A quantitative PCR method for assessing the presence of Pasteurella testudinis DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

Pasteurella testudinis has been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Our goal was to develop a sensitive and specific qPCR method for detecting DNA from P. testudinis in nasal lavage fluid collected from desert tortoises in the field. Probes for 16S ribosomal RNA and RNA polymerase ß-subunit (rpoB) genes were designed. A standard curve generated with DNA extracted from known numbers of bacterial cells determined by flow cytometry revealed a lower detection limit of 50 fg/ml (10 bacteria/ml). The nasal lavage fluid contained no interfering substances, and the qPCR method did not recognize normal flora DNA. The nasal lavage samples from 20 desert tortoises captured in Clark County, Nevada, USA in 2007 and housed at the Desert Tortoise Conservation Center, were all positive for P. testudinis DNA by qPCR. Another set of 19 lavage samples collected in 2010 from wild desert tortoises in the Mojave Desert were tested and 84% were positive for P. testudinis DNA. Fully validated, this qPCR method will provide a means of determining colonization rate. When used in conjunction with serological methods and clinical evaluations, both infection rate and disease rate can be determined for this potential URTD pathogen. This new assay provides an important tool for managing the threatened populations of the Mojave Desert tortoise.

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6pg ml(-1) to a high of 72,962pg ml(-1). Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.

Flow cytometric method for quantifying viable Mycoplasma agassizii, an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii).

AIMS: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. METHODS AND RESULTS: Here, we demonstrate that the nonfluorescent molecule 5-carboxyfluorescein (5-CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5-CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml(-1) can be labelled and counted in less than 1 h. Experiments using temperature-induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. CONCLUSIONS: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.