Widespread detection of highly pathogenic H5 influenza viruses in wild birds from the Pacific Flyway of the United States.

A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza surveillance in wild birds in the Pacific Flyway of the United States. A total of 4,729 hunter-harvested wild birds were sampled and highly pathogenic avian influenza virus was detected in 1.3% (n = 63). Three H5 clade 2.3.4.4 subtypes were isolated from wild birds, H5N2, H5N8, and H5N1, representing the wholly Eurasian lineage H5N8 and two novel reassortant viruses. Testing of 150 additional wild birds during avian morbidity and mortality investigations in Washington yielded 10 (6.7%) additional highly pathogenic avian influenza isolates (H5N8 = 3 and H5N2 = 7). The geographically widespread detection of these viruses in apparently healthy wild waterfowl suggest that the H5 clade 2.3.4.4 variant viruses may behave similarly in this taxonomic group whereby many waterfowl species are susceptible to infection but do not demonstrate obvious clinical disease. Despite these findings in wild waterfowl, mortality has been documented for some wild bird species and losses in US domestic poultry during the first half of 2015 were unprecedented.

Widespread detection of antibodies to Leptospira in feral swine in the United States.

As feral swine continue to expand their geographical range and distribution across the United States, their involvement in crop damage, livestock predation, and pathogen transmission is likely to increase. Despite the relatively recent discovery of feral swine involvement in the aetiology of a variety of pathogens, their propensity to transmit and carry a wide variety of pathogens is disconcerting. We examined sera from 2055 feral swine for antibody presence to six serovars of Leptospira that can also infect humans, livestock or domestic animals. About 13% of all samples tested positive for at least one serovar, suggesting that Leptospira infection is common in feral swine. Further studies to identify the proportion of actively infected animals are needed to more fully understand the risk they pose.

Identification and analysis of the first 2009 pandemic H1N1 influenza virus from U.S. feral swine.

The first case of pandemic H1N1 influenza (pH1N1) virus in feral swine in the United States was identified in Texas through the United States Department of Agriculture (USDA) Wildlife Services' surveillance program. Two samples were identified as pandemic influenza by reverse transcriptase quantitative PCR (RT-qPCR). Full-genome Sanger sequencing of all eight influenza segments was performed. In addition, Illumina deep sequencing of the original diagnostic samples and their respective virus isolation cultures were performed to assess the feasibility of using an unbiased whole-genome linear target amplification method and multiple sample sequencing in a single Illumina GAIIx lane. Identical sequences were obtained using both techniques. Phylogenetic analysis indicated that all gene segments belonged to the pH1N1 (2009) lineage. In conclusion, we have identified the first pH1N1 isolate in feral swine in the United States and have demonstrated the use of an easy unbiased linear amplification method for deep sequencing of multiple samples.

Seasonal variation in serum testosterone, testicular volume, and semen characteristics in the coyote (Canis latrans).

The coyote is a seasonally breeding mammal, with most copulations occurring between December and April (depending on location). The objective of this study was to characterize seasonal changes in serum testosterone concentrations, testicular volume, and ejaculate quantity and quality in captive male coyotes. There were seasonal differences in testicular volume, with the greatest volume (20.2+/-5.4cm2), mean+/-S.E.M.) in February, corresponding with peak breeding season. Circulating serum testosterone concentrations peaked (3.31+/-0.9 ng/mL) during January and were positively correlated (P< or =0.001, r=0.413) with testicular volume. Ejaculate volume (1.67+/-0.4 mL) and sperm concentration (549.2 x 10(6)+/-297.7 spermatozoa/mL) both peaked during January and February, consistent with the height of the breeding season. Ejaculate volume and sperm concentrations were positively correlated with testicular size (r=0.679, P< or =0.001 and r=0.499, P< or =0.001, respectively) and with serum testosterone concentrations (r=0.368, P< or =0.01 and r=0.208, P< or =0.05). Progressively motile, viable, and morphologically normal spermatozoa fluctuated seasonally, peaked (90.4+/-4.5, 84.8+/-4.1, and 87.9+/-2.9%) during the breeding season, and then subsequently declined (period of aspermatogenesis). All three of these end points were positively correlated with testicular size (r=0.589, P< or =0.001; r=0.586, P< or =0.001; and r=0.469; P< or =0.001) and serum testosterone (r=0.167, P< or =0.05; r=0.190, P< or =0.05; and r=0.221, P< or =0.01). In conclusion, there were intricate relationships among testosterone concentrations, testicular volume, and the production of both functionally intact and morphologically normal spermatozoa.

