Brucella abortus strain RB51 vaccination in elk. I. Efficacy of reduced dosage

Bovine brucellosis is a serious zoonotic disease affecting some populations of Rocky Mountain elk (Cervus elaphus nelsoni) and bison (Bison bison) in the Greater Yellowstone Area, USA. The fear that elk and/or bison may spread Brucella abortus to livestock has prompted efforts to reduce or eliminate the disease in wildlife. Brucella abortus strain RB51 (RB51) vaccine has recently been approved for use in cattle. Unlike strain 19 vaccine, RB51 does not cause false positive reactions on standard brucellosis serologic tests. If effective, it may become the vaccine of choice for wildlife. In February 1995, 45 serologically negative female elk calves were trapped and taken to the Sybille Wildlife Research and Conservation Education Unit near Wheatland, Wyoming, USA. In May 1995, 16 of these elk calves were hand-vaccinated with 1 x 10(9) colony forming units (CFU) of RB51, 16 were vaccinated with 1 x 10(8) CFU RB51 by biobullet, and 13 were given a saline placebo. The elk were bred in fall of 1996 and they were challenged with 1 x 10(7) CFU of B. abortus strain 2308 by intraconjunctival inoculation in March 1997. Thirteen (100%) control elk aborted, 14 (88%) hand-vaccinated elk aborted, and 12 (75%) biobullet vaccinated elk aborted or produced nonviable calves. These results suggest that a single dose of 1 x 10(8) to 1 x 10(9) CFU RB51 does not provide significant protection against B. abortus induced abortion in elk. However, the vaccine appears to be safe at this dose and additional study may reveal a more effective RB51 vaccine regimen for elk.     

Brucellosis in elk of eastern Idaho

Brucellosis occurs in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area, which includes portions of Idaho, Wyoming, and Montana. Brucella abortus was first detected in elk in Idaho in 1998, and from 1998 to 2002, serologic surveillance of hunter-killed elk was conducted in northeastern and southeastern Idaho. Prevalence of antibodies in these elk varied annually, but averaged between 2% and 3%. Elk were also trapped in northeastern Idaho from 1998-2002 and tested for brucellosis using serology and tissue culture. In areas where artificial feeding of elk was done, antibody prevalence ranged from 12% to 80% depending on site, age, and sex. At one feeding site (Rainey Creek), a decline in the prevalence of antibodies (from 56.8% in 1999 to 13.5% in 2002) was detected after the removal of seropositive elk over 4 yr. Seropositive elk removed from two artificial winter feeding sites (Rainey Creek and Conant Creek) were euthanized and sampled or held in captivity and allowed to calve prior to euthanasia and necropsy. At necropsy, B. abortus biovar 1 and B. abortus biovar 4 were isolated from both cows and calves; however, biovar 4 was predominant. A dual infection with both biovars was found in one calf born to a seropositive cow from which biovar 4 was isolated. Abortions (16%), stillbirths (8%), and weak calves (4%) were observed in these elk. These findings confirm the presence of brucellosis in elk in eastern Idaho and provide information on disease management options.     

The role of wild North American ungulates in the epidemiology of bovine brucellosis: a review

Published reports of Brucella abortus infections in wild North American ungulates and domestic cattle herds were reviewed to determine if infection in these species was related. Bison (Bison bison) were frequently found infected, but are probably a minor threat to livestock due to their current limited distribution. Most elk (Cervus elaphus) were free of infection except where their range was shared with infected bison or livestock. Deer (Odocoileus spp.), pronghorns (Antilocapra americana), moose (Alces alces), and bighorn sheep (Ovis canadensis) appeared to be insignificant hosts of Brucella abortus. The lack of significant wild ungulate hosts and the distribution of infected livestock herds in the United States suggests that wild ungulates are of little importance in the epidemiology of infections by B. abortus in cattle.     

