Appendix B

Bison in the Greater Yellowstone Area Draft Agenda 24-25 July 1997 Room 108, Reid Hall Bozeman, MT

24 July 1997

 

8:45

 

 

a. Opening remarks, introductions

 

Lee Paulson, Project Director

Norman Cheville, Principal Investigator

Dale McCullough, Principal Investigator

 

b. Comments

 

Dan Huff, National Park Service, Department of the Interior

Jack Rhyan, APHIS, U. S. Department of Agriculture

Bob Hillman, GYBIC

Factors in transmission

 

 

c. Serology and infection; epidemiology and pathogenesis

 

Tom Roffe

 

d. Brucella abortus and reproductive tissues

 

Jack Rhyan



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Appendix B Bison in the Greater Yellowstone Area Draft Agenda 24-25 July 1997 Room 108, Reid Hall Bozeman, MT 24 July 1997   8:45     a. Opening remarks, introductions   Lee Paulson, Project Director Norman Cheville, Principal Investigator Dale McCullough, Principal Investigator   b. Comments   Dan Huff, National Park Service, Department of the Interior Jack Rhyan, APHIS, U. S. Department of Agriculture Bob Hillman, GYBIC Factors in transmission     c. Serology and infection; epidemiology and pathogenesis   Tom Roffe   d. Brucella abortus and reproductive tissues   Jack Rhyan

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  e. Risk of transmission   Paul Nicoletti 10:15 Break 10:45 f. Relationship among serology, culture test results, and likelihood of infectiousness   Tom Roffe   g. Genetic diversity and disease resistance in bison with active disease eradication   Joe Templeton   h. Molecular biology and Brucella abortus   Peter Gogan 12:15 Lunch break 1:30 i. Modelling   Mike Miller   k. Population changes and distribution   Mary Meagher 3:15 Break 3:45 m. Population dynamics, preliminary data   Peter Gogan   n. Issues in vaccination   Fred Enright; Phil Elzer   o. Safety and effectiveness of existing vaccines   Steve Olsen 5:00 Adjourn 25 June 1997   8:45 p. Elk as a reinfection pathway for bison   Terry Kreeger   q. Outcomes for vaccination program specific to bison given the presence of Brucella abortus in elk and other wildlife   Terry Kreeger

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10:15 Break 10:45 r. Role of vaccine development for bison and elk   Can Brucella abortus be eliminated totally from the GYA by development and use of a vaccine?   What would be the theoretical tradeoffs between a vaccine-only approach and a vaccination approach combined with a test and slaughter program?   Steve Olsen Fred Enright Phil Elzer 11:45 Discussion 12:15 Lunch break 1:30 Public comments from interested parties 3:30 Adjourn

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Brucellosis in the Greater Yellowstone Area Agenda 4 August 1997 National Museum of Wildlife Art Jackson, Wyoming 8:45 Opening remarks, introductions   Lee Paulson, Project Director; Norman Cheville, Principal Investigator; Dale McCullough, Principal Investigator   Comments   Bob Schiller, National Park Service, Department of the Interior; Jack Rhyan, APHIS, U.S. Department of Agriculture; Art Reese, Wyoming Game and Fish Department 9:45 Molecular genetics   Betsy Bricker, National Animal Disease Center 10:15 Break 10:45 Experience in Montana   Keith Aune, Montana Department of Fish, Wildlife and Parks   Issues in transmission   Beth Williams, University of Wyoming   Brucellosis and wildlife research in Yellowstone and Grand Teton National Parks   Wayne Brewster, National Park Service 12:00 Lunch 1:00 Research in elk   Terry Kreeger, Wyoming Game and Fish   RB51 in elk   Phil Elzer, Louisiana State University

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  Vaccine applications at feedgrounds; habitat improvement   Scott Smith, Wyoming Game and Fish 2:30 Break 3:00 Public comment 5:00 Adjourn

