BVZS ZooMed Bulletin, September 2004

ZEBRA FOUNDATION GRANT REPORT
First published in the BVZS ZooMed Bulletin, September 2004
INVESTIGATION OF PARAMYXOVIRUS HAEMAGGLUTINATION INHIBITION TITRES FROM
PREHENSILE TAILED SKINKS (Corucia zebrata) WITHIN UK COLLECTIONS
Georgina Yates BVetMed MRCVS
Abstract
Paramyxovirus is considered the most important pathogen affecting captive snakes especially the viperids
(Jacobson et al, 1999). The purpose of this study was to look for antibodies in prehensile tailed skinks (Corucia
zebrata) from a range of UK reptile collections to determine whether exposure to ophidian paramyxoviruses
(OPMV) is confined to previously known positive specimens or is more widespread. Comparison of the reaction
of skink antibodies to a range of viruses was also undertaken. Serum was collected from 34 C. zebrata specimens
from 9 collections across the UK. Haemagglutination inhibition assays were performed using this serum against
avian paramyxovirus (APMV) serotypes -1 to -9, a Sendai virus isolate and an OPMV isolate serologically similar
to APMV 7. Six specimens had mono-specific reactions to OPMV 7, nine had negative reactions to all viruses and
the remaining specimens had weak non-specific reactions to two or more viruses.
Corucia zebrata from Birmingham Nature Centre
Introduction
Respiratory diseases account for considerable disease and death in collections of reptiles and whilst bacteria,
fungi and parasites play a part in this, ophidian paramyxovirus (OPMV) is the most important pathogen affecting captive
snakes especially Viperids (Jacobson et al, 1999). PMV has been isolated from a number of healthy lizard species, but
there has only been one report of disease due to OPMV in lizards (Ahne et al, 1999, Jacobson et al. 2001, Lloyd 2002,
Marschang et al. 2002). In a screening of 54 healthy reptiles at London Zoo, 3 Rhinoceros Iguanas (Cyclura cornuta
cornuta), 2 Blotched Blue Tongued Skinks (Tiliqua nigrolutea) and 3 Prehensile Tailed Skinks (Corucia zebrata) were
found to have positive antibody titres to an OPMV 7 (Lloyd 2002).
The aims of this study were to:
1) Look for antibodies in C. zebrata from a range of UK reptile collections to determine whether exposure to
paramyxoviruses is confined to previously known specimens or is more widespread.
2) Examine which viruses of avian PMV (APMV) -1 to -9, Sendai virus and OPMV 7 skink antibodies react to in a
serotype specific manner.
To achieve these aims serum was collected from 33 specimens in nine reptile collections across the UK.
Haemagglutination inhibition (HI) assays were used to achieve antibody titres to the named viruses. HI was chosen as
1
it is frequently used in serological analysis of OPMV outbreaks and is a quick and relatively easy test that would be
possible to complete within the three weeks available for this project (Jacobson et al, 1999).
Literature review
Background to Paramyxovirus infection of snakes
In 1972 a new reptilian virus was isolated from a Fer-de-Lance viper (Bothrops atrox) housed in a private
collection in Switzerland (Clark et al, 1979). The collection had suffered an outbreak of a fatal respiratory disease. The
paramyxo-like virus was isolated from lung tissue of one of the dead animals and named Fer-deLance virus (FDLV)
(Clark et al, 1979).
In 1980 the death of Rock Rattlesnakes (Crotalus lepidus) from a breeding colony in Florida was reported and
a PMV was isolated (Jacobson et al. 1980). The affected vipers had suffered from head tremors and loss of equilibrium
with delayed irregular tongue flickering and exudate was seen from the respiratory tract ( Jacobson et al, 1980).
In 1981 another PMV outbreak was reported by Jacobson et al in Louisiana and again only vipers were affected
(Jacobson et al, 1981). The clinical signs in this case involved mouth gaping followed the next day by convulsions then
death. Some snakes had been regurgitating food for the past 1 - 3 weeks; some died with no previous clinical signs
(Jacobson et al, 1981). On post-mortem grossly the only abnormality was a mucoid or caseous exudate within the lung.
Histologically an interstitial pneumonia was seen and a paramyxo-like virus was isolated from the lung tissue (Jacobson
et al, 1981). HI assays reacting with this isolate and with serum samples from 22 of the surviving snakes at the collection
gave antibody titres of 1/20 to 1/2560 (Jacobson et al, 1981). Positive titres were achieved from vipers and non-vipers
for at least five months post infection (Jacobson et al, 1981). The HI assays confirmed that this virus isolate was
antigenically similar to the existing Rock Rattlesnake isolate (Jacobson et al, 1981).
In subsequent outbreaks in USA, Germany, UK, and Canary Islands snakes from a number of families have
been infected including Viperidae, Crotalidae, Colubridae, Boidae (Jacobson et al, 1981, Manvell et al, 2000, Oros
et al, 2001). Only infections of adult animals have been reported and no sex predilection has been seen.
