Mycoplasma agassizii, an opportunistic pathogen of tortoises, shows very little genetic variation across the Mojave and Sonoran Deserts.

Agusto Luzuriaga-Neira,David Alvarez-Ponce,Richard L Tillett,Shalyn N Bauschlicher,C Richard Tracy,Franziska C Sandmeier,Chava L Weitzman

doi:10.1371/journal.pone.0245895

Agusto Luzuriaga-Neira, David Alvarez-Ponce + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0245895

Copy DOI

Abstract

Mycoplasma agassizii is a common cause of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii). So far, only two strains of this bacterium have been sequenced, and very little is known about its patterns of genetic diversity. Understanding genetic variability of this pathogen is essential to implement conservation programs for their threatened, long-lived hosts. We used next generation sequencing to explore the genomic diversity of 86 cultured samples of M. agassizii collected from mostly healthy Mojave and Sonoran desert tortoises in 2011 and 2012. All samples with enough sequencing coverage exhibited a higher similarity to M. agassizii strain PS6T (collected in Las Vegas Valley, Nevada) than to strain 723 (collected in Sanibel Island, Florida). All eight genomes with a sequencing coverage over 2x were subjected to multiple analyses to detect single-nucleotide polymorphisms (SNPs). Strikingly, even though we detected 1373 SNPs between strains PS6T and 723, we did not detect any SNP between PS6T and our eight samples. Our whole genome analyses reveal that M. agassizii strain PS6T may be present across a wide geographic extent in healthy Mojave and Sonoran desert tortoises.

Highlights

To avoid the false-positive identification caused by the low coverage sequencing, we only considered a variant as a single-nucleotide polymorphisms (SNPs) if it was present in more than 50% of the individuals
We extracted DNA from 86 cultures that appeared to have replicated and reached mid-log growth at the expected timescale. This resulted in 75 DNA samples from Mojave desert tortoises and 11 DNA samples from Sonoran tortoises that were subjected to Illumina sequencing
After the resulting sequencing reads were filtered using the genomes of M. agassizii and M. testudineum as reference, we obtained 214 to 688,878 Mycoplasma-like reads per sample in 38 samples, 37 of which were qPCR positive for M. agassizii (Tables 1 and 2 and S1)

Summary

Methods

We collected nasal lavage samples, as previously described [13], from free-ranging Mojave and Sonoran desert tortoises in 2011–2012. Lavage samples were filtered through a 0.45 μm filter into SP4 broth and placed on ice in the field. These samples were further filtered through a 0.1 μm filter and incubated at 30 ̊C in a portable incubator. We used the protocol described by ATCC (https://www.atcc.org/products/all/700616.aspx), and to our knowledge, other techniques have not been verified for this organism [14,17]. In 2011, culture samples were sent on ice to collaborators at the University of Nevada, Reno for which these eight samples originated [9,13,20]

Results

Discussion

Conclusion