Porcine ear necrosis syndrome: A preliminary investigation of

The Veterinary Journal xxx (2012) xxx–xxx
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The Veterinary Journal
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Porcine ear necrosis syndrome: A preliminary investigation of putative
infectious agents in piglets and mycotoxins in feed
C. Weissenbacher-Lang a,b,⇑, T. Voglmayr c, F. Waxenecker d, U. Hofstetter d, H. Weissenböck b,
K. Hoelzle e, L.E. Hoelzle f, M. Welle g, M. Ogris h, G. Bruns i, M. Ritzmann a
a
Clinic for Swine, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria
Institute of Pathology and Forensic Veterinary Medicine, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria
Traunkreis VetClinic, Grossendorf 3, 4551 Ried im Traunkreis, Austria
d
Biomin GmbH, Industriestrasse 21, 3130 Herzogenburg, Austria
e
Institute of Veterinary Bacteriology, VetSuisse Faculty, University of Zurich, Winterthurerstraße 268, 8057 Zurich, Switzerland
f
Institute of Environmental and Animal Hygiene and Veterinary Medicine, University Hohenheim, 70593 Stuttgart, Germany
g
Institute of Animal Pathology, VetSuisse Faculty Berne, Länggass-Strasse 122, 3001 Berne, Switzerland
h
Intervet GesmbH, Siemensstrasse 107, 1210 Vienna, Austria
i
Tieraerztliche Klinik Duemmerland, Bahnhofstrasse 40, 49439 Steinfeld, Germany
b
c
a r t i c l e
i n f o
Article history:
Accepted 29 May 2012
Available online xxxx
Keywords:
Porcine ear necrosis
Mycotoxins
Histopathology
Bacteriology
Virology
a b s t r a c t
The aim of this study was to identify the causative factors of porcine ear necrosis syndrome (PENS) in 72
pigs, 5.5–10 weeks in age housed on nine farms. Biopsy samples of ear pinnae were collected from all piglets for bacteriology, histopathology and in situ hybridization for porcine circovirus type 2 (PCV2). At the
same time, serum samples were taken for serological analysis and viral PCR, and feed was sampled for
mycotoxin analysis.
The initial lesion of PENS seemed to be a focal epidermal necrosis. Streptococci were isolated from 44
and staphylococci from 36 pinnae. PCV2 could not be detected by in situ hybridization or qPCR. Seven
piglets were positive for porcine reproductive and respiratory syndrome virus, and one for Mycoplasma
suis. One piglet had antibodies against Sarcoptes scabiei var. suis. No infectious agents were found in 15
samples. Positive virology and parasitology were often found alongside positive bacteriology. Deoxynivalenol, zearalenone and ergot alkaloids were detected in feed. The findings suggest that PENS is multifactorial in origin and that although infectious agents can be involved in the development of the
syndrome they are not the exclusive triggering factor.
Ó 2012 Elsevier Ltd. All rights reserved.
Introduction
Porcine ear necrosis syndrome (PENS) is characterized by large
erosive lesions at the margin or the tip of the pinna (Richardson
et al., 1984). The syndrome is usually seen in weaning piglets during the summer months (Penny and Hill, 1974) and presents with
two distinct stages: a mild form characterized by the appearance of
easily ruptured vesicles followed by a more severe form with deeper lesions caused by bacteria (Richardson et al., 1984). Many causative agents for the development of PENS have been suggested and
although potential triggering factors can be divided into infectious
(Richardson et al., 1984; Thibault et al., 1998; Molnar et al., 2002;
Thacker, 2006) and non-infectious agents (Osweiler, 2006; Schrod-
⇑ Corresponding author at: Clinic for Swine, University of Veterinary Medicine
Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. Tel.: +43 1 250 77 2413.
E-mail
address:
christiane.weissenbacher-lang@vetmeduni.ac.at
(C. Weissenbacher-Lang).
er-Petersen and Simonsen, 2001) no definitive aetiology has been
identified.
The aim of the present study was to evaluate the role of infectious agents in the initial stage of PENS, using histopathology and
bacteriology. In addition, the role of mycotoxins in the syndrome
was investigated by analysing the feed that the affected pigs were
being fed for mycotoxins.