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EVALUATION OF BRUCELLA ABORTUS VACCINE STRAIN RB51 IN BISON Philip H. Elzer1 and Donald S. Davis2 1   2   Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 and Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843. Introduction: Host Bison - American Buffalo, Bison bison Organism Brucella abortus first isolated in 1930's from the testicle of bull on National Bison Range, Moiese, MT Serological positive animals found in 1917 in Yellowstone National Park Disease Reproductive disease that causes abortions (late term) that have been documented in the wild. Problem Bison which inhabit the Greater Yellowstone area approximately 4000 animals with up to 50% seropositive for brucellosis Cattle grazing in areas adjacent to the park may be susceptible to infection - Wyoming, Montana, and Idaho. These states could lose their brucellosis-free status. Vaccine strain Brucella abortus RB51 - rough derivative of virulent Brucella abortus strain 2308. Multiple passages on Rifampin led to the loss of the O-polysaccharide side chain of the LPS. Therefore vaccination with this strain does not lead to the production of antibodies which will interfere with sero-diagnostic test for brucellosis. RB51 provides protection against virulent challenge with strain 2308 in a variety of species including cattle, goats, swine, elk and mice. In cattle, RB51 produces similar protection to that achieved with vaccination with S19 without vaccinal titers. In Pregnant animals, RB51 has also been found to be less pathogenic than S19 in that it induces fewer abortions.

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The purpose of this experiment was to evaluate the safety and pathogenesis of Brucella abortus strain RB51 in adult and young bison from a previously exposed herd. Materials and Methods: Animals North American buffalo (Bison bison) were obtained from a reactor herd in Kansas. The herd contained 3 reactor animal as measured by conventional brucellosis serology. The herd was composed of 10 adult males, 7 calves and 14 adult females. The animals were shipped to and housed at Texas A&M University, College Station, TX throughout the experiment. Vaccine Brucella abortus Strain RB51 was obtained from Colorado Serum Co. and rehydrated according to the manufacturer's instructions. Dose Adult males and calves received 1-3×1010 colony forming units subcutaneously (standard calfhood dose in cattle). Adult females received 1×109 colony forming units subcutaneously (standard adult dose in cattle). Experimental Design The adult males and calves plus 5 non-pregnant cows were divided into 2 groups; group 1 was slaughtered at 13 weeks post vaccination, and group 2 was slaughtered at 16 weeks post vaccination. Pregnant females were monitored until parturition, and delivery status was recorded. Live calves remained with the cows, and dead or weak calves were cultured for Brucella. Tissue collection The following tissues were collected aseptically at slaughter: liver, spleen, various lymph nodes, and reproductive tracts. Bacterial culture All of the tissues were homogenized in sterile distilled water, and the homogenates were plated on Brucella selective media.

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Serology Standard western blot analysis using RB51 and smooth field strain cell lysates were performed on all pre- and post-vaccination serum samples. Results Culture data Table 1. Culture data from adult males, non-pregnant females and calves at 13 and 16 weeks post vaccination with RB51. Weeks post-vaccination Brucella abortus strain recovered   Field strain* RB51 13 1 adult male+ none 16 none none * rifampin sensitive, smooth organism + animal number 3

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Results Serology Data Table 2. Western blot analysis of serum samples taken before vaccination and at slaughter using cell lysates from Brucella abortus smooth strain 2308 and rough strain RB51. Animal number Pre-vaccination Post-vaccination   2308 RB51 2308 RB51 1 - - +++ 3* +++ + +++ ++ 5 +++ + +++ ++ 8 - +/- + ++ 9 - +/- + ++ 11 - - - ++ 13 ++++ ++ ++++ +++ 16 +/- - +/- +++ 19 - + - +++ 20 - +/- - +++ 21 - - - +++ 22 - +/- - +++ 24 - +/- - ++ 25 - - - ++++ 26 - - - ++ 28 - - - +++ 29 - + - +++ 30 - ++ - + 31 - - - +/- * culture positive animal (field strain)

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Results Fetal Pathogenesis Table 3. Delivery status of female bison vaccinated with 1 × 109 colony forming units of RB51. No. animals Abortion Live birth Dystocia Pending 9 0 5 1* 3 * culture negative for RB51 and field strain Note: another pregnant animal was necropsied at 16 weeks post vaccination, and both the cow and the calf (150 days) were culture negative for RB51 and field strain. Future Studies: Determination of the vaccine efficacy of strain RB51 in female bison. Group 1. Controls - saline subcutaneously Group 2. RB51 vaccinates (this study) + another vaccination this year Group 3. RB51 vaccinates (Idaho) + another vaccination this year Group 4. RB51 vaccinates (this year) All of the animals will be vaccinated in September. The animals will be bred between October and November. Animals will be challenged in the conjunctival sac with Brucella abortus strain 2308 (1 × 107 colony forming units). Delivery and culture status will be monitored. Conclusions: Vaccination with RB51 in adult or young bison does not result in sero-conversion on standard brucellosis diagnostic tests. Vaccination with RB51 does not result in any gross pathological lesions in calves or adult males. RB51 does not appear to be pathogenic in adult males, non-pregnant females, or calves as measured by increased or prolonged colonization.