Pathology of brucellosis in bison from Yellowstone National Park

Between February 1995 and June 1999, specimens from seven aborted bison (Bison bison) fetuses or stillborn calves and their placentas, two additional placentas, three dead neonates, one 2-wk-old calf, and 35 juvenile and adult female bison from Yellowstone National Park (USA) were submitted for bacteriologic and histopathologic examination. One adult animal with a retained placenta had recently aborted. Serum samples from the 35 juvenile and adult bison were tested for Brucella spp. antibodies. Twenty-six bison, including the cow with the retained placenta, were seropositive, one was suspect, and eight were seronegative. Brucella abortus biovar 1 was isolated from three aborted fetuses and associated placentas, an additional placenta, the 2-wk-old calf, and 11 of the seropositive female bison including the animal that had recently aborted. Brucella abortus biovar 2 was isolated from one additional seropositive adult female bison. Brucella abortus was recovered from numerous tissue sites from the aborted fetuses, placentas and 2-wk-old calf. In the juvenile and adult bison, the organism was more frequently isolated from supramammary (83%), retropharyngeal (67%), and iliac (58%) lymph nodes than from other tissues cultured. Cultures from the seronegative and suspect bison were negative for B. abortus. Lesions in the B. abortus-infected, aborted placentas and fetuses consisted of necropurulent placentitis and mild bronchointerstitial pneumonia. The infected 2-wk-old calf had bronchointerstitial pneumonia, focal splenic infarction, and purulent nephritis. The recently-aborting bison cow had purulent endometritis and necropurulent placentitis. Immunohistochemical staining of tissues from the culture-positive aborted fetuses, placentas, 2-wk-old calf, and recently-aborting cow disclosed large numbers of B. abortus in placental trophoblasts and exudate, and fetal and calf lung. A similar study with the same tissue collection and culture protocol was done using six seropositive cattle from a B. abortus-infected herd in July and August, 1997. Results of the bison and cattle studies were similar.     

Experimental infection of some North American wild ruminants and domestic sheep with Mycobacterium paratuberculosis: clinical and bacteriological findings

Mycobacterium paratuberculosis originally isolated from bighorn sheep (Ovis canadensis) with spontaneous paratuberculosis was used to orally inoculate Rocky Mountain elk (Cervus elaphus nelsoni) calves, mule deer (Odocoileus hemionus) fawns, white-tailed deer (Odocoileus virginianus) fawns, bighorn X mouflon (Ovis musimon) hybrid lambs, and domestic lambs. All experimentally exposed animals became infected. During the first year of infection, hybrid and domestic sheep were able to control the infection but infection was progressive in elk and deer. Clinical paratuberculosis occurred only in mule deer.     

Brucella abortus in Bison. II. Evaluation of strain 19 vaccination of pregnant cows

Protection against Brucella abortus induced abortion and infection provided by strain 19 (S19) vaccination was evaluated in American bison (Bison bison). Forty-eight pregnant bison were manually inoculated (MI) with S19 vaccine, 44 were ballistically inoculated (BI) with an absorbable hollow pellet containing lyophilized S19, and 46 were manually injected with buffered saline as non-vaccinated controls (NVC). All bison were Brucella spp. seronegative prior to the experiment, in the second trimester of pregnancy, and were randomly assigned to experimental groups. Approximately 60 days post-vaccination, abortions were observed in the vaccinated bison. Brucella abortus strain 19 was recovered from a bison that had recently aborted, her fetus, and from 11 of 12 other aborted fetuses. Fifty-eight percent (53 of 92) of vaccinated bison aborted, and no abortions were observed in the NVC bison. One cow aborted during her second post-vaccinal pregnancy and S19 was identified from the dam and fetus indicating that chronic S19 infections can occur in bison. Positive antibody titers were present 10 mo post-vaccination in 73% (66 of 91) of the bison. Thirteen mo post-vaccination, 30 MI vaccinates, 27 BI vaccinates, and 30 NVC bison were challenged during the second trimester of pregnancy with 1 x 10(7) CFU of B. abortus strain 2308 via bilateral conjunctival inoculation. Protection against abortion was 67% (P less than or equal to 0.0001) for vaccinated bison compared to 4% in NVC. Protection against B. abortus infection was determined to be 39% (P greater than or equal to 0.001) for vaccinates and 0% (zero of 30) for NCV. Persistent antibody titers, vaccine induced abortions, and chronic S19 infections indicate that the S19 vaccine doses used in this study are not suitable for pregnant bison.     

