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Severe outbreak of disease in the southern chamois (Rupicapra pyrenaica) associated with border disease virus infection
Veterinary Microbiology 120 (2007) 33–41 www.elsevier.com/locate/vetmic
Severe outbreak of disease in the southern chamois (Rupicapra pyrenaica) associated with border disease virus infection Ignasi Marco a,*, Jorge Ramon Lopez-Olvera a, Rosa Rosell b,c, Enric Vidal b, Ana Hurtado d, Ramon Juste d, Marti Pumarola b, Santiago Lavin a a
Servei d’Ecopatologia de Fauna Salvatge, Facultat de Veterinaria, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Spain b Centre de Recerca en Sanitat Animal (CRESA), Universitat Auto`noma de Barcelona, 08193 Bellaterra, Spain c Departament d’Agricultura, Ramaderia i Pesca, Generalitat de Catalunya, 08007 Barcelona, Spain d NEIKER, Instituto Vasco de Investigacio´n y Desarrollo Agrario, Department of Animal Health, Berreaga 1, 48160 Derio, Bizkaia, Spain Received 31 July 2006; accepted 4 October 2006
Abstract An outbreak of a previously unreported disease affecting southern chamois (Rupicapra pyrenaica) in the central Pyrenees (NE Spain) was recorded in 2001 and 2002. There was a marked temporal distribution, most animals being found between February and June. After the outbreak, the population was found to have decreased by about 42%, most probably due to the disease. We examined 20 affected chamois. Clinical manifestations included depression, weakness and movement difficulties in all cases. Three chamois presented abnormal behaviour, with absence of flight reaction, and 16 showed different degrees of alopecia with skin hyperpigmentation. At necropsy cachexia was observed in all animals, four chamois had abscesses in different parts of the body, four had pneumonia, one had an extensive subcutaneous infection on the head and neck and one had severe orchitis. Microscopic lesions were found in the brain, mainly edema, gliosis, espongiosis, cariorrexis and neuronal multifocal necrosis. A perivascular mononuclear inflammatory infiltrate was present in three of them. Skin lesions included marked follicular atrophy, mild to moderate epidermal hyperplasia with orthokeratotic hyperkeratosis and follicular hyperkeratosis, and hypermelanosis. In 13 chamois there were haemosiderin deposits in the spleen, and in three individuals kidney ‘‘cloissone’’ was observed. Intraeritrocitic parasites were detected either by direct observation or PCR in 8 of 17 chamois. A pestivirus was isolated and detected by RT-PCR from 12 of 13 affected chamois and antigenic characterized as border disease virus by monoclonal antibodies. This is the first time a border disease virus has been associated with an outbreak of a highmortality disease in a wild species. # 2006 Elsevier B.V. All rights reserved. Keywords: Chamois; Rupicapra pyrenaica; Border disease virus; Mortality
* Corresponding author. Tel.: +34 93 5813445; fax: +34 93 5812006. E-mail address: [email protected] (I. Marco). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.10.007
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1. Introduction The pestivirus genus, classified within the Flaviridae family, comprises viruses that are major pathogens for cattle, sheep and pigs, with a significant economic impact worldwide. Four viral species are currently recognized: Bovine viral diarrhoea virus 1 and 2 (BVDV-1 and BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). In addition, there is one tentative species represented by a single pestivirus isolated from a giraffe (Thiel et al., 2005). However, there is considerable genetic diversity, especially within the BVDV-1 genotype (Vilcek et al., 2005). Pestiviruses are able to cross-species barriers to infect a wide range of hosts within the Artiodactyla (Becher et al., 1997). It has been reported that BVDV and BDV can infect numerous domestic and wild ungulate species, and only CSFV appears to be restricted to a single host species (Becher et al., 2003). Border disease (BD) is a congenital viral disease affecting sheep and goats. Clinical signs include abortions, barren ewes, stillbirths and small weak lambs, which can show tremors, abnormal body conformation and abnormal birthcoat. The disease in goats is rare and is characterized by abortions (Nettleton et al., 1998). In wildlife, serologic surveys have demonstrated prior infection with pestiviruses in more than 40 species. In a recent study, a high seroprevalence was reported in a population of healthy chamois from the Italian Alps, but no pestiviruses were isolated (Olde Riekerink et al., 2005). There are only a few confirmed cases of isolation of a pestivirus from disease in wild ruminants, but there is no evidence that they have significant population impact in free-ranging animals (Van Campen et al., 2001). The southern chamois (Rupicapra pyrenaica) is a small ruminant belonging to the Bovidae family and Caprinae subfamily. It is a widely distributed ungulate in the Pyrenees and its range and densities have increased substantially over the last few decades. In the Pyrenees, the population has increased from 35,000 individuals in 1997 to around 50,000 in 2001 (Weber, 2004). Its abundance makes it one of the most popular game species in the area. Throughout 2001 and 2002 an unusual mortality rate of chamois was observed in the central Pyrenees and we reported the molecular identification of a new
pestivirus associated with this outbreak of disease (Hurtado et al., 2004). In the current study we describe the epidemiology, clinical manifestations, pathology and virological analysis of this outbreak of disease associated with this BDV.
2. Materials and methods 2.1. Study area The study area in the central Pyrenees is the Alt Pallars-Aran National Hunting Reserve (428330 N, 18190 E), covering an area of approximately 100,000 ha in the Autonomous Community of Catalonia (NE Spain) that borders with France and Andorra (Fig. 1). The Reserve provides an ideal habitat for chamois. The lower part of the main valleys lies at 800–900 m above sea level, and the highest peak is the Pica d’Estats (3143 m). There is an extensive alpine zone above the timberline at about 2200–2300 m, characterized by a mixture of steep, rocky areas and alpine pastures. Thick forests – mainly Pinus sylvestris and Pinus uncinata – provide shade and cover. Chamois are found at all altitudes, but the areas where diseased chamois were found ranged from 800 to 1900 m. In the Reserve, the chamois population is regulated by hunting during the autumn. In addition to chamois, this area is inhabited by abundant wild boar (Sus scrofa), roe deer (Capreolus capreolus), less abundant red deer (Cervus elaphus) as well as
Fig. 1. Study area in NE Spain. The Alt Pallars-Aran National Hunting Reserve is shown in grey. Main rivers are shown. Black dots and triangles indicate affected chamois found in 2001 and 2002, respectively. Grey dots and triangles indicate carcasses found in 2001 and 2002, respectively.
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introduced mouflon (Ovis ammon) and fallow deer (Dama dama). During the summer, domestic sheep, goats, cattle and horses graze extensively on the alpine pastures. Some goats, cattle and horses remain in the area all year round.
was homogenised, protease K digested, denatured and loaded in a SDS-polyacrylamide gel. Electrophoretically separated proteins were then transferred to a PDVF membrane and immunolabelled with a monoclonal antibody against prion protein (6H4, 1:5000).
