J. Comp. Path. 2014, Vol. 151, 238e243
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EXPERIMENTALLY INDUCED DISEASE
The Recent European Isolate (08P178) of Equine Arteritis Virus causes Inflammation but not Arteritis in Experimentally Infected Ponies S. Vairo*, V. Saey†, C. Bombardi‡, R. Ducatelle† and H. Nauwynck* * Laboratory of Virology, Department of Virology, Parasitology and Immunology, † Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium and ‡ Department of Morphophysiology, Alma Mater Studiorum, Bologna University, Ozzano Emilia, Italy
Summary In the last two decades, outbreaks of equine viral arteritis (EVA) have been reported in Europe, but little is known about these European isolates of equine arteritis virus (EAV). EAV European strain (08P178, EU-1 clade) isolated from one of these recent outbreaks is able to cause clinical signs on experimental infection. The aim of the present study was to investigate the microscopical lesions induced by this isolate after experimental infection of ponies. Animals were killed at 3, 7, 14 and 28 days post infection (dpi). At 3 dpi, lesions were essentially restricted to the respiratory tract and intestines and were characterized by mild multifocal epithelial degeneration and associated mononuclear cell infiltration. Lesions were more severe at 7 dpi and by 14 dpi, respiratory lesions were even more severe and lymphoplasmacytic infiltrates extended to other organs. At 28 dpi, lesions were still present in the viscera. In all specimens the most prominent histological change was intraepithelial, subepithelial and perivascular lymphoplasmacytic infiltration, ranging from mild and multifocal to extensive and diffuse. No signs of arterial damage such as infarcts, haemorrhages or necrosis were found. In conclusion, infection of na€ıve animals with the European 08P178 strain of EAV is associated with inflammation, but not arteritis. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: equine viral arteritis virus; experimental infection; histopathology; lymphoplasmacytic inflammation
Introduction Equine viral arteritis (EVA) is an infectious disease first defined aetiologically in 1953, when equine arteritis virus (EAV) was isolated during an abortion outbreak in Ohio (Doll et al., 1957). Since then, serological surveys indicate that EAV is widely distributed in equine populations around the world (Moraillon and Moraillon, 1978; Huntington et al., 1990). Although only one neutralization serotype of EAV has been identified so far (McCollum, 1970; Golnik et al., 1986), considerable antigenic variation among EAV field strains was demonstrated by comparative sequence analysis of open reading
*Correspondence to: H. Nauwynck (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.008
frames (ORFs) 2 to 7 (Stadejek et al., 1999; Hornyak et al., 2005). Based on ORF 5 phylogenetic analysis, EAV isolates are clustered into two distinct clades: a North American and a European cluster (Balasuriya et al., 1995; Echeverrıa et al., 2010). The latter can be further divided into two subgroups: EU-1 and EU-2 (Zhang et al., 2010). Distinct EAV isolates vary markedly in the severity of clinical signs they induce and it is generally thought that the American strains are more virulent than the European isolates (Balasuriya and MacLachlan, 2004). Although the incidence of EVA outbreaks in Europe appears to have increased over the last two decades, with reports from Switzerland (B€ urki and Gerber, 1966), Germany (Golnik et al., 1986; Eichhorn et al., 1995), Spain (Monreal et al., 1995), the UK (Wood et al., Ó 2014 Elsevier Ltd. All rights reserved.
Equine Arteritis Virus Experimental Infection
1995), Denmark (Larsen et al., 2001), Hungary (Szeredi et al., 2005), France (Hans et al., 2008) and Belgium (Van der Meulen et al., 2001; Gryspeerdt et al., 2009), until now no in-vivo experimental studies have been performed with European isolates. Recently, we studied the outcome of experimental infection with EAV European strain 08P178 (EU-1 clade) isolated from a neonatal foal that died during the Belgian field outbreak of 2008 (Vairo et al., 2012). Eight seronegative Shetland ponies were infected experimentally and followed until 28 days post infection (dpi). After replication in the respiratory tract and associated lymphoid tissues, EAV caused a strong cell-associated viraemia from 3 to 10 dpi and reached secondary target organs at 7 dpi. The infection had resolved spontaneously at 14 and 28 dpi (Vairo et al., 2012). The experimental data confirmed the primary pathogenicity of this European strain of EAV clade-1. Nevertheless, the clinical signs and gross lesions observed with this strain were mild when compared with those reported after experimental infection with North American isolates of EAV. Therefore, the aim of the present study was to investigate, by sequential necropsy examination of experimental animals, the microscopical lesions induced by this isolate, in an attempt to explain the differences in clinical signs and gross lesions.