Yersinia enterocolitica: an unlikely cause of positive brucellosis tests in greater yellowstone ecosystem bison (Bison bison)

Yersinia enterocolitica serotype O:9 has identical O-antigens to those of Brucella abortus and has apparently caused false-positive reactions in numerous brucellosis serologic tests in elk (Cervus canadensis) from southwest Montana. We investigated whether a similar phenomenon was occurring in brucellosis antibody-positive bison (Bison bison) using Y. enterocolitica culturing techniques and multiplex PCR of four diagnostic loci. Feces from 53 Yellowstone bison culled from the population and 113 free-roaming bison from throughout the Greater Yellowstone Ecosystem (GYE) were tested. Yersinia enterocolitica O:9 was not detected in any of 53 the bison samples collected at slaughter facilities or in any of the 113 fecal samples from free-ranging bison. One other Y. enterocolitica serotype was isolated; however, it is not known to cause cross-reaction on B. abortus serologic assays because it lacks the perosamine synthetase gene and thus the O-antigens. These findings suggest that Y. enterocolitica O:9 cross-reactivity with B. abortus antigens is unlikely to have been a cause of false-positive serology tests in GYE bison and that Y. enterocolitica prevalence was low in bison in the GYE during this study.     

Validation of a Brucella abortus competitive enzyme-linked immunosorbent assay for use in Rocky Mountain elk (Cervus elaphus nelsoni)

Brucellosis caused by infection with Brucella abortus is present in some elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (parts of Wyoming, Montana, and Idaho, USA). Since 1985, the Wyoming Game and Fish Department has vaccinated elk on elk feedgrounds in northwestern Wyoming during the winter months using B. abortus strain 19 (strain 19). Analysis of this vaccination program is hampered by the inability of standard serologic tests to differentiate between strain 19 vaccinated elk and those exposed to field strain B. abortus. In 1993, a competitive enzyme-linked immunosorbent assay (cELISA) was licensed to serologically differentiate between strain 19 vaccinated cattle and cattle exposed to field strain B. abortus. Seven groups of elk sera representing various B. abortus exposure histories were used to validate the cELISA test for elk. The cELISA test differentiated strain 19 vaccinated elk from elk that were challenged with B. abortus strain 2308, a pathogenic laboratory strain. The specificity of the cELISA was 96.8% for elk vaccinated with strain 19 only and sampled between 6 mo and 2 yr post vaccination, or with no B. abortus exposure. The sensitivity of the cELISA was 100%. The cELISA test will be useful in evaluating sera collected from elk in vaccinated, brucellosis endemic herds in the Greater Yellowstone Area.     

Toxoplasmic encephalitis in a free-ranging Rocky Mountain bighorn sheep from Washington

A 4-mo-old free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from the Hells Canyon area (Washington, USA) was diagnosed with encephalitis associated with Toxoplasma gondii infection. The sheep had concurrent pneumonic pasteurellosis and resided in a geographic area with endemic Pasteurella-associated pneumonia and mortality in bighorn sheep. The brain had multifocal necrotizing and nonsuppurative encephalitis with intralesional protozoa. The protozoa were identified as T. gondii by immunohistochemistry. To our knowledge, this is the first report of T. gondii infection in a Rocky Mountain bighorn sheep.     

Brucella abortus strain RB51 vaccination in elk. II. Failure of high dosage to prevent abortion

Brucella abortus strain RB51 is used as a vaccine because it induces antibodies that do not react on standard serologic tests for brucellosis allowing differentiation between vaccination and infection. Strain RB51 was evaluated in captive elk (Cervus elaphus) to determine if vaccination protected against abortion following experimental challenge. Thirty elk were vaccinated intramuscularly with 1.0 x 10(10) colony-forming units (CFU) of strain RB51 in March 1998. Fourteen of these were given a booster dose of 1.13 x 10(10) CFU exactly 1 yr later. All vaccinated elk seroconverted via a modified dot blot assay to strain RB51 with the booster group having higher titers (P < or = 0.001). Seventeen other elk served as unvaccinated controls. All elk were bred and determined pregnant using pregnancy-specific protein B analysis. Elk were challenged in March 2000 with 1.1 x 10(7) CFU of B. abortus strain 2308 administered intraconjunctivally and all elk seroconverted to strain 2308. Fifteen of 17 control elk aborted; 16 of 16 elk given a single vaccination aborted (P = 0.44); and 13 of 14 elk given a booster aborted (P = 0.86). There were two viable calves in the control group and one in the booster group. Strain 2308 was recovered from fetuses and nonviable calves in all groups. Based on the results of this and other studies, the use of strain RB51 to prevent abortion in elk cannot be recommended.     

