This case report describes a small outbreak of neonatal foal death in Belgium due to infection with equine arteritis virus (EAV). The outbreak started with one foal suffering from acute dyspnea four days after birth. Despite intensive treatment, this foal died within a few hours. Three weeks later, another foal was born on the same farm in a healthy condition, although placental edema was found. At the age of 10 days, the foal showed acute respiratory distress and severe dyspnea and died shortly after. Post-mortem examination of both foals revealed consolidated diaphragmatic lung lobes with compensatory emphysema. Histologic lesions consisted of a mild acute interstitial pneumonia. Microscopic examination of the allantochorion from the second foal showed a focal necrotizing vasculitis, and immunolabeling demonstrated the presence of EAV antigens in chorionic vascular endothelial cells and macrophages. Upon inoculation of rabbit kidney (RK13) cell culture with a suspension of lung tissue, a cytopathic effect was observed. The agent was identified as equine arteritis virus by means of immunostainings

Chiers, Koen

Ducatelle, Richard

Govaere, Jan

Gryspeerdt, Annick

Nauwynck, Hans

Van de Walle, Gerlinde

Vercauteren, Griet

English

Ghent University Academic Bibliography

Vlaams Diergeneeskundig Tijdschrift, 2009, 78 Case report   189INTRODUCTIONEquine viral arteritis (EVA) is an infectious diseasein horses caused by the equine arteritis virus (EAV),which is an RNA virus classified in the family of Ar-teriviridae (genus Arterivirus, order Nidovirales) (Ca-vanagh, 1997). Serological surveys have shown thatEVA infections occur among horses in North andSouth America, Europe, Australia, Africa and Asia,with considerable variation in seroprevalence amongcountries and within equine populations (Timoney andMcCollum, 1993). The two most important modes of transmission areaerogenic and venereal. In utero transmission, trans-mission via secretions and transmission via indirectcontamination have also been reported (Bryans et al.,1975; Timoney and McCollum, 1993; Holyoak et al.,2008). Within 2 days after infection, the virus spreadsrapidly within the lungs and bronchial lymph nodes,followed by dissemination throughout the body via theNeonatal foal death due to infection with equine arteritis virusin BelgiumNeonatale veulensterfte ten gevolge van een infectie met het equine arteritis virusin België1A. Gryspeerdt, 2K. Chiers, 3J. Govaere, 2G. Vercauteren, 2R. Ducatelle, 1G.R. Van de Walle,1H.J. Nauwynck1Laboratory of Virology, Parasitology and Immunology2Department of Pathology, Bacteriology and Poultry Diseases 3Department of Reproduction, Obstetrics and Herd HealthFaculty of Veterinary Medicine, Ghent University,Salisburylaan 133, B-9820, Merelbeke, BelgiumAnnick.gryspeerdt@ugent.beABSTRACTThis case report describes a small outbreak of neonatal foal death in Belgium due to infection with equinearteritis virus (EAV). The outbreak started with one foal suffering from acute dyspnea four days after birth.Despite intensive treatment, this foal died within a few hours. Three weeks later, another foal was born on thesame farm in a healthy condition, although placental edema was found. At the age of 10 days, the foal showedacute respiratory distress and severe dyspnea and died shortly after. Post-mortem examination of both foalsrevealed consolidated diaphragmatic lung lobes with compensatory emphysema. Histologic lesions consisted ofa mild acute interstitial pneumonia. Microscopic examination of the allantochorion from the second foal showeda focal necrotizing vasculitis, and immunolabeling demonstrated the presence of EAV antigens in chorionicvascular endothelial cells and macrophages. Upon inoculation of rabbit kidney (RK13) cell culture with asuspension of lung tissue, a cytopathic effect was observed. The agent was identified as equine arteritis virus bymeans of immunostainings. SAMENVATTINGIn deze casuïstiek wordt een beperkte uitbraak beschreven van neonatale sterfte in België, veroorzaakt door een in-fectie met het equine arteritis virus (EAV) bij twee veulens. De uitbraak begon met een veulen dat acute dyspneu ver-toonde 4 dagen na de geboorte. Ondanks een intensieve behandeling stierf het veulen enkele uren later. Drie weken laterwerd op hetzelfde bedrijf een tweede veulen geboren dat gezond leek. Tijdens het onderzoek van de vruchtvliezenwerd een verdikt oedemateus allantochorion vastgesteld. Op de leeftijd van 10 dagen werden acute ademhalings-symptomen en ernstige dyspneu waargenomen. Het veulen stierf