CHONG, S. M., BAUMMAN,
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HALLIWELL, B. and S. K. LIM. 2000. Haptoglobin reduces renal oxidative DNA and tissue damage during
phenylhydrazine-induced hemolysis. Kidney international 58: 1033-1044.
LIPPI, G., SALVAGNO, G. L., MONTAGNANA, M., BROCCO, G and G. C. GUIDI. 2006. Influence of
hemolysis on routine clinical chemistry testing. Clinical Chemistry and Laboratory Medicine 44: 311-316.
MEINKOTH, J. H., and R. W. ALLISON. 2007. Sample collection and handling : getting accurate results.
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MARTINEZ-SUBIELA, S., TECLES, F., MONTES, A., GUTIERREZ, G. and J. J. CERON. 2002. effects
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THOMAS, L. 2001. Haemolysis as influence and interference factor. The journal of the internal federation of
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SHOENMAKERS. 2007. Correction of patient results for Beckmann Coulter LX-20 assays affected by
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WERNER, L. L. and D. R. REAVILL. 1999. The diagnostic utility of serum protein electrophoresis.
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112
Article 6: Effets de la lipémie sur la concentration en protéines totales et sur les profils
d’électrophorèse des protéines plasmatiques chez le milan noir (Milvus migrans).
Cet article a été soumis au Journal of zoo and wildlife medicine.
Résumé
De nos jours, l’électrophorèse des protéines plasmatiques est reconnue comme un outil diagnostique fiable
en médecine aviaire. Cependant, les effets d’interférences analytiques telles que la lipémie sont peu
documentés dans la littérature. Ce phénomène est fréquent lors de prélèvements sanguins chez les oiseaux.
Le but de cette étude était donc de déterminer les effets de la lipémie postprandiale sur les
électrophorégrammes aviaires. A cet effet, 21 milans noirs (Milvus migrans) ont été prélevés à deux reprises
à 24 heures d’intervalle : une première fois après un jeûne de 24 heures et une deuxième fois dans les trois
heures après un nourrissage. Un groupe de 10 milans noirs n’a pas été nourri durant les 48 heures de cette
étude afin de constituer un lot témoin non lipémique. Les plasmas lipémiques et non lipémiques obtenus ont
été utilisés pour quantifier les concentrations plasmatiques en protéines totales et en triglycérides, mesurer la
turbidité des prélèvements et déterminer les profils d’électrophorèse des protéines plasmatiques. Le fait de
nourrir les milans avant les prélèvements a entrainé une augmentation de la turbidité et de la concentration en
triglycérides du plasma, ainsi qu’une diminution de la concentration en protéines totales. La lipémie
postprandiale n’a en revanche eu aucun effet sur l’aspect des profils électrophorétiques obtenus. Ces résultats
sont en contradiction avec ceux d’une publication précédente réalisée chez les psittacidés. Dans notre cas, la
lipémie postprandiale chez les milans a probablement été causée principalement par la mise en circulation de
portomicrons. Le fait que ce type de lipémie n’ait pas entrainé de changements au niveau des profils
électrophorétiques observés est très probablement lié au fait que les portomicrons étaient de trop grande taille
pour pouvoir pénétrer dans les pores du gel. Ils sont donc restés au point de dépôt. Leur faible composition
en protéines ne leur a pas permis d’être colorés correctement par le noir amidon, ce qui explique le fait qu’ils
n’apparaissent pas sur les électrophorégrammes. L’interférence analytique liée à la lipémie d’un prélèvement
semble donc relativement difficile à appréhender et dépend très probablement de nombreux paramètres, tels
la technique de laboratoire utilisée, l’espèce d’oiseau prélevé et le type de lipoprotéine impliquée.
113
RH: LIPEMIA AND PLASMA PROTEIN ELECTROPHORESIS IN BIRDS
INTERFERENCE OF LIPEMIA IN PLASMA PROTEIN CONCENTRATION
MEASUREMENTS AND AGAROSE GEL ELECTROPHORESIS IN BLACK KITES
(MILVUS MIGRANS)
Yannick Roman, D.V.M., M.Sc., Michel Saint Jalme, Ph.D, Julie Levrier, M.Sc, Daniel ChasteDuvernoy and Marie-Claude Bomsel-Demontoy, D.V.M., Ph.D.
From the Muséum national d’Histoire naturelle (MNHN), DJBZ, Le Parc de Clères, 32
avenue du Parc, 76690, Clères, France (Roman, Levrier) ; the Muséum national d’Histoire naturelle
(MNHN), DJBZ, Ménagerie du Jardin des Plantes, 57 rue Cuvier 75005 Paris, France (BomselDemontoy), the Laboratoire Bio-VSM, 3 bis rue Pierre Mendès France, 77200, Torcy, France
(Chaste-Duvernoy) and the Muséum national d’Histoire naturelle (MNHN), EGB, UMR 5173 CERSB - MNHN, CNRS, Paris IV, 57 rue Cuvier, 75005 Paris, France (Saint Jalme)
Corresponding author: Yannick Roman, Muséum national d’Histoire naturelle (MNHN), DJBZ, Le
Parc de Clères, 32 avenue du Parc, 76690, Clères, France. E-mail : yannick.roman@cg76.fr. Tel: 33
-2 35 33 23 08. Fax: 33 -2 35 33 11 66.
114
Abstract: Protein electrophoresis is nowadays recognized as being a reliable diagnostic tool for
many pathologic conditions in birds. On the other hand, lipemia is considered to be a form of analytical
interference which can lead to potentially erroneous results. This frequently occurring phenomenon has only
once been investigated in birds. The purpose of this study was to determine the influence of postprandial
lipemia in the plasma protein concentration and agarose gel electrophoresis patterns in birds. Twenty one
black kites (Milvus migrans) were blood sampled twice, once after fasting for 24 hours, and once three hours
after being fed. Birds from a control group (10 birds) were not fed during the course of this study. Total
protein and triglyceride concentrations, and sample turbidity and plasma protein electrophoresis patterns
were determined for both lipemic and non lipemic samples. Feeding black kites resulted in an increase in
their triglyceride concentration and sample turbidity, and in a decrease in total protein concentration.
Lipemia did not have any impact on electrophoresis patterns. Our results are not in agreement with those of a
previous study dealing with psittacine plasma electrophoresis. In our case, postprandial lipemia in raptors is
thought to have been caused mainly by the presence of portomicrons in the bloodstream. No significant
impact was found on electrophoresis patterns, probably because the large size of such lipoproteins did not
permit them to enter the agarose pores. Since their protein composition is very low they may not have been
well stained by the protein specific dye amido-black, such that they did not appear in the electrophoresis
patterns. Lipemia interference is therefore quite unpredictable, and may depend on many parameters such as
the laboratory techniques used, the species sampled, and the kind of lipoproteins involved in the specific
form of lipemia.
Keywords:
Black kite, interference, lipemia, Milvus migrans, plasma proteins, protein
electrophoresis.
115
INTRODUCTION
Serum protein electrophoresis has been extensively used in human diagnostics for nearly 40 years to
evaluate, diagnose, and monitor a variety of diseases.11,25 The use of protein electrophoresis in veterinary
medicine and especially in bird medicine is far more recent, but plasma protein electrophoresis is nowadays
commonly recognized to be a very reliable diagnostic tool for many pathologic conditions in birds.9,21,39
However many factors, such as lipemia, which may potentially interfere with electrophoresis patterns, still
need to be investigated.
Lipemia is one of the main interference factors, and has been defined by Kroll and Elin as “the effect
of a substance present in the sample that alters the correct value of the result usually expressed as
concentration or activity for an analyte”.24 However, interference from lipemia is fundamentally different
from other forms of interference: chylomicrons and large lipoprotein molecules, called Very-Low-Density
Lipoproteins (VLDL), form suspended particles which produce cloudiness (through scattering), thereby
interfering with the transmission of light.1,23,38 The resulting turbidity can range in appearance from a slightly
opaque to a translucent, turbid or milky solution.10,31
Lipemia usually occurs when a blood sample has been collected too soon after a fatty meal10,29,31,
however its magnitude and duration are variable: in humans, triglycerides are present in the plasma in the
form of chylomicrons and their metabolites as early as 2 hours after intestinal absorption,31 and persist for 6
to 12 hours.2,14,36 Lipemia can also be associated with some pathological conditions affecting the lipid
metabolism, such as diabetes mellitus, hypothyroidism and acute pancreatitis, and may thus be
unavoidable.29,31 In birds, lipemia is very frequent because, in general, the need to obtain samples for
laboratory testing is not anticipated, and the birds are not fasted before blood sampling31. Furthermore, some
physiological conditions such as egg laying are associated with high lipoprotein concentrations in the
blood.17 In addition, birds are quite peculiar in that fatty acids are their primary source of fuel during long
distance flight, which implies the need for high fatty acid transport in the bloodstream.16,17
In humans, the measured total protein concentration in lipemic samples is known to indicate either falsely
low or falsely high values, depending on various factors.10,31 Although lipemia interference in plasma protein
electrophoresis has been investigated, mainly in capillary zone electrophoresis,3,5,29 data on the effects of
lipemia in agarose gel electrophoresis is very scarce. In humans, lipoproteins are of the alpha and beta type.
They migrate in respectively alpha-2 and beta fractions, and correspond respectively to High Density
Liproteins (HDL) and Low Density Lipoproteins (LDL).25 In the case of mammals, Groulade recommends to
avoid performing gel electrophoresis with non-fasting samples, since the alpha and beta fractions are mainly
comprised of carrier proteins.13
As far as we know, lipemia interference in laboratory results has been investigated only once for the case of
birds. In a recent study, Cray et al. indeed investigated this phenomenon in plasma electrophoresis with
psittacine birds.9 Lipemia is described as being responsible for an artifact located in the beta fraction,
although no details are given concerning the cause of lipemia.
116
The purpose of the study reported here was therefore to determine the effects of postprandial
lipemia on plasma protein concentration and agarose gel electrophoresis patterns in birds. Black kites
(Milvus migrans) were chosen as a model, since raptors are generally very prone to postprandial lipemia
(personal observation) and because, according to previous studies, it is preferable to use real lipemic patient
samples rather than samples supplemented with an artificial lipid emulsion such as Intralipid ©.4,32 Twenty
one black kites were blood sampled twice, after a 24 hours fasting period, and then three hours after feeding.
Non lipemic and lipemic samples obtained under these conditions were used to determine the influence of
lipemia on triglyceride and total protein concentrations, turbidity and electrophoresis patterns. Ten black
kites were not fed during the study and were used as a control group in order to verify that none of the
recorded variations were the result of hemodilution.
MATERIAL AND METHODS
Experimental animals
This study was conducted outside the breeding season, in September 2006, with 31 black kites
(Milvus migrans) held at the Académie de fauconnerie du Puy du fou (France). This group was composed of
18 males and 13 females. All these falconry birds were used to handlings.
The black kites were all fasted for 24 hours, before the beginning of the study, and were randomly
distributed into two groups. The experimental group (21 birds) was blood sampled twice: after a 24 hour
fasting period, and 3 hours after the birds had been fed with one day-old-chicks. In order to avoid potential
dilution artifacts,12,35 the two blood samples were taken at a 24 hour interval. The birds in the control group
(10 birds) were not fed at all during this study. Since the aim of the study was to investigate the influence of
lipemia on total protein concentration and protein electrophoregrams, two male birds, one from the
experimental group and one from the control group, were excluded from the study because these two birds
had lipemic plasmas while they were fasted.