OPMV particles range in size from 126 - 500 nm and are pleomorphic spheroidal or filamentous and enveloped
with a peripheral fringe of spines (Clarke et al, 1979, Homer et al, 1995, Kania et al, 2000). Classification as a
paramyxovirus was based on electron microscopic appearance, ability to haemagglutinate chick erythrocytes and to
grow in chicken eggs, sensitivity to ether and possession of an RNA nucleocapsid core (Clark et al, 1979, Homer et al,
1995, Kania et al, 2000, Lunger and Clark 1979a). The virus has been found to replicate in cells of mammalian, avian
and reptilian origin but is most efficient in reptilian cell cultures especially rattlesnake cell lines (Ahne W., Adrian J.,
and Mayr A. 1999, Clarke et al, 1979, Homer et al, 1995, Lunger and Clark 1979a and 1979b). Syncytium and plaque
formation is induced by the virus in cell culture (Clark et al, 1979, Homer et al, 1995, Kania et al, 2000). Disease was
not produced in mice up to one year after
inoculation in a number of ways with the FDLV isolate (Clark et al, 1979). Among paramvxoviruses OPMV has a
uniquely low optimum temperature range of 25-30°C and replication is severely impeded at 37°C suggesting that it is
specifically adapted to ectothermic hosts (Ahne W. Adrian J. and Mayr A. 1999, Clark et al, 1979, Homer et al, 1995,
Lunger and Clark 1979a and I979b).
In 1997 Jacobson et a1 fulfilled Koch's postulates using an OPMV originally isolated from an Aruba Island
Rattlesnake and propagated in Vera cells. Virus was inoculated into the tracheas of Aruba Island Rattlesnakes to produce
characteristic respiratory disease and lesions and the inoculated virus was subsequently isolated from the trial animals
(Jacobson et al, 1997). None of the animals produced antibody to the inoculated virus but as they all died less than 23
days post inoculation they may not have lived long enough to do so (Jacobson et al, 1997).
OPMV in lizards
In 1998, wild iguanas (Iguana iguana rhinolopha, Ctenosaura bakeri, C. similis) from Honduras were found to
have weak positive antibody titres (1/8 – 1/32) to an OPMV 7 isolate from a healthy monitor lizard (Varanus prasinus)
housed with snakes in a PMV epidemic (Gravendyck et al, 1998). HI and virus neutralisation test (VN) were used. VN
generally gave higher titres than HI (Gravendyek et al, 1998). Antibodies were not detectable, in these samples, to an
APMV 1 or OPMV l isolate by HI or VN and no virus was isolated (Gravendyck et al, 1998).
Three epidemics of infection in Caiman lizards (Dracaena guianensis) imported from Peru to USA were
reported in 2001 (Jacobson et al, 2001). The lizards died rapidly, some displaying clinical signs of depression, anorexia,
muscle wastage and possibly respiratory distress (Jacobson et al, 2001). Proliferative interstitial pneumonia was
diagnosed at necropsy (Jacobson et a1, 2001). PMV was seen in lung tissue by electron microscopy and
immunoperoxidase staining using rabbit antibodies to an Aruba Island rattlesnake OPMV isolate (Jacobson et al, 2001).
An OPMV was isolated from lung tissue. Seven months after the first epidemic, surviving lizards housed with the fatally
infected animals were blood sampled for serology. Of 17 animals, 10 had HI titres >1/20 and <1/80 showing sustained
low antibody levels (Jacobson et al, 2001).
A PMV was isolated from a cloacal swab of a healthy wild caught Mexican lizard (Xenosaurus patyceps)
(Marschang et al, 2002). Serology testing of the virus isolate with polyclonal antisera to APMV -1 to -9 gave the highest
2
titres to APMV 7 (Marschang et al, 2002). Four of the wild caught Mexican lizards in this study (X. grandis, X.
platyceps and Abronia graminea) were shown to have antibodies to the OPMV isolated with titres >1/32 (Marschang
et al, 2002). This isolate has been used in this study and by Lloyd 2002.
Investigation of PMV infections
Suspicion of OMPV infection occurs on visualisation of suggestive lesions such as epizootic proliferative
pneumonia. To confirm infection, antemortem virus isolation and/or demonstration of a rising titre of OPMV specific
antibodies using HI are used. VN can also be used. Postmortem demonstration of replicating virus particles
by electron microscopy and/or immunohistochemistry detection can be used in addition to N17irus isolation (Homer et
a1,1995, Oros et al, 2001, Jacobson et al, 2001, Jacobson et al, 1999). An enzyme linked immunosorbent staining assay
(ELISA) has been investigated to assess antibody reactivity to a number of antigens rather than just one as HI does
(Kania et al, 2000). However reagents to detect Ig from all major families of reptiles are needed before an ELISA can
be of widespread use for detection of seropositive reptiles (Kania et al, 2000). A polymerase chain reaction (PCR ) using
oral and cloacal swabs is currently being investigated for viability (E Holdsworth pers. comm. 2003).
Complement fixation tests titrating the reference FDLV isolate against guinea pig sera produced against a
number of paramyxoviruse isolates including APWV -1 to -5 but not Sendai virus produced negative results as did
titrating sera to FDLV against the other paramyxovirus isolates (Clark et al, 1979).