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RB51 does not appear to be pathogenic to adult pregnant females when administered to animals from a reactor herd. Further studies are necessary to determine the vaccine efficacy in bison. Acknowledgments: APHIS Brucellosis Research Committee Dr. Jack Rhyan - NVSL Dr. Mike Gilsdorf - USDA Dr. Steve Olsen - NADC Dr. Joe Templeton - Texas A&M University Dr. Fred Enright, Sue Hagius, Joel Walker and William Flahive - Louisiana State University

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BRUCELLOSIS IN THE GREATER YELLOWSTONE AREA: WHAT IS THE PROBLEM? Paul Nicoletti, D.V.M., M.S., Professor, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880 One would have to be the equivalent of Rip Van Winkle to not know of some of the controversies surrounding this subject. Passions and opinions are many and intense. Numerous articles have been published and the broadcast media has been busy and seen by millions. The conflict between the natural wildlife and those who wish to protect them and those of private interests, especially of ranchers, is a classic example of a problem in the United States. Whether it is overpopulation of raccoons in Pinellas County in Florida, or too many deer on Long Island, or brucellosis in Yellowstone Park bison, resolution of these conflicts is difficult. There is a direct confrontation between the concepts of doing whatever is necessary to eradicate a disease and to leave natural forces to function. My credibility to address this group is based upon a near lifetime career of specialization in brucellosis, an episode near Gainesville which also involved bison brucellosis, serving as an expert witness in trials and hearings and distance. Everyone knows that the further one is form the problem, the more expertise can be claimed. Brucellosis is characterized in natural animal hosts by abortion, retained placenta, and pathologic lesions in males. The susceptibility to infection and severity among wildlife hosts have been studied and some results are conflicting. It is quite clear that under natural conditions, brucellosis in bison is of little consequence in fecundity. Bison are not shaggy cows and their behavior, physiology, and responses to infectious agents may be unique. Control and hopeful eradication of brucellosis in domesticated livestock are based upon quarantine, vaccination, and slaughter of seropositive animals. Clearly, these methods are far more difficult to apply in wildlife hosts. The USDA and states depend upon selected surveillance systems to identify possible infected herds of cattle as part of the national brucellosis eradication program. In beef cattle, this surveillance largely relies upon blood samples which are collected at slaughter. This system provides data upon which classification of states depends. Classification of states as

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"free" of cattle brucellosis allows more freedom in cattle commerce. The most affected states by the wildlife brucellosis issue of Wyoming, Idaho, and Montana are classified free. The threat of reducing this state status if one or more cattle herds become infected causes terror among ranchers. Some states have threatened boycotts of cattle movements. These scenarios are harsh and without scientific merit. Surely, disease control officials can have more wisdom in handling disease. The surveillance system which is used to detect a problem should also be evidence of the lack of a problem and there is still no evidence that bison of the YNP have been responsible for any transmission of brucellosis to area cattle. There is anecdotal evidence of transmission from wildlife to cattle in the National Elk Refuge feeding grounds area to a few herds. The elk are known to have a high prevalence of clinical infection and seropositivity. It is important to understand the differences between seropositivity, infection, and disease. Many surveys among free-living bison have found a rather high seroprevalence of brucellosis. When specimens are examined bateriologically, only about 20 percent of those with antibodies are culture positive. Further, it is rare to isolate the bacteria from female reproductive organs. It is interesting that the highest percentage of culture positive bison is among young animals and bulls. The migration and subsequent slaughter of over 1000 bison during the severe winter of 1996-1997 caused enormous outcries among many persons and groups. A further 600 or so starved within the park boundaries. The migration is apparently assisted by snowmobile paths and it has often been suggested that snowmobiles be banned from the park or use be restricted. It seems epidemiologically correct to suggest that the elk feeding grounds in Wyoming present a far greater risk in disease prevalence and management than the bison of YNP. A project to vaccinate some of the elk with biobullets of stain 19 has been in progress for several years with reduction in seroprevalence among the elk. Some Observations It is hyperbole to suggest that if brucellosis cannot be eradicated from the GYA, that efforts to eradicate the disease from domestic animals have been wasted. Many believe that measures which would be necessary to eliminate brucellosis from the wild animals would eliminate the hosts. The work eradication and the state classification system must be modified to conform with reality.

