Reticuloendotheliosis virus (REV) is a retrovirus that causes a neoplastic disease in a wide range of avian hosts including chickens, turkeys, and ducks. In 1993, REV was detected in the endangered Attwater's prairie chicken (Tympanachus cupido attwateri), a subspecies of Tympanachus cupido. Subsequent infections of this prairie chicken have been identified at captive breeding facilities throughout Texas. The implications of these infections have severely hindered repopulation efforts by these facilities. This study focused on investigating REV infection of captive Attwater'€™s prairie chicken in order to better understand the disease affecting these endangered birds. The overall objective was to develop a means of eliminating this threat to the repopulation of the Attwater's prairie chicken. Several aspects of virus infection were investigated. Reagents capable of recognizing prairie chicken IgY and viral gag polypeptides were developed for use in assays for detection of antibody responses and titration of viral concentrations. Sequencing data of genomes collected from isolates of Texas prairie chickens and domestic chickens, as well as three REV prototype viruses, were compared to determine relationships among strains and identify the potential origin of the REV infecting Attwater'€™s prairie chicken. Additionally, a flow cytometry technique of segregating the lymphocyte population from peripheral blood mononuclear cells (PBMC) using a pan leukocyte monoclonal antibody was developed to more accurately measure changes within lymphocyte populations. This technique combined with intracellular labeling was used to deduce the target cells of REV infection. A nested polymerase chain reaction (PCR) test was developed for greater sensitivity in detecting infection in birds than the previous method of single amplification PCR. This greater sensitivity results in earlier identification of the virus in infected birds, which allows for earlier removal of infected birds to minimize transmission of the virus throughout the flock. The sensitivity of the nested PCR diagnostic test was determined in a dose response pathogenesis study, which was conducted on hybrid greater/Attwater's prairie chicken to observe the experimental development of disease in these birds. Finally, a vaccine was developed using plasmid DNA with REV encoded genes and tested on naturally infected prairie chickens to determine its efficacy in reducing viral load. Although no reduction in viral load was detected, the vaccine may be effective in providing prophylactic protection in future studies.

Reticuloendotheliosis virus (REV) is a retrovirus that causes a neoplastic disease in a wide range of avian hosts including chickens, turkeys, and ducks. In 1993, REV was detected in the endangered Attwater's prairie chicken (Tympanachus cupido attwateri), a subspecies of Tympanachus cupido. Subsequent infections of this prairie chicken have been identified at captive breeding facilities throughout Texas. The implications of these infections have severely hindered repopulation efforts by these facilities. This study focused on investigating REV infection of captive Attwater'€™s prairie chicken in order to better understand the disease affecting these endangered birds. The overall objective was to develop a means of eliminating this threat to the repopulation of the Attwater's prairie chicken. Several aspects of virus infection were investigated. Reagents capable of recognizing prairie chicken IgY and viral gag polypeptides were developed for use in assays for detection of antibody responses and titration of viral concentrations. Sequencing data of genomes collected from isolates of Texas prairie chickens and domestic chickens, as well as three REV prototype viruses, were compared to determine relationships among strains and identify the potential origin of the REV infecting Attwater'€™s prairie chicken. Additionally, a flow cytometry technique of segregating the lymphocyte population from peripheral blood mononuclear cells (PBMC) using a pan leukocyte monoclonal antibody was developed to more accurately measure changes within lymphocyte populations. This technique combined with intracellular labeling was used to deduce the target cells of REV infection. A nested polymerase chain reaction (PCR) test was developed for greater sensitivity in detecting infection in birds than the previous method of single amplification PCR. This greater sensitivity results in earlier identification of the virus in infected birds, which allows for earlier removal of infected birds to minimize transmission of the virus throughout the flock. The sensitivity of the nested PCR diagnostic test was determined in a dose response pathogenesis study, which was conducted on hybrid greater/Attwater's prairie chicken to observe the experimental development of disease in these birds. Finally, a vaccine was developed using plasmid DNA with REV encoded genes and tested on naturally infected prairie chickens to determine its efficacy in reducing viral load. Although no reduction in viral load was detected, the vaccine may be effective in providing prophylactic protection in future studies.

