A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus)

Abstract

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53–0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population.

Identification of eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) infection levels in Northern bobwhite quail (Colinus virginianus) of the Rolling Plains, TX using a mobile research laboratory: Implications for regional surveillance

“…DNA extraction protocols followed procedures outlined in Kalyanasundaram et al [9] and Kistler et al [17]. Fecal samples were weighed to 180–220 mg prior to extraction.…”

“…Infection levels were classified by comparing C t values generated for swabs to C t values generated by the standards as a limit of quantification (LOQ). Standards in this study consist of serially diluted plasmid DNA collected from eyeworm and caecal worm and are the same standards outlined in Kalyanasundaram et al [9] and Kistler et al [17]. Concentrations for the standards ranged from 10 1 to 10 5 copy numbers.…”

“…While feces is the best sample type when using qPCR to detect eyeworms and caecal worms, it has been suggested that cloacal swabs are more efficient to collect in field applications [9,10,17]. However, cloacal swab efficiency in determining infection may be dependent on the amount of feces present in the swab and thus, may affect the outcome of qPCR results.…”

Predicting seasonal infection of eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) in northern bobwhite quail (Colinus virginianus) of the Rolling Plains Ecoregion of Texas, USA

“…Feces samples weighing 0.18–0.22 g and cloacal swabs were snap-frozen in liquid nitrogen and then extracted using the QIAamp DNA Stool Mini Kit (Germany) following manufacturer's protocol as outlined in Kistler et al (2016a) with a final elution step of 50 μL per sample followed from Kalyanasundaram et al (2018b). Quantitative PCR techniques also followed Kalyanasundaram et al (2018b) methods with 20 μL mastermix volumes as follows: 10 μL Taqman Fast Advanced Mastermix (Applied Biosystems), 0.4 μL of forward and reverse eyeworm primers and eyeworm probe, 0.2 μL of forward and reverse caecal worm primers and caecal worm probe, 0.2 μL of forward and reverse quail primers and quail probe, 0.1 μL bovine serum albumin (BSA), and 7.66 μL of molecular-grade water.…”

“Weight of evidence” as a tool for evaluating disease in wildlife: An example assessing parasitic infection in Northern bobwhite (Colinus virginianus)

“…Nevertheless, a great deal of progress has been made to pave the way for future work investigating the interactions between bobwhite and parasites. Molecular techniques have been developed in order to non-lethally assess parasitic infection in bobwhite via a cloacal swab or feces sample that is evaluated by quantitative PCR (qPCR) and can detect the DNA for as little as one egg ( Kistler et al, 2016b ; Kalyanasundaram et al, 2018c ). These methods were then adapted for use at a regional scale ( Blanchard et al, 2018 ), as they are considered an effective form of parasite monitoring ( Gray et al, 2012 ) that provides a valuable supplement to traditional methods ( Archie et al, 2009 ).…”