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The excessive attention to the bison and much less attention to the elk are driven by attitudes of ranchers towards the two species. Clearly, there must be some attitudinal changes or ranchers face possible eventual loss of privileges of using public lands for cattle grazing. There is much agreement that the bison population within YNP needs more management. It remains very questionable if this should include possible vaccination to prevent brucellosis. There is no satisfactory vaccine, delivery system or evidence of a disease problem. During testimony in the rather famous Parker lawsuit, Judge Bremmer asked "Dr. Nicoletti, what would you do with the problem of brucellosis in the Greater Yellowstone Area?" My reply was "Your honor, I don't know." While I have several observations and opinions, I feel that my answer puts me among a rather large company of others. I appreciate the invitation to attend this conference and to present this paper. I trust that meetings such as this will educate and perhaps eventually, lead to some compromises and solutions to some very complex issues.

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Safety and Efficacy of Existing Vaccines to Prevent Brucellosis in Bison Steven Olsen, DVM, Ph.D., United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, IA 50010 Protection and lasting immunity against brucellosis is achieved with vaccines containing live bacteria which stimulate a strong cell-mediated immune response. Factors enhancing cell-mediated immunity following administration of live vaccines may include prolonged antigenic stimulation due to proliferation of the vaccine strain within the host and internal antigen processing with more efficient presentation with major histocompatibility antigens (Class I) associated with cellular immune responses. Mouse models of brucellosis indicate that antibodies may have a minor role in short-term protection.1,2 However, studies in cattle have demonstrated a poor correlation between the vigor of the humoral response and protection.3 This is supported by data from cattle experiments in which vaccinated animals which were seronegative prior to midgestational challenge with a virulent Brucella abortus strain were protected against infection and abortion at a time of maximum susceptibility. Additionally, it is customary for animals which abort to have very high titers against brucellosis despite having failed to mount an effective immune response which prevented localization in placental and fetal tissues. An ideal vaccine against brucellosis would persist long enough to induce good immunity without persisting into adulthood, would not cause clinical illness, and would not induce serologic responses which interfere with detection of animals infected with virulent field strains of B. abortus. Typically, vaccines against brucellosis are more efficacious in preventing abortions than preventing infection. Vaccination of wildlife with live vaccines would also have to consider potential detrimental effects on nontarget species, such as predators, which may inadvertently be infected with the vaccine strain. Studies evaluating the safety and efficacy of brucellosis vaccines in bison are limited. When B. abortus strain 19 was administered by hand (1.7 × 109 colony-forming units (CFU)) or ballistic methods (7.7 × 109 CFU) to bison heifer calves, 5% of vaccinated calves had titers on brucellosis

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serologic tests at 2 years of age.4 Following challenge with 1 × 107 CFU of virulent B. abortus strain 2308 during pregnancy, calves vaccinated with strain 19 averaged 25% abortions as compared to 30% abortions in heifers vaccinated with saline (controls). Only 9% of heifers calfhood vaccinated with strain 19 were protected against infection as compared to 17% of nonvaccinated controls. No statistical differences in abortion or infection rates were detected between bison calfhood vaccinated with strain 19 and nonvaccinated controls. A second study evaluating strain 19 as a vaccine for adult bison indicated that a high percentage (58%) of pregnant animals aborted following vaccination.5 When challenged with B. abortus strain 2308 during following pregnancy (13 months after vaccination), the percentage of abortions was less in strain 19-vaccinated bison as compared to nonvaccinated bison (33% versus 96%, respectively). In a similar manner, protection against infection was greater in strain 19 vaccinated bison as compared to nonvaccinates (39% versus 0%, respectively). In addition to its abortogenic effects, the strain 19 vaccine also induced persistent serologic titers on brucellosis surveillance tests and chronic infections in bison vaccinated as adults. Research at the National Animal Disease Center has identified a new vaccine for cattle, B. abortus strain RB51, that is efficacious in preventing abortion and infection.6,7 This vaccine does not induce antibody responses which cause positive responses on brucellosis surveillance tests8,9 and therefore does not impair the identification of Brucella-infected cattle under field conditions. Research projects to evaluate strain RB51 as a vaccine for bison have been initiated at our facility. A preliminary study to evaluate strain RB51 vaccination (1010 CFU) of bison indicated that the vaccine is clinically safe in bison calves and does not induce positive responses on brucellosis surveillance tests.10 Antibody responses against the vaccine strain were detected using a dot-blot test which has been demonstrated to have a high sensitivity and specificity in cattle.11 Adverse clinical signs were not detected following vaccination of bison with strain RB51. The vaccine strain was still present at 16 weeks after vaccination in bison whereas cattle typically clear strain RB51 from the draining lymph by 12 to 14 weeks. These bison were raised to maturity and pasture bred. Data obtained following challenge at midgestation with 1 × 107 CFU of B. abortus strain 2308 suggested that strain RB51 induces some protection in bison. However, as nonvaccinated bison were not included in the challenge portion of the study, conclusions cannot be made on the efficacy of strain RB51 in bison without additional studies.

