key: cord-0861202-x15qb4ch authors: Goncharuk, M. S.; Kerley, L. L.; Naidenko, S. V.; Rozhnov, V. V. title: Prevalence of seropositivity to pathogens in small carnivores in adjacent areas of Lazovskii Reserve date: 2012-11-16 journal: Biol DOI: 10.1134/s1062359012080067 sha: 722bbb329a5adcf122887c95618883308d12286b doc_id: 861202 cord_uid: x15qb4ch The prevalence of infectious diseases in wild and feral carnivores is poorly known in Primorsky Krai, where rare species such as the Amur tiger and the Far East leopard roam. In this study we evaluated the prevalence of seropositivity in feral (cats, dogs) and wild (raccoon dog, badger, American mink, Far Eastern wild cat) carnivores to various pathogens: distemper virus, parvovirus, feline immunodeficiency and feline leukemia virus, feline coronavirus, Toxoplasma gondii, Mycoplasma sp., and Chlamydia sp. It was shown that seropositivity occurred significantly more frequent in feral animals than in wild ones. The highest percentage of seropositive animals was observed in feral dogs. It is these diseases that can be dangerous for the Amur tiger and the Far East leopard, thus affecting their populations. Infectious diseases pose a serious threat to wild ani mals, causing depopulation of species (Alexander, Appel, 1994; Roelke Parker et al., 1996; Murray et al., 1999; Deem et al., 2001 ). An infection related popu lation decline has been documented in the lion (Pan thera leo) and the Ethiopian wolf (Canis simensis). The source of infection was feral animals, namely, dogs (Sillero Zubiri et al., 1996; Cleaveland et al., 2000) . Wild canine and feline species are susceptible to dis eases of feral cats and dogs (Sillero Zubiri et al., 1996; Deem et al., 2002) . However, little is known about the presence of infectious diseases in wild predatory mam mals, in particular, in Primorskii Krai which is the habitat for rare species such as the Siberian tiger (Pan thera tigris altaica), the Far Eastern leopard (P. pardus orientalis), and the Far Eastern Forest Cat (Prionailu rus bengalensis euptilura). Such studies are very impor tant for areas of likely introduction of the Far Eastern leopard, i.e., the Ussuriiskii and Lazovskii State Nature Reserves, Far East Branch, Russian Academy of Sciences. It is these reserves where we initiated such studies (Esaulova et al., 2009 Davydova et al., 2010; Naydenko et al., 2011) . The most important diseases of the Amur tiger and the Far Eastern leopard are feline panleukopenia, feline coronaviral enteritis, feline viral immunodefi ciency, leukemia, toxoplasmosis, mycoplasmosis, and clamidiosis. The viruses causing feline panleukopenia and canine and mustelid parvoviral enteritis behave similarly in regard to pathogenesis, targeting fast dividing host cells, which results in diarrhea, suppres sion of bone marrow activity, and a sharp decline in immunity (Parrish, 1995) . Feline coronaviral enteritis is prevalent in 80% of the feral cat population and induces infectious peritonitis. This frequently ends up with death of animals (Pedersen, 1987) . Distemper considerably diminished the lion population in 1994 in the Serengeti, killing about 1000 animals (Roelke Parker et al., 1996) . The feline viral immunodefi ciency virus strongly related to HIV was detected in a feral cat and other wild feline species (Brown et al., 1994) . It induces severe immunodeficiency in feral cats by decreasing T cell numbers (Torten et al., 1991) . The feline leukemia virus has worldwide preva lence and causes anemia, immunodeficiency, and var ious lymphoid tumors (Hoover et al., 1975; Hardy et al., 1976) . Toxoplasmosis caused by the protozoa Toxoplasma gondii is a cause of abortions, nonviable offspring, central nervous system disorders, and impairment of the lymphatic and endocrine systems in adult cats, dogs, and other animals (Meli et al., 2009) . Mycoplasmosis is primarily associated with conjunc tivitis, pneumonia, polyarthritis, gastrointestinal tract diseases, spontaneous miscarriages, and infertility (Meli et al., 2009) . Clamidiosis is an acute or chronic disease of cats, dogs, and other animals and is charac terized by body temperature elevation, conjunctivitis, coryza, pneumonia, and urogenital disorders. The infection occasionally becomes delayed and shows a chronic course. During infection