Materials and methods
Animals and husbandry
Seventy-two Large White Landrace Pietrain crossbred piglets aged 5.5–
10 weeks from nine farms were included in the study. The farms were selected
on the basis that all had had ear necrosis consistently in previous years. On these
farms pig density, temperature, and air velocity as well as carbon dioxide and
ammonia load were all within industry recommendations. Only one farm did not
use enrichment strategies. Piglets were housed on perforated slatted plastic floor
with 0.3 m2 floor space per pig. The compartments were cleaned in all farms between pigs, but only two of the farms additionally carried out disinfection. All farms
1090-0233/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tvjl.2012.05.026
Please cite this article in press as: Weissenbacher-Lang, C., et al. Porcine ear necrosis syndrome: A preliminary investigation of putative infectious agents in
piglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.org/10.1016/j.tvjl.2012.05.026
2
C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx
had under pressure ventilation systems. Feed was provided by Biomin (Biomin F4
pro aktiv, Biomin Profi 570 Gold), WiMa Mirakel Spezialfutter (Mirakel 1, Mirakel
Topzym), Sano – Moderne Tierernährung (Sano Suggi) and Schaumann (Schaumalac
F/80/M, Schaumalac Protect F90). The composition of feed corresponded to standard diets for this age class.
All piglets had been vaccinated against Mycoplasma hyopneumoniae in the first
week of life and at weaning. On one farm the sows had been vaccinated against porcine circovirus 2 (PCV2) 2 weeks before farrowing; on all other farms the piglets
had been vaccinated against PCV2 at weaning. Two of the farms had previously
diagnosed porcine reproductive and respiratory syndrome (PRRS), porcine respiratory disease complex and infections with Streptococcus spp. or Staphylococcus hyicus. These diseases had not been recorded on the other seven farms.
Blood and tissue samples were collected contemporaneously on two occasions,
1 year apart, from two different batches of 36 piglets (Table 1). Piglets were selected
for the study y on the basis of showing early clinical signs of PENS, i.e. oedematous
plaques of 5–10 mm size with thin, removable crusts (Figs. 1 and 2), irrespective of
age. Samples were not taken from animals with more severe disease. Five millimetre diameter tissue samples, containing all tissue layers of the ear, were collected
with a mechanical punching tool. For bacteriological analysis a swab was taken
from the biopsy site after tissue collection. Feed was collected three times: once
during the summer, when the symptoms occurred for the first time; once in the
winter after storage, and once again the following summer.
Biopsy samples were fixed in formalin, embedded in paraffin and sectioned.
These sections were stained with haematoxylin and eosin (H&E). Samples where
thrombosed blood vessels were suspected were stained using Weigert’s elastic
stain. All samples were subjected to in situ hybridization (ISH) for PCV2 nucleic acid
(Bukovsky et al., 2007). In order to evaluate the association between mycotoxin
concentrations and morphological features the microscopic lesions were classified
as: (1) Focal epidermal necrosis; (2) bacterial growth in the superficial cell debris;
(3) infiltration with granulocytes; (4) infiltration with histiocytes; (5) lysis of collagen; (6) vasculitis; (7) generation of granulation tissue, and (8) hyperkeratosis. Lesions 1 and 2 were the initial stages of ear necrosis, 3–6 were progressive disease,
while 7 and 8 were healing lesions.
Swabs were applied to blood agar plates immediately after collection; these
were incubated overnight at 37 °C. Staphylococci and streptococci were identified
macroscopically based on colony form and size and Gram’s stain. Bacterial quantities on solid media were classified based on evaluation of the three serial loop
smears as no growth (score , negative), doubtful (score (+), <10 colony forming
units (CFUs) in first loop smear), sparse growth (score +, <5 CFUs in second loop
smear), moderate growth (score ++, <5 CFUs in third loop smear) and pronounced
growth (score +++, P5 CFUs in third loop smear). The presence of PRSS virus,
PCV2 and Mycoplasma suis in serum were investigated by using previously described PCR procedures (Balka et al., 2008; Hoelzle et al., 2007; Lang et al., 2011).
Detection of specific serum antibodies against Sarcoptes scabiei var. suis was undertaken using a commercial Sarcoptes ELISA (AFOSA) according to the manufacturer’s
instructions.
All feed samples were analysed by high performance liquid chromatography
(HPLC) for the mycotoxins deoxynivalenol, zearalenone, T2 toxin, HT2 toxin, diacetoxyscirpenol, acetyldeoxynivalenol and nivalenol as well as the ergot alkaloids
ergosine, ergotamine, ergocornine, ergocryptine and ergocristine (Griessler et al.,
2010; Schiessl et al., 2010).
All statistical analyses were undertaken using PASW 17 (SPSS). The association
between histopathology and infectious agents was analysed using Pearson’s v2 test,
while that between histopathology and mycotoxin results was analysed using the
Student’s t test.