Materials and Methods Animals
This study was carried out on samples collected in a previous experimental study using eight Shetland ponies that were 4e8 months old (Vairo et al., 2012). Briefly, prior to the start of the experiment, an acclimatization period of 2 weeks was allowed. During this period, the ponies were tested weekly for the presence of any serum antibody to EAV by means of a complement-dependent seroneutralization (SN)-test and an immunoperoxidase monolayer assay (IPMA). The Belgian EAV strain 08P178 (passage 4th in RK13 cells) used in the experiment, was confirmed to belong to the subgroup EU-1 by partially sequencing (916 nucleotides) the ORF5 (GenBank accession number JN254761; Department of Health Care and Biotechnology, KATHO, Catholic University College of South-West Flanders, Belgium). The animals were inoculated intranasally with 20 ml of phosphate buffered saline (PBS) containing 107.6 tissue culture infectious dose 50% endpoint (TCID50) of EAV strain 08P178. Two other animals were sham inoculated with 20 ml of PBS. EAV-infected animals were killed at different time points post infection: M3 and F3 at 3 dpi; M7 and F7 at 7 dpi; M14 and F14
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at 14 dpi and M28a and M28b at 28 dpi. The two control animals were killed at the end of the experiment. The experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine of Ghent University (EC 2009/008). Sampling
Immediately after death, 44 different specimens were collected from each animal for histopathological examination. From the upper respiratory tract (URT) a proximal, intermediate and distal portion of the nasal septum, the ethmoid, the nasopharynx and a cranial and caudal segment of the trachea were sampled. In addition, lymphoid tissues associated with the URT were sampled including the tonsils (tubal nasopharyngeal, soft palate, lingual and palatine tonsils) and the satellite lymph nodes (retropharyngeal and submandibular lymph nodes). From the deep respiratory tract (DRT) sampling of each lung was based on distinction of the following parts: cranial, intermediate (including the middle and accessory lobe) and caudal lobes. The cranial lobe included the first bronchus of the dorsal bronchial system. The intermediate lobe contained the first bronchus of the lateral bronchial system and the first bronchus of the ventral bronchial system. The remaining bronchi of the dorsal, lateral and ventral bronchial systems and all bronchi of the medial bronchial system constituted the caudal lobe (Nakakuki, 1993). Each lobe was additionally divided into a superficial and a deep part. Consequently, the specimens of the lungs consisted of: left cranial lobe superficial, right cranial lobe superficial, left cranial lobe deep, right cranial lobe deep; left intermediate lobe superficial, right intermediate lobe superficial, left intermediate lobe deep, right intermediate lobe deep; left distal lobe superficial, right distal lobe superficial, left distal lobe deep and right distal lobe deep (Fig. 1). Finally, the cervical, mediastinal and bronchial lymph nodes were sampled as part of the lymphoid tissues associated with the DRT. From the abdominal cavity, the ileum, caecum, colon, adrenals and inguinal lymph nodes were collected as single specimens, while liver, spleen and kidneys were divided into one superficial and one deep portion (liver superficial and deep part, capsule and pulp of the spleen, cortex and medulla of the kidneys). Testes were also sampled. All samples were fixed in phosphate buffered formalin for 24 h. Histology
After fixation, the tissues were processed routinely and embedded in paraffin wax. Sections (4 mm)
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Fig. 1. Schematic representation of the system used to collect pulmonary tissue.
were stained with haematoxylin and eosin (HE) and evaluated with a light microscope (Olympus BX61, Hamburg, Germany) at 400 magnification. Tissue sections were examined in blinded fashion for (1) infiltration of mononuclear cells (lymphocytes, plasma cells and macrophages), (2) vascular alterations and tissue damage (congestion, oedema, hyperplasia, apoptosis, necrosis, degeneration and desquamation of epithelium) and (3) organ-specific lesions.
Results The gross lesions from these animals have been described elsewhere (Vairo et al., 2012). No significant microscopical lesions were found in the organs from the sham-inoculated control animals. Microscopical changes were observed in all organs collected from the infected animals. Over the course of the experiment, the most prominent feature was intraepithelial, subepithelial, perivascular and peribronchiolar infiltration of lymphocytes and plasma cells, the latter sometimes containing Russell bodies. The infiltration ranged from mild and multifocal to extensive and diffuse. Lymphoid tissue, liver and testis were often infiltrated by haemosiderin-laden macrophages, mainly at later time points (7, 14 and 28 dpi). At 3 dpi, the major lesions found in the URT were oedema and congestion with multifocal, mild degeneration of the epithelium presenting as degenerate swollen cells with pyknotic nuclei. There was also intraepithelial lymphocytic infiltration and subepithelial perivascular infiltration of lymphocytes and plasma cells, sometimes admixed with few neutrophils (Fig. 2). The lymphoid tissue showed hyperplasia and oedema. In the DRT, there was multifocal, mild degeneration of the bronchiolar epithelium with desquamation and intra- and subepithelial lymphocytic infiltration. In addition, there was moderate
Fig. 2. Lymphocyte infiltration with multifocal degeneration of epithelial cells in the epithelium and the lamina propria of the cranial trachea of animal M3 killed at 3 dpi. HE.
peribronchiolar and perivascular lymphocytic infiltration and mild thickening of the alveolar and interlobular septa. In some alveoli, foamy macrophages were noted. The bronchus-associated lymphoid tissue (BALT) was hyperplastic with dilation of the sinuses. At 7 dpi, lesions of the URT were similar to those at 3 dpi, but congestion and oedema were more severe and extensive. In the lungs, lesions were comparable to those present at 3 dpi, but extended to the pleura and interlobular septa, which both had evidence of oedema and perivascular mononuclear cell infiltration (Fig. 3). The intestine showed diffuse, mild infiltration of plasma cells, lymphocytes and eosinophils in the lamina propria of the villi and sometimes also in the submucosa. Mild infiltration of neutrophils was observed in the hepatic sinusoids.
