ABSTRACT We evaluated hemolyzed, bacterially contaminated, and Nobuto filter paper-derived serum, collected from 50 Rocky Mountain elk ( Cervus elaphus nelson ) in 2017 and 2019, divided into eight treatments to determine antibody retention. Serum was analyzed on Brucella abortus -specific fluorescence polarization assay utilizing plates and tubes. Reference titers and serostatus were compared to serum held at 22 C for 4, 8, 12, and 16 d; frozen clotted blood; blood with 2% and 10% elk rumen content (held for 8 d at 22 C); and serum eluted from Nobuto filter paper. Using Cohen's kappa test of agreement, plate assay serostatus agreement was substantial or outstanding in all treatments. Serostatus agreement was outstanding in all treatments utilizing tubes. The mean change in score (treatment minus reference) showed significant negative bias in serosuspect or seropositive animals in the frozen, 2% rumen, and 10% rumen treatments on the plate assay, and the day 16 and 10% rumen treatments on the tube assay, that could ultimately result in an animal being misclassified into a serosuspect or seronegative category. Serum eluted from Nobuto filter paper produced inconsistent results and is not recommended as an alternative to serum derived from blood. Although the potential for misclassification of animals with low titers exists, analyzing hemolyzed and bacterially contaminated serum from Brucella abortus nonendemic areas can increase sample size and the potential to detect seropositive animals.

In contrast to broad range expansion through translocations, many mountain goat ( Oreamnos americanus ) populations have shown signs of decline. Recent documentation of pneumonia in mountain goats highlights their susceptibility to bacterial pathogens typically associated with bighorn sheep ( Ovis canadensis ) epizootics. Respiratory pathogen communities of mountain goats are poorly characterized yet have important implications for management and conservation of both species. We characterized resident pathogen communities across a range of mountain goat populations as an initial step to inform management efforts. Between 2010 and 2017, we sampled 98 individuals within three regions of the Greater Yellowstone Area (GYA), with a smaller sampling effort in southeast Alaska, US. Within the GYA, we detected Mycoplasma ovipneumoniae in two regions and we found at least two Pasteurellaceae species in animals from all regions. Mannheimia haemolytica was the only pathogen that we detected in southeast Alaska. Given the difficult sampling conditions, limited sample size, and imperfect detection, our failure to detect specific pathogens should be interpreted with caution. Nonetheless, respiratory pathogens within the GYA may be an important, yet underappreciated, cause of mountain goat mortality. Moreover, because of the strong niche overlap of bighorn sheep and mountain goats, interspecific transmission is an important concern for managers restoring or introducing mountain ungulates within sympatric ranges.

The wildlife of the Greater Yellowstone Ecosystem carries brucellosis, which was first introduced to the area by cattle in the 19th century. Brucellosis transmission between wildlife and livestock has been difficult to study due to challenges in culturing the causative agent, Brucella abortus . We examined B. abortus transmission between American bison ( Bison bison ), Rocky Mountain elk ( Cervus elaphus nelsoni ), and cattle ( Bos taurus ) using variable number tandem repeat (VNTR) markers on DNA from 98 B. abortus isolates recovered from populations in Idaho, Montana, and Wyoming, US. Our analyses reveal interspecies transmission. Two outbreaks (2007, 2008) in Montana cattle had B. abortus genotypes similar to isolates from both bison and elk. Nevertheless, similarity in elk and cattle isolates from the 2008 outbreak suggest that elk are the likely source of brucellosis transmission to cattle in Montana and Wyoming. Brucella abortus isolates from sampling in Montana appear to be divided in two clusters: one found in local Montana elk, cattle, and bison; and another found mainly in elk and a bison from Wyoming, which is consistent with brucellosis having entered Montana via migration of infected elk from Wyoming. Our findings illustrate complex patterns of brucellosis transmission among elk, bison, and cattle as well as the utility of VNTRs to infer the wildlife species of origin for disease outbreaks in livestock.

