Comparison of 2 ELISAs for detecting exposure to Brucella ovis.

Control of Brucella ovis infection in sheep flocks in the United States depends on early detection of B. ovis antibodies via serologic testing. We used 2,276 sheep sera and various cutoff values to compare seroprevalence and agreement between 2 ELISAs: the National Veterinary Services Laboratories (NVSL) B. ovis indirect ELISA and the IDEXX B. ovis ELISA kit. A subset of 295 sera was used to compare agreement and evaluate relative sensitivity and specificity of the 2 ELISAs with an agar gel immunodiffusion (AGID) test kit. There was no significant difference in B. ovis seroprevalence between the ELISAs; however, there was poor agreement between them. When the AGID test was used as the reference test, the IDEXX ELISA with a moderate cutoff value (S/P ratio = 45%) had the highest relative sensitivity of 38.1% and specificity of 92.0%. The NVSL ELISA with a lax cutoff value (S/P ratio = 0.75) had relative sensitivity of 19.1% and specificity of 94.6%. Receiver operating characteristic analysis revealed that optimal cutoff values for the NVSL and IDEXX ELISAs were 0.091 and 16.5%, respectively. This results in sensitivity and specificity of 85.7% and 31.8% for the NVSL ELISA, and sensitivity and specificity of 81.0% and 53.6% for the IDEXX ELISA, respectively.

Comparisons of brucellosis between human and veterinary medicine.

Brucellosis is the world's most widespread zoonosis, but also ranks as one of the seven most neglected diseases, according to the World Health Organization. Additionally, it is recognized as the world's most common laboratory-acquired infection. There are a reported 500,000 incident cases of human brucellosis per year. However, true incidence is estimated to be 5,000,000 to 12,500,000 cases annually. Once diagnosed, focus is directed at treating individual patients with antibiotic regimes, yet overall neglecting the animal reservoir of disease. Countries with the highest incidence of human brucellosis are Syria (1,603.4 cases per 1,000,000 individuals), Mongolia (391.0), and Tajikistan (211.9). Surveillance on animal populations is lacking in many developed and developing countries. According to the World Animal Health Information Database, Mexico had the largest number of reported outbreaks, 5,514 in 2014. Mexico is followed by China (2,138), Greece (1,268), and Brazil (1,142). The majority of these outbreaks is Brucella abortus, the etiologic agent of bovine brucellosis. Brucellosis is an ancient disease that still plagues the world. There are still knowledge gaps and a need for better diagnostics and vaccines to make inroads towards control and eradication.

Adenoviral hemorrhagic disease in California mule deer, 1990-2014.

We reviewed case records from the California Animal Health and Food Safety (CAHFS) laboratory and the California Department of Fish and Wildlife (CDFW) spanning 25 years (1990-2014) for all deer accessions submitted to CAHFS for pathology and/or histopathology, with and without a diagnosis of adenoviral hemorrhagic disease (AHD), in order to determine the prevalence of AHD in California. We also examined spatial and temporal distribution, age, and mule deer subspecies in deer that died from AHD. Of 483 deer submitted to CAHFS for diagnostic testing in 1990-2014, 17.2% were diagnosed with confirmed AHD, and 26.5% were confirmed plus suspected cases of AHD. Columbian black-tailed deer ( Odocoileus hemionus columbianus), particularly fawns and juveniles, were most frequently affected. Deer adenovirus ( Odocoileus adenovirus 1; OdAdV-1) was detected by immunohistochemistry in archived CDFW formalin-fixed, paraffin-embedded tissues from deer that died in mortality events in 1981, 1983, and 1986-1987. OdAdV-1 is a common cause of hemorrhagic disease mortality events in California deer, and mortality as a result of AHD is documented as early as 1981.

Endemic chronic wasting disease causes mule deer population decline in Wyoming.