Fig. 3. Congestion, oedema, multifocal atelectasis and lymphocytic infiltration of the perivascular and alveolar septa in the cranial lobe of the right lung collected from F7 at 7 dpi. HE.
Equine Arteritis Virus Experimental Infection
At 14 dpi, the URT showed similar lesions to those described on days 3 and 7. In the lungs, pleural and interlobular thickening was observed, as well as hyperplastic type II pneumocytes in F14. Degeneration of the bronchiolar epithelium with intraluminal presence of desquamated cells admixed with degenerate neutrophils was observed. Foamy macrophages and neutrophils were present in the alveoli. The liver showed mild perivascular infiltration of lymphocytes and plasma cells and an increased number of neutrophils in the sinusoids. Interstitial mild lymphoplasmacytic infiltration was present in the kidneys and testes. At 28 dpi, the URT had intact epithelium with mild intra- and subepithelial lymphoplasmacytic infiltration and sometimes also neutrophils. The URT-associated lymphoid tissues showed enlarged follicles and infiltration of a few haemosiderin-laden macrophages. Lungs showed mild thickening of interalveolar septa with lymphocytes and plasma cells. Desquamation of bronchiolar epithelium with apoptotic debris and neutrophils was mild compared with these changes at 14 dpi. Focal subpleural lymphocytic infiltrates were noticed. BALT showed hyperplasia with dilation of the sinuses and, sometimes, infiltration of a few haemosiderin-laden macrophages. The intestine showed diffuse mild villus atrophy and moderate diffuse infiltration of the lamina propria by lymphocytes, plasma cells and eosinophils. Perivascular infiltration of lymphocytes and a few haemosiderin-laden macrophages was seen in the liver of one animal and a mild infiltration of neutrophils in the hepatic sinusoids of the other. The spleen showed hyperplasia, multifocal extramedullary haemopoiesis and infiltration of haemosiderinladen macrophages. Multifocal interstitial lymphoplasmacytic infiltrates were noted in the kidney and testis. In addition, atrophy of the seminiferous tubules was present in one animal (Fig. 4).
Discussion Over the past two decades, outbreaks of EVA have been reported in Europe, but little is known about the European isolates of EAV. Although extensive studies have been carried out on the pathogenesis of EVA with North American strains, microscopical lesions induced by European strains after experimental inoculation have not been reported previously. It was assumed hitherto that the two clades have similar pathogenesis (Jones et al., 1957; Del Piero, 2006). However, while the clinical manifestations induced by North American strains reflect endothelial injury and severely increased vascular permeability (Doll et al., 1957; Jones et al., 1957; Estes and Cheville, 1970; Crawford and Henson, 1972; Neu et al.,
241
Fig. 4. Testis collected at 28 dpi showing infiltration of lymphocytes, plasma cells and haemosiderin-laden macrophages. HE.
1987), we have previously demonstrated that EAV 08P178 induces mild signs, with only one animal showing transient scrotal oedema (Vairo et al., 2012). Post-mortem examination of ponies infected experimentally with EAV 08P178 strain revealed the presence of moderate amounts of exudate at the level of both thoracic and abdominal cavities, suggesting an increased permeability of the capillaries (Vairo et al., 2012). In the present study, the most prominent histological features observed were the intraepithelial, subepithelial, perivascular and peribronchiolar infiltration of lymphocytes and plasma cells, sporadic oedema and congestion. Experimental infection with the North American velogenic EAV Bucyrus strain not only results in an extensive lymphocyte and plasma cell infiltration, but also in arterial panvasculitis with oedema, thrombosis, infarcts, haemorrhages and necrosis. In contrast to the North American strains, no infarcts, extensive haemorrhages or necrosis of arterial walls was observed in the present study. The sustained presence of oedema, congestion and infiltration of lymphocytes and plasma cells induced by EAV 08P178 is similar to changes described following infection with North American strains (Doll et al., 1957; Jones et al., 1957; Neu et al., 1987). In other experimental models, inflammation and increased vascular permeability have been reported as a consequence of lymphocyte, neutrophil and macrophage infiltration of perivascular and interstitial spaces, leading to oedema (Stokes et al., 2011). Typical lesions induced by viruses with a high tropism for endothelial cells, other than EAV, consist of vasculitis, haemorrhages, thrombosis, hypoxia and secondary ischaemic degeneration (Borcher et al., 2010). Apparently, EAV 08P178 is able to alter the permeability of the blood
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242
vessels resulting in oedema, but it does not create extensive intercellular gaps with consequent severe haemorrhages as highly virulent EAV strains do. When comparing the present study with the virus titration and immunofluorescence results obtained previously (Vairo et al., 2012, 2013), there seems to be, at 3 and 7 dpi, an association between the severity of the lesions and the number of antigenpositive cells, suggesting that the histological changes may be due to virus-mediated injuries of infected cells. It was shown recently that EAV is able to activate caspase-8 and caspase-9, resulting in apoptosis of infected cells after 1 dpi (St Louis and Archambault, 2007). At later time points in the course of infection (14 and 28 dpi), virus titres dropped markedly in all tissues (except the tonsils), while lesions continued to persist. It is possible that those late histological changes were due to the inflammatory response rather than to a direct cytocidal effect of virus. Although most of the clinical features and histological lesions were typical of EVA, they were mild when compared with those obtained with North American strains. The differences may be strain dependent; however, since the course and the outcome of EAV infection vary markedly between breeds, it cannot be excluded that the use of Shetland ponies may have influenced the results of the present study. To draw firm conclusions, an experimental infection with an American virulent strain under the same experimental conditions as EAV 08P178 should be performed. In conclusion, infection of na€ıve horses with European 08P178 EAV strain is associated with inflammation, but not arteritis. The observed microscopical lesions are probably virus mediated at the early time point, but at latter stages of infection may be attributed to inflammation.
