Phenotypic effects of cattle mitochondrial DNA in American bison.

Hybridization between endangered species and more common species is a significant problem in conservation biology because it may result in extinction or loss of adaptation. The historical reduction in abundance and geographic distribution of the American plains bison (Bison bison bison) and their recovery over the last 125 years is well documented. However, introgression from domestic cattle (Bos taurus) into the few remaining bison populations that existed in the late 1800s has now been identified in many modern bison herds. We examined the phenotypic effect of this ancestry by comparing weight and height of bison with cattle or bison mitochondrial DNA (mtDNA) from Santa Catalina Island, California (U.S.A.), a nutritionally stressful environment for bison, and of a group of age-matched feedlot bison males in Montana, a nutritionally rich environment. The environmental and nutritional differences between these 2 bison populations were very different and demonstrated the phenotypic effect of domestic cattle mtDNA in bison over a broad range of conditions. For example, the average weight of feedlot males that were 2 years of age was 2.54 times greater than that of males from Santa Catalina Island. In both environments, bison with cattle mtDNA had lower weight compared with bison with bison mtDNA, and on Santa Catalina Island, the height of bison with cattle mtDNA was lower than the height of bison with bison mtDNA. These data support the hypothesis that body size is smaller and height is lower in bison with domestic cattle mtDNA and that genomic integrity is important for the conservation of the American plains bison.

Genetic population substructure in bison at Yellowstone National Park.

The Yellowstone National Park bison herd is 1 of only 2 populations known to have continually persisted on their current landscape since pre-Columbian times. Over the last century, the census size of this herd has fluctuated from around 100 individuals to over 3000 animals. Previous studies involving radiotelemetry, tooth wear, and parturition timing provide evidence of at least 2 distinct groups of bison within Yellowstone National Park. To better understand the biology of Yellowstone bison, we investigated the potential for limited gene flow across this population using multilocus Bayesian clustering analysis. Two genetically distinct and clearly defined subpopulations were identified based on both genotypic diversity and allelic distributions. Genetic cluster assignments were highly correlated with sampling locations for a subgroup of live capture individuals. Furthermore, a comparison of the cluster assignments to the 2 principle winter cull sites revealed critical differences in migration patterns across years. The 2 Yellowstone subpopulations display levels of differentiation that are only slightly less than that between populations which have been geographically and reproductively isolated for over 40 years. The identification of cryptic population subdivision and genetic differentiation of this magnitude highlights the importance of this biological phenomenon in the management of wildlife species.

Complete mitochondrial DNA sequence analysis of Bison bison and bison-cattle hybrids: function and phylogeny.

Complete mitochondrial DNA (mtDNA) genomes from 43 bison and bison-cattle hybrids were sequenced and compared with other bovids. Selected animals reflect the historical range and current taxonomic structure of bison. This study identified regions of potential nuclear-mitochondrial incompatibilities in hybrids, provided a complete mtDNA phylogenetic tree for this species, and uncovered evidence of bison population substructure. Seventeen bison haplotypes defined by 66 polymorphic sites were discovered, whereas 728 fixed differences and 86 non-synonymous mutations were identified between bison and bison-cattle hybrid sequences. The potential roles of the mtDNA genome in the function of hybrid animals and bison taxonomy are discussed.

Subcutaneous infection of a cat by Colletotrichum species.

A 13-year-old, domestic shorthair cat was presented for evaluation of a right tarsal mass. Physical examination revealed a 5 cm × 5 cm × 5 cm, soft, fluctuant, subcutaneous tarsal mass. Thoracic radiographs revealed several discrete lung parenchymal lesions. Abdominal ultrasound revealed abnormal architecture to both kidneys. Fungal culture and sensitivity from the subcutaneous mass revealed a uniform growth of Colletotrichum species that was susceptible to itraconazole. Colletotrichum species infection was confirmed in the subcutis and suspected to be disseminated. Colletotrichum species fungal infections in cats have not been previously documented.

Determination of the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin in Rhodococcus equi isolates and treatment outcome in foals infected with antimicrobial-resistant isolates of R equi.

