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.

Immune challenge-induced oxidative damage may be mitigated by biliverdin.

An effective immune response results in the elimination of pathogens, but this immunological benefit may be accompanied by increased levels of oxidative damage. However, organisms have evolved mechanisms to mitigate the extent of such oxidative damage, including the production and mobilization of antioxidants. One potential mechanism of mitigating immune challenge-induced changes in oxidative physiology is increasing biliverdin production. Biliverdin is chemically an antioxidant, but within-tissue correlations between biliverdin concentration and oxidative damage have never been directly examined. To test how biliverdin tissue concentrations are associated with physiological responses to an immune challenge, we exposed northern bobwhite quail (Colinus virginianus) to one of four treatments: injection of a non-pathogenic antigen - either lipopolysaccharide or phytohemagglutinin, control injection of phosphate-buffered saline or a sham procedure with no injection. Twenty-four hours later, we quantified oxidative damage and triglyceride concentration in the plasma, and biliverdin concentration in the plasma, liver and spleen. We found that both types of immune challenge increased oxidative damage relative to both non-injected and vehicle-injected controls, but treatment had no effects on any other metric. However, across all birds, oxidative damage and biliverdin concentration in the plasma were negatively correlated, which is consistent with a localized antioxidant function of biliverdin. Additionally, we uncovered multiple links between biliverdin concentration, change in mass during the immune challenges and triglyceride levels, suggesting that pathways associated with biliverdin production may also be associated with aspects of nutrient mobilization. Future experiments that manipulate biliverdin levels or oxidative damage directly could establish a systemic antioxidant function or elucidate important physiological impacts on body mass maintenance and triglyceride storage, mobilization or transport.

Immune challenges decrease biliverdin concentration in the spleen of northern Bobwhite quail, Colinus virginianus.

Most antioxidants have multiple functions; in addition to minimizing oxidative damage, many antioxidants have immune-modulating properties. For example, biliverdin is produced in the liver and spleen from the breakdown of heme, and has putative immune-suppressing and antioxidant properties. However, the majority of these properties have been investigated in vitro or in mammalian models, in which biliverdin reductase converts virtually all biliverdin to bilirubin. Thus, biliverdin's physiological roles remain largely untested. Here, we investigated whether biliverdin has immunomodulating roles by injecting Northern Bobwhite quail (Colinus virginianus) with either a vehicle control, lysed and rinsed pig red blood cells (pRBC) that contain erythrocyte antigens but no heme, or intact pRBC that contain both erythrocyte antigens and hemoglobin, thus increasing the amount of heme that can be converted to biliverdin. We then quantified hemagglutination and hemolysis ability, and biliverdin concentration in the liver and spleen, on 3, 6, and 9 days post-injection. We found that hemagglutination was greater in individuals that received intact pRBC, but not in those injected with heme-removed pRBC, demonstrating that biliverdin does not suppress immune function at this dosage. Biliverdin levels of liver and spleen were correlated within individuals, suggesting organism-level variation in biliverdin production. Lastly, individuals injected with intact pRBC had a reduced biliverdin concentration in the spleen, suggesting that immune challenges may reduce biliverdin production or accumulation. This initial investigation demonstrated that biliverdin may have more nuanced physiological roles than previously reported, supporting the value of further investigations into the physiology of biliverdin.

A preliminary assessment of relative sensitivities to foreign red blood cell challenges in the northern bobwhite for potential evaluation of immunotoxicity.

Many environmental toxins have been shown to suppress the immune system across taxa. The foreign red blood cell (RBC) challenge is an important part of a complement of tests used to assess immunocompetence in the laboratory because it can assess an individual's humoral response without impacting its health. This challenge is used commonly across species and measures antibody titers in response to an intraperitoneal, intravenous, or subcutaneous injection of foreign RBCs. Determination of the best appropriate foreign RBC challenge is therefore important when designing tests for evaluation of humoral responses. The northern bobwhite (Colinus virginianus) is a commonly used species for avian toxicity tests, however little is known about the relative sensitivities of its humoral responses to foreign erythrocytes. In this pilot study, we exposed adult quail to intravenous injections of 5% solutions of sheep, rat, rabbit, bovine, or chicken erythrocytes and performed antibody titers [hemagglutination assay for total immunoglobulin (Ig), IgG, and IgM] for primary and secondary responses. Although the bobwhites appeared to respond strongly to rat RBCs, high variability in responses were observed among individuals. Chicken RBCs elicited the poorest responses for both primary and secondary challenges. Sheep and bovine RBCs were adequate antigens for this test in bobwhites. We found that rabbit erythrocytes elicited the strongest responses with the least amount of variability between individuals. Rabbit RBCs, therefore, appear to be the ideal antigen for this test of the humoral response in this species.