2.2. Collection of data
2.5. Bacteriology
In the course of the outbreak, chamois suffering from clinical disease or found recently dead were collected by the Reserve gamekeepers. We examined 20 chamois, 4 young males between the age of 1 and 3 years, 16 adults, 12 males and 4 females more than 4 years old. Most of the chamois were hand-captured (15), while three were shot due to their poor body condition and two were found dead. Eight chamois were studied in 2001 and 11 in 2002. In 2003, no abnormal mortality was observed in the whole area, but one adult female with clinical signs consistent with the disease was found and is included in this study. The affected area was defined on the basis of the places where clinical cases and carcasses were found. Mortality was evaluated indirectly, through the census of the chamois population that are conducted twiceyearly (spring and autumn) in the Reserve.
Samples of skin scrapings from the skin lesions were obtained from the 16 chamois with skin lesions for routine microbial culturing. Routine bacteriological methods were also performed in the nine chamois with different infectious processes by the Microbiology Service, Department of Anatomy and Animal Health, Veterinary Faculty, Autonomous University of Barcelona.
2.3. Pathological examination Necropsies and tissue sampling were performed according to a standard protocol. Tissue samples were fixed in 10% phosphate buffered formalin. Fourmillimetre-thick tissue blocks were cut and dehydrated through increasing alcohols and xylene. Then the tissue was embedded in paraffin and 4 mm-thick sections were obtained, mounted on glass slides and stained with haematoxylin and eosin for histological evaluation. The tissues examined included skin, mediastinic and mesenteric lymph nodes, lung, gastrointestinal tract, spleen, heart, liver, kidney, adrenal gland, skeletal muscle from the femoral region and the brain.
2.6. Virology Virus isolation was performed in 13 affected chamois according to the OIE Manual of Diagnostic (2004). Briefly, the homogenates of spleen and kidney were inoculated on confluent monolayer of permanent Sheep line pestivirus-free SFT-R cells obtained from the Federal Institute for Virus Diseases of Animals (Island of Riems, Germany). The presence of virus was detected by the inmuno-peroxidase monolayer assay (IPMA) using a polyclonal home-made pestivirus specific serum. The border disease specificity was performed by the IPMA test with monoclonal antibodies WS363 (BDV specific, obtained from Central Veterinary Laboratory, Weybridge, UK), C-16 (pestivirus specific), CA-3 and CT-6 (BVDV-1 specific), obtained from the Reference Laboratory of Classical Swine Fever (Hannover, Germany). Amplification of viral RNA was performed in the same 13 affected chamois, using previously described panpestivirus primers (Pesti 3 and Pesti D) (Hofmann et al., 1994; Katz et al., 1993).
2.4. Prion protein detection
3. Results
Western blot analysis was performed following the kit supplier’s protocol (Prionics Check-Western Blotting), to rule out the presence of resistant prion protein deposition. Briefly, a sample of approximately 0.5 g form the obex region, at the caudal brain stem,
3.1. Geographical distribution, host and seasonal variables, mortality The area affected by the outbreak covered approximately 25,000 ha and included two main
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valleys (Vallferrera and Vallcardos), and to a lesser extent a third valley (Vall d’Aneu) (Fig. 1). The only affected chamois found in 2003 came from another valley (Val d’Aran), which is not part of the Reserve, and is 35 km west of the nearest border of the main outbreak area under consideration. Only chamois was found to be affected by the outbreak of disease. No abnormal mortality or disease was observed in any of the other wild and domestic ruminants that share the habitat with chamois, before, during or after this outbreak of disease. The first case in 2001 was reported on 10th February, and the last on 14th August. Between August 2001 and February 2002, no spread of the disease was observed and no other clinical cases were found or observed. However, during this period 19 chamois carcasses were found, but could not be analysed due to advanced decomposition. In 2002, the first case was reported on 26th February and the last 6th May. A total of 56 carcasses were found in the area, but advanced decomposition again prevented any diagnosis of the cause of death. In 2003, the only affected chamois was found on 28th February. The epizootic spread across an area inhabited by some 1800 chamois. Chamois mortality could not be assessed accurately because most of the animals that died would not have been recorded due to the size and remoteness of the area affected. However, the twiceyearly census (June and November) provided an indirect measure of mortality by determining population decrease. In November 2000, just before the episode of mortality, the chamois population in the Reserve totalled 3880; in November 2002, after the episode, the chamois population for the whole Reserve dropped by 31% to 2678. If we consider only the two main affected valleys, the census varied from 1883 chamois (1052 and 831 chamois in the Vallferrera and Vallcardos valleys, respectively) to 1086 chamois (636 and 450 in the Vallferrera and Vallcardos valleys, respectively) in 2002. Thus, the estimated cumulative decrease in this area would have been 42% (40% and 46%, respectively).
difficulty in moving. Only three chamois presented abnormal behaviour, with absence of flight reaction, loss of fear of humans and tameness. A total of 16 chamois showed skin lesions consisting of different degrees of alopecia and skin hyperpigmentation. The alopecia ranged from small areas on the head (lips, base of ears, periorbital) in four chamois, asymmetric patches on the head, neck and trunk in eight chamois, to almost complete alopecia in the other four chamois, which had winter coat persistence on the ears, distal part of limbs and tail (Fig. 2A and B). In the four chamois with an apparently normal coat, the hair was easily pulled out by just holding it. There was intense tick infestation in 13 chamois, the species identified being Ripicephalus bursa, Dermacentor marginatus, Haemaphysalis punctata and Haemaphysalis sulcata.
3.2. Clinical signs
Fig. 2. Southern chamois with clinical signs of infection associated with a border disease virus: (A) hand-captured chamois, with alopecia, skin hyperpigmentation, cachexia and head tick infestation; (B) chamois with extensive alopecia and marked cachexia; no skin hyperpigmentation is observed since the animal was hospitalised indoors for 2 months.
Of the 20 chamois studied, 2 were found recently dead and 18 alive. The latter chamois showed neurological signs such as depression, weakness and
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3.3. Necropsy and histopathology At necropsy, all animals were emaciated. Bodyweight ranged from 15 to 22 kg in adult males and from 13 to 17 kg in adult females, when normal bodyweight is 25–40 kg for males and 20–32 for females (Weber, 2004). Different infections were observed in nine chamois. Severe pneumonia was observed in four chamois, three adult males and one adult female. The adult female also had three abscesses in the left scapular region. A single abscess was present in three chamois, two adult male and one young male, in the submandibular, scapular and axilar regions, respectively. One adult male had an extensive subcutaneous infection on the head and neck. Another adult male had severe bilateral orquitis, with caseous material, and multiple necrotic foci in the spleen. The most consistent microscopic changes were found in the skin and the brain. Skin lesions consisted in severe alopecia with follicular atrophy and telogenisation of the remaining hair follicles. Images of flame follicles were observed, i.e. follicles with excessive trichilemmal keratinisation. The epidermis showed hyperplasia and melanosis with evident orthokeratotic hyperkeratosis. A mild mononuclear interstitial inflammatory infiltrate was present in the dermis. The brain consistently presented with oedema evidenced by perivascular spongiosis and the presence of clear eosinophyllic amorphous material within Virchow–Robin’s spaces being actively phagocyted by glial cells (Fig. 3A). Diffuse moderate spongiosis was present, being more evident in the white matter along with moderate to evident proliferation and astrocyte hypertrophy. Occasional glial nodules were detected. Neuronal degeneration and death occurred throughout the brain, but some areas, such as the hippocampus and the Purkinje cell layer in the cerebellar cortex, were affected more severely. At the neocortex the deeper layers were those most affected. Only in three cases was a discreet nonpurulent inflammatory infiltrate observed surrounding vessels at the leptomeninges and intraparenchymatously, occasionally percolating into the neuropil (Fig. 3B). In 8 out of the 20 cases a meningiomatous proliferation was observed in the pineal gland, another healthy chamois examined later also showed a similar lesion. Dystrophic calcifications and fibrosis were other features observed in the pineal glands.