voordat een behandeling kon worden ingesteld. Bijde autopsie werden bij beide veulens vast aanvoelende diafragmatische longkwabben en compensatoir emfyseem waar-genomen. De histologische letsels waren indicatief voor een acute interstitiële pneumonie. Bij het microscopisch on-derzoek van het allantochorion van het tweede veulen werd een focale necrotiserende vasculitis vastgesteld.EAV-antigenen werden met behulp van immunohistochemie aangetoond in endotheelcellen en macrofagen. Na de ino-culatie van konijnenniercellen (RK13) met longsuspensie van beide veulens werd een cytopathisch effect waargeno-men. Het agens werd met behulp van immunologische kleuringen geïdentificeerd als equine arteritis virus.190 Vlaams Diergeneeskundig Tijdschrift, 2009, 78blood circulation (McCollum et al., 1971; Henson andCrawford, 1974). A second cycle of infection and re-plication occurs in endothelial cells of internal organs,causing extensive endothelial damage (Bryans et al.,1957). Most infections are subclinical. Clinical signsduring an outbreak vary widely between individualhorses and between outbreaks (de Vries et al., 1996).In adult horses, EVA infections frequently resembleother infectious respiratory diseases, especially thosecaused by equine herpes viruses 1 and 4 and equineinfluenza virus (Timoney and McCollum, 1993). Feverand leukopenia are the most consistently observedfeatures, but others like abortion, systemic illness andpersistent infection in accessory glands of thereproductive tract of stallions have also been reported(Bryans et al., 1957; Holyoak et al., 1993; Holyoak etal., 2008). In neonates, sudden death, fever, depres-sion, edema, hemorrhagic enteritis and/or severe res-piratory distress have been described (Del Piero et al.,1997; Del Piero, 2000).In Belgium, a severe outbreak of equine viral arte-ritis abortion in an Arabian stud farm occurred in 2000(van der Meulen et al., 2001). Cases of neonatal foaldeath have not been reported in Belgium until now.CASE HISTORYThe first foal was born after a normal gestation pe-riod. Complete placenta expulsion occurred after 3hours. The foal received anti-tetanus serum (Intervet,Belgium) and prophylactic antibiotics (Cobactan®4,5%, Intervet, Belgium). The immunoglobulin con-centration in the serum of this foal was  > 8g/l 16 hoursafter birth. Two days later, the mare showed an eleva-ted temperature (39.8°C) and therefore the uterus wasflushed with saline. The next day, the foal showed se-vere dyspnea, without fever. A treatment with ipratro-piumbromide (Atrovent®, Boehringer Ingelheim NV,Belgium), ceftiofur (Excenel®, Pfizer, Belgium), oxy-gen and furosemide (Dimazon®, Intervet, Belgium)was started. No clinical improvement was seen and thefoal died a few hours later. At the same farm, a second healthy foal was bornfrom an 8-year-old Belgian Warmblood mare after 346days of pregnancy. Placenta expulsion occurred wit-hin 20 minutes. The placental weight was increased(8,950 kg) and edema was seen on the pregnant horn.No other placental abnormalities were noticed uponmacroscopic examination. Samples from the pregnanthorn, corpus and placenta at the level of the cervixwere fixed in 10% formalin for histological examina-tion. The foal received anti-tetanus serum (Intervet,Belgium) and prophylactic antibiotics (Cobactan®4.5%, Intervet, Belgium) were given for 5 days. Theconcentration of immunoglobulins, which was chec-ked 18 hours after birth, was higher than 8 g/l. Tendays later, the foal was found suffering from tachypneaand tachycardia and died before further clinical exa-mination and veterinary treatment were initiated.LABORATORY EXAMINATIONSGross examinationPost-mortem examination of both foals revealedenlarged, non-collapsed, congested and consolidatedlungs with superficial rib imprints. Alveolar, interstitialand focal bullous emphysema was present in the cra-niodorsal lobes of the second foal. Samples from thelungs were fixed in 10% formalin for histology andimmunohistochemistry. In addition, samples from thelungs and spleen were collected for bacteriologicalexamination and samples from the lungs, spleen andliver were collected for virological examination.Histological examination and immunohistochemistryFixed samples were embedded in paraffin, sectio-ned at 4 µm and stained with hematoxylin and eosin(HE) according to standard techniques.Histologically, a focal moderately necrotizing vas-culitis was found in allantochorionic villi (Figure 1a).Adjacent to this lesion, a moderate infiltrate of neu-trophils and lymphocytes was present. Multifocal co-Figure 