DNA vaccination of bison to brucellar antigens elicits elevated antibody and IFN-γ responses

Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area. During the past several years, cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Idaho, USA; Wyoming, USA; and Montana, USA. Current livestock B. abortus vaccines have not proven to be efficacious in bison (Bison bison) or elk (Cervus elaphus nelsoni). One problem with the lack of vaccine efficacy may stem from the failure to understand wildlife immune responses to vaccines. In an attempt to understand their immune responses, bison were vaccinated with eukaryotic DNA expression vectors encoding the Brucella periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). These DNA vaccines have previously been shown to be protective against Brucella infection in mice. Bison were immunized intramuscularly at weeks 0, 2, and 4 with bp26 and TF DNA vaccines plus CpG adjuvant or empty vector (control) plus CpG. Blood samples were collected before vaccination and at 8, 10, and 12 wk after primary vaccination. The results showed that bison immunized with bp26 and TF DNA vaccines developed enhanced antibody, proliferative T cell, and interferon-gamma (IFN-γ) responses upon in vitro restimulation with purified recombinant bp26 or TF antigens, unlike bison immunized with empty vector. Flow cytometric analysis revealed that the percentages of CD4(+) and CD8(+) T lymphocytes from the DNA-vaccinated groups were significantly greater than they were for those bison given empty vector. These data suggest that DNA vaccination of bison may elicit strong cellular immune responses and serve as an alternative for vaccination of bison for brucellosis.     

Brucella abortus in captive bison. I. Serology, bacteriology, pathogenesis, and transmission to cattle

Two groups of six, non-brucellosis vaccinated, brucellosis seronegative pregnant American bison (Bison bison) were individually challenged with 1 x 10(7) colony forming units (CFU) of Brucella abortus strain 2308. Three days after challenge, each bison group was placed in a common paddock with six non-vaccinated, brucellosis susceptible, pregnant domestic heifers. In a parallel study, two groups of six susceptible, pregnant cattle were simultaneously challenged with the identical dose as the bison and each group was placed with six susceptible cattle in order to compare bison to cattle transmission to that observed in cattle to cattle transmission. Blood samples were collected from bison and cattle weekly for at least 1 mo prior to exposure to B. abortus and for 180 days post-exposure (PE). Sera from the bison and cattle were evaluated by the Card, rivanol precipitation, standard plate agglutination, standard tube agglutination, cold complement fixation tube, warm complement fixation tube, buffered acidified plate antigen, rapid screening, bovine conjugated enzyme linked immunosorbent assay, bison or bovine conjugated enzyme linked immunosorbent assay, and the hemolysis-in-gel techniques for the presence of antibodies to Brucella spp. At the termination of pregnancy by abortion or birth of a live-calf, quarter milk samples, vaginal swabs, and placenta were collected from the dam. Rectal swabs were collected from live calves, and mediastinal lymph nodes, abomasal contents and lung were taken at necropsy from aborted fetuses for culture of Brucella spp. These tissues and swabs were cultured on restrictive media for the isolation and identification of Brucella spp. Pathogenesis of brucellosis in bison was studied in an additional group of six pregnant bison which were challenged with 1 x 10(7) CFU of B. abortus strain 2308. One animal was euthanatized each week PE. Tissues were collected at necropsy and later examined bacteriologically and histologically. Lesions of brucellosis in bison did not significantly differ grossly or histologically from those in cattle. There were six abortions and two nonviable calves in the bison group, as compared to nine abortions in the 12 similarly inoculated cattle. As determined by bacterial isolations, transmission of B. abortus from bison to cattle (five of 12 susceptible cattle became infected) did not differ statistically from cattle to cattle transmission (six of 12 susceptible cattle became infected) under identical conditions. No single serologic test was constantly reliable to diagnosing B. abortus infected bison for 8 wk PE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hemorrhagic disease in bighorn sheep in Arizona

Two bighorn sheep from Arizona (USA) were submitted for necropsy. One was a Rocky Mountain bighorn (Ovis canadensis canadensis) and the other was a desert bighorn (Ovis canadensis mexicana). Both had lesions consistent with those of hemorrhagic disease (HD). Epizootic hemorrhagic disease virus (EHDV) type-2 and bluetongue virus (BTV) type-17, respectively, were isolated from the sheep tissues. To our knowledge, HD caused by either EHDV or BTV infection has not been documented previously in Arizona bighorn sheep.     