OBJECTIVE: To determine the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin in Rhodococcus equi isolates and to describe treatment outcome in foals infected with antimicrobial-resistant isolates of R equi. DESIGN: Cross-sectional study. SAMPLE POPULATION: 38 isolates classified as resistant to macrolide antimicrobials or rifampin received from 9 veterinary diagnostic laboratories between January 1997 and December 2008. PROCEDURES: For each isolate, the minimum inhibitory concentration of macrolide antimicrobials (ie, azithromycin, erythromycin, and clarithromycin) and rifampin was determined by use of a concentration-gradient test. Prevalence of R equi isolates from Florida and Texas resistant to macrolide antimicrobials or rifampin was determined. Outcome of antimicrobial treatment in foals infected with antimicrobial-resistant isolates of R equi was determined. RESULTS: Only 24 of 38 (63.2%) isolates were resistant to >or= 1 antimicrobial. Two isolates were resistant only to rifampin, whereas 22 isolates were resistant to azithromycin, erythromycin, clarithromycin, and rifampin. The overall prevalence of antimicrobial-resistant isolates in submissions received from Florida and Texas was 3.7% (12/328). The survival proportion of foals infected with resistant R equi isolates (2/8 [25.0%]) was significantly less, compared with the survival proportion in foals that received the same antimicrobial treatment from which antimicrobial-susceptible isolates were cultured (55/79 [69.6%]). Odds of nonsurvival for foals infected with resistant R equi isolates were 6.9 (95% confidence interval, 1.3 to 37) times the odds for foals infected with susceptible isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Interpretation of the results emphasized the importance of microbiological culture and antimicrobial susceptibility testing in foals with pneumonia caused by R equi.

Detection of strain variation in isolates of Rhodococcus equi from an affected foal using repetitive sequence-based polymerase chain reaction.

Rhodococcus equi is an important pathogen of foals aged 1-6 months. Evidence exists that foals are exposed to a wide diversity of R. equi strains in their environment. However, limited data are available regarding the extent to which genotypic variation exists among isolates infecting individual foals. Therefore, electrophoresis of repetitive sequence-based polymerase chain reaction (rep-PCR) amplicons in an automated microfluidics chip format was used to genotype 9 virulent R. equi isolates obtained from distinct anatomic locations in a single foal. Four of the isolates were obtained from different regions of the lung, and 5 were from abscessed intra-abdominal lymph nodes (LNs). Six distinct genotypes were identified among the 9 isolates. None of the pulmonary isolates was identical; however, a pulmonary isolate was found to be identical to an isolate recovered from a small intestinal LN, and another pulmonary isolate was identical to an isolate from a mesenteric LN. These results indicate that foals can be infected with multiple strains of virulent R. equi. Furthermore, identical strains can be found in multiple, remote anatomic locations in an infected foal, and this can occur for >1 strain in the same foal. The automated system used in the current study provided a rapid, reproducible, and discriminating method for typing R. equi isolates.

Cytokine expression by neutrophils of adult horses stimulated with virulent and avirulent Rhodococcus equi in vitro.

Rhodococcus equi is an intracellular pathogen of macrophages that causes rhodococcal pneumonia in foals and immunocompromised people. Evidence exists that neutrophils play a vital role in resistance to infection with R. equi; however, the means by which neutrophils exert their effects have not been clearly defined. In addition to directly killing bacteria, neutrophils also may exert a protective effect by linking innate and adaptive immune responses. In the present study we evaluated the cytokine expression profiles of adult equine neutrophils in response to stimulation with isogenic strains of virulent and avirulent R. equi in vitro. After 2 and 4h incubation with virulent or avirulent R. equi, adult equine neutrophils expressed significantly (P<0.05) greater tumor necrosis factor alpha (TNFalpha), interleukin (IL)-12p40, IL-6, IL-8 and IL-23p19 mRNA, but not interferon gamma (IFNgamma) or IL-12p35 mRNA than unstimulated neutrophils. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA than avirulent R. equi. These results demonstrate that R. equi-stimulated neutrophils are a source of many proinflammatory cytokines. Furthermore, these results suggest that IL-23 may be preferentially expressed over IL-12 in response to exposure with R. equi, and that this response may be more strongly induced by virulent R. equi than avirulent R. equi. Collectively, the data presented herein suggest a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Patterns of genetic variation in US federal bison herds.