HI assays using two OPMV isolates from a disease outbreak in Germany and polyclonal antisera to APMV
serotypes -1 to -9 have been attempted (Blahak et al, 1991 ). The highest positive titres were to APMV 7 with a high
cross-reaction to APMV 1 with one isolate and to APMV 1 with a high cross-reaction to APMV 7 with another isolate
(Blahak et al, 1991). This has led to OPMV isolates being known as either --1 or -7 serotypes (Blahak et al, 1991). A
low cross-reaction to all the other antisera was achieved by both isolates (Blahak et al, 1991). The first OPMV strain
failed to bind monoclonal antibodies to OPMV 7 or APMV 1 but the second did
bind monoclonal antibodies to APMV 1 (Blahak et al, 1991). Serum from the snakes involved in this outbreak showed
positive HI antibody titres to both OPMV isolates, and APMV -1 but not APMV -3 or -8 (Blahak et al, 1991). An
OPMV from an Ottoman viper (Vipera xanthina xanthina) was reported to have a positive HI titre to parainfluenza
2 antisera (Homer et al, 1995). Chelodians with rhinitis have been found to have high antibody titres against Sendai
virus but no virus was isolated and so whether Sendai-like viruses actually cause disease is currently unknown
(Jackson and Needham 1983).
Sequence analysis of OPMV
The predominant problem with serodiagnosis is that multiple strains of OPMV have now been recognised to
exist so their relatedness needs to be assessed before the accuracy of serology methods can be determined (Cranfield
and Graczyk 1996, Ahne et al, 1999).
Analyses of the large (L) and haemagglutinin-neuramininidase (HN) protein sequences of 16 reptilian
paramyxoviruses by Ahne et al showed that there are two well defined subgroups/species of OPMV and a set of
intermediate isolates which may represent additional distinct species (Ahne et al, 1999). Isolates of one species were
mainly collected from outbreaks within USA and isolates of the other were mainly from Germany (Ahne et al, 1999).
Isolates within a single virus species were found in more than one snake species suggesting the virus does not have a
narrow host range (Ahne et al, 1999). It was also shown that OPMV forms a separate family of viruses more closely
related to each other than to the outgroup using Sendai virus (Ahne et al, 1999, Franke et al, 2001, Junqueira de Azevedo
et al, 2001).
Kindermann et al found similar results comparing the sequences of L and HN proteins from the same OPMV
isolates as Ahne et al. This latter study also related OPMV isolates to each other and to Sendai virus and human
parainfluenza -l, -2 and -3 isolates (Kindermann et al. 2001). The L protein and HN protein from these isolates were
found to be highly divergent from all the mammalian isolates but most closely related to the Sendai virus (62% and
47.1%) and least closely related to human parainfluenza virus 2 (40.4% and 26.1%) (Kindermann et al, 2001).
Franke et al also found similar results comparing the sequences of L protein and Fusion (F) protein in 18
OPMV isolates from Germany to those of a variety of mammalian PMV isolates (Franke et al, 2001).
Characterisation of the F protein from a PMV from the venom gland of a Fer-de-Lance viper (Bothrops jararaca)
was performed by Junqueira de Azevedo et al in 2001. The protein was found to be most related to parainfluenza
3, Sendai virus and human parainfluenza 1 when compared to sequences in the databanks that included those of
APMV -1 and -2 (Junqueira de Azevedo et al, 2001). The L gene was found to be more conserved than the HN
gene (Kindermann et al, 2001). The genetic relatedness of haemagglutinin from reptilian species to avian serotypes
has yet to be reported. However APMV and human parainfluenza 2 are classified within the Rubulavirus genus and
Sendai virus within the Respirovirus genus within the Paramyxoviridae family suggesting OPMV is not closely
related to APMV despite the serological evidence for this (van Regenmortel et al, 2000). Due to lack of sequence
data in 2000 OPMV is currently unassigned to a genus within the Paramyxoviridae family (van Regenmortel et al,
2000).
3
Prehensile tailed skinks
The Corucia zebrata is commonly known as the Solomon Island or prehensile tailed skink and is a large green arboreal
skink with a strong prehensile tail that lacks the tail fractural planes possessed by other species of skink (Wright 1993).
The C. zebrata is a herbivorous skink originating from the Solomon Islands and Papua New Guinea (CITES 2003,
Wright 1993). The average snout to vent length is over 25cm and average adult weight is between 500 and 700g making
them large enough to blood sample safely, withdrawing enough blood for a number of tests (Wright 1993).
Blood sampling a conscious prehensile tailed
skink from the ventral tail vein.
The animals are also relatively easy to handle, so blood sampling from
the ventral tail vein can be performed without anaesthesia.
The C. zebrata is a CITES appendix II animal classified as “threatened” but, it is felt that due to pressures on
wil
As a result, a UK captive breeding programme was started involving 55 animals at the last count (Basford 2001).
Most of the animals in this study are involved in that programme making this the most widespread OPMV screening of
the UK breeding population of C. zebrata to be undertaken to date. Being able accurately to assess the PMV status of
these animals is important to the breeding programme so that appropriate decisions can be made regarding transferring
positive and negative titre specimens between collections.
