Journal of Zoo and Wildlife Medicine 36(2): 192-197, 2005 Copyright 2005 by American Association of Zoo Veterinarians EXPOSURE OF FREE-RANGING MANED WOLVES (CHRYSOCYON BRACHYURUS) TO INFECTIOUS AND PARASITIC DISEASE AGENTS IN THE NOEL KEMPFF MERCADO NATIONAL PARK, BOLIVIA Sharon L. Deem, D.V.M., Ph.D., Dipl. A.C.Z.M., and Louise H. Emmons, Ph.D. Abstract: Maned wolves (Chrysocyon brachyurus) are neotropic mammals, listed as a CITES Appendix II species, with a distribution south of the Amazon forest from Bolivia, through northern Argentina and Paraguay and into eastern Brazil and northern Uruguay. Primary threats to the survival of free-ranging maned wolves include habitat loss, road kills, and shooting by farmers. An additional threat to the conservation of maned wolves is the risk of morbidity and mortality due to infectious and parasitic diseases. Captive maned wolves are susceptible to, and die from, common infectious diseases of domestic dogs (Canis familiaris) including canine distemper virus (CDV), canine parvovirus (CPV), rabies virus, and canine adenovirus (CAV). Results from this study show that free-ranging maned wolves in a remote area of Bolivia have been exposed to multiple infectious and parasitic agents of domestic carnivores, including CAV, CDV, CPV, canine coronavirus, rabies virus, Leptospira interroguns spp., Toxoplasma gondii, and Dirofiluria immitis, and may be at increased risk for disease due to these agents. Key words: Bolivia, Chrysocyon brachyurus, infectious diseases, maned wolf, parasitic diseases. INTRODUCTION Maned wolves {Chrysocyon brachyurus) are neotropic mammals with a distribution south of the Amazon forest from Bolivia, through northern Ar- gentina and Paraguay and into eastern Brazil and northern Uruguay. The maned wolf is listed as a CITES Appendix II species (http://www.cites.org), the United States Fish and Wildlife Service consid- ers it "endangered" (http://endangered.fws.gov), and the IUCN lists the species as "vulnerable" (http://www.redlist.org). There is no global popu- lation estimate, but maned wolves are absent from much of their former geographic range. The pri- mary threat to the survival of the maned wolf is considered to be habitat loss.3133 Road kills are the major source of mortality near small parks in Bra- zil, and farmers also shoot maned wolves that they believe hunt chickens.32 The risk of morbidity and mortality due to infec- tious diseases is a significant concern in the con- servation of maned wolves and wildlife in gener- al.10 Domestic dogs (Canis familiaris) are known or suspected reservoirs for agents of infectious diseas- From the Field Veterinary Program, Wildlife Conser- vation Society, 2300 Southern Boulevard, Bronx, New York 10460, USA (Deem); and the Department of System- atic Biology, NHB 390, MRC 108, Smithsonian Institu- tion, P.O. Box 37012, Washington, D.C. 20013-70128, USA (Emmons). Present address (Deem): Department of Animal Health, Smithsonian's National Zoological Park, 3001 Connecticut Avenue, Washington, D.C. 20008, USA. Correspondence should be directed to Dr. Deem. es, which have devastated populations of wild car- nivores in many areas of the world.118'2633 As hu- man populations expand and come into closer prox- imity with free-ranging maned wolves, there is in- creased risk of disease transmission from domestic carnivores to maned wolves. Although studies of exposure to disease agents of free-ranging maned wolves are lacking, captive maned wolves are known to be susceptible to im- portant infectious agents of domestic dogs includ- ing canine distemper virus (CDV), canine parvo- virus (CPV), rabies virus, and canine adenovirus (CAV). Morbidity and mortality have been reported for CDV,7-36 CPV,21316-27 rabies virus,35 and CAV.3 Maned wolves may be susceptible to all the known infectious agents of domestic dogs. Studies on the parasites of free-ranging or re- cently captive maned wolves have documented the presence of the giant kidney worm, Dioctophyme renale, which is known to destroy the right kidney in infected maned wolves.6 Parasites of the urinary tract, in addition to D. renale,5 gastrointestinal par- asites,12 and ectoparasites30 have also been docu- mented in free-ranging maned wolves. In captivity, maned wolves are also known to be susceptible to Dirofilaria immitis, the causative agent of heart- worm disease, Echinococcus granulosus, and gas- trointestinal parasites.28 The objective of this study was to determine the prevalence of exposure to select infectious and par- asitic diseases of maned wolves immobilized as part of an ecologic study in the Noel Kempff Mer- cado National Park, Bolivia (NKMNP). 