Acknowledgements The authors thank L. De Bels and L. Standaert for technical support and K. De Hert, L. Steukers and L. Szeredi for their scientific contribution, helpful suggestions and fruitful discussions. Permission to use the facilities of the department of Scienze Mediche Veterinarie, Faculty of Veterinary Medicine, Alma Mater Studiorum, Bologna University is gratefully acknowledged. S. Vairo is supported by a doctoral grant from the Special Research Fund of Ghent University (V72-0209).
Conflict of Interest Statement The authors declare that they have no conflict of interest.
References Balasuriya U, MacLachlan N (2004) The immune response to equine arteritis virus: potential lessons for other arteriviruses. Veterinary Immunology and Immunopathology, 102, 107e129. Balasuriya U, Timoney P, McCollum W, MacLachlan N (1995) Phylogenetic analysis of open reading frame 5 of field isolates of equine arteritis virus and identification of conserved and non-conserved regions in the GL envelope glycoprotein. Virology, 214, 690e697. Borcher M, Roddy P, Pablo P, Jeffs B, Alberto O et al. (2010) Factors associated with Marburg hemorrhagic fever: analysis of patient data from Uige, Angola. Journal of Infectious Disease, 12, 1909e1918. B€ urki F, Gerber H (1966) Ein virologisch gesicherter Grossansbruck von equiner Arteritis. Berliner und Munchener Tierarztliche Wochenschrift, 79, 391e395. Crawford TD, Henson JB (1972) Immunofluorescent, light-microscopic and immunologic studies of equine viral arteritis. Proceedings of the International Conference on Equine Infectious Disease, 3, 282e302. Del Piero F (2006) Equine viral arteritis: signs, lesions, pathogenesis and diagnoses. Proceedings of the Annual Meeting of the American College of Veterinary Pathologists and American Society for Veterinary Clinical Pathology: 2-6 December 2006. Edited by IVIS.org. Tuscon, Arizona. Doll E, Bryans J, McCollum W, Crowe M (1957) Isolation of a filterable agent causing arteritis of horses and abortion by mares. Its differentiation from the equine abortion (influenza) virus. Cornell Veterinarian, 47, 3e41. Echeverrıa M, Dıaz S, Metz G, Serena M, Panei C et al. (2010) Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported. Revista Argentina de Microbiologıa, 42, 11e17. Eichhorn W, Heilmann M, Kaaden O (1995) Equine viral arteritis with abortions: serological and virological evidence in Germany. Journal of Veterinary Medicine, 42, 574e576. Estes P, Cheville N (1970) The ultrastructure of vascular lesions in equine viral arteritis. American Journal of Pathology, 58, 235e253. Golnik W, Moraillon A, Golnik J (1986) Identification and antigenic comparison of equine arteritis virus isolated from an outbreak of epidemic abortion of mares. Journal of Veterinary Medicine Series B, 33, 413e417. Gryspeerdt A, Chiers K, Govaere J, Vercauteren G, Ducatelle R et al. (2009) Neonatal foal death due to infection with equine arteritis virus in Belgium. Vlaams Diergeneeskundig Tijdschrift, 78, 189e193. Hans A, Gaudaire D, Tapprest J, Guix E, MarcillaudPitel C et al. (2008) French 2007 equine viral arteritis outbreak: epidemiological and pathological features. Proceedings of the Second International Workshop on Equine Arteritis Virus, Lexington, Kentucky, p. 40. Hornyak A, Bakonyi T, Tekes G, Szeredi L, Rusvai M (2005) A novel subgroup among genotypes of equine arteritis virus: genetic comparison of 40 strains. Journal
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of Veterinary Medicine B: Infect Disease and Veterinary Public Health, 52, 112e118. Huntington P, Forman A, Ellis P (1990) The occurrence of equine arteritis virus in Australia. Australian Veterinary Journal, 67, 432e435. Jones T, Doll E, Bryans J (1957) The lesions of equine viral arteritis. Cornell Veterinarian, 47, 52e68. Larsen L, Storgaard T, Holm E (2001) Phylogenetic characterization of the G(L) sequences of equine arteritis virus isolated from semen of asymptomatic stallions and fatal cases of equine viral arteritis in Denmark. Veterinary Microbiology, 80, 339e346. McCollum W (1970) Vaccination for equine viral arteritis. Proceedings of the International Conference on Equine Infectious Disease, 2, 143e151. Monreal L, Villatoro A, Hooghuis H, Ros I, Timoney P (1995) Clinical features of the 1992 outbreak of equine viral arteritis in Spain. Equine Veterinary Journal, 27, 301e304. Moraillon A, Moraillon R (1978) Results of an epidemiological investigation on viral arteritis in France and some other European and African countries. Annales de Recherches Veterinaires, 9, 43e54. Nakakuki S (1993) The bronchial tree and lobular division of the horse lung. Journal of Veterinary Medical Science, 55, 435e438. Neu S, Timoney P, McCollum W (1987) Persistent infection of the reproductive tract in stallions experimentally infected with equine arteritis virus. Proceedings of the International Conference on Equine Infectious Disease, 5, 14. Stadejek T, Bjorklund H, Bascunana C, Ciabatti I, Scicluna M et al. (1999) Genetic diversity of equine arteritis virus. Journal of General Virology, 80, 691e699. St Louis M, Archambault D (2007) The equine arteritis virus induces apoptosis via caspase-8 and mitochondriadependent caspase-9 activation. Virology, 367, 147e155.
