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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 putativeinfectious 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, Austriab Institute of Pathology and Forensic Veterinary Medicine, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austriac Traunkreis VetClinic, Grossendorf 3, 4551 Ried im Traunkreis, Austriad Biomin GmbH, Industriestrasse 21, 3130 Herzogenburg, Austriae Institute of Veterinary Bacteriology, VetSuisse Faculty, University of Zurich, Winterthurerstraße 268, 8057 Zurich, Switzerlandf Institute of Environmental and Animal Hygiene and Veterinary Medicine, University Hohenheim, 70593 Stuttgart, Germanyg Institute of Animal Pathology, VetSuisse Faculty Berne, Länggass-Strasse 122, 3001 Berne, Switzerlandh Intervet GesmbH, Siemensstrasse 107, 1210 Vienna, Austriai Tieraerztliche Klinik Duemmerland, Bahnhofstrasse 40, 49439 Steinfeld, Germany

a r t i c l e i n f o

Article history:Accepted 29 May 2012Available online xxxx

Keywords:Porcine ear necrosisMycotoxinsHistopathologyBacteriologyVirology

1090-0233/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.tvjl.2012.05.026

⇑ Corresponding author at: Clinic for Swine, UniveVienna, Veterinaerplatz 1, 1210 Vienna, Austria. Tel.:

E-mail address: christiane.weissen(C. Weissenbacher-Lang).

Please cite this article in press as: Weissenbachepiglets and mycotoxins in feed. The Veterinary

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 72pigs, 5.5–10 weeks in age housed on nine farms. Biopsy samples of ear pinnae were collected from all pig-lets for bacteriology, histopathology and in situ hybridization for porcine circovirus type 2 (PCV2). At thesame time, serum samples were taken for serological analysis and viral PCR, and feed was sampled formycotoxin analysis.

The initial lesion of PENS seemed to be a focal epidermal necrosis. Streptococci were isolated from 44and staphylococci from 36 pinnae. PCV2 could not be detected by in situ hybridization or qPCR. Sevenpiglets were positive for porcine reproductive and respiratory syndrome virus, and one for Mycoplasmasuis. One piglet had antibodies against Sarcoptes scabiei var. suis. No infectious agents were found in 15samples. Positive virology and parasitology were often found alongside positive bacteriology. Deoxyni-valenol, zearalenone and ergot alkaloids were detected in feed. The findings suggest that PENS is multi-factorial in origin and that although infectious agents can be involved in the development of thesyndrome they are not the exclusive triggering factor.

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Introduction

Porcine ear necrosis syndrome (PENS) is characterized by largeerosive lesions at the margin or the tip of the pinna (Richardsonet al., 1984). The syndrome is usually seen in weaning piglets dur-ing the summer months (Penny and Hill, 1974) and presents withtwo distinct stages: a mild form characterized by the appearance ofeasily ruptured vesicles followed by a more severe form with dee-per lesions caused by bacteria (Richardson et al., 1984). Many caus-ative agents for the development of PENS have been suggested andalthough 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-

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r-Lang, C., et al. Porcine ear necJournal (2012), http://dx.doi.or

er-Petersen and Simonsen, 2001) no definitive aetiology has beenidentified.

The aim of the present study was to evaluate the role of infec-tious agents in the initial stage of PENS, using histopathology andbacteriology. In addition, the role of mycotoxins in the syndromewas investigated by analysing the feed that the affected pigs werebeing 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 selectedon the basis that all had had ear necrosis consistently in previous years. On thesefarms pig density, temperature, and air velocity as well as carbon dioxide andammonia load were all within industry recommendations. Only one farm did notuse enrichment strategies. Piglets were housed on perforated slatted plastic floorwith 0.3 m2 floor space per pig. The compartments were cleaned in all farms be-tween pigs, but only two of the farms additionally carried out disinfection. All farms

rosis syndrome: A preliminary investigation of putative infectious agents ing/10.1016/j.tvjl.2012.05.026

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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.

2 C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx

had under pressure ventilation systems. Feed was provided by Biomin (Biomin F4pro aktiv, Biomin Profi 570 Gold), WiMa Mirakel Spezialfutter (Mirakel 1, MirakelTopzym), Sano – Moderne Tierernährung (Sano Suggi) and Schaumann (SchaumalacF/80/M, Schaumalac Protect F90). The composition of feed corresponded to stan-dard diets for this age class.