Fig. 3. (A) Mesencephalon. Eosynophillic material is observed in Virchow–Robin’s spaces (asterisk), indicative of brain oedema. Note presence of neuropil spongiosis and gliosis. (B) Frontal cortex. Nonpurulent perivascular cuff.
Other lesions observed in the majority of cases were verminous pneumonia in the lungs with different intensities; also the presence of protozoal intrafibrous cysts (Sarcocystis spp.) was frequently observed in both skeletal and cardiac striated muscle fibres. Abundant accumulations of hematosiderous pigment were often seen in the spleen macrophages and, in three animals, crescent-shaped deposits of a brownish pigment were seen in the kidney’s Bowmann’s capsule. In addition, on blood smears we observed forms consistent with piroplasms in six chamois. 3.4. Prion protein detection and bacteriology No deposition of resistant prion protein was detected in any of the animals studied. No microorganisms were isolated from skin specimens. Several
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bacteria were isolated from the different organ specimens. Staphylococcus hyicus, Escherichia coli, Klebsiella pneumoniae were isolated from the chamois with pneumonia. Arcanobacterium pyogenes was isolated from the chamois with the subcutaneous infection and from the abscess of another chamois. Staphylococcus hyicus and positive Staphylococcus aureus coagulase were found in the abscesses of the other chamois. 3.5. Virology A BDV was isolated from the spleen and kidney in 12 of the 13 chamois studied for this purpose. All 12 isolates reacted positively with C-16 and WS363 monoclonal antibodies and were negative with CA-3 and CT-6 monoclonal antibodies. RT-PCR results were positive in the same culture-positive animals.
4. Discussion Ruminant pestiviruses are among the most widespread and successful animal viruses in the world (Nettleton and Entrican, 1995). However, this is the first time that a pestivirus has been associated with an outbreak of disease having a significant impact on a wild ruminant species population, since only sporadic cases of disease have been confirmed (Van Campen et al., 2001). The outbreak was analysed and animals with clinical signs of the disease, either captured or recently found dead, were considered. It is difficult to determine the origin of the disease. Since 2000, a decrease in the chamois population was recorded in the French side of the Pyrenees, just over the Spanish border, where the outbreak reported here took place. However, there is no epidemiological, clinical or pathological information of affected chamois. The first confirmed cases of pestivirus-associated infection in France and Andorra were in 2002, later than the first outbreak reported on the Spanish side (Arnal et al., 2004; Frolich et al., 2005). The characteristic clinical signs and gross lesions of the observed condition provided a provisional diagnosis when an affected animal was observed. The Reserve gamekeepers were able to recognize the condition and therefore provided consistent data that
allowed us to define the geographical distribution of the outbreak. Apparently, no direction of spread of the disease was observed throughout the area between the 2 years. The outbreak remained relatively localised in the initially affected area, although the chamois population is continuous and abundant in the whole mountain chain. The Aiguestortes i Estany de Sant Maurici National Park is only 7 km in a straight line from the affected area and has an abundant highdensity chamois population, but not a single affected chamois was detected. An important river running between the National Park and the affected area could act as a barrier, preventing the spread of the disease. There is also a local road, but it may not represent a barrier for chamois. Factors such as climatology, natural movement of chamois and possibly other unknown factors, may have influenced the geographical distribution. The single case diagnosed 35 km west of the main outbreak area in 2003, could be related to some of these factors. This epizootic occurred in a healthy host population enjoying good physiological condition. Since 1990, we have been monitoring this and other wild ungulate species in the area and only a few outbreaks of well-known chamois diseases, such as infectious keratoconjuntivitis, have been recorded. It seems that the disease is rather host-specific, since no other species of domestic and wild animals have been found to be affected. However, additional studies would be needed, to confirm the absence of the disease, especially in the domestic ruminant species that share the habitat with the chamois. The seasonal pattern of the disease was similar in 2001 and 2002. Most of the affected chamois studied were males and were found between late winter (February) and late spring (June). Only one adult female was found in August. A similar pattern of disease has been described in chamois with sarcoptic mange (Rossi et al., 1995). These authors found the highest number of affected chamois between February and May, and a higher prevalence of the disease in males. Factors influencing this seasonal pattern are mite biology, metabolic state of the host, food availability and behavioural characteristics of the chamois. Of these factors, the last three could have also influenced the development of the outbreak of disease reported here. The rut season for chamois is November and it represents high energy output,
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especially in males, that continually chase females and other males, thus, considerably decreasing food intake and reducing body weight. After November, in the winter season, available food resources for the animals to recover are low, leading to a worsening body condition by the end of winter and beginning of spring. As much as half of the male bodyweight can be lost during this period (Perez-Barberia et al., 1998; Weber, 2004). The lower prevalence of the disease in kids and young animals can be explained by the difficulty of observation and the quick disappearance of the carcasses. It is well known that kids and young corpses are more difficult to find since they are smaller and more quickly disposed of by scavengers—mainly the red fox in our study area (Rossi et al., 1995). Rates of morbidity and mortality could not be investigated in detail owing to the large area covered by the outbreak and the difficulty in surveying the affected chamois in a remote alpine mountain area. The chamois carcasses were found mainly at the bottom of the valleys, near trails, so most of the animals that died may not have been found or reported. In addition, all 20 chamois reported in this study were found or captured near the villages and main roads, thus also in the lower reaches of the mountains. The post-epidemic census drop can be used as an indirect estimation of the cumulative proportion of deaths. No other important outbreak of disease, deaths or mortality of chamois has been observed before or during the outbreak. Thus, we can assume that most of the variation between the pre- and post-epidemic census provides an estimation of the mortality associated with the disease. A 42% decrease in the affected area is unusual and extraordinarily high. The only disease known to cause such high – and even higher – mortality in chamois is sarcoptic mange (Rossi et al., 1995). The clinical picture of the affected chamois was not similar to any of the diseases known in this species and no previous reports have described a similar condition. It included similar signs in all chamois, except for some variation in coat and skin lesions, depending on the time of the year. The four chamois with an apparently normal coat were those found during late winter (February and early March) and those with the most extensive alopecia and skin hyperpigmentation were found in late spring.
