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Identification of a Novel Mortality-Associated Helicobacter Species in Gopher Tortoises (Gopherus polyphemus), qPCR Test Development and Validation, and an Epidemiologic Survey

Tasha Desiderio

Authors:  Tasha Desiderio, Nicole Stacy, Robert Ossiboff, Linda Archer, Amy Alexander, Darryl Heard, Sarah Purcell, Daniel Fredholm, Kyle Donnelly, Justin Rosenberg, April Childress, James Wellehan

Faculty Mentor:  James Wellehan

College:  College of Veterinary Medicine 

Abstract

The genus Helicobacter are spiral-shaped bacteria in the phylum Proteobacteria, order Campylobacteriales. Three wild gopher tortoises (Gopherus polyphemus) presented between 2012 and 2019 with nasal discharge, lethargy, and weight loss. Cytology of nasal discharge identified heterophilic rhinitis and spiral-shaped bacteria. PCR and sequencing of the 16S rRNA gene revealed this to be a novel Helicobacter species. Two died despite treatment, the third was moribund and was euthanized. Histologic examination of the nasal mucosa showed rhinitis with variable mucosal hyperplasia, erosion, and ulceration; Warthin-Starry staining highlighted the presence of spiral bacteria in the untreated tortoise. Genus-specific primers were designed, and the gyrA and groEL genes were amplified by PCR and sequenced. Phylogenetic analysis shows that this organism and other previously characterized Helicobacter from tortoises form a clade. Development and cross-validation of two qPCR diagnostic assays for the gyrA and groEL genes showed significant correlation of the results of two assays (P<0.0001). These assays were used to survey nasal wash samples from a collection of 37 gopher tortoises. Sex, medication, nasal discharge score, blood data, and other clinical data were compared with qPCR results for this bacteria. Higher mortality of tortoises was significantly correlated to higher Helicobacter loads detected by qPCR (P=0.0128). Upper respiratory disease in tortoises appears to involve complex microbial ecology; factors beyond Mycoplasmopsis (Mycoplasma) agassizii should be taken into account.

Poster

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6 Responses
  1. Michael V. Bowie

    I enjoyed your work. Were the tortoises from the same area (location)? What was observed during postmortem in the gut and in other tissues of the tortoises? Are there other agents like Mycoplasma isolated from all or some of the animals? What would be your next step if you wanted to continue to study Helicobacter in gopher tortoises?

  2. Elisabeth Osorio

    Hi Tasha,
    You did a really good job with all of your research. This is very interesting and well presented.
    I had a couple of questions:
    What did these tortoises eventually die of? Their primary symptoms were weight loss, rhinitis, and lethargy but which symptom do you believe was responsible for the mortality?
    What kind of disease is produced in other Helicobacter species known to infect gopher tortoises? What sparked interest in this Helicobacter in particular?

    Thank you for your time.

  3. Lily Silsby

    Hey Tasha! Great job on your research. I was wondering, how is the Helicobacter transmitted from tortoise to tortoise? Also, what will future work entail?

  5. Alyssa Quinn

    This is quite an interesting research paper! I love the image you put on of the spiral bacteria.

  6. Alexis Trumbull

    Hi Tasha! Excellent project!

    Regarding your determination that this bacteria is disease-causing in the tortoises, now that you know this, what are some specific ways that interaction can be prevented?