All piglets had been vaccinated against Mycoplasma hyopneumoniae in the firstweek of life and at weaning. On one farm the sows had been vaccinated against por-cine circovirus 2 (PCV2) 2 weeks before farrowing; on all other farms the pigletshad been vaccinated against PCV2 at weaning. Two of the farms had previouslydiagnosed porcine reproductive and respiratory syndrome (PRRS), porcine respira-tory disease complex and infections with Streptococcus spp. or Staphylococcus hyi-cus. 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 selectedfor the study y on the basis of showing early clinical signs of PENS, i.e. oedematousplaques of 5–10 mm size with thin, removable crusts (Figs. 1 and 2), irrespective ofage. Samples were not taken from animals with more severe disease. Five millime-tre diameter tissue samples, containing all tissue layers of the ear, were collectedwith a mechanical punching tool. For bacteriological analysis a swab was takenfrom the biopsy site after tissue collection. Feed was collected three times: onceduring the summer, when the symptoms occurred for the first time; once in thewinter 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 wherethrombosed blood vessels were suspected were stained using Weigert’s elasticstain. All samples were subjected to in situ hybridization (ISH) for PCV2 nucleic acid(Bukovsky et al., 2007). In order to evaluate the association between mycotoxinconcentrations and morphological features the microscopic lesions were classifiedas: (1) Focal epidermal necrosis; (2) bacterial growth in the superficial cell debris;(3) infiltration with granulocytes; (4) infiltration with histiocytes; (5) lysis of colla-gen; (6) vasculitis; (7) generation of granulation tissue, and (8) hyperkeratosis. Le-sions 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; thesewere incubated overnight at 37 �C. Staphylococci and streptococci were identifiedmacroscopically based on colony form and size and Gram’s stain. Bacterial quanti-ties on solid media were classified based on evaluation of the three serial loopsmears as no growth (score �, negative), doubtful (score (+), <10 colony formingunits (CFUs) in first loop smear), sparse growth (score +, <5 CFUs in second loopsmear), moderate growth (score ++, <5 CFUs in third loop smear) and pronouncedgrowth (score +++, P5 CFUs in third loop smear). The presence of PRSS virus,PCV2 and Mycoplasma suis in serum were investigated by using previously de-scribed 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 under-taken using a commercial Sarcoptes ELISA (AFOSA) according to the manufacturer’sinstructions.

All feed samples were analysed by high performance liquid chromatography(HPLC) for the mycotoxins deoxynivalenol, zearalenone, T2 toxin, HT2 toxin, diacet-oxyscirpenol, acetyldeoxynivalenol and nivalenol as well as the ergot alkaloidsergosine, ergotamine, ergocornine, ergocryptine and ergocristine (Griessler et al.,2010; Schiessl et al., 2010).

All statistical analyses were undertaken using PASW 17 (SPSS). The associationbetween histopathology and infectious agents was analysed using Pearson’s v2 test,while that between histopathology and mycotoxin results was analysed using theStudent’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

Table 1Types of samples, time of collection and methods.

Sample type Number of samples at different time points

First summer Winter Second summ

Ear tissue 36 piglets 36 piglets

Swab of lesion

Serum

Feed 9 samples 9 samples 9 samples

Please cite this article in press as: Weissenbacher-Lang, C., et al. Porcine ear necpiglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.or

piglets showed a high degree of variation and could not be associ-ated with any other observations or the obtained results. Micro-scopically, a focal epidermal necrosis was found to be the initiallesion. In 67% of the cases at this stage of disease, bacteria thatmorphologically resembled cocci were present in the necrotic epi-thelium (Fig. 3). These primary necrotic foci tended to expand tolarger areas of epidermal necrosis, which were consistently accom-panied by a moderate to severe infiltration with mixed, partly

Method

er

HistologyIn situ hybridization: PCV2Bacteriology: staphylococci, streptococci

PCR: PRRSVPCR: PCV2PCR: Mycoplasma suisELISA: Sarcoptes suis antibodies

HPLC: T2 toxin, HT2 toxin, diacetoxyscirpenolHPLC: deoxynivalenol, acetyldeoxynivalenol, nivalenol, zearalenoneHPLC: ergosine, ergotamine, ergocornine, ergocryptine, ergocristine



Fig. 4. More advanced stage of PENS. The necrotic tissue is demarcated byneutrophilic 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 hyperplasticepithelium (hyp) moving under the necrotic tissue (necr). Haematoxylin and eosinstaining, bar = 150 lm.