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Stokes K, Chi M, Sakamoto K, Newcomb D, Currier M et al. (2011) Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice. Journal of Virology, 85, 5782e5793. P Szeredi L, Horny ak A, alfi V, Moln ar T, Gl avits R et al. (2005) Study on the epidemiology of equine arteritis virus infection with different diagnostic techniques by investigating 96 cases of equine abortion in Hungary. Veterinary Microbiology, 108, 235e242. Vairo S, Favoreel H, Scagliarini A, Nauwynck H (2013) Identification of target cells of a European equine arteritis virus strain in experimentally infected ponies. Veterinary Microbiology, 167, 235e241. Vairo S, Vandekerckhove A, Steukers L, Glorieux S, Van den Broeck W et al. (2012) Clinical and virological outcome of an infection with the Belgian equine arteritis virus strain 08P178. Veterinary Microbiology, 157, 333e344. Van der Meulen K, Caij A, Nauwynck H, Pensaert M (2001) An outbreak of equine viral arteritis abortion in Belgium. Vlaams Diergeneeskundig Tijdschrift, 70, 221e222. Wood J, Chirnside E, Mumford J, Higgins A (1995) First recorded outbreak of equine viral arteritis in the United Kingdom. Veterinary Record, 136, 381e385. Zhang J, Timoney P, Shuck K, Seoul G, Go Y et al. (2010) Molecular epidemiology and genetic characterization of equine arteritis virus isolates associated with the 2006e2007 multi-state disease occurrence in the USA. Journal of General Virology, 91, 2286e2301.
July 1st, 2013 ½ Received, Accepted, April 14th, 2014
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EXPERIMENTALLY INDUCED DISEASE
The Recent European Isolate (08P178) of Equine Arteritis Virus causes Inflammation but not Arteritis in Experimentally Infected Ponies S. Vairo*, V. Saey†, C. Bombardi‡, R. Ducatelle† and H. Nauwynck* * Laboratory of Virology, Department of Virology, Parasitology and Immunology, † Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium and ‡ Department of Morphophysiology, Alma Mater Studiorum, Bologna University, Ozzano Emilia, Italy
Summary In the last two decades, outbreaks of equine viral arteritis (EVA) have been reported in Europe, but little is known about these European isolates of equine arteritis virus (EAV). EAV European strain (08P178, EU-1 clade) isolated from one of these recent outbreaks is able to cause clinical signs on experimental infection. The aim of the present study was to investigate the microscopical lesions induced by this isolate after experimental infection of ponies. Animals were killed at 3, 7, 14 and 28 days post infection (dpi). At 3 dpi, lesions were essentially restricted to the respiratory tract and intestines and were characterized by mild multifocal epithelial degeneration and associated mononuclear cell infiltration. Lesions were more severe at 7 dpi and by 14 dpi, respiratory lesions were even more severe and lymphoplasmacytic infiltrates extended to other organs. At 28 dpi, lesions were still present in the viscera. In all specimens the most prominent histological change was intraepithelial, subepithelial and perivascular lymphoplasmacytic infiltration, ranging from mild and multifocal to extensive and diffuse. No signs of arterial damage such as infarcts, haemorrhages or necrosis were found. In conclusion, infection of na€ıve animals with the European 08P178 strain of EAV is associated with inflammation, but not arteritis. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: equine viral arteritis virus; experimental infection; histopathology; lymphoplasmacytic inflammation
Introduction Equine viral arteritis (EVA) is an infectious disease first defined aetiologically in 1953, when equine arteritis virus (EAV) was isolated during an abortion outbreak in Ohio (Doll et al., 1957). Since then, serological surveys indicate that EAV is widely distributed in equine populations around the world (Moraillon and Moraillon, 1978; Huntington et al., 1990). Although only one neutralization serotype of EAV has been identified so far (McCollum, 1970; Golnik et al., 1986), considerable antigenic variation among EAV field strains was demonstrated by comparative sequence analysis of open reading
*Correspondence to: H. Nauwynck (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.008
frames (ORFs) 2 to 7 (Stadejek et al., 1999; Hornyak et al., 2005). Based on ORF 5 phylogenetic analysis, EAV isolates are clustered into two distinct clades: a North American and a European cluster (Balasuriya et al., 1995; Echeverrıa et al., 2010). The latter can be further divided into two subgroups: EU-1 and EU-2 (Zhang et al., 2010). Distinct EAV isolates vary markedly in the severity of clinical signs they induce and it is generally thought that the American strains are more virulent than the European isolates (Balasuriya and MacLachlan, 2004). Although the incidence of EVA outbreaks in Europe appears to have increased over the last two decades, with reports from Switzerland (B€ urki and Gerber, 1966), Germany (Golnik et al., 1986; Eichhorn et al., 1995), Spain (Monreal et al., 1995), the UK (Wood et al., Ó 2014 Elsevier Ltd. All rights reserved.