Results
The prevalence of ear necrosis on the farms ranged from 10%–
100% of piglets. The incidence and distribution pattern of affected
Fig. 1. Early stage of PENS. Macroscopic visible alterations of the ear.
Fig. 2. More advanced stage of PENS. Macroscopic visible alterations of the ear.
piglets showed a high degree of variation and could not be associated with any other observations or the obtained results. Microscopically, a focal epidermal necrosis was found to be the initial
lesion. In 67% of the cases at this stage of disease, bacteria that
morphologically resembled cocci were present in the necrotic epithelium (Fig. 3). These primary necrotic foci tended to expand to
larger areas of epidermal necrosis, which were consistently accompanied by a moderate to severe infiltration with mixed, partly
Table 1
Types of samples, time of collection and methods.
Sample type
Number of samples at different time points
First summer
Ear tissue
Winter
36 piglets
Method
Second summer
36 piglets
Swab of lesion
Serum
Feed
Histology
In situ hybridization: PCV2
Bacteriology: staphylococci, streptococci
PCR: PRRSV
PCR: PCV2
PCR: Mycoplasma suis
ELISA: Sarcoptes suis antibodies
9 samples
9 samples
9 samples
HPLC: T2 toxin, HT2 toxin, diacetoxyscirpenol
HPLC: deoxynivalenol, acetyldeoxynivalenol, nivalenol, zearalenone
HPLC: ergosine, ergotamine, ergocornine, ergocryptine, ergocristine
Please cite this article in press as: Weissenbacher-Lang, C., et al. Porcine ear necrosis syndrome: A preliminary investigation of putative infectious agents in
piglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.org/10.1016/j.tvjl.2012.05.026
C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx
degenerate inflammatory cells. In addition, there was superficial
cell debris with considerable bacterial growth. Particles of plant
material, most likely originating from faeces or litter, adhered to
the lesions.
The necrotic areas were demarcated from the dermis by
neutrophilic granulocytes (Fig. 4). Granulation tissue was generated subepidermally as well as in deeper areas of the dermis. In
three cases, re-epithelialization of the lesions was observed presenting as growth of regenerating epithelium beneath the necrotic
tissue (Fig. 5). Fully re-epithelialized areas were characterized by
severely hyperplastic epithelium and hyperkeratosis. Occasionally,
new necroses occurred in these re-epithelialized areas. In cases of
acute lesions, a marked emigration of neutrophilic granulocytes
from numerous medium-sized to small blood vessels could be
seen. In many cases inflammatory cells were also present in
the vascular wall, periadnexally and diffusely distributed in the
dermis. In more advanced lesions, granulation tissue and a
perivascular and periadenexal pyogranulomatous inflammation
was seen in the deep dermis. In 32% of the cases large blood vessels
showed subintimal proliferations suggestive of endarteriitis or
endophlebitis obliterans, sometimes with thrombus formation
(Fig. 6). In the majority of these cases, lesions indicative of
arteriosclerosis of small and medium-sized blood vessels,
predominantly characterized by hyperplasia of the intima and
hyaline degeneration of the blood vessel wall, were present.
The results of testing for infectious agents are summarised in
Table 2. Streptococci were found in 44 and staphylococci in 36 of
the swabs. PCV2 could not be detected by ISH or by qPCR. Seven
piglets were PRRSV positive, but only one piglet showed a positive
result for Mycoplasma suis. One piglet had antibodies against Sarcoptes scabiei var. suis. At the farm level, on one farm neither streptococci nor staphylococci were isolated from lesions, on another,
only staphylococci were isolated and on the remaining two only
streptococci were isolated. During the first sample collection, 5/9
farms were PRRSV negative. On the second occasion 7/9 farms
were PRRSV negative. Four of the five farms which were negative
at the first sample collection were negative 1 year later. The piglet
with the positive Sarcoptes scabiei var. suis diagnosis came from a
PRRSV positive farm, while the Mycoplasma suis positive one originated from a PRRSV negative farm. The different combinations of
diagnostic findings are presented in Table 3.
T2 toxin, HT2 toxin, diacetoxyscirpenol, acetyldeoxynivalenol
and nivalenol were not detected in any feed sample. The results
for the other toxins are summarised in Table 4. Eight samples
Fig. 3. Early stage of PENS. Multiple colonies of cocci are present within the necrotic
epithelium. In addition, particles of plant matter adhere to the surface. Haematoxylin and eosin staining, bar = 40 lm.
3
Fig. 4. More advanced stage of PENS. The necrotic tissue is demarcated by
neutrophilic granulocytes. Abundant plant material adheres to the surface.
