Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala

Abstract

Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.

Developing noninvasive methodologies to assess koala population health through detecting Chlamydia from scats

“…Changes in predictive (a) positive and (b) negative values of different noninvasive Chlamydia detection methods as the prevalence of Chlamydia increases (from 5% to 70%) in a population, the methods being the following: qPCR, quantitative Polymerase Chain Reaction as per Hulse et al (), qPCR Wedrowicz based on Wedrowicz et al (); DArTseq, Single Nucleotide Polymorphism using DArTseq method as per Schultz et al () and Detection Dog…”

“…This should be improved in the future by designing specific probes to target chlamydial DNA (such as in Holtz et al, ). Nonetheless, while qPCR outperformed DArTseq, probably because qPCR, contrary to DArTseq, does use Chlamydia ‐specific primers and probes (Hulse et al, ), the sensitivity of qPCR remained low. Applying an occupancy modelling approach to disease prevalence studies, Lachish et al () suggested a protocol of three repeats of qPCR to determine infection status in individuals.…”

“…In this study, molecular methods only detected C. pecorum , the most prevalent of the koala Chlamydia species (Polkinghorne et al, ). The qPCR method was designed to target both C. pecorum and C. pneumoniae sequences (Hulse et al, ). The DArTseq method would be capable of distinguishing pathogenic agents if sequence differences could be discovered using PstI and SphI as the complexity reduction system.…”

“…We classified them as either control koalas, when all swabs were negative for Chlamydia , or infected koalas, when Chlamydia was detected in any swab. Chlamydia presence in swabs was determined by qPCR (as described in Hulse et al, ) collected at three sites: ocular, urogenital (urogenital sinus in females, penile urethra in males) and rectal. Ocular and urogenital tracts are common locations of Chlamydia infection, and the gastrointestinal tract has been proposed as a site for Chlamydia persistence and reinfection of the urogenital tract (Rank & Yeruva, ), although it seems that dominant genotypes differ between gastrointestinal and urogenital tracts (Phillips et al, ).…”

“…Fluorescence data were acquired during the annealing/ extension phase. More details on qPCR methods are available in Hulse et al () and qPCR primer and probe sequences are given in Appendix S3.…”