The introduction of pen-reared Attwater's prairie-chickens (APC, Tympanuchus cupido attwateri) into the wild to supplement existing populations has met with marginal success. Flight characteristics, predator avoidance behavior, and rearing methods are possible factors contributing to post-release mortality of pen-reared birds. To evaluate flight characteristics and predator avoidance behavior of pen-reared APC's released onto the Attwater Prairie Chicken National Wildlife Refuge, flight characteristics and predator avoidance behavior of pen-reared APC's was compared to wild greater prairie-chickens (GPC, T. c. pinnatus) in Minnesota and Kansas using a radar gun and a trained dog. There was no difference (P = 0.134) in flight speed for pen-reared APC and wild GPC. However, wild GPC had greater (P < 0.001) flight distances than did pen-reared APC. Wild GPC and pen-reared APC that had survived in the wild for at least a year flushed at a greater (P < 0.001) distance from an approaching human than did pen-reared APC that had been released for less than 3 months. A trained dog was able to approach closer (P < 0.001) to APC than GPC before birds flushed, and APC did not fly as far as GPC after being flushed by the dog. Pen-reared APC displayed flight endurance deficiencies and were more approachable by humans and a dog before they flushed when compared to wild GPC, which could explain their increased mortality when released into the wild. To determine if APC chicks could be reared without daily human contact, pelleted food, and water in founts, a greenhouse was used to rear chicks in a semi-natural environment. Planted vegetation and commercial insects provided hiding cover and a food source for the APC chicks. An underground heat source provided chick warmth, and water misters and a sprinkler system simulated dew (a water source for chicks) and rain. The greenhouse provided chicks protection from predators and adverse weather conditions (before they could thermo-regulate) while exposing chicks to natural sunlight, day length, and temperature fluctuations. This technique allowed chicks to be reared in a semi-natural environment which reinforced their natural foraging behavior for food and water, and reinforced their hiding and avoidance behaviors, creating a wilder pen-reared bird.

The introduction of pen-reared Attwater's prairie-chickens (APC, Tympanuchus cupido attwateri) into the wild to supplement existing populations has met with marginal success. Flight characteristics, predator avoidance behavior, and rearing methods are possible factors contributing to post-release mortality of pen-reared birds. To evaluate flight characteristics and predator avoidance behavior of pen-reared APC's released onto the Attwater Prairie Chicken National Wildlife Refuge, flight characteristics and predator avoidance behavior of pen-reared APC's was compared to wild greater prairie-chickens (GPC, T. c. pinnatus) in Minnesota and Kansas using a radar gun and a trained dog. There was no difference (P = 0.134) in flight speed for pen-reared APC and wild GPC. However, wild GPC had greater (P < 0.001) flight distances than did pen-reared APC. Wild GPC and pen-reared APC that had survived in the wild for at least a year flushed at a greater (P < 0.001) distance from an approaching human than did pen-reared APC that had been released for less than 3 months. A trained dog was able to approach closer (P < 0.001) to APC than GPC before birds flushed, and APC did not fly as far as GPC after being flushed by the dog. Pen-reared APC displayed flight endurance deficiencies and were more approachable by humans and a dog before they flushed when compared to wild GPC, which could explain their increased mortality when released into the wild. To determine if APC chicks could be reared without daily human contact, pelleted food, and water in founts, a greenhouse was used to rear chicks in a semi-natural environment. Planted vegetation and commercial insects provided hiding cover and a food source for the APC chicks. An underground heat source provided chick warmth, and water misters and a sprinkler system simulated dew (a water source for chicks) and rain. The greenhouse provided chicks protection from predators and adverse weather conditions (before they could thermo-regulate) while exposing chicks to natural sunlight, day length, and temperature fluctuations. This technique allowed chicks to be reared in a semi-natural environment which reinforced their natural foraging behavior for food and water, and reinforced their hiding and avoidance behaviors, creating a wilder pen-reared bird.

The Attwater’s prairie-chicken (Tympanuchus cupido attwateri; APC) is a species of grouse native to Texas coastal prairies and is on the critically endangered species list as a result of habitat destruction and overhunting. All of the current populations were captively bred and released into the wild. Survivorship for released APCs is very low, and individuals seldom survive to reproduce in the wild. One factor contributing to this may be an alteration in the gut microbiota as a result of captivity. Factors potentially influencing the gut microbial composition in captivity include antibiotic therapy, stress, and a predominantly commercially formulated diet. Recent studies have begun to shed light on the importance of the host microbial endosymbionts. Antibiotic administration, stress, diet, age, genotype and other factors have been shown to influence microbial populations in the gastrointestinal tracts of many different vertebrates. Sequencing of 16S rRNA gene amplicons on the Ion Torrent™ platform was used in this study to identify groups of bacteria in the cloacas as a surrogate for the gut microbiota in the APC. Antibiotic-treated and untreated birds, wild-hatched and captive-bred birds, and individuals sampled before and after release to the wild were examined. Significant differences were found between wild-hatched and captive raised birds both pre- and post release. In addition, there was extensive variation among the populations at the lower taxonomic ranks between individuals for each group of APCs. Principal coordinate analysis based on the weighted UniFrac distance metric further exhibited some clustering of individuals by treatment. These data suggest that captive breeding may have long-term effects on the cloacal microbiota of APCs with unknown consequences to their long-term health and survivorship.