Haematoxylin and eosin staining, bar = 150 lm.
Fig. 5. Advanced stage of PENS. Re-epithelialization occurs by hyperplastic
epithelium (hyp) moving under the necrotic tissue (necr). Haematoxylin and eosin
staining, bar = 150 lm.
Fig. 6. Several cases of PENS showed prominent lesions of dermal blood vessels. An
artery shows marked subintimal proliferations leading to partial occlusion of the
lumen. Haematoxylin and eosin staining, bar = 80 lm.
Please cite this article in press as: Weissenbacher-Lang, C., et al. Porcine ear necrosis syndrome: A preliminary investigation of putative infectious agents in
piglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.org/10.1016/j.tvjl.2012.05.026
4
C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx
Table 2
Frequency of identification of infectious agents (n).
Staphylococci
All samples (n = 72)
First collection (n = 36)
Second collection (n = 36)
36
27
9
Streptococci
(+)
+
++
+++
7
1
6
13
2
11
13
4
9
3
2
1
28
13
15
PCV2 (ISH)
PCV2 (PCR)
PRRSV
Mycoplasma
suis
Sarcoptes suis
antibodies
(+)
+
++
+++
Neg.
Pos.
Neg.
Pos.
Neg.
Pos.
Neg.
Pos.
Neg.
Doubt.
Pos.
7
3
4
6
4
2
17
11
6
14
5
9
72
36
36
0
0
0
72
36
36
0
0
0
65
31
34
7
5
2
71
36
35
1
0
1
71
35
36
1
1
0
0
0
0
, negative; (+), doubtful; +, few; ++, moderate; +++, high density of colonies; neg., negative; pos., positive; doubt., doubtful.
(29.6%) exceeded a deoxynivalenol concentration of 0.200 mg/kg,
the minimum emetic dose of this mycotoxin (Forsyth et al.,
1977), and 9 (33.3%) had a zearalenone concentration of
>0.050 mg/kg, the critical dose for the establishment of symptoms
in prepubertal pigs (Bauer et al., 1987).
In Table 5 the different ergot alkaloid combinations and patterns are presented, and Table 6 shows the correlation between
mycotoxin and alkaloid concentrations and the progressive development of microscopic lesions. Deoxynivalenol was found only in
the initial phase of PENS. Ergotamine was negatively correlated
with collagen lysis, but positively associated with vasculitis in
the acute phase. Ergocryptine and ergocristine were negatively
correlated with histiocyte infiltration during the acute phase. Ergocryptine concentrations were not associated with any effect during
the healing phase.
The microscopic alterations were divided with regard to their
occurrence in superficial and deeper tissue layers. There were also
significant correlations between increased ergosine (P = 0.03),
ergotamine (P = 0.017) and ergocristine (P = 0.033) concentrations
and the microscopic alterations in the superficial tissue layers.
There was no significant difference between the three different
sample collections in the summer time, when the symptoms occurred for the first time, in the winter after storage and once again
in the following summer. The deoxynivalenol concentrations of the
three consecutive sample collections were 362, 219 and 173 mg/kg
on average, and the zearalenone values were 42, 36 and 43 mg/kg.
The ergot alkaloid concentrations in the three batches were as
follows: ergosine: 62, 9 and 52 mg/kg; ergotamine: 18, 11 and
12 mg/kg; ergocornine: 0, 2 and 0 mg/kg; ergocryptine: 113, 0
and 178 mg/kg; ergocristine: 403, 330 and 452 mg/kg.
Discussion
In the present study, alterations in piglet ear tissue in the initial
stage of PENS were analysed histopathologically in an attempt to
define the time course of the disease. As this was a preliminary
study, only piglets with ear necrosis were investigated to obtain
as much information as possible about this syndrome. Focal epidermal necrosis was seen as the initial lesion. Such necrosis can
either be the consequence of a vascular alteration resulting in local
ischaemia as leading to epidermal necrosis and subsequent ulceration or of an outside event such as trauma or bacterial toxins.
Based on the data, a definitive answer about the causative factor
cannot be given. The selection of piglets for sample collection was
Table 4
Mycotoxin and ergot alkaloid concentrations (mg/kg).
Toxin
Maximum
Mean
Median
SD
Deoxynivalenol
Zearalenone
Ergosine
Ergotamine
Ergocornine
Ergocryptine
Ergocristine
1.243
0.126
0.486
0.048
0.015
0.822
2.015
0.251
0.040
0.041
0.014
0.001
0.097
0.395
0.150
0.028
0.000
0.015
0.000
0.000
0.023
0.254
0.039
0.118
0.015
0.003
0.232
0.620
Table 5
Combinations of ergot alkaloids (number of feed samples = 27).