The purpose of this project was to explore the use of HI assays to detect carrier animals in UK collections.
APMVs 1-9, Sendai virus and OPMV 7 were used as antigens.
Materials and Method
Samples
Blood samples were collected by the veterinary surgeon working for each collection. Blood was taken from
the ventral tail vein of conscious specimens and spun down to remove the serum. Serum was stored at -20 °C until
needed.
Preparation of day old chicken blood
The day old chicken blood (TCS Biosciences Ltd) was washed three times with Phosphate Buffered Saline
solution (Oxiod). For haemagglutination, the washed cells were diluted in Phosphate Buffered Saline (PBS) to produce
a 1% chick erythrocyte suspension. The blood and 1% suspension were refrigerated when not in use.
Preabsorption
Before testing, serum had to undergo preabsorption to remove any antigens to chicken red blood cells from
the samples (Cross 2002, R. Manvell pers. comm. 2003). 100μ1 samples of the serum were added to 251 packed
washed chicken erythrocytes. These were mixed and left to stand for one hour at room temperature. The suspension
was then centrifuged at 800g for 2 minutes and the antisera removed for use. These were stored in aliquots at -20
°
C when not in use.
Haemagglutination assays
Haemagglutination assays (HA) were performed on each virus to calculate the titre of the virus and from this,
4 Haemagglutination Units (HAU), to be used in the HI Assays. The viruses used were APMV serotypes -1, -2, -3, -4,
4
-6, -7, -8, -9 supplied by R. Manvell (VLA, Weybridge). These stocks had been grown in chicken eggs and inactivated
with β-propiolactone before use (Russell 1989). Also used was a live Sendai virus (murine parainfluenza 1) grown in
chicken eggs, originally supplied by M-J. Gething (ICRF, London). A formalin inactivated OPMV isolate (VLA 186/00)
grown in chicken embryo fibroblasts, supplied by R. Manvell (VLA, Wevbridge), completed the panel. This OPMV
was isolated from a cloacal swab of a healthy Mexican Lizard (Xenosaurus platvseps) using Terrapene heart cells at
28˚C (Marschang et al,2002). HI titres were found of 1/8 and 1/128 to chicken antiserum to APMV -1 and -7 respectively
meaning the isolate cross matches with APMV 7 (R. Gough pers. comm. 2003, Marschang et al, 2002).
To perform the HA, 25μl PBS was placed in each well on a V-bottomed microtiter plate. An additional 20μ1
PBS and 5μl virus isolate were added to the first column well of each row. This was double diluted across the row
discarding the last 25μ1. 25μ1 PBS as a volume blank and 25μ1 1% washed chick erythrocyte suspension were then
added to each well. In the final row of each plate, 50μ1 PBS and 25μ1 1 % erythrocyte suspension were added to each
well as a negative control. The plate was left at room temperature for 45min. To read, the plate was stood at an angle to
allow streaming of non-haemagglutinated red blood cells.
Each virus was tested in triplicate and the HA titre assessed from the average result over all three rows. 1
Haemagglutinating Unit (HAU) was defined as the highest dilution to cause 100% agglutination of chicken red blood
cells. The virus was then diluted with PBS to give a 4 HAU solution. The viruses were stored at -70°C in their neat
state until needed.
Haemagglutination inhibition assays
Once 4HAU had been determined for each virus, haemagglutination inhibition assays (HI) could be
performed using the preabsorbed sera. 25μ1 PBS were added to each well on a V-bottomed microtiter plate. 20μ1
PBS and 5μ1 of serum were added to the first column well of each row and double diluted across the row to produce
l/10 to 1/1280 dilutions. The last 25μ1 from each row was discarded. 25μ1 of 4HAU virus solution was added to
every row. In an additional row, an antigen titration of 4HAU virus was double diluted across five wells to produce
dilutions from 4HAU to 1/4HAU in 50μl volumes. 25μ1 PBS were added to each of these wells as a volume blank.
The plate was left at room temperature for 30min. 25μ1 1% chicken erythrocyte suspension was added to every well
on the plate. In the final row, as a chicken erythrocyte control, 50μl PBS and 25μl 1% chicken erythrocyte suspension
were added to every well. The plate was then tapped to mix the reagents and left at room temperature for 45min.
Haemagglutination inhibition assay plate showing
buttoning and haemagglutination of chicken
erythrocytes
To read, the plate was stood at an angle to allow streaming and the HI titre was defined as the highest dilution
to prevent agglutination of erythrocytes. On each plate 4 to 5 sera were tested against one virus serotype. Each sera
was tested in duplicate and the HI titre was taken as the average of the results from both in the event that they provide
different titres.
The plates were read by two methods:
1) Taking streaming only as a true haemagglutination inhibition. Streaming was defined as where the
erythrocytes pooled to the bottom of the well leaving clear fluid until the plate was tipped when the cells stream in a
line towards the bottom edge of fluid.
2) Taking both streaming and buttoning as true haemagglutination inhibition. Clear buttoning was defined as
where the erythrocytes had pooled but no streaming was seen when the plate was raised to an angle.