1. a: Necrotizing vasculitis of arteriole (arrow) inthe chorion of the placenta. HE, bar 50 µm. b: Acute al-veolar damage in lungs: hyaline membranes (largearrow) in bronchioli, hyperplastic type 2 pneumocytes(arrowhead) and activated pulmonary alveolar macro-phages (small arrow). Bar 250 µm. Vlaams Diergeneeskundig Tijdschrift, 2009, 78 191agulation necrosis of placental trophoblasts was ob-served upon microscopic examination. In the lungs ofboth foals, multifocal necrosis of alveolar and bron-chiolar epithelial cells was present. Alveoles wereoften lined by hyaline membranes (Figure 1b). Over-all, the number of alveolar macrophages was increa-sed. Type II pneumocytes were hypertrophied andhyperplastic (Figure 1b). Occasionally, necrosis of thetunica media of medium-sized arteries was observed.Paraffin sections from lungs and placenta werestained by immunohistochemistry using a mixture ofthree mouse monoclonal antibodies (mAbs): twomAbs were directed against the Gl protein and onemAb was directed against the M protein of EAV (vander Meulen et al., 2001). Staining was performedusing Envision-mouse (Dako, Denmark). In addition,samples of liver and lungs were immunostained withbiotinylated anti-EHV1 polyclonal antibodies (vander Meulen et al., 2003), followed by Envision+sys-tem-HRP (DAB) (Dako, Denmark) for exclusion ofequine herpesvirus type 1 (EHV1) infection. Immu-nolabelling for EAV antigens was positive in thelungs of both foals and the placenta of the secondfoal. Positive staining in allantochorion was restric-ted to patchy areas of vascular endothelium and ma-crophages in the lamina propria (Figure 2a). In thelungs, multifocal, ad random distri buted areas of EAVpositive cells were mainly localized in the endothe-lium of alveolar capillaries and occasionally in mo-derately-sized arteries, as well as in the alveolarmacrophages (Figure 2b). EHV1 antigens were notdemonstrated.Bacteriological examinationStandard bacteriological and mycological exami-nations were performed on the lungs and spleen ofboth foals. Pathogenic bacteria were not isolated fromany of the samples.Virological examinationSuspensions (20%) of the lungs, spleen and liverwere incubated for 1 hour with monolayers of RK 13cells at 37°C in an atmosphere containing 5% CO2. Af-terwards, the inoculum was replaced by medium andFigure 3. a: Cytopathic effect of EVA in RK13 cells at 7days post inoculation. Rounding and vacuolization of thecells were observed. b: EVA-positive RK13 cells as re-vealed by immunofluorescence.Figure 2. a: Positive immunolabeling for EVA in endo-thelial cells (arrow) and macrophages (arrowhead) inchorion of the placenta. Bar 50 µm. b: Positive immu-nolabeling for EVA in endothelial cells of alveolar capil-laries (arrow) and pulmonary alveolar macrophages(arrowhead) in lungs. Bar 50 µm.192 Vlaams Diergeneeskundig Tijdschrift, 2009, 78the cells were further incubated. Rounding and vacuo-lization of the RK13 cells were observed after 4 daysof incubation, and after 7 days most of the cells weredetached from their support (Figure 3a). No suchchanges were observed in the negative control cells.An indirect immunofluorescence staining was per-formed on virus-infected RK13 cells and lung tissueusing the three anti-EAV mouse mAbs, as describedabove. In a second step, tissues and cells were incuba-ted with a goat anti-mouse antibody labeled with FITC(Molecular Probes, Eugene, OR, USA). EAV-positiveantigens were found in the cytoplasm of virus-infectedRK13 cells (Figure 3b) and in the cytoplasm of lungcells.DISCUSSIONAlthough equine viral arteritis (EVA) has a world-wide distribution, few clinical cases have been repor-ted in Belgium. In 2000, a severe outbreak of abortionoccurred in a Belgian stud (van der Meulen et al.,2001). The present study describes the first neonatalfoal death caused by EAV in Belgium.How the outbreak started remains elusive, as bothfoals appeared normal at birth. In the first case, themare showed fever two days post-partum. The mostcommon cause for fever after partus is an endometri-tis. However, in this case the placenta was completelyexpulsed and no macroscopic remains could be foundduring uteral lavage with saline, although the reliabi-lity of macropscopic evaluation can be questioned. Nofurther examination of the placenta was performed. Inthe second case, the placenta was edematous and clo-ser examination revealed a focal moderately necroti-zing vasculitis in the allantochorionic villi. Suchlesions suggest an infectious cause, and an in utero in-fection of the foal (Del Piero et al., 1997). The