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Additional studies have been completed evaluating calfhood vaccination of bison with 1010 CFU of strain RB51. These studies have provided further evidence that strain RB51 persists longer in bison when compared to cattle but does not appear to cause adverse clinical signs. Data from these studies suggests that strain RB51 localizes in lymphatic tissues and induces cell-mediated immune responses. Data from biosafety experiments have indicated that the strain RB51 vaccine is not shed from bison following vaccination. The strain RB51 vaccine may have similar problems in adult bison as the strain 19 vaccine. When administered to pregnant bison at a 109 CFU dosage, strain RB51 appears to induced abortion in some animals.12 This dosage is safe in pregnant cattle.13 Ongoing studies will determine if adverse clinical or biosafety effects may limit the use of strain RB51 in adult bison bulls. At the present time, the strain RB51 vaccine is the most likely candidate for use to prevent brucellosis in bison. Continued research efforts will be required to verify the efficacy of strain RB51 to prevent brucellosis in bison. Addition research will also be required to develop delivery methods and guidelines for the use of strain RB51 in management programs to reduce or eliminate Brucella infections in bison. References 1. Winter, A.J., Duncan, J.R., Santisteban, C.G., Douglas, J.T., Adams, L.G. Capacity of passively administered antibody to prevent establishment of Brucella abortus infection in mice. Infect. Immun. 57: 3438-3444, 1989. 2. Cloeckaert, A., Jacques, I., Bosseray, N., Limet, J.N., Bowden, R., Dubray, G., Plommet, M. Protection conferred on mice by monoclonal antibodies directed against outer-membrane-protein antigens of Brucella. J. Med. Microbiol. 34: 175-180, 1991. 3. Nicoletti, P. Vaccination. In: Animal Brucellosis. K. Nielsen and J.R. Duncan eds. 1990. pp 283-300. 4. Davis, D.S., Templeton, J.W., Ficht, T.A., Huber, J.D., Angus, R.D., Adams, L.G. Brucella abortus in bison. II. Evaluation of strain 19 vaccination of pregnant cows. J Wildlife Dis 1991; 27:258-264. 5. Davis, D.S. Summary of Bison/Brucellosis Research conducted at Texas A&M University 1985-1993. Proceedings of North American Public Bison Herds Symposium. July 27-29, 1993, Lacrosse, WI, pp 347-161. 6. Cheville, N.F., Stevens, M.G., Jensen, A.E., Tatum, F.M., Halling, S.M. Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus.