Equine Arteritis Virus Experimental Infection
1995), Denmark (Larsen et al., 2001), Hungary (Szeredi et al., 2005), France (Hans et al., 2008) and Belgium (Van der Meulen et al., 2001; Gryspeerdt et al., 2009), until now no in-vivo experimental studies have been performed with European isolates. Recently, we studied the outcome of experimental infection with EAV European strain 08P178 (EU-1 clade) isolated from a neonatal foal that died during the Belgian field outbreak of 2008 (Vairo et al., 2012). Eight seronegative Shetland ponies were infected experimentally and followed until 28 days post infection (dpi). After replication in the respiratory tract and associated lymphoid tissues, EAV caused a strong cell-associated viraemia from 3 to 10 dpi and reached secondary target organs at 7 dpi. The infection had resolved spontaneously at 14 and 28 dpi (Vairo et al., 2012). The experimental data confirmed the primary pathogenicity of this European strain of EAV clade-1. Nevertheless, the clinical signs and gross lesions observed with this strain were mild when compared with those reported after experimental infection with North American isolates of EAV. Therefore, the aim of the present study was to investigate, by sequential necropsy examination of experimental animals, the microscopical lesions induced by this isolate, in an attempt to explain the differences in clinical signs and gross lesions.
Materials and Methods Animals
This study was carried out on samples collected in a previous experimental study using eight Shetland ponies that were 4e8 months old (Vairo et al., 2012). Briefly, prior to the start of the experiment, an acclimatization period of 2 weeks was allowed. During this period, the ponies were tested weekly for the presence of any serum antibody to EAV by means of a complement-dependent seroneutralization (SN)-test and an immunoperoxidase monolayer assay (IPMA). The Belgian EAV strain 08P178 (passage 4th in RK13 cells) used in the experiment, was confirmed to belong to the subgroup EU-1 by partially sequencing (916 nucleotides) the ORF5 (GenBank accession number JN254761; Department of Health Care and Biotechnology, KATHO, Catholic University College of South-West Flanders, Belgium). The animals were inoculated intranasally with 20 ml of phosphate buffered saline (PBS) containing 107.6 tissue culture infectious dose 50% endpoint (TCID50) of EAV strain 08P178. Two other animals were sham inoculated with 20 ml of PBS. EAV-infected animals were killed at different time points post infection: M3 and F3 at 3 dpi; M7 and F7 at 7 dpi; M14 and F14
239
at 14 dpi and M28a and M28b at 28 dpi. The two control animals were killed at the end of the experiment. The experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine of Ghent University (EC 2009/008). Sampling
Immediately after death, 44 different specimens were collected from each animal for histopathological examination. From the upper respiratory tract (URT) a proximal, intermediate and distal portion of the nasal septum, the ethmoid, the nasopharynx and a cranial and caudal segment of the trachea were sampled. In addition, lymphoid tissues associated with the URT were sampled including the tonsils (tubal nasopharyngeal, soft palate, lingual and palatine tonsils) and the satellite lymph nodes (retropharyngeal and submandibular lymph nodes). From the deep respiratory tract (DRT) sampling of each lung was based on distinction of the following parts: cranial, intermediate (including the middle and accessory lobe) and caudal lobes. The cranial lobe included the first bronchus of the dorsal bronchial system. The intermediate lobe contained the first bronchus of the lateral bronchial system and the first bronchus of the ventral bronchial system. The remaining bronchi of the dorsal, lateral and ventral bronchial systems and all bronchi of the medial bronchial system constituted the caudal lobe (Nakakuki, 1993). Each lobe was additionally divided into a superficial and a deep part. Consequently, the specimens of the lungs consisted of: left cranial lobe superficial, right cranial lobe superficial, left cranial lobe deep, right cranial lobe deep; left intermediate lobe superficial, right intermediate lobe superficial, left intermediate lobe deep, right intermediate lobe deep; left distal lobe superficial, right distal lobe superficial, left distal lobe deep and right distal lobe deep (Fig. 1). Finally, the cervical, mediastinal and bronchial lymph nodes were sampled as part of the lymphoid tissues associated with the DRT. From the abdominal cavity, the ileum, caecum, colon, adrenals and inguinal lymph nodes were collected as single specimens, while liver, spleen and kidneys were divided into one superficial and one deep portion (liver superficial and deep part, capsule and pulp of the spleen, cortex and medulla of the kidneys). Testes were also sampled. All samples were fixed in phosphate buffered formalin for 24 h. Histology
After fixation, the tissues were processed routinely and embedded in paraffin wax. Sections (4 mm)