Ergot alkaloids
Number of samples with this
combination
Ergotamine
Ergocryptine/Ergocristine
Ergosine/Ergotamine/Ergocryptine/
Ergocristine
Ergotamine/Ergocristine
Ergosine/Ergotamine/Ergocristine
Ergosine/Ergotamine
Ergosine/Ergocristine
Ergocornine/Ergocristine
Ergocristine
Ergosine
5
4
3
3
3
2
1
1
1
1
made when the first macroscopic lesions were visible, and the very
early stage of vesiculation as documented by Richardson et al.
(1984) could not be observed in the present field study although
the histological pattern of more severe cases was comparable. Nevertheless, the lack of cases at the early stage of disease in the present study is due to the timing of sample collection and does not
suggest different aetiological causes.
The most frequent ear lesions found by Richardson et al. (1984)
were hyperkeratosis, acanthosis and intraepidermal abscesses as
well as a hyperplastic perivascular dermatitis (Mirt, 1999).
Depending on the duration of the lesions, the necrotic dermis
was replaced by granulation tissue, as in the present cases. Vascular changes could also be observed in both studies. The occlusion of
the lumen of affected vessels by thrombi has already been described by Richardson et al. (1984).
Table 3
Number of specimens with combinations of selected infectious agents.
Combinations of
No infectious agent
PRRSV
Streptococci/staphylococci
Streptococci/staphylococci/Sarcoptes antibodies
Streptococci/staphylococci/PRRSV
All samples (n = 72)
First collection (n = 36)
Second collection (n = 36)
n
%
n
%
n
%
15
2
50
1
4
20.8
2.8
69.4
1.4
5.6
10
2
21
1
1
27.8
5.6
58.8
2.8
2.8
5
0
29
0
1
14.0
0.0
81.2
0.0
2.8
Please cite this article in press as: Weissenbacher-Lang, C., et al. Porcine ear necrosis syndrome: A preliminary investigation of putative infectious agents in
piglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.org/10.1016/j.tvjl.2012.05.026
5
C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx
Table 6
Correlation of mycotoxin and ergot alkaloid concentrations with the different morphological features of microscopic lesions.
1. Focal
epidermal
necrosis
2. Bacterial growth in the
superficial cell debris
3. Infiltration
with granulocytes
4. Infiltration
with histiocytes
5. Lysis of
collagen
6. Vasculitis in the
acute stadium
7. Generation of
granulation tissue
8.
Hyperkeratosis
Deoxynivalenol
0.298
P 0.014
0.409
<0.001
0.216
0.073
0.235
0.047
0.194
0.105
0.131
0.324
0.176
0.140
0.203
0.090
Zearalenone
q 0.140
P 0.255
0.098
0.414
0.052
0.672
0.100
0.403
0.035
0.770
0.232
0.077
0.047
0.696
0.032
0.794
Ergosine
0.030
P 0.807
0.190
0.109
0.130
0.285
0.106
0.375
0.169
0.158
0.083
0.530
0.117
0.326
0.058
0.634
Ergotamine
0.079
P 0.521
0.227
0.056
0.148
0.221
0.193
0.104
0.248
0.037
0.721
0.038
0.016
0.894
0.035
0.774
Ergocryptine
0.008
P 0.948
0.162
0.173
0.025
0.839
0.259
0.028
0.013
0.914
0.381
0.003
0.263
0.026
0.316
0.007
Ergocristine
0.010
P 0.936
0.042
0.727
0.064
0.598
0.326
0.005
0.172
0.150
0.124
0.350
0.075
0.530
0.140
0.245
q
q
q
q
q
q, Spearman’s coefficient of variation; P, level of significance 0.05 (bold = significant P-values).
As only one sample was positive for ergocornine, the calculation of correlation was not possible.
In the present study, staphylococci and streptococci could be
seen in various disease stages. Streptococci were found in higher
concentrations, as well as in a higher percentage of cases. Richardson et al. (1984) isolated Staphylococcus hyicus from both early and
advanced lesions, whereas they only isolated streptococci from
ulcerated lesions. The colonization of the lesions by bacteria is
viewed as important in the breakdown of the epidermis, the extension of lesions into deeper tissues and the development of vascular
thrombosis, ischaemia and necrosis (Richardson et al., 1984).