C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx 3

degenerate inflammatory cells. In addition, there was superficialcell debris with considerable bacterial growth. Particles of plantmaterial, most likely originating from faeces or litter, adhered tothe lesions.

The necrotic areas were demarcated from the dermis byneutrophilic granulocytes (Fig. 4). Granulation tissue was gener-ated subepidermally as well as in deeper areas of the dermis. Inthree cases, re-epithelialization of the lesions was observed pre-senting as growth of regenerating epithelium beneath the necrotictissue (Fig. 5). Fully re-epithelialized areas were characterized byseverely hyperplastic epithelium and hyperkeratosis. Occasionally,new necroses occurred in these re-epithelialized areas. In cases ofacute lesions, a marked emigration of neutrophilic granulocytesfrom numerous medium-sized to small blood vessels could beseen. In many cases inflammatory cells were also present inthe vascular wall, periadnexally and diffusely distributed in thedermis. In more advanced lesions, granulation tissue and aperivascular and periadenexal pyogranulomatous inflammationwas seen in the deep dermis. In 32% of the cases large blood vesselsshowed subintimal proliferations suggestive of endarteriitis orendophlebitis obliterans, sometimes with thrombus formation(Fig. 6). In the majority of these cases, lesions indicative ofarteriosclerosis of small and medium-sized blood vessels,predominantly characterized by hyperplasia of the intima andhyaline degeneration of the blood vessel wall, were present.

The results of testing for infectious agents are summarised inTable 2. Streptococci were found in 44 and staphylococci in 36 ofthe swabs. PCV2 could not be detected by ISH or by qPCR. Sevenpiglets were PRRSV positive, but only one piglet showed a positiveresult for Mycoplasma suis. One piglet had antibodies against Sar-coptes scabiei var. suis. At the farm level, on one farm neither strep-tococci nor staphylococci were isolated from lesions, on another,only staphylococci were isolated and on the remaining two onlystreptococci were isolated. During the first sample collection, 5/9farms were PRRSV negative. On the second occasion 7/9 farmswere PRRSV negative. Four of the five farms which were negativeat the first sample collection were negative 1 year later. The pigletwith the positive Sarcoptes scabiei var. suis diagnosis came from aPRRSV positive farm, while the Mycoplasma suis positive one orig-inated from a PRRSV negative farm. The different combinations ofdiagnostic findings are presented in Table 3.

T2 toxin, HT2 toxin, diacetoxyscirpenol, acetyldeoxynivalenoland nivalenol were not detected in any feed sample. The resultsfor the other toxins are summarised in Table 4. Eight samples

Fig. 3. Early stage of PENS. Multiple colonies of cocci are present within the necroticepithelium. In addition, particles of plant matter adhere to the surface. Haematox-ylin and eosin staining, bar = 40 lm.

Fig. 6. Several cases of PENS showed prominent lesions of dermal blood vessels. Anartery shows marked subintimal proliferations leading to partial occlusion of thelumen. 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 inpiglets and mycotoxins in feed. The Veterinary Journal (2012), http://dx.doi.org/10.1016/j.tvjl.2012.05.026


Table 2Frequency of identification of infectious agents (n).

Staphylococci Streptococci PCV2 (ISH) PCV2 (PCR) PRRSV Mycoplasmasuis

Sarcoptes suisantibodies

� (+) + ++ +++ � (+) + ++ +++ Neg. Pos. Neg. Pos. Neg. Pos. Neg. Pos. Neg. Doubt. Pos.

All samples (n = 72) 36 7 13 13 3 28 7 6 17 14 72 0 72 0 65 7 71 1 71 1 0First collection (n = 36) 27 1 2 4 2 13 3 4 11 5 36 0 36 0 31 5 36 0 35 1 0Second collection (n = 36) 9 6 11 9 1 15 4 2 6 9 36 0 36 0 34 2 35 1 36 0 0

�, negative; (+), doubtful; +, few; ++, moderate; +++, high density of colonies; neg., negative; pos., positive; doubt., doubtful.

Table 4Mycotoxin and ergot alkaloid concentrations (mg/kg).