Examination of the dietary methionine requirements of breeding northern bobwhite, Colinus virginianus.

Adult Northern bobwhite were used to test the hypothesis that dietary methionine levels recommended by the NRC for breeding quail are excessive for wild bobwhite. We tested the hypothesis by comparing immunocompetence, reproductive performance, and chick viability of Northern bobwhite hens fed diets containing low (0.31%), moderate (0.39%), or high (0.47%) concentrations of methionine. Chick viability was determined by assessing immunocompetence, including evaluating the ability of hens to passively transfer immunity to their chicks. Hens were fed the experimental diets for 6 wk on an ad libitum basis. After 6 wk, methionine treatment had no measurable effect (P > or = 0.20) on hen phytohemagglutinin wing web indices, organ weights, or serum anti-Pasteurella multocida titer indices. Mean egg weight, percentage egg production, total cumulative egg production, yolk weight, yolk volume, and percentage fertile and percentage hatch of fertile eggs did not differ (P > or = 0.12) among diet treatments. Amount of albumen in eggs produced by hens fed the high methionine diet averaged 0.27 g more (P = 0.003) than eggs of hens fed the low methionine diet. Anti-P. multocida titer of yolks from eggs in Week 6 were not different (P = 0.36) between birds fed the high and the low methionine diets. The mortality rate of chicks after challenge with 23 cfu of P. multocida was not different (P > or = 0.05) among diets. Chicks hatched from eggs laid by vaccinated hens during Weeks 2 and 3, however, had lower (P < 0.05) mortality than chicks of unvaccinated hens. It appears a dietary methionine concentration of 0.3% may be sufficient for wild Northern bobwhite to produce viable chicks.

A pathogenic challenge model for adult northern bobwhite (Colinus virginianus) using a vaccine strain of Pasteurella multocida type 3.

A pathogenic challenge model causing approximately 50% mortality was developed in adult Northern bobwhite (Colinus virginianus) using Avichol, a live vaccine containing the Clemson University (CU) strain of Pasteurella multocida Type 3. A dose of 2300 or 3000 colony-forming units (CFU) of Avichol injected intramuscularly resulted in 30 to 75% mortality, whereas a dose of 230 CFU or less resulted in no mortality, and 58,720 CFU or more resulted in death in all birds challenged. Primary and secondary vaccination of Northern bobwhite with a formalinized anaculture of Avichol -derived P. multocida resulted in protection against challenge in three separate experiments. Dexamethasone treatment of birds during vaccination resulted in decreased protection against challenge exposure.

Proliferative responses of splenocytes from wild and domestic northern bobwhites (Colinus virginianus) to T- and B-cell mitogens.

Baseline information on the functional responses expected for assays used to assess immunocompetence in the Northern bobwhite (Colinus virginianus) are largely unavailable. Our primary objective was to develop an in vitro lymphoproliferative response assay for assessing cell-mediated immunocompetence in the Northern bobwhite. Culture conditions were optimised for domestic Northern bobwhites and field tested on splenocytes from wild-caught quail. Results indicated that increasing cell concentration and media volume in culture, as well as decreasing concentrations of serum in media, improved splenocyte responses to Con A stimulation. Optimum culture conditions were attained with 1 million cells per well cultured in 200 microliters of AIM-V serum-free media for 72 h. Five micrograms concanavalin A (Con A) or 2.5 micrograms Salmonella typhimurium mitogen (STM) per well provided maximum stimulation as measured by 3H-thymidine incorporation. Stimulation indices of splenocyte cultures of wild-caught Northern bobwhites to 5 micrograms Con A were approximately four-fold greater than levels observed for domestic quail (P = 0.0055). Alternatively, stimulation indices of splenocyte cultures obtained from wild-caught and domestic Northern bobwhites to 2.5 micrograms STM per well were not different (P = 0.3938).