192 DEEM AND EMMONS?DISEASE AGENTS IN FREE-RANGING MANED WOLVES 193 MATERIALS AND METHODS Study period and site description From February 2000 to October 2003, four maned wolves were immobilized in the NKMNP as part of a radiotelemetry study. NKMNP includes the Serrania de Huanchaca escarpment and the ad- jacent lowlands between the Rio Itenez (Guapore) and the Rfo Paragua and lies between 13?31'- 15?05'S and 60?14'-61o49'W. The park is at the interface of Amazonian forest with grassland eco- systems and includes a number of distinct ecosys- tems including broadleaf semievergreen forest, dry forest, inundated forest, dry savanna (Cerrado), and inundated savanna.24 Sample and data collection and analyses All maned wolves were immobilized using 100 mg tiletamine plus zolazepam (Telazol?, Fort Dodge Laboratories, Fort Dodge, Iowa 50501, USA; 3.5?4.5 mg/kg, i.m.) delivered through Tel- inject? (Telinject USA Inc., Agua Dulce, California 91390, USA) plastic darts, using a Telinject? pistol. When necessary, anesthesia supplementation was provided with ketamine (Ketaset?, Fort Dodge; 25- 50 mg increments, i.v.). Blood was collected by venipuncture of the jugular vein or the lateral sa- phenous vein. Blood was immediately placed in se- rum separator tubes (Corvac Sherwood Medical, St. Louis, Missouri 63103, USA) for all the maned wolves. For two of the maned wolves (CYB 1 and CYB 2), the sample tubes were placed in a cool place until clot formation and then sera were sep- arated by centrifugation (Mobilespin, Vulcan Tech- nologies, Grandview, Missouri 64040, USA) at 3,000 g for 15 min and stored in liquid nitrogen. Blood of the other two maned wolves (CYB 3 and CYB 4) was allowed to clot at ambient temperature, and the serum was then decanted and kept cool for 48 hr before storage in a ? 20?C freezer. Fecal samples were collected manually from the rectum and preserved in 10% formalin. Ectopara- sites were collected and stored in 70% isopropanol from all four maned wolves. Urine was collected from CYB 1 and CYB 2 by cystocentesis using a 22 g, 1.5 inch needle and 12 cc syringe. Urine sam- ples were divided into aliquots for freezing and for- malin fixation. The remaining urine was immedi- ately centrifuged at 3,000 g for 5 min. Urine sedi- ment was immediately examined by direct micro- scopic examination in the field. Samples were transported to the United States of America for laboratory testing. Blood and urine samples were transported on dry or wet ice. Fecal and urine samples were transported in 10% buff- ered formalin. Ticks were transported in 70% iso- propanol. Formalin and alcohol were removed be- fore air travel and refilled on arrival in the United States. All appropriate export and import permits accompanied the samples during transport. Serologic testing for antibodies to CAV, Brucella canis, canine coronavirus, CDV, canine herpesvirus (CHV), CPV, D. immitis, Toxoplasma gondii, and leptospirosis antibody testing was conducted at the New York State Veterinary Diagnostic Laboratory (Cornell University, Ithaca, New York 14853, USA). The 18 Leptospira interrogans serovars test- ed included L. ballum, L. wolffi, L. autumnalis, L. tarassovi, L. pomona, L. hardjo, L. grippophytosa, L. bataviae, L. canicola, L. icterolCOP, L. austral- is, L. pyrogenes, L. bratislava, L. sejroe, L. icte- rohaemorrhagialicterohaemorrhagia, L. javanica, L. szwajizak, L. saxkoebing. Serologic testing for rabies virus was performed at Kansas State Veter- inary Diagnostic Laboratory (Kansas State Univer- sity, Manhattan, Kansas 66506, USA) using the rapid fluorescent focus inhibition test. Three of the maned wolves, CYB 1, CYB 2, and CYB 3, were tested for all the infectious agents listed above, whereas CYB 4 was only tested for the presence of antibodies to CAV, CDV, and CPV. Fecal samples were examined by direct micro- scopic examination, sodium nitrate flotation, and sedimentation methods at the New York State Vet- erinary Diagnostic Laboratory. Adult ticks were identified on the basis of external morphology, us- ing the keys of Jones et al.22. Frozen and formalin-fixed urine samples were analyzed at the School of