We evaluated bighorn sheep ( Ovis canadensis ) ewes and their lambs in captivity to examine the sources and roles of respiratory pathogens causing lamb mortality in a poorly performing herd. After seven consecutive years of observed December recruitments of <10%, 13 adult female bighorn sheep from the remnant Gribbles Park herd in Colorado, US were captured and transported to the Thorne-Williams Wildlife Research Center in Wyoming in March 2013. Ewes were sampled repeatedly over 16 mo. In April 2014, ewes were separated into individual pens prior to lambing. Upon death, lambs were necropsied and tested for respiratory pathogens. Six lambs developed clinical respiratory disease and one lamb was abandoned. Pathology from an additional six lambs born in 2013 was also evaluated. Mycoplasma ovipneumoniae , leukotoxigenic Mannheimia spp., leukotoxigenic Bibersteinia trehalosi , and Pasteurella multocida all contributed to lamb pneumonia. Histopathology suggested a continuum of disease, with lesions typical of pasteurellosis predominating in younger lambs and lesions typical of mycoplasmosis predominating in older lambs. Mixed pathology was observed in lambs dying between these timeframes. We suspected that all the ewes in our study were persistently infected and chronically shedding the bacteria that contributed to summer lamb mortality.

Brucellosis (caused by the bacterium Brucella abortus ) is a zoonotic disease endemic in wild elk ( Cervus canadensis ) of the Greater Yellowstone Ecosystem, US. Because livestock and humans working with elk or livestock are at risk, validated tests to detect the B. abortus antibody in elk are needed. Using the κ-statistic, we evaluated the buffered, acidified plate antigen (BAPA) assay for agreement with the results of the four serologic tests (card test [card], complement fixation test [CF], rivanol precipitation plate agglutination test [RIV], standard plate agglutination test [SPT]) that are approved by the US Department of Agriculture for the detection of the B. abortus antibody in elk. From 2006 to 2010, serum samples collected from elk within B. abortus –endemic areas ( n = 604) and nonendemic areas ( n = 707) and from elk culture-positive for B. abortus ( n = 36) were split and blind tested by four elk serum diagnostic laboratories. κ-Values showed a high degree of agreement for the card (0.876), RIV (0.84), and CF (0.774) test pairings and moderate agreement for the SPT (0.578). Sensitivities for the BAPA, card, RIV, CF, and SPT were 0.859, 0.839, 0.899, 1.00, and 0.813, whereas specificities were 0.986, 0.993, 0.986, 0.98, and 0.968, respectively. The positive predictive values and the negative predictive values were calculated for 2.6%, 8.8%, and 16.2% prevalence levels. These findings suggest the BAPA test is a suitable screening test for the B. abortus antibodies in elk.

Bighorn sheep ( Ovis canadensis ) sinus tumors are hyperplastic to neoplastic, predominantly stromal masses of the paranasal sinuses that expand the sinus lining and obstruct the sinus cavities. Obstruction of the sinus cavities and disruption of normal sinus lining anatomy may interfere with clearance of bacterial pathogens from the upper respiratory tract. To examine this possibility, we explored whether the presence of sinus tumor features (tumor score) affected the likelihood of detecting potentially pathogenic bacteria from upper respiratory sinus lining tissues in bighorn sheep. We developed or used existing PCR assays for the detection of leukotoxigenic Pasteurellaceae and Mycoplasma ovipneumoniae in sinus lining tissues collected from 97 bighorn sheep in Colorado, US from 2009 to 2012. With the use of logistic regression analyses we found that tumor score was a good predictor of the probability of detecting potentially pathogenic bacteria in sinus lining tissues; we were more likely to detect potentially pathogenic bacteria from samples with high tumor scores. These findings add to our understanding of possible mechanisms for the maintenance and shedding of bacterial agents from the upper respiratory tracts of bighorn sheep.