Chronic wasting disease (CWD) is a fatal transmissible spongiform encephalopathy affecting white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemionus), Rocky Mountain elk (Cervus elaphus nelsoni), and moose (Alces alces shirasi) in North America. In southeastern Wyoming average annual CWD prevalence in mule deer exceeds 20% and appears to contribute to regional population declines. We determined the effect of CWD on mule deer demography using age-specific, female-only, CWD transition matrix models to estimate the population growth rate (λ). Mule deer were captured from 2010-2014 in southern Converse County Wyoming, USA. Captured adult (≥ 1.5 years old) deer were tested ante-mortem for CWD using tonsil biopsies and monitored using radio telemetry. Mean annual survival rates of CWD-negative and CWD-positive deer were 0.76 and 0.32, respectively. Pregnancy and fawn recruitment were not observed to be influenced by CWD. We estimated λ = 0.79, indicating an annual population decline of 21% under current CWD prevalence levels. A model derived from the demography of only CWD-negative individuals yielded; λ = 1.00, indicating a stable population if CWD were absent. These findings support CWD as a significant contributor to mule deer population decline. Chronic wasting disease is difficult or impossible to eradicate with current tools, given significant environmental contamination, and at present our best recommendation for control of this disease is to minimize spread to new areas and naïve cervid populations.

Comparison of the humoral response between sheep vaccinated with a killed-virus vaccine and those vaccinated with a modified-live virus vaccine against bluetongue virus serotype 17.

OBJECTIVE To compare the humoral response between sheep vaccinated with a killed-virus (KV) vaccine and those vaccinated with a modified-live virus (MLV) vaccine against bluetongue virus (BTV) serotype 17. DESIGN Randomized clinical trial followed by a field trial. ANIMALS 30 yearling crossbred ewes (phase 1) and 344 sheep from 7 Wyoming farms (phase 2). PROCEDURES In phase 1, ewes seronegative for anti-BTV antibodies received sterile diluent (control group; n = 10) or an MLV (10) or KV (10) vaccine against BTV-17 on day 0. Ewes in the KV group received a second dose of the vaccine on day 21. Ewes were bred 5 months after vaccination and allowed to lamb. Anti-BTV antibodies were measured in ewes at predetermined times after vaccination and in their lambs once at 5 to 10 days after birth. In phase 2, 248 commercial sheep were screened for anti-BTV antibodies and vaccinated with a KV vaccine against BTV-17 on day 0. Sheep seronegative for anti-BTV antibodies on day 0 (n = 90) underwent follow-up serologic testing on day 365 along with 96 unvaccinated cohorts (controls). RESULTS In phase 1, all vaccinated ewes developed anti-BTV antibodies by 14 days after vaccination and remained seropositive for 1 year; all of their lambs were also seropositive. All control ewes and lambs were seronegative. In phase 2, the prevalence of vaccinated sheep with anti-BTV antibodies 1 year after vaccination was 93% and 76% as determined by a serum neutralization assay and competitive ELISA, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Both vaccines induced antibodies against BTV-17 that persisted for at least 1 year and provided passive immunity for lambs and may be a viable option to protect sheep against disease.

Chronic Wasting Disease Drives Population Decline of White-Tailed Deer.

Chronic wasting disease (CWD) is an invariably fatal transmissible spongiform encephalopathy of white-tailed deer, mule deer, elk, and moose. Despite a 100% fatality rate, areas of high prevalence, and increasingly expanding geographic endemic areas, little is known about the population-level effects of CWD in deer. To investigate these effects, we tested the null hypothesis that high prevalence CWD did not negatively impact white-tailed deer population sustainability. The specific objectives of the study were to monitor CWD-positive and CWD-negative white-tailed deer in a high-prevalence CWD area longitudinally via radio-telemetry and global positioning system (GPS) collars. For the two populations, we determined the following: a) demographic and disease indices, b) annual survival, and c) finite rate of population growth (λ). The CWD prevalence was higher in females (42%) than males (28.8%) and hunter harvest and clinical CWD were the most frequent causes of mortality, with CWD-positive deer over-represented in harvest and total mortalities. Survival was significantly lower for CWD-positive deer and separately by sex; CWD-positive deer were 4.5 times more likely to die annually than CWD-negative deer while bucks were 1.7 times more likely to die than does. Population λ was 0.896 (0.859-0.980), which indicated a 10.4% annual decline. We show that a chronic disease that becomes endemic in wildlife populations has the potential to be population-limiting and the strong population-level effects of CWD suggest affected populations are not sustainable at high disease prevalence under current harvest levels.

BAYESIAN ANALYSIS TO EVALUATE TESTS FOR THE DETECTION OF MYCOBACTERIUM BOVIS INFECTION IN FREE-RANGING WILD BISON (BISON BISON ATHABASCAE) IN THE ABSENCE OF A GOLD STANDARD.