Experimental Brucella abortus infection in wolves

Four juvenile male wolves (Canis lupus) each received an oral dose of 1.6-1.7 x 10(12) colony-forming units of Brucella abortus biovar 1 isolated from a bison (Bison bison) in Wood Buffalo National Park (Canada), and two others served as negative controls. Infected wolves did not show clinical signs of disease but did develop high Brucella antibody titers. Small numbers of B. abortus were excreted sporadically in feces until day 50 postinoculation (PI). Very small numbers of the bacterium were isolated from urine of only one wolf late on the same day that it was infected, and very small numbers of colonies of B. abortus were obtained from buccal swabs of three wolves for up to 48 hr PI. Two infected wolves euthanized 6 mo after the start of the experiment had no lesions, and colonies of B. abortus were isolated from thymus and most major lymph nodes. The other two infected wolves euthanized 12 mo after the start of the experiment had no lesions, and smaller numbers of brucellae were recovered from fewer lymph nodes compared with the wolves killed 6 mo earlier. The sporadic excretion of very small numbers of brucellae by the wolves was insignificant when compared with the infective dose for cattle. Brucella abortus, brucellosis, Canis lupus, pathogenesis, serology, wolf.     

Safety of Brucella abortus strain RB51 vaccine in non-target ungulates and coyotes

Brucellosis is endemic in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area (GYA; USA). It is possible that an oral brucellosis vaccine could be developed and disseminated in the GYA to reduce disease transmission. Should this occur, non-target species other than elk and bison may come in contact with the vaccine resulting in morbidity or mortality. To assess biosafety, bighorn sheep (Ovis canadensis; n = 10), pronghorn (Antilocapra americana; n = 9), mule deer (Odocoileus hemionus; n = 11), moose (Alces alces shirasi; n = 10), and coyotes (Canis latrans; n = 24) were given a single oral dose of at least 1.0 x 10(10) colony-forming units of Brucella abortus strain RB51 vaccine (RB51). Animals were randomly divided into vaccinated and control groups. Ungulates were captured, blood sampled, and swabs taken from the nares, rectum, and vagina for bacterial culture on day 0, 42, and 84 post-inoculation (PI). On day 42, the vaccinated group became a control group and vice versa in a crossover design. Blood and swab samples were taken from coyotes on days 0, 14, 28, and 42 PI. There was no crossover for the coyote study. Two coyotes from each group were also euthanized and cultured for RB51 on days 42, 84, 168, and 336 PI. Blood samples were analyzed for hematologic changes and antibodies to RB51 using a modified dot-blot assay. No morbidity or mortality as a result of vaccination was observed in any animal. There were no differences in hematologic parameters at any time for ungulate species; vaccinated coyotes had higher hematocrit, hemoglobin, and eosinophil counts (P < or = 0.006). All individuals, except some moose, seroconverted to RB51. Strain RB51 was cultured from oropharyngeal lymph nodes from one coyote 42 days PI and from a moose 117 days PI. This study suggested that a single oral dose of RB51 was safe in these species.     

Susceptibility of two Rocky Mountain bighorn sheep to experimental infection with Anaplasma ovis.

In North America, the role of wild ruminants in the epidemiology of anaplasmosis has not been clearly defined. Such information is particularly meager in regard to bighorn sheep. We report the susceptibility of two Rocky Mountain bighorn sheep (Ovis canadensis canadensis) to experimental infection with a well characterized field isolate of Anaplasma ovis obtained from domestic sheep in Idaho. Both bighorn sheep developed infection resulting in severe clinical disease, with relatively high parasitemias, icterus and anemia. One animal required tetracycline therapy and responded well to treatment, while the other recovered uneventfully without treatment. Both bighorn sheep were spleen-intact, a condition that in A. ovis-exposed domestic sheep typically is associated with mild infection. The results indicate that bighorn sheep may be adversely affected if exposed to the organism in nature.     

