Luis R. Padilla

SURVEY FOR HAEMOPROTEUS SPP., TRICHOMONAS GALLINAE, CHLAMYDOPHILA PSITTACI, AND SALMONELLA SPP. IN GALAPAGOS ISLANDS COLUMBIFORMES

Abstract Endemic free-ranging Galapagos doves (Zenaida galapagoensis) and introduced rock doves (Columba livia) were surveyed in several islands of the Galapagos archipelago to establish sample prevalence of hemoparasites, Trichomonas gallinae, Chlamydophila psittaci, and Salmonella species. A Haemoproteus sp., the only hemoparasite identified, was found in 89% of the Galapagos doves sampled but not in the rock doves. Trichomonas gallinae was detected by polymerase chain reaction in 44% of rock doves from San Cristobal but in none of the Galapagos doves. Chlamydophila psittaci was detected from cloacal swabs in 6% of the Galapagos doves but in none of the rock doves sampled. All positive cases of C. psittaci occurred on Española, where the crude sample prevalence was 24%. A polymerase chain reaction–based Salmonella test failed to show evidence of this organism from any birds sampled.

HEMATOLOGY, PLASMA CHEMISTRY, SEROLOGY, AND CHLAMYDOPHILA STATUS OF THE WAVED ALBATROSS (PHOEBASTRIA IRRORATA) ON THE GALAPAGOS ISLANDS

Abstract Venipuncture was performed on 50 adult, free-ranging waved albatrosses (Phoebastria irrorata) on Española, Galapagos Islands, Ecuador, to establish hematologic and plasma biochemistry reference ranges and to determine the prevalence of exposure to important domestic avian pathogens. Weights and plasma creatine phosphokinase activities differed significantly between males and females. Serum was tested for evidence of exposure to avian influenza, avian paramyxoviruses 1, 2, and 3, avian cholera, adenovirus groups 1 and 2, avian encephalomyelitis, Mareks disease, infectious bursal disease, and infectious bronchitis virus (Connecticut and Massachusetts strains). Of 44 birds, 29 (66%) seroreacted to adenovirus group 1, and four seroreacted to avian encephalomyelitis. Cloacal swabs were negative for Chlamydophila psittaci DNA.

Comparison of Pathogens in Broiler and Backyard Chickens on the Galápagos Islands: Implications for Transmission to Wildlife

Abstract As the human population and tourism increase in the Galápagos Islands, increased poultry production raises risks of pathogen spillover into native avian populations. Here, we characterize the disease risks to Galápagos avifauna of different types of poultry farming by comparing health status and serosurvey results between broiler and backyard chickens (Gallus gallus domesticus). Backyard chickens were more frequently diseased than broilers, and were more likely to be seropositive for several pathogens (Mycoplasma gallisepticum, infectious laryngotracheitis virus, infectious bronchitis virus, avian reovirus, and Mareks disease virus). Seroprevalence for other pathogens (avian paramyxovirus-1, infectious bursal disease, avian encephalomyelitis virus, and avian adenovirus) was relatively high among all chickens. Preliminary serological results from wild birds revealed no evidence of previous exposure to these diseases, which suggests that transmission of disease from poultry to wildlife is currently not detectable with the sample sizes and tests employed, and that wildlife are likely not the source of exposure to poultry. Our results suggest that backyard chickens may pose a greater threat to Galápagos avifauna because they are more likely to be infectious, have a high seroprevalence for numerous pathogens, and interact directly with wild birds or wild bird habitat, with no biosecurity measures employed. The broiler industry has greater potential for importation of pathogens into the islands and indirect transmission of diseases to wildlife (e.g., through use of poultry litter on agricultural land). Regulatory and management decisions should focus on minimizing the poultry–wildlife interface, reducing infectious diseases in backyard chickens, and preventing importation of poultry diseases.

Pharmacokinetics of a single intramuscular injection of ceftiofur crystalline-free acid in American black ducks (Anas rubripes)

OBJECTIVE To determine the pharmacokinetic properties of 1 IM injection of ceftiofur crystalline-free acid (CCFA) in American black ducks (Anas rubripes). ANIMALS 20 adult American black ducks (6 in a preliminary experiment and 14 in a primary experiment). PROCEDURES Dose and route of administration of CCFA for the primary experiment were determined in a preliminary experiment. In the primary experiment, CCFA (10 mg/kg, IM) was administered to ducks. Ducks were allocated into 2 groups, and blood samples were obtained 0.25, 0.5, 1, 2, 4, 8, 12, 48, 96, 144, 192, and 240 hours or 0.25, 0.5, 1, 2, 4, 8, 24, 72, 120, 168, and 216 hours after administration of CCFA. Plasma concentrations of ceftiofur free acid equivalents (CFAEs) were determined by use of high-performance liquid chromatography. Data were evaluated by use of a naive pooled-data approach. RESULTS The area under the plasma concentration versus time curve from 0 hours to infinity was 783 h•μg/mL, maximum plasma concentration observed was 13.1 μg/mL, time to maximum plasma concentration observed was 24 hours, terminal phase half-life was 32.0 hours, time that concentrations of CFAEs were higher than the minimum inhibitory concentration (1.0 μg/mL) for many pathogens of birds was 123 hours, and time that concentrations of CFAEs were higher than the target plasma concentration (4.0 μg/mL) was 73.3 hours. CONCLUSIONS AND CLINICAL RELEVANCE On the basis of the time that CFAE concentrations were higher than the target plasma concentration, a dosing interval of 3 days can be recommended for future multidose CCFA studies.

