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

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.

Life-cycle of Oxyspirura petrowi (Spirurida: Thelaziidae), an eyeworm of the northern bobwhite quail (Colinus virginianus).

BACKGROUND: Oxyspirura petrowi (Spirurida: Thelaziidae), a heteroxenous nematode of birds across the USA, may play a role in the decline of the northern bobwhite (Colinus virginianus) in the Rolling Plains Ecoregion of West Texas. Previous molecular studies suggest that crickets, grasshoppers and cockroaches serve as potential intermediate hosts of O. petrowi, although a complete study on the life-cycle of this nematode has not been conducted thus far. Consequently, this study aims to improve our understanding of the O. petrowi life-cycle by experimentally infecting house crickets (Acheta domesticus) with O. petrowi eggs, feeding infected crickets to bobwhite and assessing the life-cycle of this nematode in both the definitive and intermediate hosts. METHODS: Oxyspirura petrowi eggs were collected from gravid worms recovered from wild bobwhite and fed to house crickets. The development of O. petrowi within crickets was monitored by dissection of crickets at specified intervals. When infective larvae were found inside crickets, parasite-free pen-raised bobwhite were fed four infected crickets each. The maturation of O. petrowi in bobwhite was monitored through fecal floats and bobwhite necropsies at specified intervals. RESULTS: In this study, we were able to infect both crickets (n = 45) and bobwhite (n = 25) with O. petrowi at a rate of 96%. We successfully replicated and monitored the complete O. petrowi life-cycle in vivo, recovering embryonated O. petrowi eggs from the feces of bobwhite 51 days after consumption of infected crickets. All life-cycle stages of O. petrowi were confirmed in both the house cricket and the bobwhite using morphological and molecular techniques. CONCLUSIONS: This study provides a better understanding of the infection mechanism and life-cycle of O. petrowi by tracking the developmental progress within both the intermediate and definitive host. To our knowledge, this study is the first to fully monitor the complete life-cycle of O. petrowi and may allow for better estimates into the potential for future epizootics of O. petrowi in bobwhite. Finally, this study provides a model for experimental infection that may be used in research examining the effects of O. petrowi infection in bobwhite.

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.

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 analysis of Northern bobwhite (Colinus virginianus) cytokines and TLR expression to eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) glycoproteins.

Helminth parasites have been a popular research topic due to their global prevalence and adverse effects on livestock and game species. The Northern bobwhite (Colinus virginianus), a popular game bird in the USA, is one species subject to helminth infection and has been experiencing a decline of > 4% annually over recent decades. In the Rolling Plains Ecoregion of Texas, the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) helminths are found to be highly prevalent in bobwhite. While there have been increasing studies on the prevalence, pathology, and phylogeny of the eyeworm and caecal worm, there is still a need to investigate the bobwhite immune response to infection. This study utilizes previously sequenced bobwhite cytokines and toll-like receptors to develop and optimize qPCR primers and measure gene expression in bobwhite intramuscularly challenged with eyeworm and caecal worm glycoproteins. For the challenge experiments, separate treatments of eyeworm and caecal worm glycoproteins were administered to bobwhite on day 1 and day 21. Measurements of primary and secondary immune responses were taken at day 7 and day 28, respectively. Using the successfully optimized qPCR primers for TLR7, IL1ß, IL6, IFNα, IFNγ, IL10, and ß-actin, the gene expression analysis from the challenge experiments revealed that there was a measurable immune reaction in bobwhite in response to the intramuscular challenge of eyeworm and caecal worm glycoproteins.

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.

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.

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 Ecoregion, Texas.

The Northern bobwhite quail (Colinus virginianus) is an economically significant gamebird that has experienced a decline throughout the Rolling Plains ecoregion of Texas. Recent surveys of this area have revealed a high prevalence in eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) infection that may contribute to this decline. In order to further understand these parasites role in bobwhite populations, a time-, and cost-effective multiplex quantitative PCR (qPCR) assay was developed in this study to detect eyeworm and caecal worm infection through egg detection using the ITS2 and COX1 gene region, respectively. Method validation for the qPCR involved bobwhite fecal samples from the Rolling Plains as well as samples spiked with eyeworm, caecal worm, and bobwhite DNA. Results showed an observed increasing qPCR parasite egg detection with increasing worm burdens. Future uses with this assay can also provide insight to seasonal parasite infection and the life cycles of eyeworm and caecal worm.

Molecular identification and characterization of partial COX1 gene from caecal worm (Aulonocephalus pennula) in Northern bobwhite (Colinus virginianus) from the Rolling Plains Ecoregion of Texas.

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.