Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi).

BACKGROUND: The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection. METHODOLOGY: In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper. RESULTS: Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils. CONCLUSION: Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites.

IMPLICATIONS OF TREATING PARASITIC INFECTION IN NORTHERN BOBWHITE (COLINUS VIRGINIANUS) ON OVERALL HELMINTH LIFE CYCLE.

This study was conducted to determine how reducing the parasite burden in a definitive host may affect the prevalence in intermediate hosts. Here we used the eyeworm Oxyspirura petrowi and cecal worm Aulonocephalus pennula as model species. Northern bobwhite quail (Colinus virginianus) were provided an anthelmintic medicated feed in wild systems because of convincing evidence that these parasites were suppressing their populations. Eyeworm and cecal worm prevalence were measured in Orthopterans, which act as intermediate hosts, using polymerase chain reaction. Individuals were collected from a control site, a site treated for 2 yr, and a site treated for 5 yr. Orthopteran community composition was significantly different among the sites; however, an interaction between subfamily and site was not significant for the eyeworm. There was a significant reduction in eyeworm-infected Orthopterans on the 5-yr site compared with the other 2, suggesting that treatment of a definitive host may indeed affect the prevalence of eyeworms during other life-cycle stages. There was an interaction between the Orthopteran subfamily and the site for the cecal worm, so results were analyzed within each subfamily. A significant reduction in the prevalence of cecal worms was only found in the Cyrtacanthacridinae subfamily on the 5-yr site when compared with the other sites. However, the greatest prevalence in the Gomphocerinae and Oedipodinae subfamilies across all 3 sites was 4.1%. This indicates an unknown degree of cecal worm host specificity. Therefore, conclusions could not be made through the simple assessment of prevalence.

Validation of an enzyme immunoassay for the detection of corticosterone metabolites from northern bobwhite (Colinus virginianus) feces.

Interest in the effects of stressors on wildlife has grown substantially over the past few decades. As this interest has grown, so has the need for minimally invasive and reliable methods for estimating differences in the levels of stress hormones. An enzyme immunoassay using standardized methods was validated for detecting concentrations of corticosterone (cort) metabolites from northern bobwhite fecal samples. Two physiological challenges and one biological challenge were applied to 18 northern bobwhites (nine males and nine females), and the fecal cort metabolite concentrations were compared to baseline levels. The interactions of sex and treatment, treatment and time and sex and time were all significant. Thus, the methods and tools used here were sensitive enough to detect expected changes to the hypothalamo-pituitary-adrenal axis of northern bobwhite.

MOLECULAR IDENTIFICATION OF INSECT INTERMEDIATE HOSTS WITH POTENTIAL PARASITE TRANSMISSION TO THE DEFINITIVE HOST NORTHERN BOBWHITE (COLINUS VIRGINIANUS).

Grassland birds have been declining substantially for the past several years. Habitat loss, degradation, and fragmentation as well as climate change are all thought to be the main drivers of the decline. However, as the declines continue to accelerate, it is becoming imperative to examine other factors that may contribute to population fluctuations. The nematodes Oxyspirura petrowi, Aulonocephalus pennula, and Physaloptera sp. are commonly found infecting northern bobwhite (Colinus virginianus), a game species of economic importance, and all 3 nematodes use insects as an intermediate host. Here we used polymerase chain reaction techniques to determine the occurrence of the 3 nematodes in 7 insect orders to uncover epidemiological patterns of the greatest potential for transmission to northern bobwhite. Insects were collected from March through September using sweep nets and pitfall traps. An R × C chi-squared test with Monte Carlo simulation was used to determine differences in the occurrence of the parasites across taxa and time. The results of the statistical analysis showed the nematodes are predominantly found in the order Orthoptera, and A. pennula and Physaloptera sp. showed epidemiological patterns in insects. However, no such pattern was observed with O. petrowi. An explanation for the lack of epidemiological pattern in O. petrowi is proposed and the diversity of known insect hosts of the 3 nematodes is increased.