Veterinary Medicine (University of Pennsylvania, Philadelphia, Pennsyl- vania 19104, USA); these results will be reported elsewhere. RESULTS Table 1 summarizes the infectious disease agent serologic tests performed, methods used, level of titers defined as positive, and results for each test. All four maned wolves tested had positive antibody results to CAV and CPV and two had positive an- tibody results to CDV. Of the infectious diseases for which CYB 1, CYB 2, and CYB 3 were tested, none of them had antibodies to B. canis and CHV. All three of these maned wolves were antibody pos- itive for one or more L. interrogans serovars in- cluding L. ballum, L. grippophyfosa, L. icterohae- morrhagicalicterohaemorrhagica, and L. szwajizak. One maned wolf (CYB 1) was antigen positive for D. immitis and one (CYB 3) had antibodies to co- ronavirus, rabies virus, and T. gondii. Gastrointestinal parasites were identified in the 194 JOURNAL OF ZOO AND WILDLIFE MEDICINE Table 1. Disease agent serologic tests performed, methods used, level of titers defined as positive, and results in the study for select infectious and parasitic disease agents in four free-ranging maned wolves (Chrysocyon brachyurus) in Noel Kempff Mercado National Park, Bolivia. Disease agent Positive (method used)3 titer CYB 1 CYB 2 CYB 3 CYB 4 Canine adenovirus (SN) 1:4 Positive 1:512" Positive 1:512 Positive 1:384 Positive 1:512 Brucella canis NA Negative Negative Negative NA (SlideAGG/AGID II) Canine distemper virus 1:8 Negative Positive 1:12 Negative Positive 1:12 (SN) Canine herpesvirus (SN) 1:8 Negative Negative Negative NA Canine parvovirus (HAI) 1:10 Positive 1:10 Positive 1:10 Positive 1:10 Positive 1:20 Coronavirus (SN) 1:8 Negative Negative Positive 1:32 NA Dirofilariu immitis (oc- NA Positive Negative Negative NA cult) Rabies virus (RFFIT) 1:5 Negative Negative Positive 1:13 NA Toxoplasma gondii (IHA) 1:64 Negative Negative Positive 1:128 NA Leptospira interrogans 1:100 L. szwujizak L. ballum and L. ictero- L. grippo Positive NA 18 serovars (microag- Positive 1:100 hemorrhagiali ctero- 1:400; L. icterohe- glutination) hemorrhagia Positive morrhugialicterohe- 1:200 morrhugia Positive 1:100 3 SN, serum neutralization; Slide AGG/AGID II, slide agglutination/agar gel immunodiffusion test II; HAI, hemagglutination inhibi- tion; RFFIT, rapid fluorescent focus inhibition test; IHA, indirect hemagglutination; NA, not applicable. b Positive titer. feces of all four maned wolves and included An- cylostoma sp., A. caninum, Capillaria sp., C. aero- philia, Gnathostoma sp., Isospora sp., Physaloptera sp., Strongyles spp., Toxocara canis, Trichuris sp., and Uncinaria sp. Ticks collected from all four maned wolves were Ambylomma spp. The most prevalent species, A. ti- grinum, was present on all four maned wolves. A few A. ovale ticks were found on CYB 1 and CYB 2. Of the two maned wolf urine samples evaluated in the field, one (CYB 1) was observed to have D. renale ova on urine sedimentation. The other (CYB 2) was negative for this parasite. DISCUSSION The serologic tests used in this study document exposure but not disease by the detection of anti- bodies to infectious agents and not the agents them- selves. The occult heartworm test is an exception because the test detects the causative agent, D. im- mitis. Therefore, the serologic portion of this study was used to determine whether these maned wolves were exposed to a select number of infectious and parasitic agents known to be of concern for wild carnivore conservation.17 Disadvantages to serology are the possibility of false positives, because of cross-reaction with other agents, and false nega- tives. Moreover, the tests used in this study have not been validated for use in maned wolves because there is possible inaccuracy or cross-reacting sub- stances in the host.21 Currently, there are no sero- logic tests validated for maned wolves, but the tests developed for domestic dogs, which were used in this study, are widely used in the testing of captive maned wolves. Results suggest that free-ranging maned wolves in the NKMNP have been exposed to pathogens, which are known to cause high morbidity and mor- tality in captive maned wolves and other carni- vores. This is of particular interest because the NKMNP is located in a relatively isolated area of northeastern Bolivia, far from urban centers and large populations of domestic dogs. We believe