The goals of captive breeding programs for endangered species include preserving genetic diversity and avoiding inbreeding. Typically this is accomplished by minimizing population mean kinship; however, this approach becomes less effective when errors in the pedigree exist and may result in inbreeding depression, or reduced survival. Here, both pedigree- and DNA-based methods were used to assess inbreeding depression in the critically endangered Attwater’s prairie-chicken (Tympanuchus cupido attwateri). Less variation in the pedigree-based inbreeding coefficients and parental relatedness values were observed compared to DNA-based measures suggesting that errors exist in the pedigree. Further, chicks identified with high parental DNA-based relatedness exhibited decreased survival at both 14- and 50-days post-hatch. A similar pattern was observed in later life stages (> 50 days post-hatch) with birds released to the wild; however, the pattern varied depending on the time post-release. While DNA-based inbreeding coefficient was positively correlated with mortality to one month post-release, an opposite pattern was observed at nine months suggesting purging of deleterious alleles. I also investigated whether immunocompetence, or the ability to produce a normal immune response, was correlated with survival; however, no significant correlation was observed suggesting that inbreeding was a more important factor influencing survival. Pairing individuals for breeding by minimizing DNA-based parental relatedness values resulted in a significant increase in chick survival. This study highlights the importance of using DNA-based methods to avoid inbreeding depression when errors exist in the pedigree.

Unambiguous gender identification (ID) is needed to assess parameters in studies of population dynamics, behavior, and evolutionary biology of Caribbean Flamingo (Phoenicopterus ruber ruber) and other birds. Due to its importance for management and conservation, molecular (DNA-based) avian gender ID assays targeting intron-size differences of the Chromosome Helicase ATPase DNA Binding (CHD) gene of males (CHD-Z) and females (CHD-W) have been developed. Male (ZZ) and female (WZ) genotypes are usually scored as size polymorphisms through agarose or acrylamide gels. For certain species, W-specific restriction sites or multiplex polymerase chain-reaction (PCR) involving CHD-W specific primers are needed. These approaches involve a minimum of three steps following DNA isolation: PCR, gel electrophoresis, and photo-documentation, which limit high throughput scoring and automation potential. In here, a short amplicon (SA) High-resolution Melting Analysis (HRMA) assay for avian gender ID is developed. SA-HRMA of an 81-Base Pair (bp) segment differentiates heteroduplex female (WZ) from homoduplex male (ZZ) genotypes by targeting Single-nucleotide Polymorphisms (SNPs) instead of intron-size differences between CHD-Z and CHD-W genes. To demonstrate the utility of the approach, the gender of Caribbean Flamingo (P. ruber ruber) (17 captive from the Dallas Zoo and 359 wild from Ria Lagartos, Yucatan, Mexico) was determined. The assay was also tested on specimens of Lesser Flamingo (P. minor), Chilean Flamingo (P. chilensis), Saddle-billed Stork (Ephippiorhynchus senegalensis), Scarlet Ibis (Eudocimus ruber), White-bellied Stork (Ciconia abdimii), Roseate Spoonbill (Platalea ajaja), Marabou Stork (Leptoptilos crumeniferus), Greater Roadrunner (Geococcyx californianus), and Attwater's Prairie Chicken (Tympanuchus cupido attwateri). Although the orthologous 81 bp segments of Z and W are highly conserved, sequence alignments with 50 avian species across 15 families revealed mismatches affecting one or more nucleotides within the SA-HRMA forward or reverse primers. Most mismatches were located along the CHD-Z gene that may generate heteroduplex curves and thus gender ID errors. For such cases, taxon and species-specific primer sets were designed. The SA-HRMA gender ID assay can be used in studies of avian ecology and behavior, to assess sex-associated demographics and migratory patterns, and as a proxy to determine the health of the flock and the degree by which conservation and captive breeding programs are functioning.