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Development of a Multiplex Quantitative PCR Assay for Eyeworm ( Oxyspirura petrowi ) and Caecal worm ( Aulonocephalus pennula ) Detection in Northern Bobwhite quail ( Colinus virginianus ) of the Rolling Plains Eco-Region, Texas

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... Nonlethal procedures are made even more difficult given no reported evidence of eyeworm or caecal worm expulsion in the feces of bobwhite for morphological identification. Consequently, Kalyanasundaram et al. [9] successfully developed an accurate and precise multiplex quantitative PCR (qPCR) method to detect eyeworm and caecal worm egg presence in bobwhite feces, which has been utilized in a mobile research laboratory [10]. This progress has provided an opportunity to nonlethally assess parasitic infection in bobwhite and also provides a platform for Regular surveillance of parasitic infection is vital as it can track the efficiency of mitigation efforts after rounds of treatment [11,12]. ...
... 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. ...
... 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. ...
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Over the last few decades, there has been a decline in Northern bobwhite quail (Colinus virginianus) throughout their native range. While there are various factors that may be influencing this decline, it is suggested that parasites should be taken into consideration as a potential contributor in the Rolling Plains Ecoregion. High prevalence of the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) in bobwhite of this region, coupled with a continuous decline, creates a need to assess infection through alternative methods for regional surveillance. Previous studies have developed a qPCR method and mobile research laboratory as an option for nonlethal procedures. However, there is still a need for standardization of these techniques. Therefore, this study builds on previous protocols to develop an application that considers factors that may influence qPCR results. In this study, cloacal swabs are collected from bobwhite in three locations throughout the Rolling Plains and scaled based on amount of feces present on the swab. This data is compared to qPCR standards as a limit of quantification for both eyeworm and caecal worm to define parasitic infection levels. Binary logistic regressions confirm that the probability of detection increases for both eyeworm (Odds Ratio: 2.3738; 95% Confidence Interval: [1.7804, 3.1649]) and caecal worm (Odds Ratio: 2.8516; 95% Confidence Interval: [2.2235, 3.6570]) as swab score increases. Infection levels for eyeworm and caecal worm are based on the generated cycle threshold value averages of qPCR standards. Based on the results of this study, this method can be applied in the mobile research laboratory to quantitatively assess regional parasitic infection in bobwhite throughout the Rolling Plains.
... Quantitative PCR (qPCR) can be used to indicate parasite reproduction based on parasite egg presence in fecal matter (Kalyanasundaram et al., 2018b) whereas necropsies can provide worm counts for parasite intensity. In this study, we use fecal samples, cloacal swabs, and average worm counts per bobwhite collected between March and October of 2014 through 2017 to perform predictive analyses on parasite reproduction and intensity in relation to temperature and precipitation. ...
... Feces samples weighing 0.18-0.22 g and cloacal swabs were snapfrozen 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. ...
... g and cloacal swabs were snapfrozen 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. Primer and probe sequences used in this study are outlined in Table 1. ...
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The northern bobwhite quail (Colinus virginianus) is a popular gamebird in the Rolling Plains Ecoregion of West Texas. However, there has been a population decline in this area over recent decades. Consistent reports indicate a high prevalence of the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula), which may be of major influence on the bobwhite population. While research has suggested pathological consequences and genetic relatedness to other pathologically significant parasites, little is known about the influence of climate on these parasites. In this study, we examined whether seasonal temperature and precipitation influences the intensity of these parasites in bobwhite. We also analyzed quantitative PCR results for bobwhite feces and cloacal swabs against temperature and precipitation to identify climatic impacts on parasite reproduction in this region. Multiple linear regression analyses were used for parasite intensity investigation while binary logistic regression analyses were used for parasite reproduction studies. Our analyses suggest that caecal worm intensity, caecal worm reproduction, and eyeworm reproduction are influenced by temperature and precipitation. Temperature data was collected 15, 30, and 60 days prior to the date of collection of individual bobwhite and compared to qPCR results to generate a temperature range that may influence future eyeworm reproduction. This is the first preliminary study investigating climatic influences with predictive statistics on eyeworm and caecal worm infection of northern bobwhite in the Rolling Plains.
... Mobile laboratories have previously been executed for various facets of research including atmospheric pollutant measurements (Bukowiecki et al., 2002), Marburg virus research in Angola (Grolla et al., 2011), and human specimen collection and processing for biosafety level two research in Germany (Lermen et al., 2014). Using cloacal swabs in lieu of euthanasia and a highly sensitive multiplex quantitative PCR (qPCR) assay developed by Kalyanasundaram et al. (2018b) and Kistler et al. (2016), the WTL's mobile laboratory can utilize the presence of parasite eggs detected in bobwhite fecal matter to analyse when reproduction is occurring. It is a useful technique that allows large sample sizes to be tested in a time-efficient and cost-effective manner. ...
... The qPCR protocol for this study follows Kalyanasundaram et al. (2018b). Standards for O. petrowi and A. pennula were used from Kistler et al. (2016) and Kalyanasundaram et al. (2018b), respectively. ...