5
Results
Samples were collected from:
1) Battersea Park Children's Zoo
2) West Midland Safari & Leisure Park Ltd, Bewdley
3) Birmingham Nature Centre
4) Bristol Zoo Gardens
5) Chessington World of Adventures
6) North of England Zoological Society, Chester
7) Cotswold Wildlife Park
8) Durrell Wildlife Conservation Trust Jersey
9) Zoological Society of London
A total of 33 samples were collected, 25 of these are included in the stud book for this species, 6 have been
moved into collections since the stud book was last updated and 2 are from a collection which does not participate in
the breeding programme. All of the animals were reported to be healthy apart from one at Chessington that has been
severely underweight since being rescued by the RSCPA in November 2001.
There were large variations in colour and consistency of the serum samples, and the appearance of each is noted in
table 1 along with specimen details. Many of the samples had coagulated into a gel with variable amounts of fluid
surrounding the gel. As only the fluid was usable for HI, some samples with only small volumes of fluid remaining
could not be tested against all the virus isolates.
6
7
8
9
Discussion
Analysis of results
When the results from the two methods of reading the HI plates are compared, the second method of counting
buttons as positive results leads to a high number of sera showing variable titres to different viruses. The first method
of just counting streaming samples as positives, gives much fewer positive and generally lower titres. This result agrees
with information from R. Manvell that only streaming wells should be regarded as true positives (R. Manvell pers.
10
comm. 2003). Due to the highly variable results from the second method with no coherent pattern of reaction, these
results offer no convincingly valid conclusions. The results from the first method of reading plates are however specific
enough to draw conclusions from.
These results can be divided into weak specific positives, non-specific reactions and negative reactions. There
are serum samples that only produced a mono-specific positive result to one virus from the panel, those that non/polyspecific positive titres to a number of isolates and those that produced no positive titres at all. A breakdown of these
results is given in Table 7 below. Six specimens out of the 33 sampled (18%) currently have low levels of antibodies to
OPMV 7 (186/00).
Of the six specimens with mono-specific positive titres of OPMV 7, two from London (nos.31, 32) also had
similar positive titres last year to the same OPMV 7 isolate as shown in Table 1 (VLA 186/00 (Lloyd 2002, Marschang
et al, 2002). Maintaining positive titres to OPMV 7, that are neither rising nor falling, could mean that the animals
have been exposed to OPMV infection in the past and have a long lived antibody response to it. Antibody levels, in
reptiles, can be mantained over long periods. Caiman lizards have been shown to have antibody levels of up to 1/80
to OPMV seven months after exposure (Jacobson et al, 2001). Antibody levels in snakes after an outbreak in
Louisiana, USA were high, declining to low values from 3 to 7 months after the initial outbreak (Jacobson et al,
1992). Alternatively, the two skinks could be carrying the virus, so antibody levels ar e maintained, or multiple
exposures could have occurred giving coincidentally similar titres. No other London specimens have become
positive over the year so there is no evidence for seroconversion by the positive specimens but it is unknown how
much contact occurs between animals.
Analysis of haemagglutination inhibition assays
Whilst low HI titres suggest low levels of antibody in the serum, a titre is not a quantitative measure but a
measure of the efficiency of neutralisation of haemagglutination (Jacobson 2002). The result is dependent on the
antibody affinity for antigen, the availability of the antibodies and proper preservation of the samples (Jacobson 2002).
11
Unfortunately, the samples sent from one collection were not refrigerated for five days due to being sent over a bank
holiday. This could have denatured antibodies present in the samples. However mono-specific positive titres were
produced to APMV 7 by three of these samples (nos. 9, 10, 12). No. 12 has a relatively high titre of 1/80, so denaturing
appears not to have occurred, but without re-testing these animals it is not possible to know whether these titres would
have been any different or specific to OPMV 7 had the samples been chilled throughout. As little is known about the
full nature of OPMV strains, their cross reactivity and reptile immune responses to them, the specificity and sensitivity
of HI assays for antibodies to the virus are as yet unknown.
There was a problem with some of the serum samples which coagulated into a gel form preventing use of a
large part of the collected sample. This could have been avoided by collecting plasma rather than serum (Jacobson
2002). Another potential problem is that false negatives caused by toxins or bacteria in the serum can lyse chicken
erythrocytes so that they cannot bind to the antigen or causing agglutination (Jacobson 2002). Antisera present in the
samples against antigens on the erythrocytes could also cause agglutination (Jacobson 2002). Preabsorption should have
prevented these occurring by removing agents reacting with the erythrocytes, however the clear buttons could be a sign
that some remained (Jacobson 2002). Repeating preabsoption twice is occasionally done if this is suspected but there
was insufficient time available for this to be done in this study (R. Manvell pers. comm. 2003).
Reptile immunity involves immunoglobulin (Ig)M and IgY (reptilian equivalent of IgG), but it is currently
unknown which of these are measured by HI assay for PMV (Jacobson 2002, Ridi 1992). Much higher levels of IgM
are present in the blood than IgY and IgM in mammals, fish and presumably reptiles is poly-specific (Avrameas 1992,
Ridi 1992). Therefore if predominately IgM reacts in HI assays to PMV this could account for the non-specific results
seen. More investigation of this is required to assess whether investigation of just IgY levels to OPMV would give more
specific results.