that the most likely route of pathogen exposure for these maned wolves stems from domestic dogs, which live in villages and on ranches surrounding the park. Alternatively, these disease agents may be self-sustaining in free-ranging maned wolves and other carnivores in the NKMNP. Evidence suggests two possible disease epidem- ics in the wild carnivore populations of the NKMNP during the years we have been working in the park. The first maned wolf (CYB 1) died 8 mo after it was radiocollared. Its remains were found at a time when fox activity was appreciably diminished in the park. In 2003, three pups and an unmarked mother maned wolf disappeared between July and September 2003. Based on reports from DEEM AND EMMONS?DISEASE AGENTS IN FREE-RANGING MANED WOLVES 195 park guards, road kill, shooting, and habitat chang- es were ruled out as causes of their disappearance. Disease is the most likely cause of the disappear- ance of CYB 1 and the other four maned wolves that disappeared. It is noteworthy that maned wolf CYB 3, antibody positive for CAV, CPV, corona- virus, rabies virus, T. gondii, and L. interrogans spp., was sampled in September 2003 after the dis- appearance of this family group, with which it was known to be in contact. Genetic evaluation of the maned wolf population in the NKMNP has yet to be finished, but it is known that CYB 1 was unre- lated to the other maned wolves in this study and that CYB 2-CYB 4 were all related and in docu- mented close contact with each other. The evidence of exposure in these maned wolves to CDV (two of four) and CPV (four of four) is of greatest concern. CDV has caused serious epidem- ics and population declines in wild carnivores11 and is known to cause mortality in captive maned wolves.7-36 It is most commonly spread by close contact with infectious carnivores through aerosol- ized respiratory secretions but can remain viable in the environment for weeks under the proper con- ditions.19 Parvovirus has been reported as a cause of morbidity and mortality in a number of free- ranging canid species4 and has caused mortality in captive maned wolves.1316-27 Transmitted by the fe- cal-oral route, CPV can survive for months in the environment and does not require close contact for transmission. All four of the maned wolves tested were posi- tive for CAV. CAV likewise has been shown to cause mortality in captive neonatal maned wolves,3 although it is probably of less pathogenic signifi- cance than CDV and CPV. Only one of the maned wolves was antibody pos- itive to coronavirus, rabies virus, and T. gondii. All these agents can cause morbidity and mortality in free-ranging canids and have been implicated as a threat to the conservation of a number of carnivore populations.17 Rabies virus has caused disease in captive maned wolves.36 Although not a significant pathogen alone, coronavirus infections concurrent with other viral or bacterial agents are likely to in- crease morbidity and mortality.14-29 Exposure to T. gondii in this maned wolf was most likely through the ingestion of raw meat (i.e., eating small mam- mals), and thus, probably does not suggest trans- mission from domestic dogs or other carnivores in the region. Leptospirosis is a zoonotic bacterial disease com- monly associated with fever, sepsis, kidney failure, and reproductive abnormalities in a number of an- imal species and humans.20 Many free-ranging ca- nids are seropositive to various L. interrogans ser- ovars without showing illness or functioning as im- portant reservoirs.23 The significance of the findings of positive antibodies to a few L. interrogans ser- ovars in the maned wolves is not known. One of the three maned wolves tested was pos- itive for D. immitis, the causative agent of canine heartworm. This is a potentially fatal, mosquito- borne disease of domestic and wild carnivores. In captivity, maned wolves are often maintained on a heartworm prophylactic because of the devastating effect of this parasite. The role of D. immitis in morbidity and mortality of free-ranging maned wolves is not known. The finding of D. renale ova was not surprising because this parasite is cited as a common pathogen of recently captive maned wolves.25 Dioctophyme renale is often associated with a hypoplastic right kidney in infested maned wolves and could con- tribute to mortality of wolves especially with con- current disease.25-28 Free-ranging canid species