Mannheimia haemolytica consistently causes severe bronchopneumonia and rapid death of bighorn sheep ( Ovis canadensis ) under experimental conditions. However, Bibersteinia trehalosi and Pasteurella multocida have been isolated from pneumonic bighorn lung tissues more frequently than M. haemolytica by culture-based methods. We hypothesized that assays more sensitive than culture would detect M. haemolytica in pneumonic lung tissues more accurately. Therefore, our first objective was to develop a PCR assay specific for M. haemolytica and use it to determine if this organism was present in the pneumonic lungs of bighorns during the 2009–2010 outbreaks in Montana, Nevada, and Washington, USA. Mannheimia haemolytica was detected by the species-specific PCR assay in 77% of archived pneumonic lung tissues that were negative by culture. Leukotoxin-negative M. haemolytica does not cause fatal pneumonia in bighorns. Therefore, our second objective was to determine if the leukotoxin gene was also present in the lung tissues as a means of determining the leukotoxicity of M. haemolytica that were present in the lungs. The leukotoxin-specific PCR assay detected leukotoxin gene in 91% of lung tissues that were negative for M. haemolytica by culture. Mycoplasma ovipneumoniae , an organism associated with bighorn pneumonia, was detected in 65% of pneumonic bighorn lung tissues by PCR or culture. A PCR assessment of distribution of these pathogens in the nasopharynx of healthy bighorns from populations that did not experience an all-age die-off in the past 20 yr revealed that M. ovipneumoniae was present in 31% of the animals whereas leukotoxin-positive M. haemolytica was present in only 4%. Taken together, these results indicate that culture-based methods are not reliable for detection of M. haemolytica and that leukotoxin-positive M. haemolytica was a predominant etiologic agent of the pneumonia outbreaks of 2009–2010.

We used 16S rRNA sequencing and leukotoxin gene ( lktA ) screening via PCR assay to clarify phylogenetic and epidemiologic relationships among Pasteurellaceae isolated from bighorn sheep ( Ovis canadensis ). Only six of 21 bighorn isolates identified as “Mannheimia haemolytica” in original laboratory reports appeared to be isolates of M. haemolytica sensu stricto based on 16S rRNA sequence comparisons; the remainder grouped with M. glucosida ( n =8) or M. ruminalis ( n =7). Similarly, 16S rRNA sequence comparisons grouped only 16 of 25 trehalose-fermenting bighorn isolates with reference strains of Bibersteinia trehalosi ; nine other trehalose-fermenting bighorn isolates formed a clade divergent from B. trehalosi reference strains and may belong to another species. Of the 16 bighorn isolates identified as B. trehalosi by 16S rRNA sequences, only nine carried detectable lktA and thus seemed likely pathogens; none of the Bibersteinia clade isolates yielded detectable lktA despite reportedly showing β hemolysis in culture. Our findings suggest that traditional metabolism-based methods for identifying Pasteurellaceae isolates lack sufficient accuracy and resolution for reliably discerning bacterial causes of respiratory disease in bighorn sheep. Consequently, these traditional methods should minimally be augmented by molecular techniques to improve epidemiologic relevance. Streamlined surveillance approaches focused primarily on detecting pathogenic Pasteurellaceae (e.g., M. haemolytica sensu stricto and lktA-positive B. trehalosi ) and other select pathogens may be most informative for investigating and managing bighorn respiratory disease.

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.

Brucella abortus is the causative agent of brucellosis, a disease enzootic in populations of free-ranging elk ( Cervus elaphus ) and bison ( Bison bison ) in the Greater Yellowstone Ecosystem, USA. We define the distribution of the disease in elk throughout Wyoming, USA, using three epidemiologic/geographic classifications based on winter-foraging opportunity. Antibody prevalence for >3,300 yearling and adult, female elk, sampled from supplemental feedgrounds in western Wyoming, USA, since 1985, was 21.9%. Surveillance of nearly 3,800 winter free-ranging, adult, female elk, sampled from herd units adjacent to feedgrounds from 1991 to 2008, indicated a total antibody prevalence of 3.7%. In contrast, none of the 1,930 male and female elk sampled from 1991 to 2008 in herd units distant from feedgrounds in Wyoming, USA, demonstrated exposure to B. abortus . In comparison to elk, bison had a higher incidence of brucellosis. Surveillance of 321 cow bison harvested within the Absaroka and Jackson bison herds in western Wyoming, USA, from 1992 to 2008 yielded an antibody prevalence of 63.9%.