Evaluation and significance of tetracycline stability in rabies vaccine baits.

Tetracycline is widely used as a biomarker for bait consumption by wildlife; tetracycline is incorporated into bones and teeth and can be detected by fluorescence microscopy several weeks postconsumption. During 2003, the United States Department of Agriculture distributed more than 10 million tetracycline-containing rabies-vaccine baits to control the spread of wildlife vectored rabies to humans, pets, and livestock. To estimate the percentage of target species consuming the baits, raccoons and skunks were collected in baited areas and teeth were analyzed for the presence of the biomarker. Several incidents of low biomarker detection rates prompted an investigation of the stability of the biomarker in the baits. Baits were collected at several points along the manufacturing and distribution chain. Baits were analyzed for free and polymer-bound tetracycline and the less active isomer epitetracycline. Results indicated that a portion of the tetracycline was converted to epitetracycline. Additionally, significant quantities of both compounds were trapped in the polymer, which is homogeneously distributed throughout the bait. The results of this study suggest that approximately 40% of the target quantity of tetracycline was unavailable for absorption. This situation could contribute to low biomarker detection rates and suggests that formulation modification should be considered.

Assessment of cabergoline as a reproductive inhibitor in coyotes (Canis latrans).

The efficacy of three oral formulations (gelatin capsule, tablet, oil base) and five dosages (50, 100, 250, 500, 1000 microg) of cabergoline to disrupt reproduction in coyotes (Canis latrans) was evaluated. The type of formulation used had no effect on plasma progesterone and prolactin concentrations or on mean litter size. No adverse side effects (for example, vomiting, anorexia, diarrhoea) were observed despite the use of doses of up to 20 times the therapeutic dose used for domestic dogs and cats. All coyotes treated with 50, 100, 250 and 500 microg cabergoline whelped, but plasma progesterone concentrations in these coyotes were lower (P < or = 0.07) than in control animals at day 7 after treatment. Ten of 11 females treated with 1000 microg cabergoline whelped, but progesterone concentrations in these coyotes were lower than in control animals up to day 14 after treatment (P < or = 0.04). Dosages of 1000 microg cabergoline decreased blood serum prolactin (P < or = 0.10) and progesterone (P < or = 0.06) concentrations, but apparently failed to decrease progesterone below the threshold necessary to maintain pregnancy in all but one animal. However, progressive inhibition of prolactin and progesterone with increasing doses of cabergoline indicated that higher dosages might be effective in coyotes. Survival of pups born to cabergoline-treated females was not different (P < 0.001) from that of pups born to control females, but mean litter size was smaller for females treated with cabergoline (P < or = 0.073) than for the control females. Although all cabergoline treatments in this study were ineffective at preventing reproduction in coyotes, progressive inhibition of prolactin and progesterone with increasing dosages of cabergoline indicates that higher doses might be effective in preventing reproduction in coyotes. However, the physiological differences from other canine species in dopamine D2 receptors and mechanisms of luteal support may ultimately prevent the use of cabergoline for reproductive control in coyotes.

Determination of cabergoline by electrospray ionization tandem mass spectrometry: picogram detection via column focusing sample introduction.

An electrospray ionization tandem mass spectrometric method was developed for low-picogram detection of an ergot alkaloid, cabergoline, in coyote plasma extracts. Cabergoline is under investigation as an abortifacient in canid species. Central to the successful development of this method was the ability to introduce relatively large sample volumes into the mass spectrometer. This was achieved by focusing the analyte on a conventional high-performance liquid chromatography guard column prior to elution into the spectrometer. Volumes up to at least 900 microL could be injected onto the guard column using a 100% aqueous mobile phase. Cabergoline retained on the column was eluted as a discreet band into the mass spectrometer by the rapid addition of methanol (30%) to the mobile phase. As compared to flow injection sample introduction, the ability to inject larger sample volumes led to a greatly lowered detection limit. Using this technique and a modification of a previously reported extraction procedure, cabergoline could be determined in coyote plasma at concentrations as low as 9 pg of cabergoline/mL of plasma.