Samples
Blood samples were taken from the right jugular vein, using 23 G needles and 5 ml syringes
(respectively: Terumo Neolus 23 G, and Terumo Syringe 5ml, Terumo Europe N.V., Leuven, 3001,
Belgium). Two millilitres of blood were drawn from each bird, collected on lithium heparin (Venosafe,
Terumo Europe N.V., Leuven, 3001, Belgium), and centrifuged at 3000 g for 5 minutes. Analyses were
carried out on the resulting plasma, since it is commonly accepted that plasma is preferable to serum, for
protein electrophoresis in birds. Plasma is less prone to haemolysis than serum, and contains fibrinogen
which is a protein characteristic of the acute phase.8,19 The plasma samples were then stored in cryotubes (20°C; -4°F) until they were analysed (1.4 ml U-Tubes, Micronic B.V., Lelystad, 8211, Holland). They were
117
then thawed and re-homogenised, by gentle mixing, one hour before analysis. All of the blood samples were
collected on the same day, and therefore underwent similar freezing conditions.
Total protein and triglyceride concentration and turbimetry measurements
Total protein concentration was determined by the Biuret reaction, using a Roche Integra 400© wet
chemistry analyser (Roche Integra 400©, Roche diagnostics, Meylan, 38242, France). Readings were
performed at a wavelength of 552 nm. Triglyceride concentrations were determined with the same analyser,
using Trinder’s reaction (GOP/PAP), and the readings were taken at 512 nm. Turbidity measurements were
carried out with the same analyser at 659 nm, since other forms of interference (such as hemolysis) are
known to be low at this wavelength.27 Light of a known intensity “I0” was passed through the sample, on the
other side of which the transmitted intensity “I” (“I0” less the absorbed light) was measured. The turbidity τ
= ln (I0/I) was reported in nephelometric turbidity units (NTU).23,33
Protein electrophoresis
Agarose gel electrophoresis of plasma proteins was performed with a Hydrasys© semiautomated
system (Hydrasys©, Sebia, Evry, 91008, France), using the Hydragel protein 15/30© set (Hydragel protein
15/30©, Sebia, Evry, 91008, France). It was operated according to the manufacturer’s instructions, using
version 7.00 F0.1 of the system software.
Ten microlitres of plasma samples were manually distributed onto the sample template, and were allowed to
diffuse for a period of 5 minutes in a wet chamber. Application of the samples to the gel, electrophoresis and
drying of the gel, were all performed automatically in the migration compartment of the instrument. The
temperature was maintained at 20°C (68°F) using a Peltier device during the whole migration process, and
drying was obtained by heating the gels to 65°C. Electrophoretic separation was obtained on 8 g/l agarose
gels in a Tris-barbital buffer (pH 9.2), at constant power 20W, until 33 Vh had accumulated.
Once dried, the gels were manually transferred to the staining compartment of the instrument where amidoblack staining, destaining and drying were performed automatically.
Once dried, the gels were read with a Sebia Preference© densitometer (Preference©, Sebia, Evry, 91008,
France), which enabled us to acquire electrophoresis patterns, define protein fractions and measure Areas
under the Curves (AUC). Albumin was identified to have the strongest anodal peak. The beta peak was
identified as fibrinogen.34 Globulins were divided into 5 fractions. The Alfa fractions were located between
the albumin and beta peaks, and the gamma fractions were located beyond the beta peak. As previously
described in other studies of avian protein electrophoresis, the A/G ratio was calculated by dividing the
albumin content by the sum of the globulin fractions.8,26 The influence of lipemia on electrophoresis patterns
was studied by comparing the areas under the curves, in addition to the protein concentrations of each
fraction, in order to avoid possible errors in total protein dose.10,31
118
Statistical analysis
Systat 7.0 software (Systat 7.0, Systat Software Inc., London, TW4 6JQ, United Kingdom) was used
for all analyses. In view of the small size of the studied population, Wilcoxon’s signed rank test and
Spearman’s rank correlation were used.
RESULTS
The black kite electrophoresis patterns were divided into six fractions (Figure 1). None of these
showed any pre-albumin fraction.
Figure 1: example of plasma electrophoresis patterns of a Black kite (Milvus migrans).
In the experimental group, all plasma derived from postprandial blood samples had a turbid to milky
visual appearance. Blood samples drawn after the birds were fed showed significant increases in both
triglyceride concentrations and turbidity (Table 1). However, the turbidity and triglyceride levels were not
directly related, as shown by the Spearman rank correlation coefficient (n = 20, Rs = 0.416, p > 0.05). In the
control group, no significant difference was found in either turbidity or triglyceride concentration. In the
experimental group, the total protein concentration appeared to decrease significantly after feeding (Table 1),
whereas no significant difference was found between the two blood samples in the control group.
The authors observed no significant difference in the AUC of any fraction, between the first and second
blood samples, neither in the experimental group nor in the control group. Postprandial lipemia thus appears
to have no impact on the electrophoresis patterns of back kites.
119
In the experimental group, the albumin, alpha 1, beta and gamma 2 fraction concentrations decreased
significantly after the meal, as a result of the decrease in total protein concentration (Table 1). No significant
difference in concentration of the fractions was found in the control group, between the first and second
blood samples.
Finally, no significant difference was found between males and females for any of the parameters measured
in this study, in both the experimental and control groups.
Table 1: total protein and triglyceride concentrations, and turbidity and electrophoresis patterns of black kites
(Milvus migrans) from the experimental group (n = 20), 24 h after fasting and 3 h after a fatty meal. Results
are expressed as mean ± SD.
Postprandial
Wilcoxon Z
sample
values
35.13 ± 4.55
33.68 ± 4.22
-2.632
0.008
Triglyceride concentration (g/l)
0.38 ± 0.09
1.86 ± 1.08
3.92
0
Turbidity (NTU)
0.03 ± 0.03
0.26 ± 0.1
2.504
0.012
Albumin (g/l)
15.31 ± 1.83
14.83 ± 1.80
-2.053
0.040
Alpha 1 (g/l)
11.64 ± 1.69
11.12 ± 1.70
-2.427
0.015
Alpha 2 (g/l)
0.76 ± 0.23
0.72 ± 0.19
-1.829
0.067
Beta (g/l)
4.79 ± 2.36
4.54 ± 2.28
-2.576
0.010
Gamma 1 (g/l)
0.76 ± 0.30
0.73 ± 0.25
-0.373
0.709
Gamma 2 (g/l)
1.86 ± 0.58
1.72 ± 0.52
-2.875
0.04
A/G (g/l)
0.8 ± 0.13
0.8 ± 0.13
1.867
0.062
Parameter
Fasting sample
Total protein concentration (g/l)
p
DISCUSSION
Our study shows that lipemic samples had a lower total protein concentration than non lipemic ones.
Postprandial lipemia did not appear to have any effect in plasma electrophoresis patterns. However, the
decrease in total protein concentration related to lipemia had repercussions on fraction concentrations. Such a
decrease may not be clinically relevant for the practitioner, because the inter-individual variability of these
parameters was higher than changes related to lipemia. Therefore, in the work reported here, postprandial
lipemia in raptors does not appear to be an important impact factor in total protein concentration and plasma
protein electrophoresis.
Feeding black kites with one-day-old chicks resulted in significant increases in both triglyceride
concentration and turbidity, showing that these birds can be good models for the study of lipemia
interference. Indeed, this kind of diet contains a high level of fat. Dietary lipids are the primary energy
120
source for carnivorous birds such as raptors, to such an extent that the capacity of their liver to synthesise
fatty acids is low, compared with grain-consuming birds.22 Contrary to mammals, the intestinal lymphatic
system is poorly developed in birds. Lipoproteins are therefore secreted directly into the portal system from
enterocytes, and are termed portomicrons instead of chylomicrons.17,18 These portomicrons are released into
the bloodstream and cleared by the liver, which repackages dietary lipids and adds lipids synthesized by
hepatocytes to give Very Low Density Lipoproteins (VLDL) which are secreted into the blood stream.22
Portomicrons and VLDL in chickens are very similar in size and density to chylomicrons and VLDL in
mammals.15,16,20 Their role in light scattering may therefore be quite similar to that described in mammals.23
Lipemia in mammals is known to interfere with laboratory results in three different ways:
1. Optical interference. Turbidity interferes by scattering light in all directions, thereby decreasing the
intensity of the light reaching the detector of the spectrophotometer.23 The light scattering strength is
affected by the number and size of particles suspended in the solution, by the refractive index of the
solution depending itself on the particle concentration, and by the wavelength of the scattered light.23
2. Volume depletion effect. After centrifugation, lipids are concentrated in the upper phase of the sample.14
Since the volume occupied by lipoproteins in plasma or serum is included in the calculation of analyte
concentration, the lipids decrease the apparent value of the latter, by reducing the available water in the
sample volume.14,23 When the lipoproteins are not homogeneously distributed in the serum or plasma, the
concentration of an analyte dissolved in the aqueous phase is lower in the upper layer than in the lower
phase of the sample.14
3. Interference by physico-chemical mechanisms. A constituent that is extracted by lipoproteins may not be
accessible to the reagent. In the same way, electrophoretic procedures may be affected by lipoproteins
present in the sample.14,23
In our study, the total protein concentration was measured with a Biuret blanked method, which was known
to be only weakly affected by sample lipemia (Cobas integra 400, User’s Manual 2006-10 V1 FR). However,
the postprandial total protein levels appeared to be significantly lower than in fasting samples. This may be
related mainly to the volume depletion effect, since optical interference may have been reduced by the
blanking procedure. In any case, the clinician must be fully aware of the fact that lipemia interference is
quite difficult to predict, and may be highly dependant on the analyser or even on the sample itself.23 In any
case, changes observed in the experimental group may not be related to hemodilution artifacts, nor to the
stressed condition of the birds following blood sampling, since none of the parameters measured in the
control group showed any significant difference between the two blood samples.
In the present study, turbidity did not appear to be correlated to triglyceride concentrations, as reported in
previous articles relevant to humans.4,23,37 This is probably related to the fact that portomicrons and VLDL
particles can vary in size, number and triglyceride content.16,17 Therefore, direct triglyceride content is not a
good tool for predicting lipemia interference in birds.
Lipemia interference in plasma protein electrophoresis has been investigated mainly in capillary
zone electrophoresis in humans and dogs, where it has been reported to produce an interference peak in the
121
alpha 2 fraction.3,5,29 However, capillary electrophoresis makes use of UV detection for direct protein
quantification, via the peptide bonds. Consequently, any substance in the serum, which absorbs light at this
measurement wavelength, has the potential to produce an artifact, contrary to conventional gel-based
methods in which quantification of the protein fractions is based on dye binding (Amido-black in our case),
which has a great affinity for proteins.5
Our results do not match those of a recent study dealing with protein electrophoresis of psittacine plasma.9
The authors assessed the effects of lipemia in plasma agarose gel electrophoresis, using five psittacine birds.
They clarified lipemic samples by ultracentrifugation, and added the resulting supernatant lipid layer to the
clarified samples, in order to mimic heavy lipemia. The sample lipemia was showed to result in an increase
of the beta fraction. However, in this particular study, no information was provided concerning the lipemia’s
origin, although (for instance) lipemia resulting from egg laying, which corresponds mainly to circulating
VLDL, may be different from postprandial lipemia.17 As previously described, in the case of the present
study the presence, within hours after a fatty meal, of postprandial lipemia in raptors such as black kites may
have been induced largely by the presence of portomicrons in the bloodstream, even though some of their
metabolites may have been present.22 One hypothesis is that the large size of these lipoproteins (150 nm)15
may have prevented them from entering the agarose pores of the electrophoretic gels. The portomicrons may
have remained where the samples were deposited onto the gels. Since their protein composition is very low
(1-2%),15,16 they may not have been well stained by the protein specific dye amido-black, such that they did
not appear in the electrophoresis patterns. Furthermore, in our study, postprandial lipemia was obtained
under physiological conditions, and may therefore be less significant than in the study reported by Cray et
al.9 Further studies should be carried out in order to investigate the effects of lipemia resulting from different
kinds of lipoproteins.
