<p><a>The
hemotrophic mycoplasma<i>, Mycoplasma ovis</i>,
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 <i>M. ovis</i>-specific
assays for detection of this hemoplasma, and to better understand infection
dynamics within pregnant ewes and lambs. </a>The first study describes the
development and validation of a SYBR<sup>®</sup> Green quantitative PCR (qPCR) assay,
which was subsequently used to determine the prevalence of <i>M. ovis</i> 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 <i>M. ovis</i> ranged from 1.05 x 10<sup>3</sup>
to 1.85 x 10<sup>5 </sup>copies/mL of blood with a mean of 1.31 x 10<sup>4 </sup>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 <i>M. ovis</i> 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 10<sup>5 </sup>copies/mL.</p><p> The second project utilized a
combination of cross-sectional and longitudinal studies to estimate the
prevalence of <i>M. ovis</i> infection from
a cohort of naturally-infected pregnant ewes, assess changes in their bacterial
loads, and determine the incidence of <i>M.
ovis</i> in lambs pre- and post-weaning. The prevalence of <i>M. ovis</i> 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 10<sup>4 </sup>to 10<sup>9 </sup>copies/mL
of blood, with the median bacterial load at 10<sup>5</sup> 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 <i>M. ovis</i>
loads may be higher in pregnant sheep, particularly in ewes half-way through
pregnancy. Most of the <i>M. ovis</i> infections in the study lambs
were detected post-weaning which suggests that transplacental or transmammary
infection of <i>M. ovis</i> are unlikely
routes.</p><p> In the third study, a subset of <i>M. ovis</i> 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 <i>M.
ovis </i>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 <i>M. ovis</i> from different geographical
origins, within a herd the discrimination power is low, which may hamper its
usefulness in transmission studies. </p><p> The fourth and final study was the
development of a loop-mediated isothermal amplification (LAMP) assay targeting
the dnaK gene of <i>M. ovis</i>, 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. <i>Mycoplasma ovis</i>
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 <i>M. ovis</i>.
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 <i>M. ovis</i> isolate or
stain discrimination to investigate transmission or disease spread in an
outbreak.</p><p>
</p><p> In conclusion, three new molecular
tools for the detection of <i>M. ovis</i> 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 <i>M. ovis</i> 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 <i>M.
ovis</i> and investigate strain variations. Most importantly, the scheme is
sufficiently robust to allow direct genotyping of <i>M. ovis</i> 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 <i>M.
ovis</i> in pregnant sheep and lambs. It is shown herein that despite the high
prevalence and sometimes high bacterial loads in pregnant ewes, <i>M. ovis</i> 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. </p><br>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/8044946 |
| Date | 10 June 2019 |
| Creators | Kathy Ann Johnson (6615560) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Assay_Development_and_Characterization_of_i_Mycoplasma_ovis_i_/8044946 |
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