kittens show acute clinical signs, which tends to end up with death (Meli et al., 2009) . The main objective of this work was to carry out a serological study aimed at determining the prevalence of pathogens in populations of wild and feral predatory mammals that inhabit the Lazovskii State Nature Reserve and adjacent territories. Sampling was done across the territory of the Lazovskii State Nature Reserve and adjacent territories from March 2008 to September 2009. The reserve is located in the territory of the similarly named district in the southeastern part of Primorskii krai on the slopes of the Sikhote Alin mountain range that overlook the Sea of Japan. The total reserve area is 1210 km 2 . The reserve is the habitat for the fox (Vulpes vulpes), raccoon dog (Nyctereutes procyonoides), Amur badger (Meles leucurus), sable (Martes zibellina), yellow throated marten (Lamprogale flavigula), yellow weasel (Mustela sibirica), the least weasel (M. nivalis), com mon weasel (M. erminea), otter (Lutra lutra), Ameri can mink (Neovison vision), Himalayan black bear (Ursus thibetanus), Amur tiger, lynx (Lynx lynx), and the Far Eastern wild cat. There are 9 major settlements near the reserve. There is a buffer zone occupied by game husbandries with an area of 150 km 2 between settlements and the reserve. Rearing dogs without a leash and cats outdoor as well as illegal dog hunting make it possible for feral animals to come into contact with wild ones. Amur tigers are distributed throughout the reserve and in adjacent areas, regularly coming in close proximity with settlements or even invading them. Blood was sampled from stray and feral nonvacci nated cats and dogs. Sampling sites are mapped in Fig. 1 . To rule out false positive serological results due to maternal antibodies, only animals 6 months of age or older were bled. To avoid errors due to prior vaccina tion, owners of cats and dogs were polled before bleed ing. Dogs were bled without immobilization using man ual fixation. When bled cats were immobilized, inject ing intramuscularly tiletamine/zolesepam 10 µg/kg of body weight (Zoletil, Virbak, France). Wild animals were captured in live animal traps from welded mesh (Havahart Traps, Woodstream Corp., United States) and home made cages. Fresh fish and chicken were used as bait. Traps were placed in 6 locations ( Fig. 1 ) along roads and pathways every 250 m in the vicinity of reserve cordons. Traps were monitored daily. Trapped animals were immobilized by injecting Zoletil. Raccoon dogs and badgers were injected with 5 µg/kg of this medicine, Far Eastern wild cats were injected with 10 µg/kg, and American minks were injected with 20 µg/kg. Animals were weighed, photo graphed, labeled using microchips, clinically evalu ated, and bled. In addition, wool and fecal samples were collected, including ectoparasites. After recovery the animal was released at its capture site. All collected blood samples were stored for 2 h and centrifuged at 3000 rpm for 10 min, followed by serum harvest and storage in liquid nitrogen. Sera in liquid nitrogen were transported to the labo ratory. All samples were tested by the Russian Cher nogolovka Center for Scientific Research, Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences. All samples were evaluated for seropositiv ity to parvovirus, distemper virus, chlamydia, Myco plasma, and toxoplasma. All feline sera were tested for antibodies to feline leukemia virus, feline immunodefi ciency virus, and feline enteric coronavirus. Seropositivity to parvoviruses (canine parvovirus, parvovirus of mustelids, and feline panleukopenia) and distemper virus was evaluated by qualitative and quanti tative enzyme linked immunosorbent assay kits (ELISA), produced by Hema Medika (Russia, Mos cow) following the manufacturer's instructions. The two parvoviruses (canine parvovirus, parvovirus of mus telids, and feline panleukopenia) are strongly related, and host antibodies cross react with both viruses with high affinity. Seropositivity to toxoplasma, Chlamydia, and Mycoplasma was also assessed by ELISA commer cial kits of the same company without quantitative anal Lazovskii Reserve Note that seropositivity (occurrence of antibodies) does not imply an infection, but shows prior exposure of an animal to a specific antigen. In addition, animals