Interspecific differences may also have played an important role. This study is only the second to have dealt
with lipemia interference in birds. Further studies should investigate the impact of postprandial lipemia in
other avian species, in particular non carnivorous ones such as pigeons or chickens, in which de novo fatty
acid synthesis in the liver is mainly based on the use of dietary carbohydrates and in which postprandial
lipemia may mainly resulting from circulating VLDL.22
At the present time, it is too early to suggest detailed guidelines for sample management, since as
previously described, effects of lipemia may depend on the kind of lipoproteins involved. Samples should
always be collected in transparent containers, so that the practitioner can estimate the sample’s lipemia after
centrifugation. If possible, lipemic samples should preferably be rejected and taken again later, once the
animal has been fasted.31 If such a procedure is not possible, lipemia may be successfully reduced by high
speed centrifugation (> 10000 g).4,10,38 Although clearing agents such as lipoclear © may be useful in
determining total protein concentration,38 the impact of this type of reagent should be investigated in further
studies before it is used for plasma protein electrophoresis.
122
Acknowledgements: We wish to thank JL Liegeois from the Académie de fauconnerie du Puy du
fou (France) for the black kite blood samples he provided, P Trolliet from Sebia for his invaluable technical
support, and the Muséum national d’Histoire naturelle and the Conseil Général de Seine Maritime (France)
for their financial support. We also appreciate the help provided by Glenn Lund from Techtrans consulting,
for proofreading this document.
LITTERATURE CITED
1. Alleman, A.R. 1990. The effects of hemolysis and lipemia on serum biochemical constituents.
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Hematological
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39. Werner, L.L., and D.R. Reavill. 1999. The diagnostic utility of serum protein electrophoresis.
Vet. Clin. North. Am. Exot. Anim. Pract. 2: 651-662.
126
CHAPITRE IV : Comparaison de techniques électrophorétiques
Article 7: L’électrophorèse des protéines plasmatiques chez les oiseaux: comparaison d’une
méthode semi-automatique d’électrophorèse en gel d’agarose, Hydrasys©, avec une méthode
automatisée d’électrophorèse capillaire de zone, Capillarys2©.
Cet article a été soumis à la revue Veterinary Clinical Pathology.
Résumé
L’électrophorèse des protéines plasmatiques est de nos jours reconnu comme étant un examen
complémentaire fiable pour le diagnostic des maladies des oiseaux. Durant les 10 dernières années, de
nouvelles techniques électrophorétiques telles que l’électrophorèse capillaire de zone (CZE) ont vu le jour
dans le domaine de la médecine humaine. Cependant, l’utilisation de telles techniques n’a jamais été abordée
chez l’oiseau. Le but de cet article est donc d’étudier la CZE chez les oiseaux et de comparer les profils
obtenus en CZE avec ceux obtenus en électrophorèse en gel d’agarose (AGE), technique la plus utilisée à ce
jour. Cette étude a été conduite sur 30 coqs (Gallus gallus), 20 milans noirs (Milvus migrans) et 10 pigeons
domestiques (Columba livia). Les plasmas obtenus ont permis de comparer les profils électrophorétiques
obtenus en AGE et en CZE, ainsi que de calculer la répétabilité et la reproductibilité de ces deux techniques.
Dans les trois espèces étudiées, les valeurs des fractions obtenus par AGE et CZE étaient significativement
différentes, bien que les fractions albumine, beta et gamma soient apparues bien corrélées entre les deux
techniques. Les valeurs des fractions alpha-3 chez le coq, alpha-1 chez le milan noir et alpha chez le pigeon
obtenues par AGE se sont révélées très bien corrélées avec les fractions pré-albumines obtenues en CZE. La
répétabilité et la reproductibilité des résultats étaient meilleures pour la CZE que pour l’AGE. Bien que
l’interprétation des profils électrophorétiques obtenus en CZE paraisse relativement similaire à celle des
profils électrophorétiques obtenus par AGE, le praticien doit prendre en compte le fait que certaines
protéines des fractions alpha en AGE migrent au niveau de la fraction pré-albumine en CZE. Chez le pigeon
le phénomène est si important, pour les profils électrophorétiques obtenus par CZE, qu’il pourrait facilement
conduire le praticien à prendre la fraction pré-albumine pour une fraction albumine. Bien que la CZE
requière la mise en place d’intervalles de références propres à cette méthode, cette technique présente des
avantages par rapport à l’AGE, tels qu’une meilleure répétabilité et reproductibilité, et une cadence d’analyse
plus importante.
127
128
Plasma protein electrophoresis in birds: comparison of Hydrasys©, a semiautomated agarose
gel electrophoresis system, with Capillarys2©, an automated capillary electrophoresis system
RH: Agarose gel and capillary plasma protein electrophoresis in birds
Yannick ROMAN, Marie-Claude BOMSEL-DEMONTOY, Julie LEVRIER, Daniel CHASTEDUVERNOY and Michel SAINT JALME.
From the Museum national d’Histoire naturelle (MNHN), DJBZ, Le Parc de Clères, Clères, France (Roman,
Levrier) ; the Museum national d’Histoire naturelle (MNHN), EGB, UMR 5173 - CERSB - MNHN, CNRS,
Paris IV, Paris, France (Saint Jalme), the Laboratoire Bio-VSM, Torcy, France (Chaste-Duvernoy) and the
Museum national d’Histoire naturelle (MNHN), DJBZ, Ménagerie du Jardin des Plantes, Paris, France
(Bomsel-Demontoy). Corresponding author: Yannick Roman (yannick.roman@cg76.fr).
129
Background: Plasma agarose gel electrophoresis (AGE) is nowadays recognised to be a very reliable
diagnostic tool in avian medicine. Within the last 10 years, new electrophoresis techniques such as capillary
zone electrophoresis (CZE) have emerged in the field of human laboratory medicine. Such techniques have
never been investigated in birds. Objective: The purpose of this study was to investigate the use of CZE in
birds and to compare it with AGE. Methods: The study was conducted on 30 roosters (Gallus gallus), 20
black kites (Milvus migrans) and 10 racing pigeons (Columba livia). Plasma obtained from these samples
was used to carry out both AGE and CZE. Results: Fraction values obtained by AGE and CZE were
significantly different, although for the three species studied they appeared to be well correlated for albumin,
beta and gamma fraction values. With AGE, alpha-3 fraction values in the rooster, alpha-1 fraction values in
the black kite and alpha fraction values in the pigeon were very well correlated with the prealbumin fraction
values obtained using CZE. Repeatability and reproducibility appeared to be higher with CZE than with
AGE. Conclusions: Although the interpretation of CZE electrophoresis patterns seems to produce results
quite similar to those obtained with AGE, the practitioner must take into account the fact that some proteins
present in the alpha fraction, measured with AGE, migrate to the prealbumin fraction found with CZE. In
pigeon CZE patterns, this phenomenon is so significant that the practitioner could mistake the prealbumin
fraction for the albumin fraction. Although it requires the use of specific reference ranges, CZE has many
advantages when compared to AGE, including better repeatability and reproducibility, and higher analysis
throughput.
Keywords: Agarose gel electrophoresis, capillary zone electrophoresis, plasma proteins, protein
electrophoresis, bird.
130
In human medicine, serum protein electrophoresis techniques have been used for five decades.1-3
Although the use of protein electrophoresis in avian medicine is far more recent and is still in its infancy,
publications made during the last 15 years show that it is a very reliable diagnostic test in birds.4-6 Indeed,
plasma proteins have many functions in health and disease. In the case of an inflammatory condition acute
phase protein plasma levels increase, and are of clinical interest in the diagnosis of various diseases in
birds.7,8 Within the last 10 years, new electrophoresis techniques, such as Capillary Zone Electrophoresis
(CZE) have emerged in the field of human medicine.2,9 Such advances should also be investigated in birds.
Since Tiselius’s pioneering work, human laboratory clinical medicine has always closely followed
advances in protein electrophoresis.10 Nowadays, Agarose Gel Electrophoresis (AGE) is the most currently
used technique.3,11 In 1997, Sebia developed a semi-automated electrophoresis system: Hydrasys©. This
system further improved the reproducibility of agarose gel electrophoresis by automating most steps of the
procedure, including sample application, migration and staining.12
However, despite such semi-automated systems, AGE remains quite labour-intensive, resulting in limited
analytical performance and throughput.13
Over the last decade, CZE has emerged as a powerful diagnostic tool in the field of human diagnostics, and
represents a major advance in electrophoresis technology for clinical applications.9,13-15 This technique, first
introduced by Hjerten in 1967, consists in the separation of charged molecules in small capillary tubes. Since
heat can readily radiate from the tubes, extremely high voltages can be used without overheating the
samples.16 The use of such high voltages shortens the analysis times.17 Protein analysis is performed in a free
buffered solution, and with this system, the electro-osmotic velocity exceeds the electrophoretic mobility of
the proteins, which therefore migrate towards the cathode instead of the anode. Real time detection and
quantification of protein fractions is based on the direct detection of peptide bonds by way of UV light
absorption through the capillary wall.9,13-15,17
The first automated capillary electrophoresis system, the Paragon CZE2000© (Beckman) was
commercialized in 1994. In 2001, Sebia developed its own CZE system, Capillarys©. Such systems are
particularly helpful in clinical laboratories which have to deal with a large daily workload of serum protein
electrophoresis (up to 100/h with Sebia Capillarys2©).12 CZE systems are indeed fully automated and require
only minimal human intervention. In human medicine, various publications have dealt with the topic of CZE
and compared its technique with that of AGE. These studies show good correlations between the results
obtained with both techniques, with the exception of alpha-1 and beta fractions. The reproducibility of CZE
appears to be superior to that of AGE.2,9,11,13,15 CZE is at least equivalent to AGE, and the interpretation of its
electrophoregrams is similar to that for AGE.15
In birds, various publications have documented the use of paper, cellulose acetate or agarose gel
electrophoresis for research purposes or as diagnostic tools.18-29 Comparative studies of these techniques have
also been made.18 However, our review of the literature has failed to reveal any study in which the use of
CZE has been studied for birds.
131
The objective of the present study was therefore to describe the use of Sebia Capillarys2© CZE for
birds, and to compare its results with those obtained using Sebia Phoresis© AGE. The study was also
designed to produce the first CZE reference values for birds, and to measure the reproducibility of this
technique. To this end, plasma samples from 30 roosters (Gallus gallus), 20 black kites (Milvus migrans) and
10 pigeons (Columba livia) were analysed using Sebia AGE and CZE systems.
Material and methods
Experimental animals
The study was conducted on 30 SY33 line roosters (Gallus gallus), held at the INRA Tours-Nouzilly
research station (France). These birds were three years old and were housed in individual cages. In order to
broaden the scope of our investigation of capillary electrophoresis in birds, 20 black kites (Milvus migrans),
held at the Académie de fauconnerie du Puy du fou (France), and 10 rock doves (Columba livia), held at the
zoological park of Clères (France), were included in the study. Both the pigeons and the black kites were
housed in aviaries. All birds were clinically examined, and found to be healthy.
Samples
Blood samples were taken from the right jugular vein of the roosters and black kites, and from the brachial
vein of the rock doves, using 23 G needles and 5 ml syringes (respectively: Terumo Neolus 23 G, and
Terumo Syringe 5ml, Terumo Europe N.V., Leuven, Belgium). Two millilitres of blood were drawn from
each bird, collected on lithium heparin (Venosafe vacutainers, Terumo, Leuven, Belgium), and centrifuged at
3000 g for 5 minutes. Analyses were carried out on the resulting plasma, since it is commonly accepted that
it is preferable to serum, for protein electrophoresis in birds. Plasma is indeed less prone to hemolysis than
serum and contains fibrinogen, a protein characteristic of the acute phase of inflammatory conditions.4,30
Samples did not show any hemolysis nor lipemia. Two aliquots of each plasma sample were then stored in
cryotubes (-20°C; -4°F) until they were analysed (Micronic systems, Lelystad, Holland). They were then
thawed and re-homogenised, by gentle mixing, one hour before analysis. All blood samples were collected
on the same day, and were therefore exposed to similar freezing conditions.