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Chronic wasting was a common feature in all affected chamois. It has been described as one of the commonest causes of death in experimental sheep infected with border disease virus (Nettleton et al., 1992). Also, this condition has been described as one of the levels of pestivirus persistence in the host animal in nature (Evermann and Barrington, 2005). Symptoms of reduced flight behaviour and loss of natural shyness observed in the three chamois that were easily hand-captured, have also been reported in wild boar with classical swine fever (Hofmann et al., 1999). In sheep, border disease is characterized by embryonic or foetal death, abortion and the birth of dead or weak lambs, with poor growth (Loken, 2000). However, it is not known whether this condition also obtains in the chamois, since abortions and postnatal diseases are very difficult to study in free-ranging wildlife. In sheep, PI animals reach a maximum age of 5 years. Thus, the danger of creating lines of PI sheep, as suggested in cattle, would appear to be remote (Nettleton et al., 1992). In the chamois under study, an adult female kept in captivity for more than 1 month, was demonstrated to be a PI animal; 12 other chamois between 1 and 12 years of age were seronegative and antigen positive at one sampling time (Hurtado et al., 2004). Thus, it is very likely that lines of PI chamois enable the maintenance of pestivirus transmission in the chamois population. Ruminant pestivirus pathogenesis is uniquely complex, as indicated by a wide variety of clinical signs and the variable results following intrauterine infections (Nettleton and Entrican, 1995). In the chamois under study, the main lesions were observed in the brain and skin. Brain lesions suggested a viral (i.e. nonpurulent) encephalitis only in a minority of cases, otherwise a discyclia causing brain oedema was the main feature. Disruption of blood vessel patency would presumably have caused the glial reaction and the inflammation as well as the oedema-induced hypoxia giving rise to the neuronal death described. The irregular distribution of neuronal damage in the brain might be due to differential susceptibility to hypoxia. Skin lesions could be explained by the replication of viruses in hair follicle cells. However, hair abnormalities were not similar to those described for sheep (Nettleton, 2000). Haemorrhagic disease, as
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seen in other pestivirosis (Van Campen et al., 2001) has not been detected. Border disease strongly compromises cellmediated immunity and causes immune suppression in PI animals, making them highly susceptible to intercurrent infectious and parasitic diseases (Loken, 2000). The high prevalence of different infections leads us to presume that a similar immunosuppression could have happened in the chamois under study. However, specific studies will be needed to investigate this condition in affected chamois. Also, a multifactorial wasting syndrome, of which pestivirus would be one of the components, should be considered. Piroplasms were detected previously in two of the affected chamois and Anaplasma phagocitophilum in three (Hurtado et al., 2004). Forms consistent with piroplasms were observed on blood smears in six other chamois. Lesions in spleen and kidney consistent with haemolytic anaemia such as hemosiderosis or pigment deposition in glomeruli (kidney ‘‘cloisonne´’’) observed in 14 chamois were probably related to piroplasm infection. Thus, pestivirus infection could have triggered the development of piroplasmosis in these chamois. On blood smears from healthy chamois from other areas in the Pyrenees, we have observed similar forms of piroplasms (unpublished data), so it could be a normal parasite in this species, as has been the case in the Spanish ibex studied in Catalonia (Ferrer et al., 1998). However, further studies of this process in chamois would be needed. In PI domestic animals a virus-host equilibrium exists, pathogenicity usually being reasserted following reinfection with a cytopathogenic strain. However, unusually high pathogenic strains of pestivirus have been described in sheep, giving rise to severe outbreak of disease (Nettleton and Entrican, 1995). The chamois under study concur with the definition of PI animals, but we do not know whether it is a case of infection with a high pathogenic strain or a reinfection with another cytopathogenic strain. Direct contact between PI and susceptible animals is the most important transmission and infection route, and the key to controlling the disease in domestic animals is to prevent foetal infections in early gestation (Moen et al., 2005). It is plausible that in chamois the same thing happens. However, control of the disease would be very difficult in the chamois population living free in such an extensive and remote
area as the Pyrenees, whereas in domestic animals control is achieved through vaccination, all in-all out management and/or other zootechnical measures. Taking into account the different infections found in the studied chamois, where evidence of bacteria and parasitic infections occurred, it may be feasible that these concomitant processes influence the final outcome of pestivirus infection towards the development of an outbreak with high mortality. Other reasons for the high mortality rate, such as virulence of the virus, immunosuppression, population density, health of chamois and climatological conditions need to be investigated in detail. The data reported in this paper associate a severe decrease in the free-ranging chamois population to a disease associated to a new pestivirus. The clinical picture reported was different from the diseases that are known to affect this species. A pestivirus was previously detected in the affected chamois, either by RT-PCR, a capture antigen ELISA test and/or immunohistochemistry (Hurtado et al., 2004). In this study, we have isolated a BDV in 12 chamois. Lack of isolation and PCR amplification in one affected chamois was probably due to sample degradation. Altogether, the presence of pestivirus was demonstrated in 19 of the 20 chamois analysed, either with one or more diagnostic virological techniques. In conclusion, the similar clinical signs, pathological and virological findings strongly suggest the role of this BDV in the outbreak of mortality. However, more studies are needed to confirm that this virus is indeed the primary cause of the disease.
Acknowledgements We are grateful to the rangers and staff of the Alt Pallars-Aran National Hunting Reserve for the capture of the chamois. This research was supported by the Direccio´ del Medi Natural del Department de Medi Ambient de la Generalitat de Catalunya.
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I. Marco et al. / Veterinary Microbiology 120 (2007) 33–41 Becher, P., Orlich, M., Shannon, A.D., Horner, G., Konig, M., Thiel, H.-J., 1997. Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J. Gen. Virol. 78, 1357–1366. Becher, P., Avalos-Ramirez, R., Orlich, M., Cedillo-Rosales, S., Konig, M., Schweizer, M., Stalder, H., Schirrmeier, H., Thiel, H.-J., 2003. Genetic characterization of novel pestivirus genotypes: implications for classification. Virology 311, 96–104. Evermann, J.F., Barrington, G.M., 2005. Clinical features of bovine viral diarrhea virus. In: Goyal, S.M., Ridpath, J.F. (Eds.), Bovine Viral Diarrhea Virus: Diagnosis, Management and Control. Blackwell Publishing, Ames, pp. 105–119. Ferrer, D., Castella, J., Gutierrez, J.F., Lavin, S., Marco, I., 1998. Seroprevalence of Babesia ovis in Spanish ibex (Capra pyrenaica) in Catalonia, northeastern Spain. Vet. Parasitol. 75, 93–98. Frolich, K., Jung, S., Ludwig, A., Lieckfeldt, D., Gibert, P., Gauthier, D., Hars, J., 2005. Detection of a newly described pestivirus of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in France. J. Wildl. Dis. 41, 606–610. Hofmann, M.A., Brechtbu¨hl, K., Sta¨uber, N., 1994. Rapid characterization of new pestivirus strains by direct sequencing of PCRamplified cDNA from the 50 noncoding region. Arch. Virol. 139, 217–219. Hofmann, M.A., Thu¨r, B., Vanzetti, T., Schleiss, W., Schmidt, J., Griot, C., 1999. Klassische Schweinepest beim Wildschwein in der Schweiz. Schweiz Arch. Tierheilk. 141, 185–190. Hurtado, A., Aduriz, G., Gomez, N., Oporto, B., Juste, R.A., Lavin, S., Lopez-Olvera, J., Marco, I., 2004. Molecular identification of a new pestivirus associated to an outbreak of disease in the Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain. J. Wildl. Dis. 40, 796–800. Katz, J.B., Ridpath, J.F., Bolin, S.R., 1993. Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach. J. Clin. Microbiol. 31, 565–568. Loken, T., 2000. Border diseases in goats. In: Tempesta, M. (Ed.), Recent advances in goat diseases, International Veterinary Information Service. On line: http://www.ivis.org/advances/Disease_Tempesta/Loken/chapter_frm.asp?LA=1. Moen, A., Sol, J., Sampimon, O., 2005. Indication of transmission of BVDV in the absence of persistently infected (PI) animals. Prev. Vet. Med. 72, 93–98.