We estimated the sensitivity and specificity of the caudal-fold skin test (CFT), the fluorescent polarization assay (FPA), and the rapid lateral-flow test (RT) for the detection of Mycobacterium bovis in free-ranging wild wood bison (Bison bison athabascae), in the absence of a gold standard, by using Bayesian analysis, and then used those estimates to forecast the performance of a pairwise combination of tests in parallel. In 1998-99, 212 wood bison from Wood Buffalo National Park (Canada) were tested for M. bovis infection using CFT and two serologic tests (FPA and RT). The sensitivity and specificity of each test were estimated using a three-test, one-population, Bayesian model allowing for conditional dependence between FPA and RT. The sensitivity and specificity of the combination of CFT and each serologic test in parallel were calculated assuming conditional independence. The test performance estimates were influenced by the prior values chosen. However, the rank of tests and combinations of tests based on those estimates remained constant. The CFT was the most sensitive test and the FPA was the least sensitive, whereas RT was the most specific test and CFT was the least specific. In conclusion, given the fact that gold standards for the detection of M. bovis are imperfect and difficult to obtain in the field, Bayesian analysis holds promise as a tool to rank tests and combinations of tests based on their performance. Combining a skin test with an animal-side serologic test, such as RT, increases sensitivity in the detection of M. bovis and is a good approach to enhance disease eradication or control in wild bison.

Prevalence of and risk factors associated with ovine progressive pneumonia in Wyoming sheep flocks.

OBJECTIVE: To determine the prevalence of antibodies against small ruminant lentivirus (SRLV), the causative agent of ovine progressive pneumonia (OPP), and to identify risk factors associated with OPP in Wyoming sheep flocks. DESIGN: Cross-sectional study. ANIMALS: 1,415 sheep from 54 flocks in Wyoming. PROCEDURES: Flocks were surveyed as part of the National Animal Health Monitoring System (NAHMS) 2011 sheep study. Serum samples obtained from sheep in Wyoming were analyzed for anti-SRLV antibodies by use of a competitive-inhibition ELISA. The prevalence of seropositive animals overall and within each flock was calculated. Respective associations between flock OPP status and various demographic and management variables were assessed. RESULTS: The estimated prevalence of sheep seropositive for anti-SRLV antibodies and OPP-infected flocks in Wyoming was 18.0% and 47.5%, respectively. Within OPP-infected flocks, the prevalence of seropositive sheep ranged from 3.9% to 96%. Flocks maintained on nonfenced range were more likely to be infected with OPP than were flocks maintained on fenced range (OR, 3.4; 95% confidence interval, 1.1 to 10.7). The estimated prevalence of OPP-infected flocks in Wyoming did not vary substantially from that at the regional or national level reported in the NAHMS 2001 sheep study. Compared with results of the NAHMS 2011 sheep study, Wyoming producers were more familiar with OPP than were other US sheep producers, but only 61% of Wyoming producers surveyed reported being very or somewhat familiar with the disease. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that OPP is prevalent in many Wyoming sheep flocks, which suggested that continued efforts are necessary to increase producer knowledge about the disease and investigate practices to minimize economic losses associated with OPP.

Canine dysautonomia in a litter of Havanese puppies.

Canine dysautonomia is a sporadic, generally fatal disease that rarely affects groups of related animals. Four 10-week-old Havanese puppies from a litter of 5 developed clinical signs of canine dysautonomia. The 4 affected dogs were exposed to an outdoor environment, whereas the fifth littermate was not exposed to the outdoors and remained clinically healthy. Clinical signs of dysautonomia developed 10-16 days after going outside the house. An unrelated dog also developed dysautonomia after exposure to 1 of the affected Havanese littermates. All 5 dogs had morphological changes consistent with dysautonomia (widespread neuronal degeneration in autonomic ganglia, select brainstem nuclei, and ventral horn motor neurons). Differential diagnoses were excluded through negative toxicological evaluation, fecal parasite screening, negative Canine distemper virus reverse transcription polymerase chain reaction, fluorescent antibody testing, attempted virus isolation, and electron microscopy. The 5 affected dogs were in the Kansas City, Missouri area, where there is a high incidence of dysautonomia.

Outbreak investigation and control case report of brucellosis: experience from livestock research centre, Mpwapwa, Tanzania.