Measurement of total protein concentration
Total protein concentration was determined by the Biuret reaction, using a Roche Integra 400 wet chemistry
analyser (Roche diagnostics GmbH, Mannheim, Germany). Readings were made at a wavelength of 552 nm.
Agarose gel electrophoresis (AGE)
Agarose gel electrophoresis of plasma proteins was carried out using a Hydrasys© semi-automated system
(Sebia, Evry, France), using the Hydragel protein 15/30© set (Sebia, Evry, France). It was operated
according to the manufacturer’s instructions, using version 7.00 F0.1. of the system software. Ten microlitres
of plasma samples were manually distributed onto the sample template, and were allowed to diffuse for a
132
period of 5 minutes in a wet chamber. Application of the samples to the gel, electrophoresis and drying of the
gel, were all performed automatically in the migration compartment of the instrument. The temperature was
maintained at 20°C (68°F) using a Peltier device during the complete migration process, and drying was
obtained by heating the gels to 65°C. Electrophoretic separation was obtained on 8 g/l agarose gels in a Trisbarbital buffer (pH 9.2), at a constant power level of 20W, until 33 V-h had been accumulated. Once dried,
the gels were manually transferred to the staining compartment of the instrument where amido-black
staining, destaining and drying were performed automatically. Once these operations had been completed,
the gels were scanned with a high resolution Epson perfect V700 photo scanner (Epson France, Nanterre,
France). Electrophoretic curves and dosages of the different fractions were acquired using Phoresis©
software, version 5.50 (Sebia, Evry, France). Protein fractions were determined by referring to other
publications.24,27,29 By convention, the A/G ratio in birds is calculated by dividing the sum of the prealbumin
and albumin fractions by the sum of the globulin fractions.4,31
Capillary zone electrophoresis (CZE)
Capillary zone electrophoresis of plasma proteins was carried out on a Capillarys2© automated system
(Sebia, Evry, France). This system was operated according to the manufacturer’s instructions under version
6.00 of the supplied Software.
Sample tubes were manually installed into racks (up to 13 racks of 8 sample tubes). A minimum volume of
140 µl was used. All of the following steps were performed automatically. The samples were diluted to a 1:5
ratio with the migration buffer in dilution segments (40 µl of plasma to a final volume of 200 µl). The
samples were then hydrodynamically injected using an anodic depression of 80 millibars for 4 s (< 1 nl,
representing less than 1% of the total volume of the capillary tube). With the Capillarys protein 6© reagent
set (Sebia, Evry, France), electrophoretic separation of the protein fractions was obtained by applying a 7.5
kV voltage, for about 4 minutes, to eight fused-silica capillaries (17.5 cm in length, 15.5 cm in effective
length; 25 µm internal diameter) in a pH 10 buffer. The temperature was maintained at 35.5 °C (95.9°F)
using a Peltier controller.
Real time detection and quantification of protein fractions was based on the direct detection of peptide
bonds, resulting from UV light absorption through the capillary wall. Detection was performed at 200 nm, in
an optical cell placed at the cathodic end of the capillary tube and connected to the detector by means of
eight optical fibers. Electrophoretic curves and dosages of the different fractions were acquired using version
6.00 of the Phoresis© software (Sebia, Evry, France).
Repeatability and reproducibility
One rooster sample was chosen at random, and was used to investigate the repeatability and reproducibility
of AGE and CZE.
133
For AGE, repeatability within a run was estimated by interpreting the plasma patterns of 30 aliquots of the
same sample run on the same gel. Inter-run reproducibility was estimated by interpreting the plasma patterns
of 8 aliquots of the same sample run on separated gels.
For CZE, repeatability within a run was estimated by interpreting the plasma patterns of 8 aliquots of the
same sample run on the same rack. Inter-run reproducibility was estimated by interpreting the plasma
patterns of 6 aliquots of the same sample run on different racks.
The mean, SD and CV were calculated for each fraction.
Statistical analysis
Systat 7.0 © software (SPSS inc. © 1997) was used for all analyses but Passing-Bablok and Bland-Altman
tests. The study of AGE and CZE accuracy was made by calculating the coefficients of variations (CV).
Other authors have indeed stated that CV analysis is the recommended approach for the assessment
repeatability and reproducibility in chemistry tests.6 In view of the small size of the studied population,
Wilcoxon’s signed rank was used to compare the two different electrophoresis techniques. The normality of
the rooster data was tested with the Kolmogorov-Smirnov procedure. As the data was found to be normally
distributed, it was decided to use Pearson’s correlation matrix to test correlations between the two
techniques, in all of the studied species. Passing-Bablok regression analysis and Bland-Altman difference
plot were carried on with the Medcalc 9.5.2 © software (Medcalc software, Mariakerke, Belgium). These
tests were used to compare the discrepancies between AGE and CZE for the fraction which appeared to be
well correlated.
Results
Protein electrophoresis patterns
The rooster’s AGE patterns were divided into eight fractions, which were then classified into one
prealbumin fraction, one albumin fraction, three alpha fractions, two beta fractions and one gamma fraction
(Figure1, Table 1). The electrophoresis patterns obtained with CZE were found to be quite similar to those
obtained with AGE, and fractions were defined the same way (Figure 1, Table 1). The values obtained, using
both CZE and AGE for the albumin, alpha-1, beta-1, beta-2 and gamma fractions, were found to be well
correlated (respectively: n = 30, R = 0.939, p < 0.01; n = 30, R = 0.555, p < 0.01 ; n = 30, R = 0.815, p <
0.01 ; n = 30, R = 0.670, p < 0.01 ; n = 30, R = 0.784, p < 0.01). On the other hand, although the alpha-3
fractions obtained with AGE were only weakly correlated with the alpha-3 fraction values obtained with
CZE (n = 30, R = 0.420, p < 0.05), they were very well correlated with the prealbumin values obtained using
CZE (n = 30, R = 0.656, p < 0.01). With the exception of the gamma-1 fraction, although the values obtained
with AGE and CZE for the remaining fractions were correlated, they were found to be significantly different
(Wilcoxon’s test: p = 0).
Passing-Bablok regression analysis and Bland-Altman difference plot were carried on to compare the
discrepancies between AGE and CZE for the fraction which appeared to be well correlated (Table 2). No
134
significant deviation from linearity was found, whatever the fraction may be. Both techniques appeared to
differ significantly for the quantification of alpha-1, alpha-3, and gamma fractions. Except for the alpha-1,
alpha-3, and gamma fractions, the differences between the two techniques did not vary in any systematic
way over the range of measurement. For alpha-1 and gamma fractions, the discrepancy between the two
studied techniques increased with the magnitude of the measurement.
The A/G ratios obtained from the two techniques were slightly different (average A/G ratio of 0.62 ± 0.08
for AGE and 0.76 ± 0.11 for CZE), but were well correlated (n = 30, R = 0.952, p < 0.01).
The AGE and CZE patterns of the black kites were divided into six fractions (Figure1, Table 1).
These were classified into one prealbumin fraction, one albumin fraction, two alpha fractions, one beta
fraction and one gamma fraction. The albumin fraction was identified as having the strongest anodal peak.
However, with AGE, the electrophoresis patterns of the black kites were characterised by the presence of a
strong alpha-1 peak, similar in intensity to the albumin peak. Such a strong peak was not observed in CZE
patterns (Figure 1, Table 1). The values of the albumin, beta and gamma fractions determined using CZE and
AGE were found to be very well correlated (respectively: n = 20, R = 0.723, p < 0.01; n = 20, R = 0.701, p <
0.02; n = 20, R = 0.912, p < 0.01). The values of the alpha-1 fraction determined using AGE were found to
be correlated both with those of the CZE alpha-1 fraction (n = 20, R = 0.571, p < 0.01), and with those of the
CZE prealbumin fraction (n = 20, R = 0.780, p < 0.01). Even when the values of the fractions obtained using
AGE and CZE were correlated, they were found to be significantly different (Wilcoxon’s test: p = 0).
Passing-Bablok regression analysis and Bland-Altman difference plot were carried on to compare the
discrepancies between AGE and CZE for the fraction which appeared to be well correlated (Table 3). No
significant deviation from linearity was found, whatever the fraction may be. Both techniques appeared to
differ significantly for the quantification of alpha-1 and gamma fractions. For albumin and beta fractions, the
differences between the two techniques did not vary in any systematic way over the range of measurement.
For alpha-1 and gamma fractions, the discrepancy between the two studied techniques increased with the
magnitude of the measurement.
The average of the A/G ratios was 0.63 ± 0.04 with AGE and 1.56 ± 0.16 with CZE, with no direct
relationship between the values obtained by the two techniques.
The AGE patterns of the pigeons were divided into six fractions (Figure 1, Table 1). These were then
classified into one prealbumin fraction, one albumin fraction, one alpha fraction, two beta fractions and one
gamma fraction. The albumin fraction was identified as having the strongest anodal peak. As for the case of
the black kites with AGE, the electrophoresis patterns of the pigeons were characterised by the presence of a
second anodic peak at the alpha-1 position, comparable in intensity with the albumin peak (Figure 1).
The CZE electrophoresis patterns of the pigeons were divided into nine fractions (Figure 1, Table 1). These
electrophoresis patterns were then also found to present two intense anodic peaks. By comparing the height
of the peaks obtained using AGE and CZE, we chose to class the most anodic peak with the prealbumins,
and the second peak with the albumin. The correlations confirmed this choice, since the resulting albumin
fraction was found to be very well correlated with that determined by AGE (n = 10, R = 0.951, p < 0.01).
135
The prealbumin fraction obtained using CZE was found to be very well correlated with the alpha fraction
obtained with AGE (n = 10, R = 0.944, p < 0.01; n = 10). The beta and gamma fractions were named by
comparison with the electrophoresis patterns obtained on agarose gel. The beta-1 and beta-2 fractions were
found to be very well correlated with the beta-1 and beta-2 fractions obtained on agarose gel (n = 10, R =
0.922, p = 0.01; n = 10, R = 0.927, p < 0.01). The sum of the gamma-1 and gamma-2 fractions was found to
be very well correlated with the gamma fraction obtained on agarose gel
(n = 10, R = 0.941, p <
0.01).Although the values of the fractions obtained using AGE and CZE were correlated, they were all found
to be significantly different (Wilcoxon’s test: p = 0).
Passing-Bablok regression analysis and Bland-Altman difference plot were carried on to compare the
discrepancies between AGE and CZE for the fraction which appeared to be well correlated (Table 4). No
significant deviation from linearity was found, whatever the fraction may be. Both techniques appeared to
differ significantly for the quantification of the beta-2 fraction. For all of the fractions, the differences
between the two techniques did not vary in any systematic way over the range of measurement. For the beta2 fraction, the discrepancy between the two studied techniques increased with the magnitude of the
measurement.
The average value of the A/G ratios was 0.62 ± 0.07 for AGE and 2.51 ± 0.22 % for CZE. The ratios
obtained with these two techniques are thus markedly different, and were not found to be correlated to each
other.
136
Figure 1. Agarose gel electrophoresis tracings of a rooster (A), a Black kite (B), and a racing pigeon (C).
Capillary zone electrophoresis tracings of a rooster (D), a Black kite (E), and a racing pigeon (F).
137
Table 1: Plasma protein electrophoresis fraction values in roosters, black kites and racing pigeons obtained by Agarose Gel Electrophoresis (AGE) and
Capillary Zone Electrophoresis (CZE). Results are expressed as median and ranges of the fraction values.