Investigation into the prevalence of OPMV is still in its infancy especially as there is no gold standard test. HI
is a useful test as it is quick and simple to perform, but with the recent developments in characterisation of the many
virus isolates it is increasingly obvious that the choice of isolates used is vital to its accuracy. Without full knowledge
of the different serotypes of OPMV, and the relatedness of their haemagglutinin, it is currently not possible to be certain
of our ability to screen animals for OPMV. Testing samples against more strains of OPMV especially an OPMV 1 would
have been a useful adjunct to this study but none were available. HI is still an invaluable tool for testing for a specific
strain of OPMV, for example, to determine potential carriers in the face of an outbreak.
Serum neutralisation assays involve incubating the virus with serial dilutions of serum, then assessing for
residual infectivity on cell culture or another biological substrate. Serum neutralisation is considered the gold standard
test for serological diagnosis of viral infection and is more sensitive but also more laborious than HI (Jacobson 2002).
Although both HI and serum neutralisation can show antibody titres to PMV strains considered suspicious or positive
for PMV, they cannot prove if virus is still present even with paired samples. Had it been possible, virus isolation or an
ELISA to show shedding would have been a useful adjunct to this study.
Comparison of APMV and OPMV
When examining the cross reactions seen in this experiment it is helpful to look at known cross reactions
between the APMV viruses themselves. A common cross reaction is between APMV 1 and APMV 3 but lesser cross
reactions are also known to occur within two groups with APMV -1, -3, -4 , -7 and -9 in one group and APMV -2, -6
and -8 in the other (Alexander 1980, Russell 1989). In this study however, the main cross reactions occurred between
OPMV 7 and APMV 2 that are in separate grouping so are not expected to cross react (Russell 1989). Also some of
these samples show a higher HI titre to APMV 2 than OPMV 7 which is the opposite of what could be expected. This
suggests that these results are non-specific reactions and therefore the positive titre to OPMV 7 cannot always be relied
upon to indicate that there are specific antibodies present in that specimen to OPMV. The positive results to just one
isolate are more likely to be specific, especially as two of them (nos. 31, 32) correlate with previous results (Lloyd
2002). The one specimen (no. 17) showing a cross reaction between just OPMV 7 and APMV 7 is also likely to be
correct as there is a known cross reaction between them. It would be expected for the APMV to give a lower titre than
the OPMV rather than being the same, but these titres are very low, making differences harder to distinguish.
When comparing pathogenesis information regarding OPMV and APMV some distinct similarities appear.
Alexander describes AMPV 1 as having four distinguishable forms that vary in severity from causing greater than 90%
mortality in outbreaks to mild or inapparent disease (Alexander 1980). The severest forms can cause death without
previous clinical signs; slightly more mild forms give respiratory and nervous signs as seen with OPMV (Alexander
1980). However sequencing has shown no relatedness of OPMVs to the Rubulavirus family containing APMVs
(Kindermann et al, 2001). This raises questions as to the reliabilty of cross reactions between OPMV and APMV.
Hopefully further sequence analysis will improve understanding of how cross reactions are occurring and which, if any,
are specific.
Significance of OPMV
12
Recent findings of OPMV antibodies in healthy reptiles from wild populations reduces the chance that this is
an emerging infectious disease that, if released into wild populations, could cause mass mortalities and even extinctions
(Daszak et al, 2001). However, if different strains do have differing severities, possibly depending on the species
infected, this is still a risk and should be a major consideration when planning programmes to breed reptiles for release
into the wild (Daszak et al, 2001). Jacobson recommends that reptiles bred in captivity for release into the wild be
isolated away from the main collection to reduce infiltration of pathogens to the release group, and that these breeding
programmes should be established within the geographical range of the species (Jacobson 1993). Isolation of breeding
groups may be possible in some collections, but many will not have the facilities to allow this. Whilst breeding animals
in the vicinity of where they will eventually be released should be the objective, this again will not be possible in many
situations. Therefore, collections need to do as much as possible to eliminate entrance of pathogens to the collection as
a whole.
While it is unlikely that animals with weak positive titres pose a risk to collections, there is a small chance they
are carrying the virus and could infect other specimens. Combining serology with virus isolation from cloacal swabs,
ELISAs for OPMV antigens or PCRs for OPMV related sequences (once developed and validated) would allow this
risk to be better assessed. It is still unknown how OPMV is spread. though other PMVs are generally spread by aerosol
or faeces; it is possible fomites or even parasites are involved (Jacobson et al, 1999, van Regenmortel et al, 2000). This
makes it difficult to recommend anything less than total isolation of carriers and infected animals (Jacobson et al, 1999).
All collections should already have a good quarantine protocol and should practice isolation of any sick specimens.
With more information on the natural history of this virus, collections will be able to make better decisions about
precautions regarding OPMV.