Brucellosis screening was conducted between 2005 and 2010 at the National Livestock Research Institute headquarters, Mpwapwa, Tanzania, following an abortion storm in cattle. The initial screening targeted breeding herds; 483 cattle were screened using the Rose Bengal Plate Test (RBPT) followed by the Competitive Enzyme-linked Immunosorbent Assay (c-ELISA) as a confirmatory test. The seropositivity on c-ELISA was 28.95% in 2005; it subsequently declined to 6.72%, 1.17%, 0.16% and 0.00% in 2006, 2007, 2009 and 2010, respectively. Brucella seropositivity was not detected in goats. Seropositivity declined following institution of stringent control measures that included: gradual culling of seropositive animals through slaughter; isolation and confinement of pregnant cows close to calving; proper disposal of placentas and aborted foetuses; the use of the S19 vaccine; and restricted introduction of new animals. It was thought that the source of this outbreak was likely to have been from the introduction of infected animals from another farm. Furthermore, humans were found with brucellosis antibodies. Out of 120 people screened, 12 (10%) were confirmed seropositive to brucella antigen exposure by c-ELISA analysis. The majority of the seropositive individuals (80%) were milkers and animal handlers from the farm. Nine individuals had clinical signs suggestive of brucellosis. All cases received medical attention from the district hospital. This achievement in livestock and human health showed that it is possible to control brucellosis in dairy farms, compared to pastoral and agro-pastoral farms, thus providing evidence to adopt these strategies in dairy farms thought to be at risk.

Wildlife-livestock interactions in a western rangeland setting: quantifying disease-relevant contacts.

Disease transmission between wild ungulates and domestic livestock is an important and challenging animal health issue. The potential for disease transmission between wildlife and livestock is notoriously difficult to estimate. The first step for estimating the potential for between-species disease transmission is to quantify proximity between individuals of different species in space and time. This study estimates second-order statistics of spatio-temporal location data from radio-collared free-ranging deer, elk and cattle in northeast Oregon. Our results indicate, that when observed simultaneously, elk and cattle occur in closer proximity to each other than what would be expected based on general space use of these species. The same is true for deer and elk but not for deer and cattle. Our analysis also demonstrates that average distances between cattle and elk are largely driven by rare events of close co-mingling between the species, which extend over several hours. Behavioral causes for these co-mingling events are currently unknown. Understanding the causes for such events will be important for designing grazing practices that minimize wildlife-livestock contacts.

Agreement between the caudal fold test and serological tests for the detection of Mycobacterium bovis infection in bison.

The objective of this study was to estimate agreement between the caudal fold test (CFT) and different serological tests for the detection of Mycobacterium bovis infection in bison by using prevalence-adjusted bias-adjusted kappa (PABAK). A total of 212 of wild wood bison from Wood Buffalo National Park were tested with the CFT as well as several serological tests: fluorescent polarization assay (FPA), multiantigen print immunoassay (MAPIA), rapid lateral-flow test (RT) and dual path platform test (DPP). For RT, 3 variations were conducted using 30µl of serum (RT 30), 20µl of serum (RT 20) and 20µl of serum considering only a strong reaction as positive (RT 20 ST). The McNemar's χ(2) test was conducted to assess whether the proportion of positive test results to 2 different tests differed. Two measures of agreement between pair of tests were estimated: the Cohen's kappa statistic and PABAK. The apparent prevalence of tuberculosis in the sampled animals varied depending on the diagnostic test from 6.1% (FPA and DPP) to 47.2% (CFT). The prevalence estimated by CFT differed from the prevalence estimated by the other tests, whereas the prevalence estimated by FPA, MAPIA, RT 20 ST and DPP were not significantly different. The kappa and PABAK estimates calculated between CFT and the rest of the tests suggested poor to slight agreement between tests (k and PABAK<0.25 in all cases). The PABAK estimates for the pairwise combinations among serological tests were numerically greater than the kappa estimates (and significantly greater when FPA was compared to the rest of serological tests), and suggested substantial to almost perfect agreement (PABAK>0.75 in all cases). The disagreement between the skin and serological tests for the detection of M. bovis infection could be partly because the tests measure different immunological responses (cell-mediated vs. humoral) that are predominant at different stages of the infection, and partly due to inaccuracy of the tests. Further research is needed to evaluate the accuracy of diagnostic tests in order to establish a reliable case definition, combining different tests, to be used in the surveillance and control of tuberculosis in free-ranging bison populations.