Species
Rooster
(n = 30)
Rooster
(n = 30)
Black kite
(n = 20)
Black kite
(n = 20)
Pigeon
(n = 10)
Pigeon
(n = 10)
138
Technique
AGE
CZE
AGE
CZE
AGE
CZE
Pre albumin
Albumin
α1-globulin
α2-globulin
α3-globulin
β1-globulin
β2-globulin
γ1-globulin
γ2-globulin
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
0.77
13.40
0.74
1.21
6.31
9.73
2.8
2.38
[0.26-1.90]
[10.52-16.81]
[0.51-1.78]
[0.87-1.86]
[5.57-7.66]
[6.56-14.68]
[1.59-3.97]
[1.65-4.48]
1.99
13.98
2.19
1.08
3.51
7.69
2.55
4.60
[1.29-2.80]
[10.92-18.02]
[1.62-3.33]
[0.84-1.50]
[2.84-5.15]
[4.61-11.54]
[1.43-4.10]
[2.29-8.02]
0.49
14.07
12.54
0.98
5.16
5.09
[0.25-0.84]
[10.78-18.43]
[9.16-15.91]
[0.80-1.38]
[4.09-6.31]
[3.23-6.79]
5.88
16.91
3.70
1.31
4.25
5.89
[3.60-8.66]
[13.14-21.97]
[2.46-5.02]
[1.01-1.73]
[3.02-5.38]
[3.39-8.44]
0.73
9.44
10.81
3.33
1.56
1.52
[0.56-1.01]
[8.08-12.89]
[9.04-14.08]
[2.66-4.54]
[1.23-2.52]
[0.67-1.83]
11.22
8.47
1.00
1.18
0.78
2.52
0.71
0.50
1.13
[9.11-14.52]
[7.30-11.77]
[0.73-1.26]
[1.00-1.99]
[0.46-0.93]
[1.68-3.48]
[0.52-1.42]
[0.40-0.68]
[0.42-1.90]
Table 2: Slopes and intercepts calculated by the Passing-Bablok regression analysis, and Mean
differences calculated by the Bland-Altman difference plot, in roosters for the fractions which values
assessed by AGE and CZE appeared to be correlated.
Albumin
Alpha-1
Alpha-3
Beta-1
Beta-2
Gamma
Slope B
0.90
0.45
1.29
1.15
1.19
0.44
Confidence
[0.82 to 1.00]
[0.23 to 0.75]
[0.95 to 1.60
[0.99 to 1.48]
[0.92 to 1.51]
[0.31 to 0.56]
Intercept A
0.7890
-0.2129
1.9595
1.3068
-0.4139
0.5068
Confidence
[-0.53 to 1.88]
[-0.85 to 0.28]
[0.95 to 3.06]
[-1.35 to 2.38]
[-1.30 to 0.33]
[-0.00 to 1.02]
-0.65
-1.40
2.90
2.18
0.12
-2.09
[0.37 to -1.67]
[-1.92 to -0.89]
[2.27 to 3.53]
[0.74 to 3.63]
[-0.56 to 0.81]
[-3.88 to -0.31]
interval 95%
interval 95%
Mean
difference
Confidence
interval 95%
Table 3: Slopes and intercepts calculated by the Passing-Bablok regression analysis, and Mean
differences calculated by the Bland-Altman difference plot, in black kites for the fractions which
values assessed by AGE and CZE appeared to be correlated.
Albumin
Alpha-1
Beta
Gamma
Slope B
0.90
3.17
0.88
0.65
Confidence
[0.75 to 1.06]
[2.10 to 4.70]
[0.68 to 1.21]
[0.50 to 0.76]
Intercept A
-1.0903
0.6012
1.4090
1.2741
Confidence
[-3.85 to 1.57]
[-5.06 to 4.90]
[-0.02 to 2.13]
[0.66 to 1.97]
-2.93
8.8
0.87
-0.83
[-4.30 to -1.56]
[6.2 to 11.5]
[0.29 to 1.44]
[-1.94 to 0.28]
interval 95%
interval 95%
Mean
difference
Confidence
interval 95%
139
Table 4: Slopes and intercepts calculated by the Passing-Bablok regression analysis, and Mean
differences calculated by the Bland-Altman difference plot, in pigeons for the fractions which values
assessed by AGE and CZE appeared to be correlated.
Albumin
Beta-1
Beta-2
Gamma
Slope B
1.09
1.06
1.93
0.87
Confidence
[0.97 to 1.28]
[0.74 to 1.48]
[1.22 to 10.72]
[0.55 to 1.15]
Intercept A
0.26
0.71
0.16
-0.02
Confidence
[-1.28 to 1.49]
[-0.31 to 1.46]
[-6.29 to 0.70]
[-0.57 to 0.45]
1.14
0.87
0.95
-0.31
[0.65 to 1.64]
[0.44 to 1.30]
[0.43 to 1.47]
[-0.64 to 0.02]
interval 95%
interval 95%
Mean
difference
Confidence
interval 95%
Repeatability and reproducibility
The results of the intra-run repeatability and inter-run reproducibility, obtained for both AGE and
CZE, are given in Table 5. The coefficients of variations (CV) ranged from 1.8 to 9.9% for AGE, and
0.9 to 4.2 % for CZE. The accuracy therefore appeared to be higher in the case of CZE than with
AGE. With each technique, both repeatability and reproducibility were very good for strong and well
defined fractions. On the other hand, they were lower for badly defined fractions, i.e. alpha-1, alpha-2,
beta-2 and gamma fractions in AGE, and alpha fractions in CZE. The repeatability and reproducibility
achieved for the fractions of weak intensity were also generally weak, as was the case for example
with the prealbumin and alpha-2 fractions obtained with both techniques.
140
Table 5: Intra-run repeatability and inter-run reproducibility of Agarose Gel Electrophoresis (AGE) and Capillary Zone Electrophoresis (CZE) in roosters. For
AGE, repeatability within a run was estimated by interpreting the plasma patterns of 8 aliquots of the same sample run on the same rack. Inter-run
reproducibility was estimated by interpreting the plasma patterns of 6 aliquots of the same sample run on different racks.
For CZE, repeatability within a run was estimated by interpreting the plasma patterns of 8 aliquots of the same sample run on the same rack. Inter-run
reproducibility was estimated by interpreting the plasma patterns of 6 aliquots of the same sample run on different racks.
Agarose gel electrophoresis
Fraction
Capillary zone electrophoresis
Intra-run
Intra-run repeatability
Inter-run
Inter-run reproducibility
Intra-run
Intra-run repeatability
Inter-run
Inter-run reproducibility
Mean (g/l)
*
CV (%)
Mean (g/l)
*
CV (%)
Mean (g/l)
*
CV (%)
Mean (g/l)
CV* (%)
(n = 30)
(n = 30)
(n = 8)
(n = 8)
(n = 8)
(n = 8)
(n = 6)
(n = 6)
Prealbumin
0.51
9.1
0.5
8.3
1.81
4.2
1.87
4.6
Albumin
12.53
1.8
12.65
2.2
13.89
0.9
13.75
1.1
α1-globulin
0.59
8
0.58
9.1
2.3
2.6
2.34
2.8
α2-globulin
1.00
9.9
1.08
11.1
1.59
3.4
1.6
2.9
α3-globulin
6.74
1.7
6.75
1.5
1.11
3.3
1.14
4.5
β1-globulin
9.78
2.5
9.35
3.9
7.15
0.8
7.17
1.5
β2-globulin
3.04
7.5
3.35
8.9
3.4
1.7
3.46
4.1
γ-globulin
3.01
6.5
2.94
6.3
5.94
1.9
5.88
2.4
A/G ratio
0.54
2.7
0.55
3.2
0.73
1.9
0.72
2.1
*
Coefficient of variations
141
Discussion
This study is, as far as we know, the first to have dealt with plasma protein capillary
electrophoresis in birds. Our data therefore represents the first set of baseline information relative to
the use of CZE in avian medicine.
Generally speaking, for the three studied species we found a good degree of correlation between the
results obtained from the two techniques, in particular for the albumin, beta and gamma fractions.
These correlations do not mean that these fractions are exactly the same, but that some of the most
abundant proteins constituting them are the same. The interpretation of CZE electrophoresis patterns
therefore seems to be quite similar to that of AGE patterns. However, the results from correlations
between fractions indicate that, for the three species studied, some of the alpha fraction proteins in
AGE could migrate to the prealbumin fraction in CZE. Indeed, we observed a high degree of
correlation between the alpha-3 fraction values in AGE and those corresponding to the prealbumin
fraction in CZE for the rooster, and between the alpha-1 fraction values and those corresponding to the
prealbumin fraction in CZE for the black kites. This phenomenon is even more obvious in the pigeon
with the alpha fraction. In this species, the strenght of the resulting prealbumin peak obtained with
CZE can lead to confusion. Such an intense anodic fraction could in effect be mistakenly identified as
the fraction corresponding to albumin. Such a phenomenon has, as far as we know, never been
documented in humans, as AGE and CZE patterns are considered to be very similar.2,9,11,13,15 Indeed,
for human laboratory medicine, buffered solutions have been specially designed so that CZE patterns
have the same general aspect as AGE patterns.17 Additional studies should be undertaken in birds in
order to identify the protein(s) present in the alpha fraction of AGE. Their identification would lead to
an improved understanding of avian electropherograms, using both AGE and CZE.
For some fractions, such as the alpha-2 fraction in roosters and black kites, the values obtained with
AGE and CZE were not correlated. This is probably due to the fact that manual separation of such
poorly defined fractions of such weak intensity is highly subjective for the technician.6,32 As a
consequence, for such fractions the resulting relative error margin is significant.
Passing-Bablok regression analysis and Bland-Altman difference plot show significant discrepancies
between the two techniques, for alpha, beta and gamma fraction values, depending on the species. The
numerical values for the CZE fractions are furthermore in general very different to those obtained with
AGE, thus exposing the need for specific reference intervals for the correct interpretation of capillary
electropherograms. As previously discussed, such differences could be related to differences in
electrophoretic mobility between certain proteins, depending on which technique is used. They could
also be related to differences in the affinity of certain proteins for the dyes used in AGE. Indeed, in
human laboratory medicine, comparisons between CZE, immunonephelometry and colorimetry
suggest that direct quantification of albumin and alpha-1 globulin by UV absorption is more accurate
than protein staining.2.9 In the alpha-1 fraction, for instance, the high sialic acid content of the alpha-1
142
acid glycoprotein has been shown to interfere with the binding of dyes in gel-based methods, whereas
UV absorption used in CZE is not affected by these sugar moieties.33
Due to the higher resolution of CZE compared with AGE,2 some fractions were separated by CZE and
not identified in AGE. The interpretation of such fractions is currently unknown and would be
investigated in further studies.
By convention, the A/G ratio in birds is defined as the sum of the prealbumin and albumin fractions
divided by the sum of the globulin fractions.4,31 Taking into account the localisation at the level of the
prealbumin fraction in CZE, of proteins initially present in the AGE alpha fraction, the A/G ratios
defined for AGE can not be compared with those obtained with CZE. It may therefore be more
appropriate to define the A/G ratio in birds as that given by the albumin fraction divided by the sum of
the globulin fractions, including the prealbumin in the globulins.6
The study reported here stresses that in roosters, the repeatability and reproducibility of CZE
are found to be higher than those of AGE. This is consistent with publications concerning human
laboratory medicine.2,9,33 As reported by Bienvenu in humans, the resolution achieved with CZE is
higher, and the clarity of the electrophoresis bands is better than with classical methods.2 The
improved repeatability and reproducibility may furthermore be related to the complete automation of
the analytical procedure, and to the fact that in CZE, protein absorbance is measured directly by UV
absorption, instead of being determined indirectly from the amount of dye adsorbed onto the protein.33
Further studies should however assess more thoroughly repeatability and reproducibility of CZE in
pathological avian samples.
For both techniques, the repeatability and reproducibility figures determined in this study are generally
poorer than those given in publications related to human electrophoresis using the same
equipment,12,13,33 or in the user’s manuals of the Protein 6© and Hydragel protein© kits published by
Sebia (Hydragel 7, 15 and 30 protein – 2005/5; Capillarys protein 6 – 2007/06). This is probably
related to the fact that the fractions are better separated from one another on the human
electropherograms, than on those obtained from birds, reported in our study. The weak and poorly
separated fractions are also those determined with the lowest accuracy. Manual separation of such
fractions by the technician may indeed be more subjective, and the smaller the fraction is, the
comparatively more significant the error becomes.6,32 This may explain why, in contrast, the
repeatability and reproducibility of strong and well-defined fractions such as albumin are excellent.