S. Vairo et al.
240
Fig. 1. Schematic representation of the system used to collect pulmonary tissue.
were stained with haematoxylin and eosin (HE) and evaluated with a light microscope (Olympus BX61, Hamburg, Germany) at 400 magnification. Tissue sections were examined in blinded fashion for (1) infiltration of mononuclear cells (lymphocytes, plasma cells and macrophages), (2) vascular alterations and tissue damage (congestion, oedema, hyperplasia, apoptosis, necrosis, degeneration and desquamation of epithelium) and (3) organ-specific lesions.
Results The gross lesions from these animals have been described elsewhere (Vairo et al., 2012). No significant microscopical lesions were found in the organs from the sham-inoculated control animals. Microscopical changes were observed in all organs collected from the infected animals. Over the course of the experiment, the most prominent feature was intraepithelial, subepithelial, perivascular and peribronchiolar infiltration of lymphocytes and plasma cells, the latter sometimes containing Russell bodies. The infiltration ranged from mild and multifocal to extensive and diffuse. Lymphoid tissue, liver and testis were often infiltrated by haemosiderin-laden macrophages, mainly at later time points (7, 14 and 28 dpi). At 3 dpi, the major lesions found in the URT were oedema and congestion with multifocal, mild degeneration of the epithelium presenting as degenerate swollen cells with pyknotic nuclei. There was also intraepithelial lymphocytic infiltration and subepithelial perivascular infiltration of lymphocytes and plasma cells, sometimes admixed with few neutrophils (Fig. 2). The lymphoid tissue showed hyperplasia and oedema. In the DRT, there was multifocal, mild degeneration of the bronchiolar epithelium with desquamation and intra- and subepithelial lymphocytic infiltration. In addition, there was moderate
Fig. 2. Lymphocyte infiltration with multifocal degeneration of epithelial cells in the epithelium and the lamina propria of the cranial trachea of animal M3 killed at 3 dpi. HE.
peribronchiolar and perivascular lymphocytic infiltration and mild thickening of the alveolar and interlobular septa. In some alveoli, foamy macrophages were noted. The bronchus-associated lymphoid tissue (BALT) was hyperplastic with dilation of the sinuses. At 7 dpi, lesions of the URT were similar to those at 3 dpi, but congestion and oedema were more severe and extensive. In the lungs, lesions were comparable to those present at 3 dpi, but extended to the pleura and interlobular septa, which both had evidence of oedema and perivascular mononuclear cell infiltration (Fig. 3). The intestine showed diffuse, mild infiltration of plasma cells, lymphocytes and eosinophils in the lamina propria of the villi and sometimes also in the submucosa. Mild infiltration of neutrophils was observed in the hepatic sinusoids.
Fig. 3. Congestion, oedema, multifocal atelectasis and lymphocytic infiltration of the perivascular and alveolar septa in the cranial lobe of the right lung collected from F7 at 7 dpi. HE.
Equine Arteritis Virus Experimental Infection
At 14 dpi, the URT showed similar lesions to those described on days 3 and 7. In the lungs, pleural and interlobular thickening was observed, as well as hyperplastic type II pneumocytes in F14. Degeneration of the bronchiolar epithelium with intraluminal presence of desquamated cells admixed with degenerate neutrophils was observed. Foamy macrophages and neutrophils were present in the alveoli. The liver showed mild perivascular infiltration of lymphocytes and plasma cells and an increased number of neutrophils in the sinusoids. Interstitial mild lymphoplasmacytic infiltration was present in the kidneys and testes. At 28 dpi, the URT had intact epithelium with mild intra- and subepithelial lymphoplasmacytic infiltration and sometimes also neutrophils. The URT-associated lymphoid tissues showed enlarged follicles and infiltration of a few haemosiderin-laden macrophages. Lungs showed mild thickening of interalveolar septa with lymphocytes and plasma cells. Desquamation of bronchiolar epithelium with apoptotic debris and neutrophils was mild compared with these changes at 14 dpi. Focal subpleural lymphocytic infiltrates were noticed. BALT showed hyperplasia with dilation of the sinuses and, sometimes, infiltration of a few haemosiderin-laden macrophages. The intestine showed diffuse mild villus atrophy and moderate diffuse infiltration of the lamina propria by lymphocytes, plasma cells and eosinophils. Perivascular infiltration of lymphocytes and a few haemosiderin-laden macrophages was seen in the liver of one animal and a mild infiltration of neutrophils in the hepatic sinusoids of the other. The spleen showed hyperplasia, multifocal extramedullary haemopoiesis and infiltration of haemosiderinladen macrophages. Multifocal interstitial lymphoplasmacytic infiltrates were noted in the kidney and testis. In addition, atrophy of the seminiferous tubules was present in one animal (Fig. 4).