Staphylococci are adapted for residence on normal pig skin and
can be considered an aetiologic agent of early skin lesions (L’Ecuyer, 1967), whereas the invasion of streptococci must be preceded
by damage of the skin due to infection or trauma (Wannamaker,
1970). As seen in the present study, both kinds of bacteria can appear at different stages of disease independent of their pathogenesis, thus underlining a multifactorial aetiology.
Particles of plant material originating from faeces or litter stuck
to the lesions emphasized the assumption of local trauma as a
starting point, with secondary colonization of those wounds by
bacteria underlying the development of severe necrotizing lesions.
A significant association between ear scratches and an increased
risk of mild ear necrosis was also shown by Busch et al. (2008).
Mirt (1999) isolated staphylococci twice as frequently from lesions
as from healthy skin and assumed that infection with staphylococci began at sites of slight trauma, but definitely ruled out an
exclusively traumatic origin. Maddox et al. (1973) mainly detected
streptococci in samples from necrotic ear margins, which presented as thromboarteriitis. The lesions healed after treatment
with ampicillin.
Since trials with antibiotic treatment of ear necrosis against
staphylococci (Hansen and Busch, 2008) resulted in no significant
clinical effect, it seems unlikely that bacteria alone can cause this
clinical pattern, which further supports other infectious or noninfectious agents being involved in this apparently multifactorial
disease. Comparable microscopic lesions were also observed in
association with a cutaneous necrotizing vasculitis in weaning pigs
even when the lesions did not remain limited to the margins of the
ears (Thibault et al., 1998). Aside from the distribution of necrotic
lesions, the only difference from the present study was the pres-
ence of eosinophils, which could have resulted from an immunemediated inflammation. The initial causative agent of this cutaneous necrotizing vasculitis could not be determined, but the authors
suggested that these lesions probably developed spontaneously
when a specific combination of predisposing events, including
PRRSV infection, occurred. In the present study PRRSV was detected in some cases, but there were no histopathological differences compared with other samples.
The association between PRRSV and the microscopic findings
was not significant, but a contribution to the development of the
lesions cannot be completely ruled out, particularly in the cases
with bacterial and PRRSV co-infections. The detection of PCV2 in
various tissues of pigs with symptoms of postweaning multisystemic wasting syndrome or porcine dermatitis and nephropathy
syndrome is generally possible (Molnar et al., 2002; Zlotowski
et al., 2008), but PCV2 has not been proven to be a causative agent
for necrotizing vasculitis in the context of PENS. Pejsak et al. (2011)
showed reductions in both prevalence and severity of ear necrosis
in weaners after sow vaccination against PCV2, even though factors
such as the herd immune status or co-infections were not considered in this study.
All of our piglets were vaccinated either actively or passively
against PCV2 and the virus could not be detected by ISH or PCR.
Nevertheless, a high prevalence of ear necrosis was recorded on
some farms. So, in contrast to the findings of Papatsiros (2011), a
direct association between PCV2 and the development of PENS
could not be demonstrated. Because vaccination of piglets and
sows against PCV2 is widely used, the relevance of this pathogen
in relation to the development of ear necrosis has to be questioned.
A similar conclusion could also be made for Mycoplasma suis and
Sarcoptes scabiei var. suis.
Mycotoxins do not necessarily and exclusively cause ear necrosis, but because they have dermonecrotic potential (Osweiler,
2006), their concentration was determined in feed. T2 toxin, HT2
toxin, diacetoxyscirpenol, acetyldeoxynivalenol and nivalenol
were of no importance. Only deoxynivalenol and zearalenone
could be detected in biologically significant concentrations
(Forsyth et al., 1977; Bauer et al., 1987). Combinations of different
mycotoxins may potentiate the action of each other or at least
Please cite this article in press as: Weissenbacher-Lang, C., et al. Porcine ear necrosis syndrome: A preliminary investigation of putative infectious agents in
piglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.org/10.1016/j.tvjl.2012.05.026
6
C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx
exert an additive effect (Huff et al., 1988). The co-existence of
zearalenone with other mycotoxins such as nivalenol or
deoxynivalenol has already been described by some authors (IARC,
1993) due to some compounds being produced by the same
Fusarium species. On the other hand, different biological and
climate conditions are necessary for the production of the common
mycotoxins and these rarely appear in the same grain at a specific
time point. Therefore, concurrent production of mycotoxins could
be relatively uncommon, and there is presently little evidence that
common mycotoxins act synergistically (Osweiler, 2006).