With regard to the poor accuracy with which the prealbumin fraction is determined using AGE, it was
found that in certain cases, despite the care taken to clean the gels before curve acquisition, the
electrophoresis curve did not completely return to the baseline, thus leading to over-estimation of this
fraction. This phenomenon was not noticed with CZE.
The analysis of electrophoresis patterns obtained from both techniques has finally enabled
significant inter-species differences to be revealed. The electrophoresis patterns of the three species
chosen for this study are in effect very different. Some authors have already discussed these significant
143
inter-species differences for classical techniques,4,26,34,35 and all practitioners should be aware that the
electropherogram of an individual must always be interpreted by comparing it with a normal
electrophoregram for the given species, or better still with personal reference values.
Further studies should be carried out in order to analyse plasma protein electrophoresis in other bird
taxa with CZE, in particular for the case of species regularly seen in veterinary practices or zoological
gardens. Further studies should also investigate the effects of hemolysis and lipemia as interference
factors, and the effects of sample conservation on avian capillary electrophoregrams, like it has been
already assessed in humans.9
Although the use of CZE requires establishing reference ranges for each species, it allows
considerable advantages over traditional gel-based methods. Capillary zone electrophoresis doesn’t
require any matrix for protein separation. Apart from its improved repeatability and reproducibility,
when compared with agarose gel or cellulose acetate methods, it can therefore offer considerable
technological progress to the laboratories. CZE systems such as Capillarys2© are indeed fully
automated and thus require almost no technical manipulation (bar-code identification of the samples,
automation of all analytical steps). Such automated machines allow very high analysis rates to be used,
and are thus very well suited to laboratories having to deal with high work loads.
Indeed, in our study, the Capillarys2© had a throughput of 80 samples / h when the samples were
analysed in a single batch, whereas in the case of the Hydrasis© system, processing of the first series
took 45 min, and 25 min for the following series, with 30 plasma samples being simultaneously
analyzed per batch.
Capillary electrophoresis nevertheless suffers from some drawbacks. It requires a bigger sample size
than AGE (14 fold more in our case), which can be a major disadvantage when dealing with small
birds. Furthermore, because it makes use of UV detection for direct protein quantification, via the
peptide bonds, any substance in the plasma, such as a radio-opaque agent or sulfamethoxazole, which
absorbs light at this wavelength, has the potential to produce an artifact. This is not the case with
conventional gel-based methods, where quantification of the protein fractions is based on dye binding
(Amido-black in our case), which has a great affinity for proteins.33,36,37 In the case of birds, it would
be highly interesting to verify whether the supplementation of certain species held in captivity, such as
flamingos or the Scarlet Ibis, using synthetic carotenoids such as canthaxanthin, is not responsible for
the presence of artefacts found with CZE.
144
Acknowledgements
We wish to thank the INRA in Tours-Nouzilly for the rooster blood samples they provided, Mr. JL
Liegeois from the Académie de fauconnerie du Puy du fou (France) for the black kite blood samples
he provided, Sebia for all the CZE analyses they performed, and P Trolliet from Sebia for his
invaluable technical support, the team from bio VSM, Mrs. K Naudin from bio VSM who carried out
total protein measurements. We are grateful to the Muséum National d’Histoire Naturelle and the
Conseil Général de Seine Maritime (France) for their financial support. We also appreciate the help
provided by Glenn Lund from Techtrans consulting, for proofreading this document.
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DISCUSSION GENERALE : apport de nos travaux à la
compréhension globale des électrophorégrammes aviaires
I.
DIVERSITE TAXONOMIQUE DES OISEAUX
Dans la totalité des études que nous avons menées sur plusieurs espèces, nous avons pu
constater que la diversité taxonomique de la classe des oiseaux se traduisait par une grande diversité
des électrophorégrammes obtenus. De telles variations interspécifiques dans l’aspect des profils
électrophorétiques des oiseaux ont d’ores et déjà été signalées par de nombreux auteurs (Sibley &
Hendrickson, 1970; Cray & Tatum 1998; Zaias et al., 2000; Blanco & Hofle, 2003). Elles sont un des
principaux obstacles à l’utilisation de l’électrophorèse des protéines plasmatiques dans le diagnostic
des maladies des oiseaux.
Certaines particularités telles que la présence d’un pic important dans la région des alphaglobulines chez certains taxons comme les Colombiformes, les Falconiformes, les Strigiformes, les
Psittaciformes, les Phénicoptériformes, les Charadriiformes ou les Ciconiiformes que nous avons pu
observer lors de l’utilisation courante de l’électrophorèse à des fins diagnostiques sont particulièrement
surprenantes. L’importance de ce pic en alpha est variable et semble être une caractéristique d’espèce.
Cependant, mis à part Tatum et al. (2000) qui mentionnent chez les strigiformes et les falconiformes la
présence d’un pic monoclonal accolé au pic d’albumine, dans la région des alpha-1 globulines, aucune
autre publication à l’heure actuelle n’en fait clairement état. Tout au plus, certains auteurs publient
chez les rapaces diurnes ou le pigeon des valeurs de références mettant en exergue une fraction alpha
pouvant atteindre 30% des protéines totales (autant que l’albumine). Ils ne font aucun commentaire au
sujet d’un pic important dans cette zone (Blanco & Hofle, 2003; Gayathri & Hegde, 2006). L’étude par
spectrométrie de masse de portions de gels excisées correspondant au pic monoclonal présent dans la
région des alpha-globulines du pigeon domestique (Columba livia), du milan noir (Milvus migrans) et
de l’amazone aourou (Amazona amazonica) nous a permis d’identifier ce pic comme étant constitué
d’apolipoproteines A-I chez ces trois espèces. Cette apolipoprotéine joue un rôle capital dans le
transport et le transport « reverse » de cholestérol et de phospholipides sous la forme des High Density
Lipoproteines (HLD) (Blue et al., 1981 ; Klasing, 2000 ; Kiss et al., 2001). L’abondance de cette
protéine chez certaines espèces pourrait être liée au métabolisme particulier des graisses chez les
oiseaux, pour qui les lipides constituent la principale source d’énergie utilisée lors de vols sur de
longues distances (Butler, 1991 ; Butler & Bishop, 2000). Des publications ultérieures permettront
d’approfondir cette étude en élargissant l’éventail des espèces et des statuts physiologiques étudiés
(hyperphagie prémigratoire, migration, ponte, mue, etc.).
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Le fait que l’apolipoprotéine A-I soit présente en quantité aussi importante au niveau de la
fraction alpha chez certaines espèces d’oiseaux doit être pris en considération lors de l’interprétation de
leurs électrophorégrammes plasmatiques. En effet, la présence d’un pic d’une telle intensité peut être
une source d’erreur diagnostique grave, puisque jusqu’à présent, la plupart des auteurs s’accordaient à
dire que la fraction alpha était principalement composée de protéines de la phase aiguë de
l’inflammation, telles que l’alpha-1 antitrypsine ou l’alpha-2 macroglobuline, dont l’augmentation du
taux plasmatique indique la présence d’un phénomène inflammatoire (Cray, 1997 ; Cray & Tatum ,
1998 ; Werner & Reavill, 1999). Or il a été démontré chez l’homme et chez le poulet que
l’apolipoproteine A-I est, tout comme l’albumine, une protéine négative de la phase aiguë de
l’inflammation, puisque son taux plasmatique diminue lors d’un phénomène inflammatoire (Wicher et
al., 1991 ; Monnet et al., 2002 ; Upragarin, 2005). Ceci doit être pris en compte, tant pour
l’interprétation des profils électrophorétiques que pour le calcul du rapport albumine / globulines
(A/G). Chez les espèces ayant des pics en alpha important, un rapport défini par la somme des fractions
albumine et alpha 1 divisée par le reste des globulines serait en effet probablement plus puissant dans
le diagnostic d’une inflammation, que le rapport A/G classique.
Comme nous l’avons dit précédemment, notre utilisation courante de l’électrophorèse des
protéines plasmatiques, ainsi que les travaux d’autres auteurs ont permis de mettre en évidence de
nombreuses variations interspécifiques des électrophorégrammes chez les oiseaux (Sibley &
Hendrickson, 1970; Cray & Tatum 1998; Zaias et al., 2000; Blanco & Hofle, 2003). Nous avons donc
entrepris d’étudier de manière plus approfondie ces différences inter-taxonomiques en nous basant sur
des espèces d’oiseaux phylogénétiquement éloignées, telles que le paon bleu (Pavo cristatus), l’oie à
tête barrée (Anser indicus), le pigeon domestique (Columba livia), le milan noir (Milvus migrans) et
l’amazone aourou (Amazona amazonica). La mesure des distances de migration sur gel d’agarose de
l’albumine et du fibrinogène chez ces espèces nous a permis d’aborder de manière quantifiable les
disparités pouvant exister entre les profils électrophorétiques de ces cinq taxa. La comparaison des
distances de migration de l’albumine a montré que cette protéine migre à des endroits différents en
fonction de l’espèce concernée. Ceci avait déjà été décrit chez la perruche ondulée (Nymphicus
hollandicus) et le poulet (Gallus gallus), et attribué à des différences de répartition des charges de
surface de cette protéine en fonction de l’espèce concernée (Archer & Battison, 1997). La
comparaison des distances de migration du fibrinogène nous a également permis de scinder les cinq
espèces étudiées en deux groupes distincts correspondant à la classification phylogénétique des
oiseaux proposée par Lecointre et Le Guyader (2001). Dans l’infraclasse des Galloanserae
comprenant dans notre cas l’oie à tête barrée et le paon bleu, le fibrinogène migre significativement
plus loin que dans l’infraclasse des Neoaves comprenant ici le pigeon, le milan noir et l’amazone
aourou. Dans chacune des deux infraclasses, aucune différence interspécifique de la distance de
migration du fibrinogène n’a pu être mise en évidence. La structure ou la répartition des charges de
surface du fibrinogène semblerait ainsi être différente dans les deux infraclasses. Il sera nécessaire de
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tester cette hypothèse dans des études ultérieures basées sur un nombre plus important d’espèces
appartenant aux deux groupes taxinomiques.
Le fibrinogène est l’une des principales protéines de la phase aiguë de l’inflammation (Hawkey &
Hart, 1988). Tous les auteurs s’accordent à dire qu’il migre en position beta sur les
électrophorégrammes des oiseaux (Kaneko, 1997 ; Cray & Tatum, 1998 ; Werner and Reavill 1999 ;
Rosenthal, 2000 ; Fudge and Speer, 2001). Nous proposons donc que cette protéine, facile à identifier
par la comparaison des profils d’électrophorèse plasmatique et sérique puisse servir de repère pour
nommer les autres fractions de manière conventionnelle chez les oiseaux. Le pic correspondant au
fibrinogène pourrait ainsi être nommé «beta », tandis que les globulines présentes entre l’albumine et
le fibrinogène seraient nommées alpha, et les globulines situées en position cathodique par rapport au
fibrinogène, gamma.
II. APPORTS A LA COMPREHENSION DES EFFETS DE LA PONTE
ET DE LA MUE
Bien que la mue soit indéniablement la cause de changements radicaux au niveau du
métabolisme protéique des oiseaux, son impact sur les électrophorégrammes aviaires n’est pas apparu
comme très important. Les changements observés chez les oies à tête barrée (Anser indicus) en periode
de mue se sont en effet révélés significatifs, mais peu importants par rapport aux variations
interindividuelles. Le modèle utilisé dans cette étude avait de plus été choisi en raison de la brièveté et
de l’intensité du phénomène de mue au cours duquel la repousse simultanée des rémiges impose à
l’oiseau de rester au sol pendant 35 à 40 jours (Delacour, 1964; Todd, 1996). Il est donc très probable
que, dans d’autres taxons où la mue s’étale sur plusieurs mois, comme chez les falconiformes, les
psittaciformes, ou les gruiformes (Mirande et al., 1994 ; Brown & Amadon, 1989 ; Forshaw &
Cooper, 1989), l’impact de la mue soit négligeable.