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Nettleton, P.F., Gilmour, J.S., Herring, J.A., Sinclair, J.A., 1992. The production and survival of lambs persistently infected with border disease virus. Comp. Immun. Microbiol. Infect. Dis. 15, 179–188. Nettleton, P.F., Entrican, G., 1995. Ruminant pestiviruses. Br. Vet. J. 151, 615–642. Nettleton, P.F., Gilray, J.A., Russo, P., Dlissi, E., 1998. Border disease of sheep and goats. Vet. Res. 29, 327–340. Nettleton, P.F., 2000. Border disease. In: Martin, W.B, Aitken, I.D. (Eds.), Diseases of Sheep. Blackwell Science, London, pp. 95–102. OIE, 2004. Border disease. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Available at: http:// www.oie.int/eng/normes/mmanual/A_00131.htm. Olde Riekerink, R.G.M., Dominici, A., Barkema, H.W., de Smit, A.J., 2005. Seroprevalence of pestivirus in four species of alpine wild ungulates in the High Valley of Susa, Italy. Vet. Microbiol. 108, 297–303. Perez-Barberia, F.J., Mutuberria, G., Nores, C., 1998. Reproductive parameters, kidney fat index, and grazing activity relationships between the sexes in Cantabrian chamois Rupicapra pyrenaica parva. Acta Theriol. 43, 311–324. Rossi, L., Meneguz, P.G., De Martin, P., Rodolfi, M., 1995. The epizootiology of sarcoptic mange in chamois, Rupicapra rupicapra, from the Italian eastern Alps. Parassitologgia 37, 233–240. Thiel, H., Collet, M.S., Gould, E.A., Heinz, F.X., Houghton, M., Meyers, G., Purcell, R.H., Rice, C.M., 2005. Family Flaviviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy. Eight Report of the International Committee on Taxonomy Viruses. Elsevier Academic Press, New York, pp. 981–998. Van Campen, H., Fro¨lich, K., Hofmann, M., 2001. Pestivirus infections. In: Williams, E.S., Barker, I.K. (Eds.), Infectious Diseases of Wild Mammals. Manson Publishing Veterinary Press, London, pp. 232–237. Vilcek, S., Durkovic, B., Kolesarova, M., Paton, D.J., 2005. Genetic diversity of BVDV: consequences for classification and molecular epidemiology. Prev. Vet. Med. 72, 31–35. Weber, E., 2004. El rebeco norten˜o y meridional. Omega, Barcelona.
Severe outbreak of disease in the southern chamois (Rupicapra pyrenaica) associated with border disease virus infection Ignasi Marco a,*, Jorge Ramon Lopez-Olvera a, Rosa Rosell b,c, Enric Vidal b, Ana Hurtado d, Ramon Juste d, Marti Pumarola b, Santiago Lavin a a
Servei d’Ecopatologia de Fauna Salvatge, Facultat de Veterinaria, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Spain b Centre de Recerca en Sanitat Animal (CRESA), Universitat Auto`noma de Barcelona, 08193 Bellaterra, Spain c Departament d’Agricultura, Ramaderia i Pesca, Generalitat de Catalunya, 08007 Barcelona, Spain d NEIKER, Instituto Vasco de Investigacio´n y Desarrollo Agrario, Department of Animal Health, Berreaga 1, 48160 Derio, Bizkaia, Spain Received 31 July 2006; accepted 4 October 2006
Abstract An outbreak of a previously unreported disease affecting southern chamois (Rupicapra pyrenaica) in the central Pyrenees (NE Spain) was recorded in 2001 and 2002. There was a marked temporal distribution, most animals being found between February and June. After the outbreak, the population was found to have decreased by about 42%, most probably due to the disease. We examined 20 affected chamois. Clinical manifestations included depression, weakness and movement difficulties in all cases. Three chamois presented abnormal behaviour, with absence of flight reaction, and 16 showed different degrees of alopecia with skin hyperpigmentation. At necropsy cachexia was observed in all animals, four chamois had abscesses in different parts of the body, four had pneumonia, one had an extensive subcutaneous infection on the head and neck and one had severe orchitis. Microscopic lesions were found in the brain, mainly edema, gliosis, espongiosis, cariorrexis and neuronal multifocal necrosis. A perivascular mononuclear inflammatory infiltrate was present in three of them. Skin lesions included marked follicular atrophy, mild to moderate epidermal hyperplasia with orthokeratotic hyperkeratosis and follicular hyperkeratosis, and hypermelanosis. In 13 chamois there were haemosiderin deposits in the spleen, and in three individuals kidney ‘‘cloissone’’ was observed. Intraeritrocitic parasites were detected either by direct observation or PCR in 8 of 17 chamois. A pestivirus was isolated and detected by RT-PCR from 12 of 13 affected chamois and antigenic characterized as border disease virus by monoclonal antibodies. This is the first time a border disease virus has been associated with an outbreak of a highmortality disease in a wild species. # 2006 Elsevier B.V. All rights reserved. Keywords: Chamois; Rupicapra pyrenaica; Border disease virus; Mortality
* Corresponding author. Tel.: +34 93 5813445; fax: +34 93 5812006. E-mail address: [email protected] (I. Marco). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.10.007
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1. Introduction The pestivirus genus, classified within the Flaviridae family, comprises viruses that are major pathogens for cattle, sheep and pigs, with a significant economic impact worldwide. Four viral species are currently recognized: Bovine viral diarrhoea virus 1 and 2 (BVDV-1 and BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). In addition, there is one tentative species represented by a single pestivirus isolated from a giraffe (Thiel et al., 2005). However, there is considerable genetic diversity, especially within the BVDV-1 genotype (Vilcek et al., 2005). Pestiviruses are able to cross-species barriers to infect a wide range of hosts within the Artiodactyla (Becher et al., 1997). It has been reported that BVDV and BDV can infect numerous domestic and wild ungulate species, and only CSFV appears to be restricted to a single host species (Becher et al., 2003). Border disease (BD) is a congenital viral disease affecting sheep and goats. Clinical signs include abortions, barren ewes, stillbirths and small weak lambs, which can show tremors, abnormal body conformation and abnormal birthcoat. The disease in goats is rare and is characterized by abortions (Nettleton et al., 1998). In wildlife, serologic surveys have demonstrated prior infection with pestiviruses in more than 40 species. In a recent study, a high seroprevalence was reported in a population of healthy chamois from the Italian Alps, but no pestiviruses were isolated (Olde Riekerink et al., 2005). There are only a few confirmed cases of isolation of a pestivirus from disease in wild ruminants, but there is no evidence that they have significant population impact in free-ranging animals (Van Campen et al., 2001). The southern chamois (Rupicapra pyrenaica) is a small ruminant belonging to the Bovidae family and Caprinae subfamily. It is a widely distributed ungulate in the Pyrenees and its range and densities have increased substantially over the last few decades. In the Pyrenees, the population has increased from 35,000 individuals in 1997 to around 50,000 in 2001 (Weber, 2004). Its abundance makes it one of the most popular game species in the area. Throughout 2001 and 2002 an unusual mortality rate of chamois was observed in the central Pyrenees and we reported the molecular identification of a new
pestivirus associated with this outbreak of disease (Hurtado et al., 2004). In the current study we describe the epidemiology, clinical manifestations, pathology and virological analysis of this outbreak of disease associated with this BDV.