Summary
Six C. zebrata specimens were found to have weak mono-specific HI titres to OPMV 7. All of these specimens were
from collections where OPMV has been previously recognised (S. Thornton pers. comm.. 2003). Positive titres were
also produced to some APMV serotypes but not with any consistent pattern. Without being able to look for virus as well
as antibodies it is difficult accurately to assess the risk posed by these positive specimens to others in the collection.
More research is needed into the relatedness of OPMV strains to each other and to APMVs before the specificity of HI
assays for OPMV can be accurately assessed.
References
Ahne W., Adrian J. and Mayr A. (1999) Replication of reptilian paramyxovirus in avian host systems. Journal of Veterinary
Medicine 46(1), 57-62
Ahne W., Batts W., Kurath G. and Winton J. (1999) Comparative sequence analyses of sixteen reptilian paramyxoviruses. Virus
Research 63(1-2), 65-7
Alexander D. (1980) Avian paramyxoviruses. Veterinary Bulletin 50(9), 757 - 752
Avrameas F. (1992) In: Roitt and Delves Encyclopaedia of Immunity Pub Academic Press Pp 1135
Basford L. (2001) JMSP Studbook Report 2001 Prehensile Tailed Skink Corucia zebrata. Birmingham City Council
Blahak S., Gobel T., Alexander D., and Manvell R. (1991) Some investigations of the occurrence of paramyxoviruses in snakes of
German zoos. Proceedings of the Imernational Colloquium on Pathology ofReptiles and Amphibians 4, 17-24
Clark H., Lief F., Lunger P., Waters D., Leloup P., Foelsch D. and Wyler R. (1979) Fer de Lance virus (FDLV): a probable
paramyxovirus isolated from a reptile. Journal of General Virology. 44(2), 405-418
Cranfield M. and Graczyk T. (1996) Ophidian Paramyxovirus In: Mader Reptile Medicine and Surgery. Pub Saunders. Pp 392- 394
Cross G. (2002) Haemagglutination Inhibition Assays. Seminars in Avian and Exotic Pet Medicine 11(1), 15-18
CITES website (2003) www.cites.org
Daszak P., Cunningham A. and Hyatt A. (2001) Anthropogenic environmental change and the emergence of infectious disease in
wildlife. Acta Tropica 78, 103ll6
Fanke J., Essbauer S., Ahne W. and Blahak S. (2001) Identification and molecular characterization of 18 paramyxoviruses isolated
from snakes. Virus Research 80, 67-74
Gravendyck NL, Ammermann P., Marschang R. and Kaleta E. (1998) Paramyxoviral and reoviral infections of iguanas on Honduran
Islands. Journal of Wildlife Diseases 34(l), 33-38
Homer B., Sundberg J., Gaskin J., Schumacher J. and Jacobson E. (1995) Immunoperoxidase detection of ophidian paramyxovirus
in snake lung using a polyclonal antibody. Journal of Veterinary Diagnostic Investigation 7(1), 72-77
Cranfield M. and Graczyk T. (1996) Ophidian Paramyxovirus In: Mader Reptile Medicine and Surgery. Pub Saunders. Pp 392- 394
Cross G. (2002) Haemagglutination Inhibition Assays. Seminars in Avian and Exotic Pet Medicine 11(1), 15-18
CITES website (2003) www.cites.org
Daszak P., Cunningham A. and Hyatt A. (2001) Anthropogenic environmental change and the emergence of infectious disease in
wildlife. Acta Tropica 78, 103ll6
Fanke J., Essbauer S., Ahne W. and Blahak S. (2001) Identification and molecular characterization of 18 paramyxoviruses isolated
from snakes. Virus Research 80, 67-74
Gravendyck NL, Ammermann P., Marschang R. and Kaleta E. (1998) Paramyxoviral and reoviral infections of iguanas on
Honduran Islands. Journal of Wildlife Diseases 34(l), 33-38
13
Homer B., Sundberg J., Gaskin J., Schumacher J. and Jacobson E. (1995) Immunoperoxidase detection of ophidian paramyxovirus
in snake lung using a polyclonal antibody. Journal of Veterinary Diagnostic Investigation 7(1), 72-77
Jackson O. and Needham J. (1983) Rhinitis and virus antibody titres in chelodians. Journal of Small Animal Practitioners 24, 31
- 36
Jacobson E. Gaskin J. Simpson C. Terrell T. (1980) Paramyxo-like virus infection in a Rock Rattlesnake. JAVMA 177(9),
796- 799
Jacobson E., Gaskin J., Page D., Iverson W. and Johnson J. (1981) Illness associated with Paramyxo-like virus infection in a
zoologic collection of snakes. JAVMA 179(11), 1227-1230
Jiacobson E., Gaskin J., Flanagan J. and Andrew Odum R (1991). Antibody Responses of Western Diamondback Rattlesnakes
(Crotalu,s atrox) to Inactivated Ophphidian Paramyxovirus Vaccines. Journal of Zoo and Wildlife Medicine 22(2): S-4-190
Jacobson E., Gaskin J., Wells S., Bowler K. and Schumacher J. (1992) Epizootic of Ophidian Paramyxovirus in a zoological
collection: Pathological microbiological and serological findings. Journal of Zoo and Wildlift Medicine 23 (3), 318-327
Jacobson E. (1993) Implication of infectious diseases for captive propagation and introduction programs of threatened/endangered
reptiles. Journal of Zoo and
Wildlife Medicine 24(3), 245-255
Jacobson E,, Adams H., Geisbert T., Tucker S., Hall B. and Homer B. (1997)
Pulmonary lesions in experimental ophidian paramyxovirus pneumonia of Aruba
Island Rattlesnakes, Crotalus unicolor. Veterinary Pathology 34(5), 450-409
Jacobson E., Flanagan J., Rideout B., Ramsey E. and Morris P.(1999) Ophidian Paramvxovirus. Round table discussion.