Les oiseaux en mue ont présenté une diminution de la concentration en protéines totales,
principalement liée à une diminution des fractions albumine, alpha-2 et gamma. Parallèlement, durant
la même période, nous avons observé une augmentation de la fraction pré-albumine. Autour de la
période de mue, les valeurs des fractions alpha-1 étaient plus élevées qu’avant ou après la mue. Les
seules modifications décrites par les études précédentes concernaient les protéines totales et les
fractions albumine et alpha-2 (Driver, 1981; Gildersleeve et al., 1983; De Graw & Kern, 1985; Work,
1996). Notre étude a donc permis pour la première fois la mise en évidence de variations des fractions
préalbumine, alpha-1 et gamma chez l’oiseau. Chez l’oiseau en mue, l’accélération du turn-over des
protéines (Murphy & Todd, 1995) permet une plus grande souplesse dans la mobilisation des acides
aminés contenus dans des protéines comme l’albumine, ce qui permet de favoriser la pousse des
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plumes composées à 95% de protéines (Murphy & King, 1992). Ceci, ainsi que l’augmentation de la
volémie liée à l’expansion du système circulatoire autour des follicules plumeux (Chilgren & De
Graw, 1977; De Graw & Kern, 1985) a très vraisemblablement contribué à la diminution des taux
plasmatiques des fractions albumine, alpha-2 et gamma. De plus, l’augmentation des taux de triiodothyronine lors de la mue s’accompagne d’une diminution de l’activité du système immunitaire à
médiation humorale (Bourgeon & Raclot, 2007) expliquant la diminution des taux plasmatiques de la
fraction gamma. L’augmentation des taux plasmatiques d’hormones thyroïdiennes s’accompagne
également de l’augmentation de la synthèse de protéines présentes dans la fraction alpha-1, et de
protéines de transport de la thyroxine, comme la transthyrétine (Grieninger, 1978; Chamanza et al.
1999, Lanzarot et al., 2005), ce qui explique respectivement l’augmentation que nous avons observée
chez les oiseaux en mue pour les fractions alpha-1 et pré-albumine.
Les effets de la ponte sur les électrophorégrammes aviaires se sont révélés plus importants que
ceux liés à la mue. Ils sont donc plus susceptibles d’induire le praticien en erreur. Le modèle choisi
dans notre étude, pour évaluer l’impact de la ponte sur l’électrophorèse des protéines plasmatiques
chez l’oiseau est la poule pondeuse (Gallus gallus). Ce modèle nous a permis de pouvoir observer
simultanément un grand nombre d’oiseaux en ponte et de nous soustraire aux effets potentiels de la
couvaison, puisque les œufs étaient ramassés quotidiennement. Les profils électrophorétiques des
oiseaux en ponte se sont caractérisés par une augmentation des fractions alpha-1, beta-1 et beta-2 et
une diminution de la fraction alpha-3. L’augmentation de la fraction beta était liée à l’apparition, chez
les pondeuses, d’une bande supplémentaire dans cette zone, d’aspect monoclonale et de mobilité
électrophorétique relativement variable. La très forte corrélation entre cette fraction et le taux de
triglycéride chez les oiseaux en ponte laisse à penser que cette fraction est principalement constituée
de VLDLy, principale forme de transport des lipides synthétisés dans le foie et à destination des
follicules en croissance (Hermier et al.,1989; Walzem et al., 1994; Klasing, 2000). La diminution
de la concentration en HDL, décrite par certains auteurs chez les oiseaux en ponte (Hermier et al.,
1989; Cho & Park, 1991; Hermann et al., 2003) est très probablement responsable de la
diminution de la fraction alpha-3, comme l’indique la très forte corrélation entre les valeurs de cette
fraction et le taux de cholestérol HDL dans cette étude. L’identité de la protéine présente dans la
fraction alpha-1, et dont la concentration plasmatique augmente lors de la ponte reste inconnue, mais
pourrait correspondre aux vitellogénines, complexes moléculaires de densité supérieure aux HDL
(Kudzma et al., 1979; Chapman, 1980).
L’étude de l’influence de la ponte sur l’aspect des profils électrophorétiques des oiseaux met en
évidence qu’il est capital, lors du recueil des commémoratifs, d’apprécier l’état physiologique d’un
oiseau, pour pouvoir interpréter correctement son électrophorégramme. L’apparition du pic de VLDLy
chez un oiseau en ponte peut, en effet être interprétée à tort par un praticien non averti comme un pic
de fibrinogène signant l’existence d’un processus inflammatoire.
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III. APPORTS A LA COMPREHENSION DES EFFETS DE
L’HEMOLYSE ET DE LA LIPEMIE
Notre utilisation courante de l’électrophorèse des protéines plasmatiques à des fins
diagnostiques chez les oiseaux, a permis de mettre en évidence que des interférences analytiques telles
que l’hémolyse ou la lipémie sont fréquentes. Elles représenteraient respectivement 4,55 et 2,87 % des
prélèvements réalisés (Fudge, 2000). Cependant, l’impact de telles interférences analytiques n’a
jamais été étudié spécifiquement.
Dans notre étude, les effets de l’hémolyse sur les profils électrophorétiques des oiseaux ont été
analysés chez deux espèces phylogénétiquement éloignées, l’oie à tête barrée (Anser indicus), et le
milan noir (Milvus migrans). Il est apparu que la présence d’hémoglobine libre dans le prélèvement
s’accompagnait de l’augmentation de la fraction gamma. Cette observation correspond à la description
succincte des effets de l’hémolyse sur l’électrophorèse des protéines effectuée par Werner & Reavill
(1999) et Cray et al. (2007) chez les psittacidés. Cependant, il semble qu’en fonction des espèces, la
présence d’hémoglobine dans le prélèvement s’accompagne également d’une augmentation de la
fraction beta. En effet, bien que l’hémoglobine migre indubitablement en gamma, chez certaines
espèces, comme le milan noir dans notre cas, le voisinage immédiat sur le gel d’agarose de la fraction
beta et de la bande correspondant à l’hémoglobine entraine une augmentation artéfactuelle de la
fraction beta lors de l’hémolyse, ce qui n’est pas le cas chez l’oie à tête barrée. L’interférence de
l’hémolyse sur les profils électrophorétiques est donc bien différente de celle qui est décrite chez les
mammifères où l’hémoglobine libre est connue pour migrer en beta-1, tandis que les complexes
hemoglobine-haptoglobine migrent en alpha-2 (Bossuyt et al., 1998b; Benlakehal et al., 2000; Thomas,
2001). Chez l’oiseau, la différence réside probablement dans le fait que l’haptoglobine ne semble pas
exister dans la sous-classe des neognathae, où elle est remplacée par une autre protéine porteuse de
l’hémoglobine, la protéine PIT 54 (Wicher & Fries, 2006).
Les effets de la lipémie sur les profils électrophorétiques des oiseaux sont plus complexes à
aborder. La nature des lipoprotéines mises en jeu peut en effet être très variable en fonction de son
origine (posprandiale ou ponte) (Hermier et al. 1989, Klasing, 2000), ou en fonction du moment où le
prélèvement a été réalisé (Klasing, 2000). Le modèle choisi ici pour l’étude des interférences liées à la
lipémie postprandiale est le milan noir (Milvus migrans). Il est en effet facile chez la plupart des
rapaces d’obtenir une lipémie postprandiale dans les deux heures après un nourrissage (observation
personnelle). De notre étude, il ressort que celle-ci n’a aucun effet sur les profils d’électrophorèse des
protéines plasmatiques chez l’oiseau. Elle entraine tout au plus une diminution des protéines totales
par un effet de dilution de ces dernières par l’afflux de triglycérides (Guder et al., 2002 ; Kroll , 2004),
cette diminution des protéines totales se répercutant significativement sur les valeurs brutes des
différentes fractions. Mais ces variations sont peu significatives d’un point de vue clinique, car
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inférieures à la variabilité interindividuelle. Les lipides sont la source d’énergie la plus importante
pour les rapaces, à tel point que leur capacité de synthèse hépatique des acides gras est faible, comparé
aux oiseaux granivores (Klasing, 2000). Les oiseaux ont d’une manière générale un système
lymphatique peu développé. Les lipoprotéines issues de la digestion sont donc directement sécrétés
dans la circulation sanguine sous la forme de portomicrons, proches, sur le plan structurel des
chylomicrons des mammifères (Hermier, 1989 ; Klasing, 2000). Ces portomicrons sont ensuite
progressivement captés par le foie qui les reconditionne sous la forme de Very Low Density
Lipoproteins (VLDL) (Hermier, 1997 ; Klasing, 2000). Dans notre cas, la lipémie postprandiale a donc
probablement été principalement le fait de la mise en circulation massive de portomicrons. Or, la
grande taille de ces lipoprotéines (150 nm) n’a très probablement pas permis leur pénétration et leur
migration dans le gel d’agarose. Ces portomicrons seraient donc restés au point de dépôt, sans être
colorés correctement par le noir amidon, en raison de leur très faible teneur en protéines (1-2%)
(Hermier et al., 1985, 1988). Il importera dans des études ultérieures de déterminer les effets de la
lipémie sur les électrophorégrammes d’oiseaux granivores. En effet, leur capacité de synthèse
hépatique d’acides gras est principalement basée sur l’utilisation des hydrates de carbones de la ration
et résulte donc en une lipémie liée majoritairement à la présence de VLDL dans le plasma (Klasing,
2000).
Revenons pour finir à l’importance de l’origine de la lipémie. Les effets liés à la lipémie de
ponte ont déjà été développés dans la partie précédente traitant des changements liés à des
phénomènes physiologiques. Or, ils sont beaucoup plus importants que ceux liés dans notre étude à la
lipémie postprandiale, puisqu’ils entrainent chez la poule pondeuse une augmentation des fractions
alpha-1 et beta et une diminution de la fraction alpha-3. Ces modifications des profils
électrophorétiques correspondent à celles évoqués par Werner & Reavill (1999) ou par Cray et al.
(1999) dans le cadre plus général de leur étude de la lipémie. Elles illustrent parfaitement que les effets
d’un artéfact comme la lipémie sont largement dépendants des lipoprotéines mises en jeu (VLDLy
dans le cas de la ponte). Dans la mesure où il parait difficile de prévoir les perturbations observées sur
les électrophorégrammes aviaires sur la base de la seule observation de l’état lipémique d’un
prélèvement, il semble plus raisonnable d’écarter systématiquement les prélèvements lipémiques.
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IV. COMPARAISON DE L’UTILISATION, DES DEUX TECHNIQUES
LES PLUS UTILISEES ACTUELLEMENT EN LABORATOIRE DE
DIAGNOSTIC MEDICAL : L’ELECTROPHORESE EN GEL
D’AGAROSE ET L’ELECTROPHORESE CAPILLAIRE DE ZONE
L’électrophorèse en gel d’agarose (AGE) est actuellement la technique la plus utilisée en
diagnostic de laboratoire (Daunizeau, 2003 ; Lissoir et al., 2003). Elle a cependant tendance à être peu
à peu supplantée dans les gros laboratoires par l’électrophorèse capillaire de zone (CZE). Nous avons
étés les premiers à utiliser cette technique chez les oiseaux. Notre étude a été réalisée chez le coq
domestique (Gallus gallus), le milan noir (Milvus migrans) et le pigeon domestique (Columba livia).
Les profils obtenus en CZE ont paru, au premier abord, assez similaires à ceux obtenus par AGE.