In the present study, higher deoxynivalenol concentrations
were correlated with microscopic alterations in the initial stage
of the disease. A synergistic effect with infectious agents or an impact as a precursor has not been documented, but cannot be ruled
out. Ergot alkaloids cause gangrenous ergotism as a result of combined vasoconstriction and endothelial damage leading to prolonged ischaemia of appendages and eventually dry gangrene
(Osweiler, 2006). The contractile response of veins depends not
only on the concentration of alkaloids, but also on their types
due to their chemical diversity as well as on interactions between
different alkaloid types (Klotz et al., 2008). As described by other
European authors (Uhlig et al., 2007), ergocristine dominated the
alkaloid spectrum of most extracts in the present study, followed
by ergotamine, ergosine and ergocryptine. Ergocornine was of secondary importance. Ear and tail necrosis have rarely been described in the context of alkaloid concentrations and have been
observed after intake of a combination of ergotamine, ergocristine,
and ergonovine (10 mg/kg grain) (Lopez et al., 1997). For this reason, their relevance as well as the required dose for the development of ear necrosis still need to be investigated.
Conclusions
Our study clearly showed that multiple infectious agents could
be involved in the development of PENS, but also that none of the
agents we investigated were the exclusive triggering factor. This
does not mean that there is not one factor – indeed other infectious
agents that we did not investigate here could be of primary importance in the aetiology of PENS. Exclusion of more bacterial, viral
and parasitic diseases as well as non-infectious factors is necessary. Further investigation should include challenge trials with different infectious agents, with a focus on the resulting
histopathology. In addition, the potential synergistic effect of ergot
alkaloids needs to be considered as do other non-infectious agents,
such as endotoxin. Finally, a comparison between diseased and
healthy piglets is required.
Conflict of interest statement
None of the authors of this paper has a financial or personal
relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
Acknowledgments
This study was supported by the Dres. Jutta und Georg-BrunsStiftung. The bacteriological analysis of the feed samples was carried out at the Futtermittellabor Rosenau (Chamber of Agriculture
Lower Austria, Petzenkirchen, Austria), the mycotoxin analysis at
ROMER Labs Diagnostic GmbH (Tulln, Austria).
References
Balka, G., Hornyak, A., Balint, A., Kiss, I., Kecskemeti, S., Bakonyi, T., Rusvai, M., 2008.
Genetic diversity of porcine reproductive and respiratory syndrome virus
strains circulating in Hungarian swine herds. Veterinary Microbiology 127,
128–135.
Bauer, J., Heinritzi, K., Gareis, M., Gedek, B., 1987. Changes in the genital tract of
female swine after feeding with practice-relevant amounts of zearalenone.
Tierärztliche Praxis 15, 26–33.
Bukovsky, C., Schmoll, F., Revilla-Fernandez, S., Weissenböck, H., 2007. Studies on
the aetiology of non-suppurative encephalitis in pigs. Veterinary Record 161,
552–558.
Busch, M.E., Dedeurwaerdere, A., Wachmann, H., 2008. The development and the
consequences of ear necrosis in one herd. In: Proceedings of the 20th IPVS
Congress, Durban, South Africa, p. 278.
Forsyth, D.M., Yoshizawa, T., Morooka, N., Tuite, J., 1977. Emetic and refusal activity
of deoxynivalenol to swine. Applied and Environmental Microbiology 34, 547–
552.
Griessler, K., Rodrigues, I., Handl, J., Hofstetter, U., 2010. Occurrence of mycotoxins
in Southern Europe. World Mycotoxin Journal 3, 301–309.
Hansen, K.K., Busch, M.E., 2008. Antibiotic treatment as an intervention against ear
necrosis in one herd. In: Proceedings of the 20th IPVS Congress, Durban, South
Africa, p. 594.
Hoelzle, L.E., Helbling, M., Hoelzle, K., Ritzmann, M., Heinritzi, K., Wittenbrink, M.M.,
2007. First LightCycler real-time PCR assay for the quantitative detection of
Mycoplasma suis in clinical samples. Journal of Microbiological Methods 70,
346–354.
Huff, W.E., Kubena, L.F., Harvey, R.B., Doerr, J.A., 1988. Mycotoxin interactions in
poultry and swine. Journal of Animal Science 66, 2351–2355.
IARC, 1993. Toxins derived from Fusarium graminearum, F. culmorum and F.
crookwellense: Zearalenone, deoxynivalenol, nivalenol and fusarenone X. In:
IARC Monographs on the Evaluation of Carcinogenic Risks on Humans: Some
Naturally Occurring Substances, Food Items and Constituents, Heterocyclic
Aromatic Amines and Mycotoxins. IARC Lyon 56, pp. 445–466.