Toxin Maximum Mean Median SD

Deoxynivalenol 1.243 0.251 0.150 0.254Zearalenone 0.126 0.040 0.028 0.039Ergosine 0.486 0.041 0.000 0.118Ergotamine 0.048 0.014 0.015 0.015Ergocornine 0.015 0.001 0.000 0.003Ergocryptine 0.822 0.097 0.000 0.232Ergocristine 2.015 0.395 0.023 0.620

Table 5Combinations of ergot alkaloids (number of feed samples = 27).

Ergot alkaloids Number of samples with thiscombination

Ergotamine 5Ergocryptine/Ergocristine 4Ergosine/Ergotamine/Ergocryptine/

Ergocristine3

Ergotamine/Ergocristine 3Ergosine/Ergotamine/Ergocristine 3Ergosine/Ergotamine 2Ergosine/Ergocristine 1Ergocornine/Ergocristine 1Ergocristine 1Ergosine 1


(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 symptomsin prepubertal pigs (Bauer et al., 1987).

In Table 5 the different ergot alkaloid combinations and pat-terns are presented, and Table 6 shows the correlation betweenmycotoxin and alkaloid concentrations and the progressive devel-opment of microscopic lesions. Deoxynivalenol was found only inthe initial phase of PENS. Ergotamine was negatively correlatedwith collagen lysis, but positively associated with vasculitis inthe acute phase. Ergocryptine and ergocristine were negativelycorrelated with histiocyte infiltration during the acute phase. Ergo-cryptine concentrations were not associated with any effect duringthe healing phase.

The microscopic alterations were divided with regard to theiroccurrence in superficial and deeper tissue layers. There were alsosignificant correlations between increased ergosine (P = 0.03),ergotamine (P = 0.017) and ergocristine (P = 0.033) concentrationsand the microscopic alterations in the superficial tissue layers.There was no significant difference between the three differentsample collections in the summer time, when the symptoms oc-curred for the first time, in the winter after storage and once againin the following summer. The deoxynivalenol concentrations of thethree consecutive sample collections were 362, 219 and 173 mg/kgon average, and the zearalenone values were 42, 36 and 43 mg/kg.The ergot alkaloid concentrations in the three batches were asfollows: ergosine: 62, 9 and 52 mg/kg; ergotamine: 18, 11 and12 mg/kg; ergocornine: 0, 2 and 0 mg/kg; ergocryptine: 113, 0and 178 mg/kg; ergocristine: 403, 330 and 452 mg/kg.

Discussion

In the present study, alterations in piglet ear tissue in the initialstage of PENS were analysed histopathologically in an attempt todefine the time course of the disease. As this was a preliminarystudy, only piglets with ear necrosis were investigated to obtainas much information as possible about this syndrome. Focal epi-dermal necrosis was seen as the initial lesion. Such necrosis caneither be the consequence of a vascular alteration resulting in localischaemia as leading to epidermal necrosis and subsequent ulcer-ation or of an outside event such as trauma or bacterial toxins.

Based on the data, a definitive answer about the causative factorcannot be given. The selection of piglets for sample collection was

Table 3Number of specimens with combinations of selected infectious agents.

Combinations of All samples (n = 72)

n %

No infectious agent 15 20.8PRRSV 2 2.8Streptococci/staphylococci 50 69.4Streptococci/staphylococci/Sarcoptes antibodies 1 1.4Streptococci/staphylococci/PRRSV 4 5.6


made when the first macroscopic lesions were visible, and the veryearly stage of vesiculation as documented by Richardson et al.(1984) could not be observed in the present field study althoughthe histological pattern of more severe cases was comparable. Nev-ertheless, the lack of cases at the early stage of disease in the pres-ent study is due to the timing of sample collection and does notsuggest different aetiological causes.

The most frequent ear lesions found by Richardson et al. (1984)were hyperkeratosis, acanthosis and intraepidermal abscesses aswell as a hyperplastic perivascular dermatitis (Mirt, 1999).Depending on the duration of the lesions, the necrotic dermiswas replaced by granulation tissue, as in the present cases. Vascu-lar changes could also be observed in both studies. The occlusion ofthe lumen of affected vessels by thrombi has already been de-scribed by Richardson et al. (1984).

First collection (n = 36) Second collection (n = 36)

n % n %

10 27.8 5 14.02 5.6 0 0.0

21 58.8 29 81.21 2.8 0 0.01 2.8 1 2.8



Table 6Correlation of mycotoxin and ergot alkaloid concentrations with the different morphological features of microscopic lesions.