2. Materials and methods 2.1. Study area The study area in the central Pyrenees is the Alt Pallars-Aran National Hunting Reserve (428330 N, 18190 E), covering an area of approximately 100,000 ha in the Autonomous Community of Catalonia (NE Spain) that borders with France and Andorra (Fig. 1). The Reserve provides an ideal habitat for chamois. The lower part of the main valleys lies at 800–900 m above sea level, and the highest peak is the Pica d’Estats (3143 m). There is an extensive alpine zone above the timberline at about 2200–2300 m, characterized by a mixture of steep, rocky areas and alpine pastures. Thick forests – mainly Pinus sylvestris and Pinus uncinata – provide shade and cover. Chamois are found at all altitudes, but the areas where diseased chamois were found ranged from 800 to 1900 m. In the Reserve, the chamois population is regulated by hunting during the autumn. In addition to chamois, this area is inhabited by abundant wild boar (Sus scrofa), roe deer (Capreolus capreolus), less abundant red deer (Cervus elaphus) as well as
Fig. 1. Study area in NE Spain. The Alt Pallars-Aran National Hunting Reserve is shown in grey. Main rivers are shown. Black dots and triangles indicate affected chamois found in 2001 and 2002, respectively. Grey dots and triangles indicate carcasses found in 2001 and 2002, respectively.
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introduced mouflon (Ovis ammon) and fallow deer (Dama dama). During the summer, domestic sheep, goats, cattle and horses graze extensively on the alpine pastures. Some goats, cattle and horses remain in the area all year round.
was homogenised, protease K digested, denatured and loaded in a SDS-polyacrylamide gel. Electrophoretically separated proteins were then transferred to a PDVF membrane and immunolabelled with a monoclonal antibody against prion protein (6H4, 1:5000).
2.2. Collection of data
2.5. Bacteriology
In the course of the outbreak, chamois suffering from clinical disease or found recently dead were collected by the Reserve gamekeepers. We examined 20 chamois, 4 young males between the age of 1 and 3 years, 16 adults, 12 males and 4 females more than 4 years old. Most of the chamois were hand-captured (15), while three were shot due to their poor body condition and two were found dead. Eight chamois were studied in 2001 and 11 in 2002. In 2003, no abnormal mortality was observed in the whole area, but one adult female with clinical signs consistent with the disease was found and is included in this study. The affected area was defined on the basis of the places where clinical cases and carcasses were found. Mortality was evaluated indirectly, through the census of the chamois population that are conducted twiceyearly (spring and autumn) in the Reserve.
Samples of skin scrapings from the skin lesions were obtained from the 16 chamois with skin lesions for routine microbial culturing. Routine bacteriological methods were also performed in the nine chamois with different infectious processes by the Microbiology Service, Department of Anatomy and Animal Health, Veterinary Faculty, Autonomous University of Barcelona.
2.3. Pathological examination Necropsies and tissue sampling were performed according to a standard protocol. Tissue samples were fixed in 10% phosphate buffered formalin. Fourmillimetre-thick tissue blocks were cut and dehydrated through increasing alcohols and xylene. Then the tissue was embedded in paraffin and 4 mm-thick sections were obtained, mounted on glass slides and stained with haematoxylin and eosin for histological evaluation. The tissues examined included skin, mediastinic and mesenteric lymph nodes, lung, gastrointestinal tract, spleen, heart, liver, kidney, adrenal gland, skeletal muscle from the femoral region and the brain.
2.6. Virology Virus isolation was performed in 13 affected chamois according to the OIE Manual of Diagnostic (2004). Briefly, the homogenates of spleen and kidney were inoculated on confluent monolayer of permanent Sheep line pestivirus-free SFT-R cells obtained from the Federal Institute for Virus Diseases of Animals (Island of Riems, Germany). The presence of virus was detected by the inmuno-peroxidase monolayer assay (IPMA) using a polyclonal home-made pestivirus specific serum. The border disease specificity was performed by the IPMA test with monoclonal antibodies WS363 (BDV specific, obtained from Central Veterinary Laboratory, Weybridge, UK), C-16 (pestivirus specific), CA-3 and CT-6 (BVDV-1 specific), obtained from the Reference Laboratory of Classical Swine Fever (Hannover, Germany). Amplification of viral RNA was performed in the same 13 affected chamois, using previously described panpestivirus primers (Pesti 3 and Pesti D) (Hofmann et al., 1994; Katz et al., 1993).
2.4. Prion protein detection
3. Results
Western blot analysis was performed following the kit supplier’s protocol (Prionics Check-Western Blotting), to rule out the presence of resistant prion protein deposition. Briefly, a sample of approximately 0.5 g form the obex region, at the caudal brain stem,
3.1. Geographical distribution, host and seasonal variables, mortality The area affected by the outbreak covered approximately 25,000 ha and included two main
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valleys (Vallferrera and Vallcardos), and to a lesser extent a third valley (Vall d’Aneu) (Fig. 1). The only affected chamois found in 2003 came from another valley (Val d’Aran), which is not part of the Reserve, and is 35 km west of the nearest border of the main outbreak area under consideration. Only chamois was found to be affected by the outbreak of disease. No abnormal mortality or disease was observed in any of the other wild and domestic ruminants that share the habitat with chamois, before, during or after this outbreak of disease. The first case in 2001 was reported on 10th February, and the last on 14th August. Between August 2001 and February 2002, no spread of the disease was observed and no other clinical cases were found or observed. However, during this period 19 chamois carcasses were found, but could not be analysed due to advanced decomposition. In 2002, the first case was reported on 26th February and the last 6th May. A total of 56 carcasses were found in the area, but advanced decomposition again prevented any diagnosis of the cause of death. In 2003, the only affected chamois was found on 28th February. The epizootic spread across an area inhabited by some 1800 chamois. Chamois mortality could not be assessed accurately because most of the animals that died would not have been recorded due to the size and remoteness of the area affected. However, the twiceyearly census (June and November) provided an indirect measure of mortality by determining population decrease. In November 2000, just before the episode of mortality, the chamois population in the Reserve totalled 3880; in November 2002, after the episode, the chamois population for the whole Reserve dropped by 31% to 2678. If we consider only the two main affected valleys, the census varied from 1883 chamois (1052 and 831 chamois in the Vallferrera and Vallcardos valleys, respectively) to 1086 chamois (636 and 450 in the Vallferrera and Vallcardos valleys, respectively) in 2002. Thus, the estimated cumulative decrease in this area would have been 42% (40% and 46%, respectively).
difficulty in moving. Only three chamois presented abnormal behaviour, with absence of flight reaction, loss of fear of humans and tameness. A total of 16 chamois showed skin lesions consisting of different degrees of alopecia and skin hyperpigmentation. The alopecia ranged from small areas on the head (lips, base of ears, periorbital) in four chamois, asymmetric patches on the head, neck and trunk in eight chamois, to almost complete alopecia in the other four chamois, which had winter coat persistence on the ears, distal part of limbs and tail (Fig. 2A and B). In the four chamois with an apparently normal coat, the hair was easily pulled out by just holding it. There was intense tick infestation in 13 chamois, the species identified being Ripicephalus bursa, Dermacentor marginatus, Haemaphysalis punctata and Haemaphysalis sulcata.
