Assay Development and Characterization of Mycoplasma ovis
The hemotrophic mycoplasma, Mycoplasma ovis, is found in sheep and goats throughout the world. This pathogenic bacterium is capable of causing an acute, life-threatening infection as well as chronic or subclinical infections in these animals. The purposes of the present studies were to develop M. ovis-specific assays for detection of this hemoplasma, and to better understand infection dynamics within pregnant ewes and lambs. The first study describes the development and validation of a SYBR® Green quantitative PCR (qPCR) assay, which was subsequently used to determine the prevalence of M. ovis infection within a population of goats and to evaluate risk factors for infection. This highly sensitive and specific assay consistently detected as few as 10 copies of plasmid/reaction. Convenience-based sampling of 362 goats from 61 farms located in Indiana revealed a prevalence of infection of 18% (95% confidence interval (CI), 14% to 22%). Bacterial loads of M. ovis ranged from 1.05 x 103 to 1.85 x 105 copies/mL of blood with a mean of 1.31 x 104 copies/mL of blood. The only risk factor associated with hemoplasma infection was the production use of the goat; dairy goats had a 3.3 fold increase compared with the prevalence in goats used for meat. This study not only demonstrates that M. ovis infection is common in goats in Indiana, but shows the variability of bacterial loads that can be found in chronically-infected animals. While sub-clinically infected goats may have a bacteremia, levels are characteristically less than 2.0 x 105 copies/mL.
The second project utilized a combination of cross-sectional and longitudinal studies to estimate the prevalence of M. ovis infection from a cohort of naturally-infected pregnant ewes, assess changes in their bacterial loads, and determine the incidence of M. ovis in lambs pre- and post-weaning. The prevalence of M. ovis infection in ewes was not found to be significantly different during pregnancy, and before and after weaning of the lambs, with prevalence estimates of 45% (95% CI, 23.1 – 68.5), 36% (95% CI, 17.9 – 57.4), and 44%, (95% CI, 24.4 – 65.1), respectively. Bacterial loads of the ewes from the cross-sectional study ranged from 104 to 109 copies/mL of blood, with the median bacterial load at 105 copies/mL of blood. While higher bacterial loads are typical of an acute infection, none of the ewes in this study had overt clinical signs. The data suggest that M. ovis loads may be higher in pregnant sheep, particularly in ewes half-way through pregnancy. Most of the M. ovis infections in the study lambs were detected post-weaning which suggests that transplacental or transmammary infection of M. ovis are unlikely routes.
In the third study, a subset of M. ovis genes for use in a multi-locus sequence typing assay (MLST) were evaluated. Next-generation sequencing was performed to generate data from pooled DNA amplicons in order to identify single nucleotide polymorphisms (SNPs) of M. ovis from five genes. Evaluation of the quality and depths of coverage for the reads and SNPs indicated that the pooled DNA amplicons produced reads and SNPs having high quality and sufficient depth. This pooling technique is a cost-effective alternative to whole-genome sequencing. While the MLST has good discriminatory power and may be used to identify genetically distant and divergent clusters of M. ovis from different geographical origins, within a herd the discrimination power is low, which may hamper its usefulness in transmission studies.
The fourth and final study was the development of a loop-mediated isothermal amplification (LAMP) assay targeting the dnaK gene of M. ovis, with comparison of the assay to conventional PCR (cPCR). The metal ion indicator hydroxynaphthol blue (HNB) was added prior to the reaction, which allowed for visual detection of LAMP-positive samples as indicated by a color change from violet to sky blue. Mycoplasma ovis was consistently detected in 45 minutes with the LAMP assay at a reaction temperature of 64°C, with more infected sheep being detected than by cPCR. Therefore, the LAMP assay is fast and reliable in the detection of M. ovis. The developed LAMP assay may have applications in diagnostics, surveillance and disease management as well as prevalence studies. However, a more robust molecular technique is necessary for M. ovis isolate or stain discrimination to investigate transmission or disease spread in an outbreak.
In conclusion, three new molecular tools for the detection of M. ovis in goats and sheep were developed as results of these studies. We have shown that the qPCR assay is an efficient tool for detection and quantification of M. ovis loads in blood from both of these species. On the other hand, the value of the LAMP assay is for reliable detection of infection (not quantification), especially in resource-limited situations. The five-locus MLST protocol developed herein, a typing assay based on the polymorphism of five gene sequences, is a laborious technique requiring DNA extraction, PCR amplification, purification and sequencing of target loci. The value of this technique is not as a routine diagnostic, but rather it may be used to better understand the genetic diversity of M. ovis and investigate strain variations. Most importantly, the scheme is sufficiently robust to allow direct genotyping of M. ovis in total blood DNA extracts without culture isolation. The MLST approach may prove useful as a tool for future investigations of transmission and disease spread. These studies have also expanded our understanding of the infection dynamics of M. ovis in pregnant sheep and lambs. It is shown herein that despite the high prevalence and sometimes high bacterial loads in pregnant ewes, M. ovis does not appear to be transmitted to the lambs in utero or during the perinatal period. The lambs become infected mostly after weaning; this may suggest a protective effect during the pre-weaning period and/or subsequent exposure/infection from their environment.