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Am. J. Vet. Res. 54: 1591-1597, 1993. 7. Cheville, N.F., Olsen, S.C., Jensen, A.E., Stevens, M.G., Palmer, M.V. Effects of age at vaccination on efficacy of Brucella abortus strain RB51 to protect cattle against brucellosis. Am. J. Vet Res 57: 1153-1156, 1996. 8. Stevens, M.G., Hennager, S.G., Olsen, S.C., Cheville, N.F.. Serologic responses in diagnostic tests for brucellosis in cattle vaccinated with Brucella abortus strain 19 or RB51. J. Clin. Microbiol. 32: 1065-1066, 1994. 9. Olsen, S.C., Evans, D., Hennager, S.G., Cheville, N.F., Stevens, M.G. Serologic Responses of Calfhood-Vaccinated Cattle to Brucella abortus strain RB51. J. Vet. Diagn. Invest. 8: 451-454, 1996. 10. Olsen, S.C., Cheville, N.F., Kunkle, R.A., Palmer, M.V., Jensen, A.E. Bacterial survival, lymph node changes, and immunologic responses of bison (Bison bison) vaccinated with Brucella abortus strain RB51. J. Wildlife Dis. 33: 146-151, 1997. 11. Olsen, S. C., Stevens, M.G., Cheville, N.F., Schurig, G. Experimental use of a dot-blot assay to measure serologic responses of cattle vaccinated with Brucella abortus strain RB51. J. Vet. Diagn. Invest. (In Press) 12. Palmer, M.V., Olsen, S.C., Jensen, A.E., Gilsdorf, M.J., Philo, L.M., Clarke, P.R., Cheville, N.F. Abortion and placentitis in pregnant bison (Bison bison) induced by the vaccine candidate Brucella abortus strain RB51. Am. J. Vet. Res. 57: 1604-1607, 1996. 13. Palmer, M.V., Olsen, S.C., Cheville, N.F. Safety and immunogenicity of Brucella abortus strain RB51 vaccine in pregnant cattle. Am. J. Vet. Res. 58: 472-477, 1997.

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Lesions and Sites of Tissue Localization of Brucella abortus in Female Bison from Yellowstone National Park: Preliminary Results Jack C. Rhyan,1 Keith Aune,2 Thomas J. Roffe,3 Thomas Gidlewski,1 Darla R. Ewalt,1 and Michael Philo4 1   U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, P.O. Box 844, Ames, IA 50010; 2   Montana Department of Fish Wildlife and Parks, Research and Technical Services Bureau, Montana State University Campus, Bozeman, MT 59717; 3   U.S. Department of the Interior, U.S. Geological Survey, Biological Resources Division, National Wildlife Health Center, Bozeman Station, Montana State University Campus, Bozeman, MT 59717; 4   U.S Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Western Region, 9439 Owl Way, Bozeman, MT 59715 Introduction Brucella abortus produces abortions in cattle, bison (Davis et al., 1990; Rhyan et al., 1994; Williams et al., 1993) and elk (Thorne et al., 1978). Metritis and retained placentas have also been associated with the infection in cattle and bison (Corner and Connell, 1958; Williams et al., 1993). Seminal vesiculitis, orchitis, and epididymitis have been observed with B. abortus infection in male cattle and bison (Corner and Connell, 1958; Creech, 1930; Tunnicliff and Marsh, 1935; Williams et al., 1993; Rhyan et al., 1997). In a recent study, B. abortus was isolated from two or more tissues from six of seven young bison bulls that had recently seroconverted (Rhyan et al., 1997). The purpose of this study was to determine the most frequent sites of tissue localization of B. abortus in female bison from Yellowstone National Park (YNP). Materials and Methods Between February 1995 and January 1997, specimens were collected from 26 seropositive adult female bison. Twenty-five of the animals were killed after leaving YNP, and one animal was killed by YNP personnel because it had a retained placenta and was in close proximity to the northern border of YNP. The cow had recently aborted as evidenced by

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the early date (March of 1995) and the lack of mammary gland development. No fetus or calf was found. Additionally, specimens were collected from a term fetus and placenta that were found near Gardiner, Montana, in April of 1996. Tissue specimens were collected from all animals for culture in accordance with the recommendations published by the Greater Yellowstone Interagency Brucellosis Committee (GYIBC, 1996). Additionally, portions of the uterus and placenta from the cow killed in YNP and portions of lung and placenta from the fetus found near Gardiner, Montana, were fixed in 10 percent neutral buffered formalin and routinely processed for histopathologic examination. Selected tissues were also stained using a previously described immunohistochemical technique (Rhyan et al., 1997) that employs a polyclonal antibody developed against B. abortus (Palmer et al., 1996). Tissues were cultured using a previously described technique (Rhyan et al., 1997) in which each piece of tissue was individually minced, macerated with an equal volume of PBS in a stomacher, and further processed in a glass tissue grinder. The resulting slurry was then poured in aliquots onto the following media: tryptose agar with five percent bovine serum and antibodies (TSA), TSA with ethyl violet, Ewalts medium, and Farrel's medium. Plates were incubated with added CO2 at 37 C for 2 weeks. Cultures were identified and biotyped using the techniques of Alton et al. 1988). Sero status of the animals was initially determined using the card test and was confirmed with the following tests: standard plate, standard tube, rivanol, complement fixation (CF), buffered acidified plate antigen (BAPA), and particle concentrate fluorescence immunoassay (PCFIA). All animals chosen for this study were positive on multiple serologic tests. Results At present, cultures have been completed on 16 of the adult bison and on the fetus. Brucella abortus was isolated from tissues of 7 of the 16 animals. The most common culture positive tissues were the supramammary lymph nodes (7/7), retropharyngeal lymph nodes (5/7), and iliac lymph nodes (5/7). Brucella abortus was isolated from 15 specimens including the placenta and feces from the bison with the retained placenta. The organism was also isolated from 15 sites cultured from the term fetus and placenta found near Gardiner. Histologically, lesions from both placentas and the fetus consisted of necropurulent placentitis and mild pleocellular bronchointerstitial pneumonia. Immunohistochemical staining