Discussion Over the past two decades, outbreaks of EVA have been reported in Europe, but little is known about the European isolates of EAV. Although extensive studies have been carried out on the pathogenesis of EVA with North American strains, microscopical lesions induced by European strains after experimental inoculation have not been reported previously. It was assumed hitherto that the two clades have similar pathogenesis (Jones et al., 1957; Del Piero, 2006). However, while the clinical manifestations induced by North American strains reflect endothelial injury and severely increased vascular permeability (Doll et al., 1957; Jones et al., 1957; Estes and Cheville, 1970; Crawford and Henson, 1972; Neu et al.,
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Fig. 4. Testis collected at 28 dpi showing infiltration of lymphocytes, plasma cells and haemosiderin-laden macrophages. HE.
1987), we have previously demonstrated that EAV 08P178 induces mild signs, with only one animal showing transient scrotal oedema (Vairo et al., 2012). Post-mortem examination of ponies infected experimentally with EAV 08P178 strain revealed the presence of moderate amounts of exudate at the level of both thoracic and abdominal cavities, suggesting an increased permeability of the capillaries (Vairo et al., 2012). In the present study, the most prominent histological features observed were the intraepithelial, subepithelial, perivascular and peribronchiolar infiltration of lymphocytes and plasma cells, sporadic oedema and congestion. Experimental infection with the North American velogenic EAV Bucyrus strain not only results in an extensive lymphocyte and plasma cell infiltration, but also in arterial panvasculitis with oedema, thrombosis, infarcts, haemorrhages and necrosis. In contrast to the North American strains, no infarcts, extensive haemorrhages or necrosis of arterial walls was observed in the present study. The sustained presence of oedema, congestion and infiltration of lymphocytes and plasma cells induced by EAV 08P178 is similar to changes described following infection with North American strains (Doll et al., 1957; Jones et al., 1957; Neu et al., 1987). In other experimental models, inflammation and increased vascular permeability have been reported as a consequence of lymphocyte, neutrophil and macrophage infiltration of perivascular and interstitial spaces, leading to oedema (Stokes et al., 2011). Typical lesions induced by viruses with a high tropism for endothelial cells, other than EAV, consist of vasculitis, haemorrhages, thrombosis, hypoxia and secondary ischaemic degeneration (Borcher et al., 2010). Apparently, EAV 08P178 is able to alter the permeability of the blood
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vessels resulting in oedema, but it does not create extensive intercellular gaps with consequent severe haemorrhages as highly virulent EAV strains do. When comparing the present study with the virus titration and immunofluorescence results obtained previously (Vairo et al., 2012, 2013), there seems to be, at 3 and 7 dpi, an association between the severity of the lesions and the number of antigenpositive cells, suggesting that the histological changes may be due to virus-mediated injuries of infected cells. It was shown recently that EAV is able to activate caspase-8 and caspase-9, resulting in apoptosis of infected cells after 1 dpi (St Louis and Archambault, 2007). At later time points in the course of infection (14 and 28 dpi), virus titres dropped markedly in all tissues (except the tonsils), while lesions continued to persist. It is possible that those late histological changes were due to the inflammatory response rather than to a direct cytocidal effect of virus. Although most of the clinical features and histological lesions were typical of EVA, they were mild when compared with those obtained with North American strains. The differences may be strain dependent; however, since the course and the outcome of EAV infection vary markedly between breeds, it cannot be excluded that the use of Shetland ponies may have influenced the results of the present study. To draw firm conclusions, an experimental infection with an American virulent strain under the same experimental conditions as EAV 08P178 should be performed. In conclusion, infection of na€ıve horses with European 08P178 EAV strain is associated with inflammation, but not arteritis. The observed microscopical lesions are probably virus mediated at the early time point, but at latter stages of infection may be attributed to inflammation.
Acknowledgements The authors thank L. De Bels and L. Standaert for technical support and K. De Hert, L. Steukers and L. Szeredi for their scientific contribution, helpful suggestions and fruitful discussions. Permission to use the facilities of the department of Scienze Mediche Veterinarie, Faculty of Veterinary Medicine, Alma Mater Studiorum, Bologna University is gratefully acknowledged. S. Vairo is supported by a doctoral grant from the Special Research Fund of Ghent University (V72-0209).
Conflict of Interest Statement The authors declare that they have no conflict of interest.
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July 1st, 2013 ½ Received, Accepted, April 14th, 2014