1. Focalepidermalnecrosis

2. Bacterial growth in thesuperficial cell debris

3. Infiltrationwith granulocytes

4. Infiltrationwith histiocytes

5. Lysis ofcollagen

6. Vasculitis in theacute stadium

7. Generation ofgranulation tissue

8.Hyperkeratosis

Deoxynivalenolq 0.298 0.409 0.216 0.235 0.194 0.131 0.176 0.203P 0.014 <0.001 0.073 0.047 0.105 0.324 0.140 0.090

Zearalenoneq 0.140 0.098 0.052 �0.100 �0.035 0.232 0.047 0.032P 0.255 0.414 0.672 0.403 0.770 0.077 0.696 0.794

Ergosineq �0.030 �0.190 �0.130 �0.106 �0.169 0.083 0.117 0.058P 0.807 0.109 0.285 0.375 0.158 0.530 0.326 0.634

Ergotamineq �0.079 �0.227 �0.148 �0.193 �0.248 0.721 0.016 0.035P 0.521 0.056 0.221 0.104 0.037 0.038 0.894 0.774

Ergocryptineq 0.008 0.162 �0.025 �0.259 0.013 0.381 �0.263 �0.316P 0.948 0.173 0.839 0.028 0.914 0.003 0.026 0.007

Ergocristineq 0.010 �0.042 �0.064 �0.326 �0.172 0.124 �0.075 �0.140P 0.936 0.727 0.598 0.005 0.150 0.350 0.530 0.245

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.

C. Weissenbacher-Lang et al. / The Veterinary Journal xxx (2012) xxx–xxx 5

In the present study, staphylococci and streptococci could beseen in various disease stages. Streptococci were found in higherconcentrations, as well as in a higher percentage of cases. Richard-son et al. (1984) isolated Staphylococcus hyicus from both early andadvanced lesions, whereas they only isolated streptococci fromulcerated lesions. The colonization of the lesions by bacteria isviewed as important in the breakdown of the epidermis, the exten-sion of lesions into deeper tissues and the development of vascularthrombosis, ischaemia and necrosis (Richardson et al., 1984).Staphylococci are adapted for residence on normal pig skin andcan be considered an aetiologic agent of early skin lesions (L’Ecu-yer, 1967), whereas the invasion of streptococci must be precededby damage of the skin due to infection or trauma (Wannamaker,1970). As seen in the present study, both kinds of bacteria can ap-pear at different stages of disease independent of their pathogene-sis, thus underlining a multifactorial aetiology.

Particles of plant material originating from faeces or litter stuckto the lesions emphasized the assumption of local trauma as astarting point, with secondary colonization of those wounds bybacteria underlying the development of severe necrotizing lesions.A significant association between ear scratches and an increasedrisk of mild ear necrosis was also shown by Busch et al. (2008).Mirt (1999) isolated staphylococci twice as frequently from lesionsas from healthy skin and assumed that infection with staphylo-cocci began at sites of slight trauma, but definitely ruled out anexclusively traumatic origin. Maddox et al. (1973) mainly detectedstreptococci in samples from necrotic ear margins, which pre-sented as thromboarteriitis. The lesions healed after treatmentwith ampicillin.

Since trials with antibiotic treatment of ear necrosis againststaphylococci (Hansen and Busch, 2008) resulted in no significantclinical effect, it seems unlikely that bacteria alone can cause thisclinical pattern, which further supports other infectious or non-infectious agents being involved in this apparently multifactorialdisease. Comparable microscopic lesions were also observed inassociation with a cutaneous necrotizing vasculitis in weaning pigseven when the lesions did not remain limited to the margins of theears (Thibault et al., 1998). Aside from the distribution of necroticlesions, the only difference from the present study was the pres-


ence of eosinophils, which could have resulted from an immune-mediated inflammation. The initial causative agent of this cutane-ous necrotizing vasculitis could not be determined, but the authorssuggested that these lesions probably developed spontaneouslywhen a specific combination of predisposing events, includingPRRSV infection, occurred. In the present study PRRSV was de-tected in some cases, but there were no histopathological differ-ences compared with other samples.