Like many wide-ranging mammals, American bison (Bison bison) have experienced significant range contraction over the past two centuries and are maintained in artificially isolated populations. A basic understanding of the distribution of genetic variation among populations is necessary to facilitate long-term germplasm preservation and species conservation. The 11 herds maintained within the US federal system are a critically important source of germplasm for bison conservation, as they include many of the oldest herds in the USA and have served as a primary resource for the establishment of private and public herds worldwide. In this study, we used a panel of 51 nuclear markers to investigate patterns of neutral genetic variation among these herds. Most of these herds have maintained remarkably high levels of variation despite the severe bottleneck suffered in the late 1800s. However, differences were noted in the patterns of variation and levels of differentiation among herds, which were compared with historical records of establishment, supplementation, herd size, and culling practices. Although some lineages have been replicated across multiple herds within the US federal system, other lineages with high levels of genetic variation exist in isolated herds and should be considered targets for the establishment of satellite herds. From this and other studies, it is clear that the genetic variation represented in the US federal system is unevenly distributed among National Park Service and Fish and Wildlife Service herds, and that these resources must be carefully managed to ensure long-term species conservation.

A comprehensive evaluation of cattle introgression into US federal bison herds.

Genetic introgression, especially from interspecies hybridization, is a significant threat to species conservation worldwide. In this study, 11 US federal bison populations were comprehensively examined for evidence of both mitochondrial and nuclear domestic cattle (Bos taurus) introgression. Mitochondrial introgression was examined using established polymerase chain reaction methods and confirmed through analysis of D-loop sequences. Nuclear introgression was assessed in 14 chromosomal regions through examination of microsatellite electromorph and sequence differences between bison and domestic cattle. Only one population was identified with domestic cattle mitochondrial DNA introgression. In contrast, evidence of nuclear introgression was found in 7 (63.6%) of the examined populations. Historic accounts of bison transfers among populations were corroborated with evidence of introgressed DNA transmission. While neither nuclear nor mitochondrial domestic cattle introgression was detected in bison from Grand Teton National Park, Sully's Hill National Game Preserve, Wind Cave National Park, or Yellowstone National Park, adequate sample sizes were available only from the last 2 populations to allow for statistical confidence (>90%) in nuclear introgression detection limits. The identification of genetically unique and undisturbed populations is critical to species conservation efforts, and this study serves as a model for the genetic evaluation of interspecies introgression.

Variations in equid SLC11A1 (NRAMP1) genes and associations with Rhodococcus equi pneumonia in horses.

Rhodococcus equi is an important intracellular pathogen of horses, most commonly causing chronic, suppurative bronchopneumonia in foals. Although most foals likely are exposed to environmental R. equi within the 1st few days of life, only some develop R. equi pneumonia, and the basis of differences in susceptibility among foals currently is unknown. In this study, we investigated solute carrier family 11 member 1 (SLC11A1) gene sequences in the 5' untranslated region, exon 1, and a portion of intron 1 for variations in 3 equid species (horse, donkey, zebra) and compared variants within 3 independent horse breeding farms for associations with R. equi pneumonia by use of an age-matched case-control design. Seven novel variants in the 5'untranslated region were identified as specific for one or both of the non-horse equid species sampled. In addition, a single novel horse variant in the 5'untranslated region, -57C/T, was identified in 4 breeds. The -57C/T variant was found on 2 of the 3 farms with endemic R. equi pneumonia, representing 2 different horse breeds. Significant allelic and genotypic associations with susceptibility to R. equi pneumonia were observed for the -57C/T variant in foals from these farms. Although the functional impact of this novel variant remains to be determined, this study represents an important step in our understanding of natural resistance to R. equi foal pneumonia and other intracellular bacterial diseases affecting equids.

Marker genotypes and population admixture and their association with body weight, height and relative body mass in United States federal bison herds.