Ejaculate Traits and Sperm Cryopreservation in the Endangered Baird's Tapir (Tapirus bairdii)

There is little information on the reproductive biology of the male Bairds tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P < .004). Cryopreservation in either Botu-Crio or INRA 96 resulted in a decline (P < .05) in percent sperm motility and acrosomal integrity. Sperm forward progression remained unaffected immediately after thawing in INRA 96 but continued to decline over time. These results characterize, for the first time, the ejaculate traits of the tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Bairds tapir.

Cardiac Evaluation of Clinically Healthy Captive Maned Wolves (Chrysocyon brachyurus)

Abstract The objective of this project was to determine radiographic vertebral heart sizes and electrocardiographic (ECG) and echocardiographic measurements in healthy anesthetized maned wolves (Chrysocyon brachyurus). The wolves, housed at the White Oak Conservation and Smithsonian National Zoo Conservation and Research Centers, were being anesthetized for annual examinations. Cardiac auscultation, thoracic radiographs, a standard 6-lead ECG, and echocardiography were performed on the wolves while they were under general anesthesia. Thirteen maned wolves were evaluated: five males and eight females. Mean age was 6.4 ± 4.4 years (range, 2–13 years). Mean weight was 26 ± 2.95 kg (range, 22–32 kg). Low-grade systolic murmurs were auscultated in three of 13 maned wolves. Evaluation of ECGs revealed a sinus rhythm, with a QRS morphology, and mean electrical axis similar to domestic canines. Radiographic evaluation revealed a mean vertebral heart size of 8.27 ± 0.48 (range, 7.9–8.6). In addition, the cardiac silhouette was seen to elongate, with an increase in sternal contact in older wolves. Echocardiography showed that mitral valve degenerative changes and insufficiency is likely common in older wolves. Visualization of physiologic regurgitation across the mitral and pulmonary valves was common in wolves of all ages. Left ventricular measurements were similar to those reported for healthy dogs, and several variables correlated well with body weight. Two wolves were found to have one to three heartworms in the right pulmonary artery, and degenerative mitral valve disease was determined in maned wolves older than 6 years of age. All of the wolves in this study were on heartworm preventative and tested negative for heartworm antigen at their annual examinations. The results of this study provide reference information for use in the cardiac evaluation of anesthetized maned wolves.

Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii) ☆

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalskis horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalskis horse spermatozoa during liquid storage at 4°C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalskis versus domestic horse. When Przewalskis horse spermatozoa were incubated at 4°C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P<0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P>0.05) between the Przewalskis (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose-EDTA-glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4CN (CN=mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalskis and 49.3%, 24.6%, 88.9% and 25.8CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P>0.05) among treatments across species, mitochondrial membrane potential was higher (P<0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalskis stallion sperm expressed higher (P<0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalskis stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalskis and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalskis horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF.

XY MALE PSEUDOHERMAPHRODITISM IN A CAPTIVE ARABIAN ORYX (ORYX LEUCORYX)

Abstract A 2-yr-old Arabian oryx (Oryx leucoryx) was presented for evaluation of abnormal genitalia and infantile behavior. The oryx had a penis and a scrotum, but testes were not palpable within the scrotum or inguinal canal. The total serum testosterone for the individual was lower than in age-matched males of the same species. Surgical exploration showed markedly hypoplastic intra-abdominal gonads, which demonstrated both testicular and uterine tissue on histologic examination. After karyotype analysis, the individual was classified as an XY male pseudohermaphrodite. This condition resembles two human intersex syndromes: embryonic testicular regression syndrome and partial gonadal dysgenesis syndrome, which occur in familial lines.

Domestic and Peridomestic Animals in Galapagos: Health Policies and Practices

The wildlife of the Galapagos Islands faces the threat of disease due to spillover from introduced domestic and peridomestic species. Many domestic species benefit the roughly 25,000 residents and 250,000 visitors that travel to the islands every year. Although expanded human activities and the necessary agricultural and trade practices to support them are a potential concern for long-term sustainability of endemic species, the thriving economy behind this prime ecotourism destination is a possible asset and solution to protect it. Local agencies and strong, comprehensive management policies that are adaptable to rapidly changing conditions must be in place to guard against known and unknown disease threats.

CLINICAL DISEASE ASSOCIATED WITH ANAPLASMA PHAGOCYTOPHILUM INFECTION IN CAPTIVE PRZEWALSKI'S HORSES (EQUUS FERUS PRZEWALSKII)

Abstract Anaplasma phagocytophilum is a tick-borne pathogen of domestic horses and the causative agent of equine granulocytic anaplasmosis. This case series describes three confirmed cases of clinical anaplasmosis, and a fourth case of presumptive anaplasmosis in Przewalskis horses (Equus ferus przewalskii) housed at the Smithsonian Conservation Biology Institute from 2008 to 2014. Clinical signs varied among individuals with affected horses exhibiting lethargy, weakness, pyrexia, hypophagia, reluctance to move, or ataxia. Anaplasmosis cases were confirmed with a combination of identification of neutrophilic inclusions (morulae) on peripheral blood smear, positive polymerase chain reaction (PCR) testing of whole blood, or convalescent titers. All animals recovered after antimicrobial therapy with oxytetracycline. Diagnosis should be made by a combination of clinical signs plus identification of morulae or positive A. phagocytophilum PCR. Treatment is curative using oxytetracyline intramuscularly or intravenously followed by daily therapy with oxytetracyline or minocycline for 14–30 days. The authors recommend that A. phagocytophilum infection be included on any differential list for Przewalskis horses presenting with fever or ataxia within or near an enzootic area.