The association between PRRSV and the microscopic findingswas not significant, but a contribution to the development of thelesions cannot be completely ruled out, particularly in the caseswith bacterial and PRRSV co-infections. The detection of PCV2 invarious tissues of pigs with symptoms of postweaning multisys-temic wasting syndrome or porcine dermatitis and nephropathysyndrome is generally possible (Molnar et al., 2002; Zlotowskiet al., 2008), but PCV2 has not been proven to be a causative agentfor necrotizing vasculitis in the context of PENS. Pejsak et al. (2011)showed reductions in both prevalence and severity of ear necrosisin weaners after sow vaccination against PCV2, even though factorssuch as the herd immune status or co-infections were not consid-ered in this study.

All of our piglets were vaccinated either actively or passivelyagainst PCV2 and the virus could not be detected by ISH or PCR.Nevertheless, a high prevalence of ear necrosis was recorded onsome farms. So, in contrast to the findings of Papatsiros (2011), adirect association between PCV2 and the development of PENScould not be demonstrated. Because vaccination of piglets andsows against PCV2 is widely used, the relevance of this pathogenin relation to the development of ear necrosis has to be questioned.A similar conclusion could also be made for Mycoplasma suis andSarcoptes scabiei var. suis.

Mycotoxins do not necessarily and exclusively cause ear necro-sis, but because they have dermonecrotic potential (Osweiler,2006), their concentration was determined in feed. T2 toxin, HT2toxin, diacetoxyscirpenol, acetyldeoxynivalenol and nivalenolwere of no importance. Only deoxynivalenol and zearalenonecould be detected in biologically significant concentrations(Forsyth et al., 1977; Bauer et al., 1987). Combinations of differentmycotoxins may potentiate the action of each other or at least




exert an additive effect (Huff et al., 1988). The co-existence ofzearalenone with other mycotoxins such as nivalenol ordeoxynivalenol has already been described by some authors (IARC,1993) due to some compounds being produced by the sameFusarium species. On the other hand, different biological andclimate conditions are necessary for the production of the commonmycotoxins and these rarely appear in the same grain at a specifictime point. Therefore, concurrent production of mycotoxins couldbe relatively uncommon, and there is presently little evidence thatcommon mycotoxins act synergistically (Osweiler, 2006).

In the present study, higher deoxynivalenol concentrationswere correlated with microscopic alterations in the initial stageof the disease. A synergistic effect with infectious agents or an im-pact as a precursor has not been documented, but cannot be ruledout. Ergot alkaloids cause gangrenous ergotism as a result of com-bined vasoconstriction and endothelial damage leading to pro-longed ischaemia of appendages and eventually dry gangrene(Osweiler, 2006). The contractile response of veins depends notonly on the concentration of alkaloids, but also on their typesdue to their chemical diversity as well as on interactions betweendifferent alkaloid types (Klotz et al., 2008). As described by otherEuropean authors (Uhlig et al., 2007), ergocristine dominated thealkaloid spectrum of most extracts in the present study, followedby ergotamine, ergosine and ergocryptine. Ergocornine was of sec-ondary importance. Ear and tail necrosis have rarely been de-scribed in the context of alkaloid concentrations and have beenobserved after intake of a combination of ergotamine, ergocristine,and ergonovine (10 mg/kg grain) (Lopez et al., 1997). For this rea-son, their relevance as well as the required dose for the develop-ment of ear necrosis still need to be investigated.

Conclusions

Our study clearly showed that multiple infectious agents couldbe involved in the development of PENS, but also that none of theagents we investigated were the exclusive triggering factor. Thisdoes not mean that there is not one factor – indeed other infectiousagents that we did not investigate here could be of primary impor-tance in the aetiology of PENS. Exclusion of more bacterial, viraland parasitic diseases as well as non-infectious factors is neces-sary. Further investigation should include challenge trials with dif-ferent infectious agents, with a focus on the resultinghistopathology. In addition, the potential synergistic effect of ergotalkaloids needs to be considered as do other non-infectious agents,such as endotoxin. Finally, a comparison between diseased andhealthy piglets is required.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

Acknowledgments

This study was supported by the Dres. Jutta und Georg-Bruns-Stiftung. The bacteriological analysis of the feed samples was car-ried out at the Futtermittellabor Rosenau (Chamber of AgricultureLower Austria, Petzenkirchen, Austria), the mycotoxin analysis atROMER Labs Diagnostic GmbH (Tulln, Austria).

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