may also seroconvert to pathogens that are not infec tious for a certain species (herein it is the case for dis temper virus in feral cats and probably in the Far East ern forest cat). Statistics was found by comparing rates. A total of 21 serum samples from feral and stray dogs, 34 serum samples from feral and stray cats, 2 serum samples from Far Eastern forest cats, 5 serum samples from Amur badgers, 3 serum samples from American minks, and 5 serum samples from raccoon dogs (in total 15 serum samples from wild predatory mam mals). Far Eastern forest cats and feral cats could be affected by all the pathogens that are infectious to a tiger (except distemper virus). Amur badgers and rac coons dogs are important prey for the Amur tiger. Serology results are given in the table. Seropositivity to distemper virus was found in 16% of the animals. A suspect result was identified in 4 feral dogs and 1 raccoon dog. Seropositivity occurred only in feral dogs (52%). According to the statistics, this difference was statistically significant at the rate of seropositivity in wild canine and mustelid species (p < 0.01). Seropositive animals were found west, south, and east of Lazovskii district (Fig. 2) . Seropositivity to parvovirus (the etiological agent of enteritis in canine and mustelid species and feline panleukopenia) occurred in 9% of the animals. The same percentage of animals (9%) was identified as sus pect (4 feral dogs and 2 raccoon dogs). Seropositivity was only found in feral dogs. The frequency of serop ositivity in feral dogs showed statistical significance over feral cats (p < 0.01). No significant difference in the frequency of seropositivity was found between wild and feral predatory mammals, feral dogs, and wild canine and mustelid species. Seropositive animals were found south and east of Lazovskii Reserve (Fig. 2) . Nineteen percent of the sera collected from feline animals tested positive for antibodies to coronavirus (n = 36). Only stray and wild cats were seropositive; two Far Eastern forest cats trapped were seronegative. Seropositive animals were found north, west, and southwest of Lazovskii Reserve (Fig. 2) . No feline leukemia virus antigen was detected in sera from feral cats. Only one feral cat was found seropositive to feline immunodeficiency virus (3%) (Fig. 3) . Toxoplasma seropositivity was detected in 16% of the animals tested. Of seropositive animals, 75% were feral dogs (38% of dogs were seropositive); however, Toxoplasma gondii antibodies were also identified in one Far Eastern forest cat and one American mink. No significant difference in frequency of seropositivity was generally seen in wild and feral carnivores, although it occurred statistically more frequently in feral dogs over wild canine and mustelid species (p < 0.05) and feral cats (p < 0.001). An overwhelming majority of seropositive animals were found east of Lazovskii Reserve; one case was observed in the south of an area close to Petrovskaya Pad (Fig. 3) . Chlamydia seropositive animals were 6%. Chlamy dia antibodies were found only in feral and stray dogs (19%) . No statistical significance in the frequency of seropositivity was observed among wild and feral car nivores, feral dogs, and wild canine and mustelid spe cies, although Chlamydia seropositivity was numeri cally more frequent in feral dogs over feral cats (p < 0.05). Seropositive animals were found east of Lazovskii Reserve (Fig. 4) . Antibodies to Micoplasma were observed in 36% of the animals tested. Of feral and stray dogs, 90% were seropositive to Mycoplasma. All raccoon dogs and 1 stray cat also showed seropositivity to Mycoplasma infection (Fig. 4) . No statistical significance in the fre quency of seropositivity was observed between wild and feral carnivores, although the frequency of serop ositivity in feral dogs statistically differed from wild canine and mustelid species (p < 0.01) and feral cats (p < 0.001). Serology results showed that feral dogs had the highest prevalence of seropositivity. Dog owners do not tend to vaccinate their dogs against distemper and parvoviral enteritis, which, by itself, poses the threat of an outbreak of these diseases. Fifty percent of seropos itivity to distemper virus and about 25% of seropositiv ity to parvovirus in feral dogs show that dogs have prior