... The qPCR protocol for this study follows Kalyanasundaram et al. (2018b). Standards for O. petrowi and A. pennula were used from Kistler et al. (2016) and Kalyanasundaram et al. (2018b), respectively. Standard concentrations used in this study ranged from 10 5 to 10 1 . ...
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Northern bobwhite quail ( Colinus virginianus ), a popular gamebird among hunters, have been declining over recent decades in the Rolling Plains ecoregion. Investigations in the past few years have revealed a high prevalence of eyeworms ( Oxyspirura petrowi ) and caecal worms ( Aulonocephalus pennula ) in this ecoregion, prompting a need to better understand their host–parasite interaction and other factors that influence infection. In this study, the efficiency of a mobile laboratory was tested by deploying it to three field sites in the Rolling Plains between July and August of 2017 and collecting cloacal swabs from bobwhites. The DNA was extracted from swabs for quantitative PCR and was run in the mobile and reference laboratory to specifically detect A. pennula and O. petrowi infection. When compared with the Wildlife Toxicology's reference laboratory, the mobile laboratory had a 97 and 99% agreement for A. pennula and O. petrowi , respectively. There were no significant differences in infection levels between field sites. Due to its efficiency, it is proposed that the mobile laboratory would be an effective way to monitor infection levels, in addition to factors that may affect infection such as climate, diapause, and intermediate host populations.
... 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 nonlethally 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). ...
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The potential of parasites to affect host abundance has been a topic of heated contention within the scientific community for some time, with many maintaining that issues such as habitat loss are more important in regulating wildlife populations than diseases. This is in part due to the difficulty in detecting and quantifying the consequences of disease, such as parasitic infection, within wild systems. An example of this is found in the Northern bobwhite quail (Colinus virginanus), an iconic game bird that is one of the most extensively studied vertebrates on the planet. Yet, despite countless volumes dedicated to the study and management of this bird, bobwhite continue to disappear from fields, forest margins, and grasslands across the United States in what some have referred to as “our greatest wildlife tragedy”. Here, we will discuss the history of disease and wildlife conservation, some of the challenges wildlife disease studies face in the ever-changing world, and how a “weight of evidence” approach has been invaluable to evaluating the impact of parasites on bobwhite in the Rolling Plains of Texas. Through this, we highlight the potential of using “weight of the evidence” to better understand the complex effects of diseases on wildlife and urge a greater consideration of the importance of disease in wildlife conservation.
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Aulonocephalus pennula is a nematode living in the caeca of the wild Northern bobwhite quail (Colinus virginianus) present throughout the Rolling Plains Ecoregion of Texas. The cytochrome oxidase 1 (COX 1) gene of the mitochondrial genome was used to screen A. pennula in wild quail. Through BLAST analysis, similarity of A. pennula to other nematode parasites was compared at the nucleotide level. Phylogenetic analysis of A. pennula COX1 indicated relationships to Subuluridae, Ascarididae, and Anisakidae. This study on molecular characterization of A. pennula provides new insight for the diagnosis of caecal worm infections of quail in the Rolling plains Ecoregion of Texas.
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Parasitic nematodes that infect quail have been understudied and long been dismissed as a problem in quail management. Within the Rolling Plains ecoregion of Texas, an area that has experienced quail population “boom and bust” cycles and ultimately a general decline, the need to determine why Northern bobwhite (Colinus virginianus) populations are diminishing has increased in priority. Previously, caecal parasites have been documented to cause inactivity, weight loss, reduced growth, inflammation to the caecal mucosa, and even death. The caecal worm Aulonocephalus pennula is an intestinal nematode parasite that is commonly found within the caecum of quail, as well as many other avian species. In the Rolling Plains ecoregion, A. pennula has been documented to have as high as a 98% prevalence in bobwhite quail samples; however, the effect it has on its host is not well understood. The present study documents A. pennula causes no pathological changes within the caeca of the Northern bobwhite. However, there is concern for disruption of digestion and the possible implications of infection for wild bobwhite quail survival are discussed.
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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.
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Debilitating ocular diseases are often reported in avian species. By and large, helminth parasites have been overlooked in avian diseases and regarded as inconsequential. The decline of Northern bobwhite quail (Colinus virginianus) in the Rolling Plains ecoregion of Texas has prompted an investigation of the factors influencing their disappearance. Infection by the eyeworm (Oxyspirura petrowi) has been documented in many avian species; however, the effect it has on its host is not well understood. Heavy eyeworm infection has been documented in Northern bobwhites throughout this ecoregion, leading to eye pathology in this host species. The present study further documents and supports the pathological changes associated with O. petrowi in bobwhites.
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The Rolling Plains have historically provided some of the best opportunities to hunt northern bobwhite (Colinus virginianus) populations anywhere. Historically, scaled quail (Callipepla squamata) have been common to abundant over much of the Rolling Plains, but the populations decreased dramatically in the late 1980s and have been slow to reclaim their historic range. Copyright © 2007 by Leonard Alfred Brennan Manufactured in the United States of America All rights reserved.
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Survey for Trichomonas gallinae and Assessment of Helminth Parasites in Northern Bobwhites from the Rollin Plains Ecoregion
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Survey for Trichomonas gallinae and Assessment of Helminth Parasites in Northern Bobwhites from the Rollin Plains Ecoregion
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