Elucidating genetic influences on bison growth and body composition is of interest, not only because bison are important for historical, cultural, and agricultural reasons, but also because their unusual population history makes them valuable models for finding influential loci in both domestic cattle and humans. We tested for trait loci associated with body weight, height, and bison mass index (BMI) while controlling for estimated ancestry to reduce potential confounding effects due to population admixture in 1316 bison sampled from four U.S. herds. We used 60 microsatellite markers to model each phenotype as a function of herd, sex, age, marker genotypes, and individual ancestry estimates. Statistical significance for genotype and its interaction with ancestry was evaluated using the adaptive false discovery rate. Of the four herds, two appeared to be admixed and two were nonadmixed. Although none of the main effects of the loci were significant, estimated ancestry and its interaction with marker loci were significantly associated with the phenotypes, illustrating the importance of including ancestry in the models and the dependence of genotype-phenotype associations on background ancestry. Individual loci contributed approximately 2.0% of variation in weight, height, and BMI, which confirms the utility and potential importance of adjusting for population stratification.

Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene.

OBJECTIVE: To evaluate sensitivity and specificity of a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Rhodococcus equi and differentiation of strains that contain the virulence-associated gene (vapA) from strains that do not. SAMPLE POPULATION: 187 isolates of R equi from equine and nonequine tissue and environmental specimens and 27 isolates of bacterial species genetically or morphologically similar to R equi. PROCEDURE: The multiplex PCR assay included 3 gene targets: a universal 311-bp bacterial 16S ribosomal RNA amplicon (positive internal control), a 959-bp R equi-specific target in the cholesterol oxidase gene (choE), and a 564-bp amplicon of the vapA gene. Duplicate multiplex PCR assays for these targets and confirmatory singleplex PCR assays for vapA and choE were performed for each R equi isolate. An additional PCR assay was used to examine isolates for the vapB gene. RESULTS: Results of duplicate multiplex and singleplex PCR assays were correlated in all instances, revealing high specificity and reliability (reproducibility) of the vapA multiplex assay. Of the pulmonary isolates from horses with suspected R equi pneumonia, 97.4% (76/78) yielded positive results for vapA. Seven of 50 (14%) human isolates of R equi yielded positive results for vapA. Six human R equi isolates and 1 porcine isolate yielded positive results for vapB. No isolates with vapA and vapB genes were detected. CONCLUSIONS AND CLINICAL RELEVANCE: The multiplex PCR assay is a sensitive and specific method for simultaneous confirmation of species identity and detection of the vapA gene. The assay appeared to be a useful tool for microbiologic and epidemiologic diagnosis and research.

Conservation genomics: disequilibrium mapping of domestic cattle chromosomal segments in North American bison populations.

Introgressive hybridization is one of the major threats to species conservation, and is often induced by human influence on the natural habitat of wildlife species. The ability to accurately identify introgression is critical to understanding its importance in evolution and effective conservation management of species. Hybridization between North American bison (Bison bison) and domestic cattle (Bos taurus) as a result of human activities has been recorded for over 100 years, and domestic cattle mitochondrial DNA was previously detected in bison populations. In this study, linked microsatellite markers were used to identify domestic cattle chromosomal segments in 14 genomic regions from 14 bison populations. Cattle nuclear introgression was identified in five populations, with an average frequency per population ranging from 0.56% to 1.80%. This study represents the first use of linked molecular markers to examine introgression between mammalian species and the first demonstration of domestic cattle nuclear introgression in bison. To date, six public bison populations have been identified with no evidence of mitochondrial or nuclear domestic cattle introgression, providing information critical to the future management of bison genetic resources. The ability to identify even low levels of introgression resulting from historic hybridization events suggests that the use of linked molecular markers to identify introgression is a significant development in the study of introgressive hybridization across a broad range of taxa.

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi.

OBJECTIVE: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. SAMPLE POPULATION: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. PROCEDURE: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. RESULTS: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.

Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3.

Amino acid replacements encoded by the prion protein gene (PRNP) have been associated with transmissible and hereditary spongiform encephalopathies in mammalian species. However, an association between bovine spongiform encephalopathy (BSE) and bovine PRNP exon 3 has not been detected. Moreover, little is currently known regarding the mechanisms of evolution influencing the bovine PRNP gene. Therefore, in this study we evaluated the patterns of nucleotide variation associated with PRNP exon 3 for 36 breeds of domestic cattle and representative samples for 10 additional species of Bovinae. The results of our study indicate that strong purifying selection has intensely constrained PRNP over the long-term evolutionary history of the subfamily Bovinae, especially in regions considered to be of functional, structural, and pathogenic importance in humans as well as other mammals. The driving force behind this intense level of purifying selection remains to be explained.