Klotz, J.L., Kirch, B.H., Aiken, G.E., Bush, L.P., Strickland, J.R., 2008. Effects of selected
combinations of tall fescue alkaloids on the vasoconstrictive capacity of
fescue-naive bovine lateral saphenous veins. Journal of Animal Science 86,
1021–1028.
L’Ecuyer, C., 1967. Exudative epidermitis in pigs. Bacteriological studies on the
causative agent. Canadian Journal of Comparative Medicine and Veterinary
Science 31, 243–247.
Lang, C., Soellner, H., Barz, A., Ladinig, A., Langhoff, R., Weissenböck, H., Kekarainen,
T., Segalés, J., Ritzmann, M., 2011. Investigation of the prevalence of swine
torque teno virus in Austria. Berliner Muenchner Tieraerztliche Wochenschrift
124, 10–15.
Lopez, T.A., Campero, C.M., Chayer, R., de Hoyos, M., 1997. Ergotism and
photosensitization in swine produced by the combined ingestion of Claviceps
purpurea sclerotia and Ammi majus seeds. Journal of Veterinary Diagnostic
Investigation 9, 68–71.
Maddox, E.T., Graham, C.W., Reynolds, W.A., 1973. Ampicillin treatment of three
cases of streptococcal auricular dermatitis in swine. Veterinary Medicine and
Small Animal Clinician 68, 1018–1019.
Mirt, D., 1999. Lesions of so-called flank biting and necrotic ear syndrome in pigs.
Veterinary Record 144, 92–96.
Molnar, T., Glavits, R., Szeredi, L., Dan, A., 2002. Occurrence of porcine dermatitis
and nephropathy syndrome in Hungary. Acta Veterinaria Hungarica 50, 5–
16.
Osweiler, G.D., 2006. Occurrence of mycotoxins in grains and feed. In: Straw, B.E.,
Zimmerman, J.J., d’Allaire, S.D., Taylor, D.J. (Eds.), Diseases of Swine, Ninth Ed.
Iowa State Press, Ames, Iowa, USA, pp. 915–929.
Papatsiros, V.G., 2011. Exploration of the connection between porcine necrotic ear
syndrome and PCV2 infection. Journal of Animal and Veterinary Advances 10,
185–187.
Pejsak, Z., Markowska-Daniel, I., Pomorska-Mól, M., Porowski, M., Kolacz, R., 2011.
Ear necrosis reduction in pigs after vaccination against PCV2. Research in
Veterinary Science 91, 125–128.
Penny, R.H., Hill, F.W., 1974. Observations of some conditions in pigs at the abattoir
with particular reference to tail biting. Veterinary Record 94, 174–180.
Richardson, J.A., Morter, R.L., Rebar, A.H., Olander, H.J., 1984. Lesions of porcine
necrotic ear syndrome. Veterinary Pathology 21, 152–157.
Schiessl, A., Daxner, B., Schandl, M., Pichler, E., 2010. New reliable method and
reference materials for the detection of ergot alkaloids. In: Proceedings of the
6th World Mycotoxin Forum, Noordwijkerhout, The Netherlands, p. 181.
Schroder-Petersen, D.L., Simonsen, H.B., 2001. Tail biting in pigs. The Veterinary
Journal 162, 196–210.
Thacker, E.L., 2006. Mycoplasmal diseases. In: Straw, B.E., Zimmerman, J.J., d’Allaire,
S.D., Taylor, D.J. (Eds.), Diseases of Swine, Ninth Ed. Iowa State Press, Ames,
Iowa, USA, pp. 701–717.
Thibault, S., Drolet, R., Germain, M.C., D’Allaire, S., Larochelle, R., Magar, R., 1998.
Cutaneous and systemic necrotizing vasculitis in swine. Veterinary Pathology
35, 108–116.
Uhlig, S., Vikoren, T., Ivanova, L., Handeland, K., 2007. Ergot alkaloids in Norwegian
wild grasses: A mass spectrometric approach. Rapid Communication in Mass
Spectrometry 21, 1651–1660.
Wannamaker, L.W., 1970. Differences between streptococcal infections of the throat
and of the skin. New England Journal of Medicine 282, 23–31.
Zlotowski, P., Correa, A.M.R., Barcellos, D.E.S.N., Driemeier, D., 2008. Presence of
PCV2 in ear lesions in the course of PCVAD in growing pigs. In: Proceedings of
the 20th IPVS Congress, Durban, South Africa, p. 555.
Please cite this article in press as: Weissenbacher-Lang, C., et al. Porcine ear necrosis syndrome: A preliminary investigation of putative infectious agents in
piglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.org/10.1016/j.tvjl.2012.05.026