Bison PRNP genotyping and potential association with Brucella spp. seroprevalence

The implication that host cellular prion protein (PrP(C)) may function as a cell surface receptor and/or portal protein for Brucella abortus in mice prompted an evaluation of nucleotide and amino acid variation within exon 3 of the prion protein gene (PRNP) for six US bison populations. A non-synonymous single nucleotide polymorphism (T50C), resulting in the predicted amino acid replacement M17T (Met --> Thr), was identified in each population. To date, no variation (T50; Met) has been detected at the corresponding exon 3 nucleotide and/or amino acid position for domestic cattle. Notably, 80% (20 of 25) of the Yellowstone National Park bison possessing the C/C genotype were Brucella spp. seropositive, representing a significant (P = 0.021) association between seropositivity and the C/C genotypic class. Moreover, significant differences in the distribution of PRNP exon 3 alleles and genotypes were detected between Yellowstone National Park bison and three bison populations that were either founded from seronegative stock or previously subjected to test-and-slaughter management to eradicate brucellosis. Unlike domestic cattle, no indel polymorphisms were detected within the corresponding regions of the putative bison PRNP promoter, intron 1, octapeptide repeat region or 3'-untranslated region for any population examined. This study provides the first evidence of a potential association between nucleotide variation within PRNP exon 3 and the presence of Brucella spp. antibodies in bison, implicating PrP(C) in the natural resistance of bison to brucellosis infection.     

Muellerius capillaris (Mueller, 1889) (Nematoda: Protostrongylidae): an unusual finding in Rocky Mountain bighorn sheep (Ovis canadensis canadensis Shaw) in South Dakota

Lungs and fecal samples from nine hunter-killed Rocky Mountain bighorn sheep were examined for lungworms. All samples contained adults and/or larvae of Muellerius capillaris (Mueller, 1889). Protostrongylus spp., the lungworms commonly reported from bighorn sheep, were not present in any samples. Larvae of M. capillaris bear a spine on the dorsal side of the posterior end and are shorter than dorsal-spined larvae of other lungworms recorded from North American ungulates. Larvae similar in shape but longer than those of Muellerius were found in free-ranging bighorn sheep in Alberta and British Columbia. In addition, dorsal-spined larvae have been found in bighorn sheep in Montana, North Dakota, and Washington. The identity of the dorsal-spined larvae is known only from sheep in South Dakota. Thus, caution must be taken when diagnosing lungworm infections in Rocky Mountain bighorn sheep.     

Paratuberculosis (Johne's disease) in bighorn sheep and a Rocky Mountain goat in Colorado.

Between May, 1972 and February, 1978, six cases of paratuberculosis (Johne's Disease) caused by Mycobacterium paratuberculosis were diagnosed in free-ranging Rocky Mountain bighorn sheep (Ovis canadensis) and one Rocky Mountain goat (Oreamnos americanus) on or near Mt. Evans in Colorado. Diagnosis of paratuberculosis was based on gross and histopathologic examination of the animals and by isolation of M. paratuberculosis from three sheep and the goat. The clinical signs and pathologic changes seen in the bighorn sheep resembled those described in cattle, while the lesions in the goat were similar to those described for domestic sheep and goats.     

Seroprevalence of Toxoplasma gondii in Rocky Mountain bighorn sheep (Ovis canadensis)

Serum samples from 697 Rocky Mountain bighorn sheep (Ovis canadensis) from North America were examined for antibodies to Toxoplasma gondii by the modified agglutination test incorporating mercaptoethanol and formalin-fixed tachyzoites. Antibodies to T. gondii were found in 25 of 697 (3.6%) sheep in titers of 1:25 (8 sheep), 1:50 (4 sheep), 1:100 (7 sheep), 1:200 (1 sheep), 1:400 (1 sheep), 1:800 (1 sheep), and 1:1,600 (3 sheep). This is the first record of T. gondii exposure in bighorn sheep.