Hormis pour certaines fractions alpha et pour la fraction pré-albumine, les fractions obtenues par AGE
et CZE étaient bien corrélées. Les résultats bruts, étaient, cependant significativement différents d’une
méthode à l’autre. Il semble donc nécessaire, pour l’interprétation des électrophorégrammes obtenus
en CZE d’établir des valeurs de références propres à cette méthode. Pour les fractions alpha et préalbumine, l’étude des corrélations entre les deux méthodes met en évidence que certaines protéines des
fractions alpha en AGE (alpha-3 chez le coq, alpha-1 chez le milan noir, alpha chez le pigeon) migrent
en position pré-albumine en CZE. Cette protéine, précédemment identifiée comme apolipoprotéine A-I
chez le pigeon et le milan semble donc avoir une mobilité électrophorétique radicalement différente
dans les conditions de l’AGE et de la CZE. Dans des espèces comme le pigeon, l’importance de la
fraction pré-albumine résultant de ce phénomène est telle, qu’elle peut conduire le praticien non averti
à se tromper dans l’identité du pic d’albumine. Le passage de l’apolipoprotéine A-I au niveau de la
fraction pré-albumine en CZE entraine de plus une différence importante dans la définition du rapport
A/G tel qu’utilisé conventionnellement chez les oiseaux. Enfin, l’électrophorèse capillaire permet la
séparation de fractions supplémentaires, dont la signification clinique reste à définir.
L’électrophorèse capillaire présente des avantages certains par rapport à l’AGE, tels une répétabilité et
une reproductibilité des résultats accrues, une cadence d’analyse supérieure et l’absence de support qui
permet la quantification directe des différentes fractions par mesure d’absorbance à travers la paroi du
capillaire. Cependant, elle présente aussi quelques inconvénients. Le volume de plasma nécéssaire
pour la CZE est beaucoup plus important pour la CZE que pour l’AGE (14 fois plus important dans le
cadre de notre étude). De plus, la quantification directe des protéines par mesure d’absorbance rend
cette méthode plus sensible à la présence artéfactuelle de molécules absorbant les mêmes longueurs
d’onde que les protéines (sulfaméthoxazole, produits de contrastes iodés, hémoglobine, lipoprotéines)
(Bossuyt et al., 1999, 2003 ; Brouwers et al., 2006). Des études supplémentaires devront donc être
menées pour estimer l’influence de telles interférences sur les électrophorégrammes aviaires. Il sera de
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même intéressant d’évaluer l’effet de pigments tels que les caroténoïdes présents, parfois en quantité
importante dans les plasmas des oiseaux, en particulier chez les flamants roses (Phoenicopterus sp.) ou
les ibis rouges (Eudocimus ruber) maintenus en captivités et supplémentés avec des caroténoïdes de
synthèse comme la canthaxanthine.
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CONCLUSION
Cette étude avait pour objectif de contribuer à valider l’électrophorèse des protéines
plasmatiques comme outil diagnostique en médecine aviaire appliquée à la gestion des élevages
conservatoires, cela en analysant l’impact de la diversité taxinomique, de phénomènes physiologiques
comme la mue ou la ponte, d’artéfacts potentiels comme l’hémolyse ou la lipémie sur les profils
obtenus. Notre étude s’est principalement basée sur l’utilisation de l’électrophorèse en gel d’agarose,
technique actuellement la plus utilisée en laboratoire médical. Ces travaux ont de plus été l’occasion
d’aborder pour la toute première fois chez l’oiseau l’utilisation de l’électrophorèse capillaire de zone,
technique émergente en médecine humaine.
Que ce soit dans le cadre de la gestion à long terme des populations ou dans un but de
réintroduction, la problématique de conservation en parc zoologique et en élevage conservatoire
nécessite de travailler avec un nombre important d’oiseaux répartis dans des taxons très variés. La
densité importante des collections animales et la promiscuité qui en découle, ainsi que les conditions
de stress inhérentes à la captivité sont favorables à la transmission de nombreuses maladies et
imposent par conséquent la mise en place de mesures de prophylaxie offensive et défensive. De même,
les échanges fréquents d’oiseaux entre institutions dans le but de minimiser les effets de la
consanguinité impliquent un contrôle sanitaire rigoureux reposant sur le respect de mesures de
quarantaine. La perspective de réintroduction in situ des animaux, à court ou à moyen terme, impose
de plus une gestion sanitaire stricte afin d’éviter de répandre de nouveaux agents pathogènes dans des
populations déjà fragilisées. Contrairement à la médecine aviaire en élevage de rente, la médecine
aviaire en élevage conservatoire est donc une médecine individuelle de pointe dans laquelle la survie
et la reproduction d’individus à forte valeur patrimoniale est capitale. La conduite des mesures de
police sanitaire génère de manière récurrente des volumes importants de prélèvements sur des espèces
différentes. Il est donc essentiel de disposer d’examens complémentaires permettant d’estimer
rapidement l’état de santé d’un individu quelle que soit son espèce.
Dans ce contexte l’électrophorèse des protéines plasmatiques présente de nombreux avantages.
Il s’agit en effet d’une technique dont l’usage en laboratoire médical est répandu, ce qui la rend par
conséquent accessibles à tout praticien. L’automatisation des systèmes disponibles sur le marché
permet de traiter simultanément un grand nombre d’échantillons et autorise des débits d’analyse
importants (jusqu’à 30 électrophorèses toutes les 20 minutes avec l’Hydrasys ©). Notre utilisation
courante de l’électrophorèse des protéines plasmatiques au Parc Zoologique de Clères, ainsi que les
études de différents auteurs (Cray & Tatum, 1998 ; Werner & Reavill, 1999 ; Cray et al., 2007)
tendent à montrer que cet examen complémentaire est fiable et permet d’orienter rapidement un
diagnostic par la mise en évidence d’un processus inflammatoire.
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Nos travaux ont mis en évidence que l’interprétation des électrophorégrammes doit tenir
compte de l’état physiologique de l’oiseau au moment du prélèvement. Certains phénomènes
physiologiques, tels que la ponte sont en effet susceptibles d’induire en erreur le praticien. L’impact
d’autres paramètres tels la croissance, la composition du régime alimentaire, l’hyperphagie prémigratoire, la couvaison, l’élevage des jeunes et le stress devront faire l’objet de recherches futures.
Nous avons montré également que la qualité du prélèvement, et par-dessus tout l’absence d’hémolyse
conditionnent la fiabilité du résultat d’analyse et de son interprétation. La diversité taxinomique de la
classe des oiseaux se reflète par une grande diversité des profils électrophorétiques. Il sera donc
important, dans des études ultérieures de poursuivre ces recherches en se basant sur un panel plus large
d’espèces, afin de dégager les grandes lignes de l’interprétation des électrophorégrammes aviaires. Il
sera enfin nécessaire d’étudier de manière systématique l’impact des différentes classes d’agents
pathogènes sur les électrophorégrammes (bactérie, virus, parasite).
Cette thèse nous a permis de jeter des bases solides qui permettront d'utiliser en routine
l’électrophorèse des protéines plasmatiques chez les oiseaux sauvages, qu'ils soient en élevage
conservatoire, en voie de réintroduction, ou vivant dans leur milieu naturel. Ces résultats ont ouvert
des perspectives intéressantes et bien sûr conduit à de nouveaux questionnements, principe même de
l’avancée de la science.
158
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LISTE DES ABRÉVIATIONS UTILISÉES
AAV: Association of Avian Veterinarians
AGE: Agarose Gel Electrophoresis
A/G (rapport): rapport Albumine / Globuline
APP : Acute Phase Protein
CZE : Capillary Zone electrophoresis
EAAV : European Association of Avian Veterinarians
EAZA : European Association of Zoo and Aquaria
EDTA : Ethylène diamine Tetra Acetate
ELISA : Enzyme-Linked ImmunoSorbent Assay
HDL : High Density Lipoprotein
LDL : Low Density Lipoprotein
PBFD : Psittacine Beak and Feather Disease
UICN : International Union for Conservation of Nature
VLDL : Very Low Density Lipoprotein
174
ILLUSTRATION DES ESPECES D’OISEAUX ETUDIEES
Milan noir
(Milvus migrans)
Amazone aourou
(Amazona amazonica)
Oie à tête barrée
(Anser indicus)
Pigeon domestique
(Columba livia)
Paon bleu
(Pavo cristatus)
175
Contribution à la validation de l’électrophorèse des protéines plasmatiques
comme outil diagnostique en médecine aviaire appliquée à la conservation
Résumé : à l’heure actuelle, les menaces pesant sur la diversité biologique in situ s’accroissent sans cesse et les
espèces doivent survivre dans des environnements de plus en plus anthropisés. La survie de certaines espèces
dont le biotope est extrêmement dégradé est actuellement assurée par des programmes de conservation ex situ.
Les parcs zoologiques font partie de ces institutions pouvant jouer un rôle important dans la conservation
d’espèces en voie de disparition, notamment via l’élevage en captivité. Cependant, dans ces élevages
conservatoires toutes les conditions de l’émergence des maladies sont réunies : grande concentration d’animaux
et sédentarité des collections. La constitution et le maintien d’effectifs d’oiseaux dans un objectif de
conservation ex-situ est par conséquent indissociable d’une gestion sanitaire efficace reposant sur des examens
complémentaires fiables. L’utilisation de l’électrophorèse des protéines plasmatiques dans le diagnostic des
phénomènes inflammatoires présente de nombreux avantages dans ce domaine. Cependant, la classe des oiseaux
est très diversifiée. En absence de repère normatif, il peut donc être difficile d’interpréter finement les résultats
obtenus. Le cycle annuel de vie d’un oiseau passe par diverses phases, comme la ponte et la mue, qui modifient
profondément leur métabolisme, mais dont l’impact sur les électrophorégrammes n’est connu que de manière
très superficielle. De même, les effets d’interférences analytiques telles que l’hémolyse et la lipémie sont mal
connus. Enfin, à l’heure actuelle, l’électrophorèse en gel d’agarose (AGE) est la technique électrophorétique la
plus utilisée en laboratoire médical. Elle tend cependant à être peu à peu remplacée par l’électrophorèse
capillaire de zone (CZE), dont l’utilisation n’a jamais été abordée chez l’oiseau. Ces problématiques ont été
successivement étudiées dans cette thèse afin d’améliorer l’utilisation de l’électrophorèse des protéines
plasmatiques pour le diagnostic des maladies des oiseaux en élevage conservatoire. De notre étude, il ressort que
certains taxons d’oiseaux, comme les psittaciformes, les falconiformes et les colombiformes se caractérisent par
la présence d’un pic important d’apolipoprotéine A-1 dans la région alpha. Le fibrinogène, dont la distance de
migration est différente dans l’infraclasse des galloanserae et des neoaves pourrait servir de point de repère dans
la dénomination des fractions électrophorétiques. Les effets de la mue sur les electrophorégrammes sont peu
significatifs d’un point de vue clinique, tandis que ceux de la ponte, impliquant notamment une augmentation de
la fraction beta liée à la mise en circulation de VLDLy peut être source d’erreur pour le praticien. La lipémie
postprandiale ne semble pas avoir d’effet notoire sur les profils électrophorétiques. L’hémolyse entraine par
contre une augmentation de la fraction gamma pouvant conduire à un diagnostic erroné d’inflammation
chronique. Enfin, les profils électrophorétiques obtenus par CZE et par AGE ne sont pas comparables, certaines
protéines de la fraction alpha en AGE migrant au niveau de la fraction pré-albumine en CZE. L’utilisation de la
CZE présente des avantages en termes de précision et de cadence d’analyse par rapport à l’AGE, mais nécessite
l’établissement d’intervalles de références propres à cette technique.
L’ensemble des connaissances acquises par le biais de nos travaux constituera une base solide permettant
d’utiliser en routine l’électrophorèse des protéines plasmatiques en élevage conservatoire.
Mots clés : électrophorèse, protéine, plasma, agarose, oiseau, inflammation.