Canine distemper outbreak in pet store puppies linked to a high-volume dog breeder.

Canine distemper is uncommon in the pet trade in the United States, in large part due to effective vaccines against Canine distemper virus (CDV). This is a report of CDV affecting 24 young dogs of multiple breeds shortly after sale by 2 pet stores in Wyoming during August-October 2010. Cases were diagnosed over 37 days. Diagnosis was established by a combination of fluorescent antibody staining, reverse transcription polymerase chain reaction, negative stain electron microscopy, and necropsy with histopathology. Viral hemagglutinin gene sequences were analyzed from 2 affected dogs and were identical (GenBank accession no. JF283477). Sequences were distinct from those in a contemporaneous unrelated case of CDV in a Wyoming dog (JF283476) that had no contact with the pet store dogs. The breeding property from which the puppies originated was quarantined by the Kansas Animal Health Department. Puppies intended for sale were tested for CDV. Canine distemper was diagnosed on site in November 2010. At that point 1,466 dogs were euthanized to eliminate dispersal of the disease through commercial channels. The investigation underscores the risks inherent in large-scale dog breeding when vaccination and biosecurity practices are suboptimal.

Evaluation of the fluorescence polarization assay for the detection of Brucella abortus antibodies in bison in a natural setting.

Bison and elk in the greater Yellowstone area are the last-known reservoir of Brucella abortus in the United States. Diagnosis of brucellosis is challenging as there is no perfect reference test. The objectives of this study were to estimate the accuracy of the fluorescence polarization assay (FPA) for the screening of B. abortus antibodies in bison in a natural setting. Serum and tissue samples were collected and analyzed from the known brucellosis-infected bison herd in Yellowstone National Park (YNP). Additionally, serum samples from privately owned bison were serologically tested for brucellosis. While the FPA and five other tests had perfect sensitivity, all tests had substantially lower specificity in the YNP herd. However, a Bayesian analysis showed that as many as 59-74% of the culture-negative animals were most-likely truly infected. A decision-tree analysis showed that the expected cost of FPA testing was comparable to the cost of other serologic tests. The FPA was shown to be highly sensitive but may not be able to differentiate culture-positive and culture-negative animals. There is a need for long-term longitudinal studies to estimate diagnostic accuracy of tests for B. abortus in bison.

Evaluation of the Western immunoblot as a detection method for Brucella abortus exposure in elk.

Brucella abortus has been an important wildlife disease issue for most of the last century, especially because wildlife species are considered to be important disease reservoirs for cattle. Diagnostic uncertainty, caused in part by cross-reactions of antibodies to environmental pathogens such as Yersinia enterocolitica O:9 on standard Brucella serology, has exacerbated the challenges of managing the disease and has highlighted the need for test validation in wildlife species. The western immunoblot was evaluated for use in detecting B. abortus exposure in elk (Cervus elaphus) and for ruling out exposure to cross-reacting bacteria. Samples collected from 2003 to 2006, including 54 female and immature elk from four different elk herds, were tested using standard Brucella serologic methods (card, rapid automated presumptive [RAP], and rivanol tests), as well as the western immunoblot. Samples (n=28) from animals known to be naturally infected with B. abortus biovar 1 served as positive controls. For presumed negative samples, sera (n=26) were collected from two elk herds in which negative serologic tests, and the absence of clinical signs of disease such as abortions, supported Brucella-negative classification. In addition to these study samples, serologic data from 12 tule elk (Cervus elaphus nannodes) were provided from the California Department of Fish and Game in order to illustrate a field application of the western blot. The western immunoblot had the highest sensitivity (1.0; % 0.899-1.0) and specificity (1.0; 0.891-1.0) among all tests used in the study. The Kappa statistic for agreement between the western blot and the card, rivanol, and RAP tests were 0.701, 0.808, and 0.921, respectively, showing good to excellent agreement with the standard diagnostic tests currently in use. Although the western immunoblot is more expensive and time intensive than other tests, in this limited study, it was shown to be reliable for establishing and confirming B. abortus disease status in elk. In addition to this study, subsequent applications of the western blot assay have been successful in detecting Yersinia sp. exposure in elk after their antibodies cross-reacted on standard Brucella serology.