3.2. Clinical signs
Fig. 2. Southern chamois with clinical signs of infection associated with a border disease virus: (A) hand-captured chamois, with alopecia, skin hyperpigmentation, cachexia and head tick infestation; (B) chamois with extensive alopecia and marked cachexia; no skin hyperpigmentation is observed since the animal was hospitalised indoors for 2 months.
Of the 20 chamois studied, 2 were found recently dead and 18 alive. The latter chamois showed neurological signs such as depression, weakness and
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3.3. Necropsy and histopathology At necropsy, all animals were emaciated. Bodyweight ranged from 15 to 22 kg in adult males and from 13 to 17 kg in adult females, when normal bodyweight is 25–40 kg for males and 20–32 for females (Weber, 2004). Different infections were observed in nine chamois. Severe pneumonia was observed in four chamois, three adult males and one adult female. The adult female also had three abscesses in the left scapular region. A single abscess was present in three chamois, two adult male and one young male, in the submandibular, scapular and axilar regions, respectively. One adult male had an extensive subcutaneous infection on the head and neck. Another adult male had severe bilateral orquitis, with caseous material, and multiple necrotic foci in the spleen. The most consistent microscopic changes were found in the skin and the brain. Skin lesions consisted in severe alopecia with follicular atrophy and telogenisation of the remaining hair follicles. Images of flame follicles were observed, i.e. follicles with excessive trichilemmal keratinisation. The epidermis showed hyperplasia and melanosis with evident orthokeratotic hyperkeratosis. A mild mononuclear interstitial inflammatory infiltrate was present in the dermis. The brain consistently presented with oedema evidenced by perivascular spongiosis and the presence of clear eosinophyllic amorphous material within Virchow–Robin’s spaces being actively phagocyted by glial cells (Fig. 3A). Diffuse moderate spongiosis was present, being more evident in the white matter along with moderate to evident proliferation and astrocyte hypertrophy. Occasional glial nodules were detected. Neuronal degeneration and death occurred throughout the brain, but some areas, such as the hippocampus and the Purkinje cell layer in the cerebellar cortex, were affected more severely. At the neocortex the deeper layers were those most affected. Only in three cases was a discreet nonpurulent inflammatory infiltrate observed surrounding vessels at the leptomeninges and intraparenchymatously, occasionally percolating into the neuropil (Fig. 3B). In 8 out of the 20 cases a meningiomatous proliferation was observed in the pineal gland, another healthy chamois examined later also showed a similar lesion. Dystrophic calcifications and fibrosis were other features observed in the pineal glands.
Fig. 3. (A) Mesencephalon. Eosynophillic material is observed in Virchow–Robin’s spaces (asterisk), indicative of brain oedema. Note presence of neuropil spongiosis and gliosis. (B) Frontal cortex. Nonpurulent perivascular cuff.
Other lesions observed in the majority of cases were verminous pneumonia in the lungs with different intensities; also the presence of protozoal intrafibrous cysts (Sarcocystis spp.) was frequently observed in both skeletal and cardiac striated muscle fibres. Abundant accumulations of hematosiderous pigment were often seen in the spleen macrophages and, in three animals, crescent-shaped deposits of a brownish pigment were seen in the kidney’s Bowmann’s capsule. In addition, on blood smears we observed forms consistent with piroplasms in six chamois. 3.4. Prion protein detection and bacteriology No deposition of resistant prion protein was detected in any of the animals studied. No microorganisms were isolated from skin specimens. Several
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bacteria were isolated from the different organ specimens. Staphylococcus hyicus, Escherichia coli, Klebsiella pneumoniae were isolated from the chamois with pneumonia. Arcanobacterium pyogenes was isolated from the chamois with the subcutaneous infection and from the abscess of another chamois. Staphylococcus hyicus and positive Staphylococcus aureus coagulase were found in the abscesses of the other chamois. 3.5. Virology A BDV was isolated from the spleen and kidney in 12 of the 13 chamois studied for this purpose. All 12 isolates reacted positively with C-16 and WS363 monoclonal antibodies and were negative with CA-3 and CT-6 monoclonal antibodies. RT-PCR results were positive in the same culture-positive animals.
4. Discussion Ruminant pestiviruses are among the most widespread and successful animal viruses in the world (Nettleton and Entrican, 1995). However, this is the first time that a pestivirus has been associated with an outbreak of disease having a significant impact on a wild ruminant species population, since only sporadic cases of disease have been confirmed (Van Campen et al., 2001). The outbreak was analysed and animals with clinical signs of the disease, either captured or recently found dead, were considered. It is difficult to determine the origin of the disease. Since 2000, a decrease in the chamois population was recorded in the French side of the Pyrenees, just over the Spanish border, where the outbreak reported here took place. However, there is no epidemiological, clinical or pathological information of affected chamois. The first confirmed cases of pestivirus-associated infection in France and Andorra were in 2002, later than the first outbreak reported on the Spanish side (Arnal et al., 2004; Frolich et al., 2005). The characteristic clinical signs and gross lesions of the observed condition provided a provisional diagnosis when an affected animal was observed. The Reserve gamekeepers were able to recognize the condition and therefore provided consistent data that
allowed us to define the geographical distribution of the outbreak. Apparently, no direction of spread of the disease was observed throughout the area between the 2 years. The outbreak remained relatively localised in the initially affected area, although the chamois population is continuous and abundant in the whole mountain chain. The Aiguestortes i Estany de Sant Maurici National Park is only 7 km in a straight line from the affected area and has an abundant highdensity chamois population, but not a single affected chamois was detected. An important river running between the National Park and the affected area could act as a barrier, preventing the spread of the disease. There is also a local road, but it may not represent a barrier for chamois. Factors such as climatology, natural movement of chamois and possibly other unknown factors, may have influenced the geographical distribution. The single case diagnosed 35 km west of the main outbreak area in 2003, could be related to some of these factors. This epizootic occurred in a healthy host population enjoying good physiological condition. Since 1990, we have been monitoring this and other wild ungulate species in the area and only a few outbreaks of well-known chamois diseases, such as infectious keratoconjuntivitis, have been recorded. It seems that the disease is rather host-specific, since no other species of domestic and wild animals have been found to be affected. However, additional studies would be needed, to confirm the absence of the disease, especially in the domestic ruminant species that share the habitat with the chamois. The seasonal pattern of the disease was similar in 2001 and 2002. Most of the affected chamois studied were males and were found between late winter (February) and late spring (June). Only one adult female was found in August. A similar pattern of disease has been described in chamois with sarcoptic mange (Rossi et al., 1995). These authors found the highest number of affected chamois between February and May, and a higher prevalence of the disease in males. Factors influencing this seasonal pattern are mite biology, metabolic state of the host, food availability and behavioural characteristics of the chamois. Of these factors, the last three could have also influenced the development of the outbreak of disease reported here. The rut season for chamois is November and it represents high energy output,
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especially in males, that continually chase females and other males, thus, considerably decreasing food intake and reducing body weight. After November, in the winter season, available food resources for the animals to recover are low, leading to a worsening body condition by the end of winter and beginning of spring. As much as half of the male bodyweight can be lost during this period (Perez-Barberia et al., 1998; Weber, 2004). The lower prevalence of the disease in kids and young animals can be explained by the difficulty of observation and the quick disappearance of the carcasses. It is well known that kids and young corpses are more difficult to find since they are smaller and more quickly disposed of by scavengers—mainly the red fox in our study area (Rossi et al., 1995). Rates of morbidity and mortality could not be investigated in detail owing to the large area covered by the outbreak and the difficulty in surveying the affected chamois in a remote alpine mountain area. The chamois carcasses were found mainly at the bottom of the valleys, near trails, so most of the animals that died may not have been found or reported. In addition, all 20 chamois reported in this study were found or captured near the villages and main roads, thus also in the lower reaches of the mountains. The post-epidemic census drop can be used as an indirect estimation of the cumulative proportion of deaths. No other important outbreak of disease, deaths or mortality of chamois has been observed before or during the outbreak. Thus, we can assume that most of the variation between the pre- and post-epidemic census provides an estimation of the mortality associated with the disease. A 42% decrease in the affected area is unusual and extraordinarily high. The only disease known to cause such high – and even higher – mortality in chamois is sarcoptic mange (Rossi et al., 1995). The clinical picture of the affected chamois was not similar to any of the diseases known in this species and no previous reports have described a similar condition. It included similar signs in all chamois, except for some variation in coat and skin lesions, depending on the time of the year. The four chamois with an apparently normal coat were those found during late winter (February and early March) and those with the most extensive alopecia and skin hyperpigmentation were found in late spring.