usually harbor enteric parasites, including those found in the maned wolves in this study.23 These parasites are not often present in high numbers and do not cause a clinical problem in adult free-ranging canids. However, in animals immunocompromised because of factors such as concurrent disease or physiologic stress re- lated to habitat or population modifications, enteric pathogens may result in disease. The lungworm Capillaria aerophila, detected in the feces of two maned wolves in the study, can cause clinical signs associated with bronchitis and pneumonia, but these animals had no overt respiratory signs. Am- blyomma spp. ticks have been collected from free- ranging maned wolves before this study;12 however, this was the first documented A. ovate record from maned wolves, as previously reported.30 Recently, disease has become a recognized threat to the long-term conservation of free-ranging wild- life.8-10'38 An increased prevalence of disease is most likely associated with global scale anthropo- genic changes, which include human population growth, habitat fragmentation and degradation, the isolation of populations of species, and an increased proximity of humans (and their domestic animals) to wildlife;10 all these appear to be present in Bo- livia. Studies to determine pathogen exposure and disease prevalence in both the threatened wildlife and the domestic animals, which share the habitats with these animals, are imperative to minimize the effect of disease on the conservation of wildlife species. For example, a study on the border of the Madidi National Park in northwestern Boliva showed a high prevalence of exposure of domestic 196 JOURNAL OF ZOO AND WILDLIFE MEDICINE dogs and cats to pathogens, which may infect wild carnivores in that park.15 The data presented in this study have been the basis for formulating a large-scale study of the dis- ease ecology of domestic dogs, sympatric crab?eat- ing foxes (Cerdocyon thous), and maned wolves in and on the perimeter of the NKMNP. The popula- tion of maned wolves in the park has been esti- mated at 120 breeding pairs.34 If so, NKMNP is one of the most important remaining protected areas for free-ranging maned wolves. Determining the risk of infectious disease exposure to the whole population will be important to help park managers as they develop policies to protect maned wolves and other carnivores in the park. Control of disease in domestic and feral domestic animals is likely to become an increasingly impor- tant part of protected area management. Although already followed by many National Park systems, we recommend that two rules be universally fol- lowed in protected areas: 1) prohibit the release of individuals of wild species from captivity into wild populations, unless the wild populations are threat- ened and in need of augmentation for survival, and strict health and genetic evaluations have been per- formed before release37 and 2) exclude all pets and other domestic animals from parks. If parks include human settlements with domestic animals, we rec- ommend that these are vaccinated and monitored for disease. These measures have been instituted in the NKMNP, elsewhere in Bolivia, and at many other sites around the world. Acknowledgments: We thank V. Greco, K. C. Kahler, and L. J. Starr for help with sample pro- cessing and travel logistics, R. G. Robbins for tick identification, and W. B. Karesh for his support of this project. The studies by LHE on maned wolves have been supported by The Douroucouli Founda- tion, The National Geographic Society, the Wildlife Conservation Society, and Amazon Conservation Association. We especially thank D. Rumiz and K. R. Rumiz for their help with logistics and permits. Able field assistance was given by F. del Aguila, V. Chavez, and M. Swarner, and G. Anez volunteered much logistic aid in the field. This project is in collaboration with the Museo de Historia Natural Noel Kempff Mercado, Universidad Autonoma "Gabriel Rene Moreno," Santa Cruz, Bolivia, and is part of their program of biodiversity studies of the Parque Nacional Noel Kempff Mercado. Final- ly, we thank Fundacion Amigos de la Natureleza for supporting and encouraging our work in the park and all the park guards of the Noel Kempff Mercado National Park for their enthusiastic help. LITERATURE CITED 1. Alexander, K. A., and M. J. G. 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