Natalie D. Halbert

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.

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.

CONSERVATION GENETIC ANALYSIS OF THE TEXAS STATE BISON HERD

Abstract The Texas State Bison Herd is directly descended from the herd assembled from 5 wild-caught bison by Charles Goodnight in the 1880s. In 1997, 36 bison were used to establish a herd at Caprock Canyons State Park. To aid in the development of a long-term genetic conservation plan for this population, we examined and analyzed allelic variation at 54 microsatellite loci representing each of the nuclear chromosomes in the bison genome. The current Texas State Bison Herd population exhibits low genetic diversity and heterozygosity levels compared with bison at Yellowstone National Park and Theodore Roosevelt National Park. Parentage analysis indicates that relatively few adults have contributed offspring in the last 5 years, leading to low effective population size estimations and a rapid increase in the average age of animals in the herd. The very limited number of original founders, multiple population bottlenecks over the last 120 years, and chronically small population size, coupled with genetic drift and inbreeding, have resulted in dangerously low levels of genetic diversity. This, in turn, has likely triggered demographic problems such as low recruitment and high calf mortality rates. Population viability analysis based on current population demography reveals that there is a 99% chance of extinction of the herd within the next 41 years. Based on these findings, the continued existence of this historically important bison population appears doubtful without the introduction of new genetic variation from another plains bison herd.

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.

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.

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.

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.

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 ∼2.0% of variation in weight, height, and BMI, which confirms the utility and potential importance of adjusting for population stratification.