exposure to the viruses; they carry the viruses, and that the viruses are widely distributed across these popula tions. Feline coronavirus was also documented across this territory. Despite the fact that the original virus is avir ulent for cats, the threat is posed by a mutant variant of the virus that causes peritonitis and death (Herrewegh et al., 1997) . This virulent mutant emerges in about 10% of infected cats (Vennema et al., 1998) . Considering that the leukemia virus is highly trans missible and no leukemia virus antigen was detected in feline sera, we suggest that this virus has no distribu tion across the territory or it is confined to cat popula tions not included in this study. The occurrence of antibodies to Mycoplasma agents is ambiguous to interpret. In terms of tiger infections, only one Mycoplasma species, Mycoplasma felis, is of importance, capable of affecting feline spe cies, whereas in this study we detected generic anti bodies to all species from the Mycoplasma genus, which includes many species. Overall, a positive result is difficult to interpret unambiguously; however, a neg ative result is much unambiguous in this case: 64.3% of the animals tested had no prior exposure to mycoplas mas, including pathogenic M. felis. Only canine ani mals were seropositive (only one stray cat showed mycoplasma seropositivity); therefore, it is suggested that M. felis is not a threat for feline species inhabiting Lazovskii Reserve and adjacent areas. Similarly, seropositivity to Chlamydia and Chlomy dophila is ambiguous. Of greatest importance for us was only Chamydophila felis. The absence of chlamy dia antibodies in feline animals shows that the animals tested may have had no exposure to pathogenic Chlamydia and Chlomydophila species. Among wild predatory mammals of Lazovskii Reserve, seropositivity was observed to two pathogens: toxoplasma (13%, American mink and Far Eastern forest cat) and mycopalsma (33%, raccoon dogs). No seropositivity to other pathogens was found. More over, the absence of antibodies cannot ensure mainte nance of a population. For a small population, a dis ease incursion into one or two animals threatens most of the population like the leukemia virus outbreak in the Iberian lynx (Lynx pardina) in the Do ana National Park (Meli et al., 2009) . In feral animals we found seropositivity to 7 out of 8 pathogens tested for. Besides toxoplasma and myco plasma, seropositivity was observed to distemper virus, parvoviruses (including feline panleukopenia), immu nodeficiency virus, enteric coronavirus, and chlamy dia. The percentage of feral animals seropositive to a number of pathogens is similar to that of wild animals. Statistical differences were only observed for distem per virus in wild and feral animals (feral animals had a statistically significant frequency). Importantly, feral cats are not affected by distemper, and no seropositive result was identified. Distemper seropositive dogs were over 50%. Dog hunting (including poaching) and rear ing dogs without a leash could increase a probability of contacts of Amur tigers with virus carries (Seredkin et al., 2010) . Panthera species are sensitive to distem per virus: there is evidence that it is capable of causing neural system disorders in Amur tigers and ultimately death of tigers (Quigley et al., 2010) . In addition, the occurrence of distemper virus in tigers is quite high (Naydenko et al., 2011) . In summary, infections pose a potential threat to Amur tigers inhabiting Lazovskii Reserve and to the Far Eastern leopard to be introduced into the reserve territory. One of the ways to resolve the problem is to mass vaccinate all feral dogs as part of programs aimed at studying and conserving the Amur tiger and the Far ñ Eastern leopard implemented by the Russian Acad emy of Sciences, the Zoological Society of London, and Wildlife Conservation Society. Note: The number of seropositive samples/the number of tested samples from a certain animal species. 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Serology testing was performed as part of the Program on study ing the Amur tiger in the Far East of Russia and the Program on studying, conserving, and restoring the population of the Far Eastern leopard in the Far East of Russia and supported by the Russian Geographic Society.