Brucellosis is endemic in elk ( Cervus elaphus nelsoni ) using winter feedgrounds of western Wyoming, USA presumably because of increased animal density, duration of attendance, and subsequent contact with aborted fetuses. However, previous research addressed antibody prevalence rather than more direct measures of transmission and did not account for elk behavior or scavenging in transmission risk. Throughout March and early April 2005–07, we monitored 48 sets of culture-negative, pseudoaborted elk fetuses, placentas, and fluids (fetal units, FUs) on one winter free-ranging (WFR) location and four sites (Feedline, High Traffic, Low Traffic, Adjacent) associated with four feedgrounds. ‘‘At-risk'' elk (total elk within 5 m of FU) and proportions of elk sniffing and contacting FUs were highest on Feedlines and decreased toward Low Traffic sites. We did not observe elk investigating FUs Adjacent to feedgrounds or on the WFR location. At-risk elk on Feedline and High Traffic sites decreased throughout the sampling period, whereas proportions of elk investigating FUs were correlated positively to at-risk elk among all sites within feedgrounds. At-risk elk and proportions of elk investigating FUs were correlated with total feedground elk density and population only on High Traffic and Low Traffic sites. Proportions of sex/age groups (female, juvenile, male) investigating FUs did not differ from background populations. Females, however, spent more time (mean [SE], 21.07 [3.47] sec) investigating FUs than juveniles (14.73 [3.53] sec) and males (10.12 [1.45] sec), with positive correlation between total investigations and time spent investigating per female. Eight species of scavengers consumed FUs, removing FUs faster on feedgrounds than WFR locations and reducing proportions of elk that investigated FUs. Our results suggest that 1) reduction of elk density and time attending feedgrounds, particularly on Feedlines; and 2) protection of scavengers on and adjacent to feedgrounds would likely reduce intraspecific transmission risk of brucellosis.

Plague is the primary cause for the rangewide decline in prairie dog ( Cynomys spp.) distribution and abundance, yet our knowledge of plague dynamics in prairie dog populations is limited. Our understanding of the effects of plague on the most widespread species, the black-tailed prairie dog ( C. ludovicianus ), is particularly weak. During a study on the population biology of black-tailed prairie dogs in Wyoming, USA, plague was detected in a colony under intensive monitoring, providing a unique opportunity to quantify various consequences of plague. The epizootic reduced juvenile abundance by 96% and adult abundance by 95%. Of the survivors, eight of nine adults and one of eight juveniles developed antibodies to Yersinia pestis . Demographic groups appeared equally susceptible to infection, and age structure was unaffected. Survivors occupied three small coteries and exhibited improved body condition, but increased flea infestation compared to a neighboring, uninfected colony. Black-tailed prairie dogs are capable of surviving a plague epizootic and reorganizing into apparently functional coteries. Surviving prairie dogs may be critical in the repopulation of plague-decimated colonies and, ultimately, the evolution of plague resistance.

Nine (four female, five male) captive adult Rocky Mountain bighorn sheep ( Ovis canadensis ) contracted brucellosis caused by Brucella abortus biovar 4 as a result of natural exposure to an aborted elk ( Cervus elaphus ) fetus. Clinical signs of infection were orchitis and epididymitis in males and lymphadenitis and placentitis with abortion in females. Gross pathologic findings included enlargement of the testes or epididymides, or both, and yellow caseous abscesses and pyogranulomas of the same. Brucella abortus biovar 4 was cultured in all bighorn sheep from a variety of tissues, including testes/epididymides, mammary gland, and lymph nodes. All bighorn sheep tested were positive on a variety of standard Brucella serologic tests. This is the first report of brucellosis caused by B. abortus in Rocky Mountain bighorn sheep. It also provides evidence that bighorn sheep develop many of the manifestations ascribed to this disease and that infection can occur from natural exposure to an aborted fetus from another species. Wildlife managers responsible for bighorn sheep populations sympatric with Brucella -infected elk or bison ( Bison bison ) should be cognizant of the possibility of this disease in bighorn sheep.

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.

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 1.0×10 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 ≤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.

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 × 10 10 colony-forming units (CFU) of strain RB51 in March 1998. Fourteen of these were given a booster dose of 1.13 × 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 ≤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 × 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.