Association of Reptilian and Amphibian Vets 9(1), 15-22
Jacobson E., Origgi F., Pessier A., Lamirande E., Walker I., Whitaker B., Stalis I., Nordhausen R., Owens J., Nichols D., Heard
D., and Homer B. (2001) Paramyxovirus infection in caiman lizards (Draecena guianensis) Journal of Veterinary Diagnostic
Investigation 13(2), 143-51
Jacobson E. (2002) Use of Serology in Reptile Medicine. Seminars in Avian and Exotic Pet Medicine. 11(1), 33-45
Junqueira de Avevedo T., Prieto da Silva A., Carmona E. and Lee Ho P. (2001) Characterization of a Paramyxovirus from a Fer
de Lance viper (Bothrops jararaca) : partial nucleotide sequence of the putative fusion protein. Archives of Virology 146,51-57
Kania S., Kennedy M., Nowlin N., Diderrich V. and Ramsey E. (2000) Development of an enzyme linked immunosorbent assay
(ELISA) for the diagnosis of ophidian paramyxovirus (OPV) Infectious Disease Review 2(4), 213-217
Kindermann J., Kübber-Heiss A., Kerschbaumer P. and Nowotny N. (2001) Phylogenetic analysis of the L and HN gene of
ophidian paramyxoviruses. Archives of Virology 146, 1021 - 1035
Lloyd C. (2002) Investigation into sero-prevalence and viral shedding of Paramyxovirus, amongst the sub-order Sauna, on
a zoological collection. Unpublished report as part of MSc in Wild Animal Health.
Lunger P. and Clark H. (1979a) Morphogenesis of Fer-de-Lance virus (FDLV) cultured at optimal (30 °C) cell growth temperature.
Journal of Comparative Pathology 89, 265-279
Lunger P and Clark H (1979b). Morphogenesis of Fer-de-Lance virus (FDLV) cultured at sub- (23°C) and supra- (36 °C) optimal
cell growth temperatures. Journal of Comparative Pathology 89(2), 281-291.
Manvell R., Geach M. and Lewis J. (2000) Isolation of ophidian paramyxovirus type 7 from a reticulated python in the UK. The
Veterinary Record 147, 696
Marschang R., Donahoe S., Manvell R. and Lemos-Espinal J. (2002) Paramyxovirus and reovirus infections in wild-caught Mexican
lizards (Xenosaurus and Abronia spp.) Journal of Zoo and Wildlife Medicine 33(4), 317-321
Oros J., Torrent A., Castro P., Deniz S., Casal A., Jacobson E. and Homer B. (2001) Ophidian paramyxovirus in snakes in the
Canary Islands: An Immunohistochemical Study. The Veterinary Record 149, 21-23
van Regenmortel M., Fauquet C., Bishop D., Carstens E., Estes M., Lemon S., Maniloff J., Mayo M., McGeoch D., Pringle
C., Wickner R. (2000) Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses.
Academic Press, New York, NY.
Ridi R. Reptilian Immune System (1992) In: Roitt and Delves Encyclopaedia of Immunity Pub Academic Press Pp 1313 - 1316
Russell P. (1989) Antibody-forming Cell Assays of Avian Paramyxoviruses: Immunization of Chickens Demonstrates Two
Serotype Groups. Journal of General Virology 70, 2645 -2651
Wright K. (1993) Medical Management of the Solomon Island Prehensile-Tailed Skink, Corucia zebrata. Association of Reptilian
and Amphibian Veterinarians 3(1), 9-17
Acknowledgements
I would like to express my thanks to Peter Russell my project supervisor and to Ruth Manvell from VLA, Weybridge for
advice and supply of viruses. Also to Sue Thornton for advice and assistance as well as collecting blood samples. In addition, I am
grateful to Andrew Mackie for technical instruction and advice on HI assays. Thank you to all the veterinary surgeons and other
staff at the reptile collections involved with sampling including: Stephanie Sanderson at Chester Zoo; Javier Lopez at Durrell
Wildlife Conservation Trust; Sharon Redrobe at Bristol Zoo Gardens; Chris Marshall at West Midlands Safari Park; Peter Aylmer
at Cotswold Wildlife Park; Tai Strike at Zoological Society of London; staff at Chessington World of Adventures and Battersea
Park Children's Zoo; and Les Basford at Birmingham Nature Centre.
14