hypo-thesis of an infectious etiology was confirmed byimmunohistochemical staining, where EAV-positivemacrophages and endothelial cells were demonstratedin the allantochorion. Transplacental infections withEAV during late gestation have been reported, but arenot very common (Vaala et al., 1992; Timoney andMcCollum, 1993). Neonatal deaths associated with na-turally acquired EVA have also been reported (Doll etal., 1957; Golnick et al., 1981), along with a sporadic,isolated case in a foal (Vaala et al., 1992). Hence, todate, only a few reports of EVA outbreaks are availa-ble, primarily affecting neonates (Del Piero et al.,1997; Del Piero, 2000). The histological findings inthe lungs and allantochorion are in agreement withthose reports.Looking in retrospect, the outbreak described inthis study probably started with an EAV infection inthe first mare, which was introduced without quaran-tine to the farm to foal. The virus was most likely thentransmitted by the respiratory route to the foal. The se-cond mare probably got infected around the same pe-riod, either via the respiratory route or via indirectcontamination, and it then passed the virus on, in utero,to its unborn foal. There were no records of otherhorses showing fever or other symptoms in the samestable at that time. Since the infections were mostlysubclinical, other adult horses on the same farm mighthave got an EVA infection without being noticed.More than 40 foals were born healthy on the farm inthe months following this outbreak.The outbreak of EVA in neonates described in thepresent report serves as a warning about the necessityto include EVA in the differential diagnosis when foalsare presented with fever, depression, edema, respira-tory distress and/or sudden death. Since the prevalenceof EVA in Belgium seems to be rising, more clinicaloutbreaks can be expected in the future. Although vac-cines are available abroad, they are not yet registeredfor use in Belgium. The most important measures inthe event of an outbreak in Belgium are managementstrategies such as quarantining new animals and eli-minating EVA-positive breeding stallions from bree-ding populations (Holyoak et al., 2008).ACKNOWLEDGEMENTSThe authors wish to thank Chris Bracke, SarahLoomans, Delphine Ameye and Christian Puttevils fortheir excellent technical assistance, as well as Profes-sor Dr. P. Rottier of the University of Utrecht for pro-viding the three mouse monoclonal antibodies.REFERENCESBryans J.T., Crowe M.E., Doll E.R., McCollum W.H.(1957). Isolation of a filterable agent causing arteritis ofhorses and abortion by mares; its differentiation from theequine abortion (influenza) virus. The Cornell Veterina-rian 47, 3-41.Cavanagh D. (1997). Nidovirales: a new order comprimi-sing Coronaviridae and Arteriviridae. Archives of Viro-logy 142, 629-633.Del Piero F., Pamela A., Wilkins, Lopez J.W., Glaser A.L.,Dubovi E.J., Schlafer D.H., Lein D.H. (1997). 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Relationship between onset of puberty and es-tablishment of persistent infection with equine arteritisvirus in the experimentally infected colt. Journal of Com-parative Pathology 109, 29-46.Vlaams Diergeneeskundig Tijdschrift, 2009, 78 193Holyoak G.R., Balasuriya U.B.R., Broaddus C.C., TimoneyP.J. (2008). Equine viral arteritis: Current status and pre-vention. Theriogenology 70, 403-414.McCollum W.H., Prickett M.E., Bryans J.T. (1971). Tem-poral distribution of equine arteritis virus in respiratoymucosa, tissues and body fluids of horses infected by in-halation. Research in Veterinary Science 12, 459-464.Timony P.J., McCollum W.H. (1993). Equine viral arteritis.Veterinary Clinics of North America – Equine Practice 9,295-309.Vaala W.E., Hamir A.N., Dubovi E.J., Timoney P.J., RuizB. (1992). Fatal, congenitally acquired infection withequine arteritis virus in a neonatal Thoroughbred. EquineVeterinary Journal 24, 155-158.van der Meulen K., Caij A., Nauwynck H., Pensaert M.(2001). An outbreak of equine viral arteritis abortion inBelgium. Vlaams Diergeneeskundig Tijdschrift 70, 221-222.van der Meulen K.M, Nauwynck H.J., Pensaert M.B.(2003). Absence of viral antigens on the surface of equineherpesvirus 1-infected blood mononuclear cells: strategyto avoid complement-mediated lysis. Journal of GeneralVirology 84, 93-99.

Neonatal foal death due to infection with equine arteritis virus in Belgium

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Neonatal foal death due to infection with equine arteritis virus in Belgium

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