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Chronic wasting was a common feature in all affected chamois. It has been described as one of the commonest causes of death in experimental sheep infected with border disease virus (Nettleton et al., 1992). Also, this condition has been described as one of the levels of pestivirus persistence in the host animal in nature (Evermann and Barrington, 2005). Symptoms of reduced flight behaviour and loss of natural shyness observed in the three chamois that were easily hand-captured, have also been reported in wild boar with classical swine fever (Hofmann et al., 1999). In sheep, border disease is characterized by embryonic or foetal death, abortion and the birth of dead or weak lambs, with poor growth (Loken, 2000). However, it is not known whether this condition also obtains in the chamois, since abortions and postnatal diseases are very difficult to study in free-ranging wildlife. In sheep, PI animals reach a maximum age of 5 years. Thus, the danger of creating lines of PI sheep, as suggested in cattle, would appear to be remote (Nettleton et al., 1992). In the chamois under study, an adult female kept in captivity for more than 1 month, was demonstrated to be a PI animal; 12 other chamois between 1 and 12 years of age were seronegative and antigen positive at one sampling time (Hurtado et al., 2004). Thus, it is very likely that lines of PI chamois enable the maintenance of pestivirus transmission in the chamois population. Ruminant pestivirus pathogenesis is uniquely complex, as indicated by a wide variety of clinical signs and the variable results following intrauterine infections (Nettleton and Entrican, 1995). In the chamois under study, the main lesions were observed in the brain and skin. Brain lesions suggested a viral (i.e. nonpurulent) encephalitis only in a minority of cases, otherwise a discyclia causing brain oedema was the main feature. Disruption of blood vessel patency would presumably have caused the glial reaction and the inflammation as well as the oedema-induced hypoxia giving rise to the neuronal death described. The irregular distribution of neuronal damage in the brain might be due to differential susceptibility to hypoxia. Skin lesions could be explained by the replication of viruses in hair follicle cells. However, hair abnormalities were not similar to those described for sheep (Nettleton, 2000). Haemorrhagic disease, as
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seen in other pestivirosis (Van Campen et al., 2001) has not been detected. Border disease strongly compromises cellmediated immunity and causes immune suppression in PI animals, making them highly susceptible to intercurrent infectious and parasitic diseases (Loken, 2000). The high prevalence of different infections leads us to presume that a similar immunosuppression could have happened in the chamois under study. However, specific studies will be needed to investigate this condition in affected chamois. Also, a multifactorial wasting syndrome, of which pestivirus would be one of the components, should be considered. Piroplasms were detected previously in two of the affected chamois and Anaplasma phagocitophilum in three (Hurtado et al., 2004). Forms consistent with piroplasms were observed on blood smears in six other chamois. Lesions in spleen and kidney consistent with haemolytic anaemia such as hemosiderosis or pigment deposition in glomeruli (kidney ‘‘cloisonne´’’) observed in 14 chamois were probably related to piroplasm infection. Thus, pestivirus infection could have triggered the development of piroplasmosis in these chamois. On blood smears from healthy chamois from other areas in the Pyrenees, we have observed similar forms of piroplasms (unpublished data), so it could be a normal parasite in this species, as has been the case in the Spanish ibex studied in Catalonia (Ferrer et al., 1998). However, further studies of this process in chamois would be needed. In PI domestic animals a virus-host equilibrium exists, pathogenicity usually being reasserted following reinfection with a cytopathogenic strain. However, unusually high pathogenic strains of pestivirus have been described in sheep, giving rise to severe outbreak of disease (Nettleton and Entrican, 1995). The chamois under study concur with the definition of PI animals, but we do not know whether it is a case of infection with a high pathogenic strain or a reinfection with another cytopathogenic strain. Direct contact between PI and susceptible animals is the most important transmission and infection route, and the key to controlling the disease in domestic animals is to prevent foetal infections in early gestation (Moen et al., 2005). It is plausible that in chamois the same thing happens. However, control of the disease would be very difficult in the chamois population living free in such an extensive and remote
area as the Pyrenees, whereas in domestic animals control is achieved through vaccination, all in-all out management and/or other zootechnical measures. Taking into account the different infections found in the studied chamois, where evidence of bacteria and parasitic infections occurred, it may be feasible that these concomitant processes influence the final outcome of pestivirus infection towards the development of an outbreak with high mortality. Other reasons for the high mortality rate, such as virulence of the virus, immunosuppression, population density, health of chamois and climatological conditions need to be investigated in detail. The data reported in this paper associate a severe decrease in the free-ranging chamois population to a disease associated to a new pestivirus. The clinical picture reported was different from the diseases that are known to affect this species. A pestivirus was previously detected in the affected chamois, either by RT-PCR, a capture antigen ELISA test and/or immunohistochemistry (Hurtado et al., 2004). In this study, we have isolated a BDV in 12 chamois. Lack of isolation and PCR amplification in one affected chamois was probably due to sample degradation. Altogether, the presence of pestivirus was demonstrated in 19 of the 20 chamois analysed, either with one or more diagnostic virological techniques. In conclusion, the similar clinical signs, pathological and virological findings strongly suggest the role of this BDV in the outbreak of mortality. However, more studies are needed to confirm that this virus is indeed the primary cause of the disease.
Acknowledgements We are grateful to the rangers and staff of the Alt Pallars-Aran National Hunting Reserve for the capture of the chamois. This research was supported by the Direccio´ del Medi Natural del Department de Medi Ambient de la Generalitat de Catalunya.
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