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revealed large numbers of brucellae in placental trophoblasts and in phagocytes present in placental and uterine exudate. Fetal lung also contained brucellar antigen in exudate in airways. Discussion: The preliminary results of this study suggest that the supramammary, iliac, and retropharyngeal lymph nodes are the most frequent sites of tissue localization of B. abortus in female bison from YNP. Additionally, the results from the cow that had recently aborted suggest widespread infection in that animal at the time of abortion. The presence of B. abortus in the feces probably resulted from ingestion of portions of the infected placenta and/or licking off the infected fetus. Similar findings in cattle have been reported. The placentitis and fetal pneumonia with large numbers of organisms in placental trophoblasts are consistent with lesions produced by B. abortus in cattle (Payne, 1959), goats (Meador et al., 1986), and captive bison (Davis et al, 1990). REFERENCES Alton GG, Jones LM, Angus RD, Verger JM: 1988, Techniques for the brucellosis laboratory. Institut National de la Recherche Agronomique, Paris, France, 190 pp. Corner AH, Connell R: 1958, Brucellosis in bison, elk, and moose in Elk Island National Park, Alberta, Canada. Can J Comp Med 22:9-20. Creech GT: 1930, Brucella abortus infection in a male bison. North Am Vet 11:35-36. Davis DS, Templeton JW, Ficht TA, et al.: 1990, Brucella abortus in captive bison I. Serology, bacteriology, pathogenesis, and transmission to cattle. J Wildlife Dis 26:360-371. Meador VP, Tabatabai LB, Hagemoser WA, Deyoe BL: 1986, Identification of Brucella abortus in formalin-fixed, paraffin-embedded tissues of cows, goats, and mice with an avidin-biotin-peroxidase complex immunoenzymatic staining technique. Am J Vet Res 47:2147-2150. Palmer MV, Cheville NF, Tatum FM: 1996, Morphometric and histopathologic analysis of lymphoid depletion in murine spleens following infection with Brucella abortus strains 2308, RB51, or an htrA deletion mutant. Vet Pathol 33:282-289.

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Payne JM: 1959, Pathogenesis of experimental brucellosis in the pregnant cow. J. Pathol Bacteriol 78:447-459. Rhyan JC, Quinn WJ, Stackhouse LL, et al.: 1994, Abortion caused by Brucella abortus biovar 1 in a free-ranging bison (Bison bison) from Yellowstone National Park. J Wildlife Dis 30:445-446. Rhyan, JC, Holland SD, Gidlewski T, et al.: 1997, Seminal vesiculitis and orchitis caused by Brucella abortus biovar 1 in young bison bulls from South Dakota. J Vet Diagn Invest 9: IN PRESS. Thorne ET, Morton JK, Blunt FM, Dawson HA: 1978, Brucellosis in elk. II. Clinical effects and means of transmission as determined through artificial infections. J Wildl Dis 14:280-291. Tunnicliff EA, Marsh H: 1935, Bang's disease in bison and elk in Yellowstone National Park and on the National Bison Range. J Am Vet Med Assoc 86:745-752. Williams ES, Thorne ET, Anderson SL, Herriges JD Jr: 1993, Brucellosis in free-ranging bison (Bison bison) from Teton County, Wyoming. J Wildlife Dis 29:118-122.