Giardia duodenalis and Cryptosporidium occurrence in Australian sea lions (Neophoca cinerea) exposed to varied levels of human interaction

AbstractGiardia and Cryptosporidium are amongst the most common protozoan parasites identified as causing enteric disease in pinnipeds. A number of Giardia assemblages and Cryptosporidium species and genotypes are common in humans and terrestrial mammals and have also been identified in marine mammals. To investigate the occurrence of these parasites in an endangered marine mammal, the Australian sea lion (Neophoca cinerea), genomic DNA was extracted from faecal samples collected from wild populations (n = 271) in Southern and Western Australia and three Australian captive populations (n = 19). These were screened using PCR targeting the 18S rRNA of Giardia and Cryptosporidium. Giardia duodenalis was detected in 28 wild sea lions and in seven captive individuals. Successful sequencing of the 18S rRNA gene assigned 27 Giardia isolates to assemblage B and one to assemblage A, both assemblages commonly found in humans. Subsequent screening at the gdh and β-giardin loci resulted in amplification of only one of the 35 18S rRNA positive samples at the β-giardin locus. Sequencing at the β-giardin locus assigned the assemblage B 18S rRNA confirmed isolate to assemblage AI. The geographic distribution of sea lion populations sampled in relation to human settlements indicated that Giardia presence in sea lions was highest in populations less than 25 km from humans. Cryptosporidium was not detected by PCR screening in either wild colonies or captive sea lion populations. These data suggest that the presence of G. duodenalis in the endangered Australian sea lion is likely the result of dispersal from human sources. Multilocus molecular analyses are essential for the determination of G. duodenalis assemblages and subsequent inferences on transmission routes to endangered marine mammal populations

Molecular detection of antibiotic-resistance determinants in Escherichia coli isolated from the endangered Australian sea lion (Neophoca cinerea)

Greater interaction between humans and wildlife populations poses significant risks of anthropogenic impact to natural ecosystems, especially in the marine environment. Understanding the spread of microorganisms at the marine interface is therefore important if we are to mitigate adverse effects on marine wildlife. We investigated the establishment of Escherichia coli in the endangered Australian sea lion (Neophoca cinerea) by comparing fecal isolation from wild and captive sea lion populations. Fecal samples were collected from wild colonies March 2009-September 2010 and from captive individuals March 2011-May 2013. Using molecular screening, we assigned a phylotype to E. coli isolates and determined the presence of integrons, mobile genetic elements that capture gene cassettes conferring resistance to antimicrobial agents common in fecal coliforms. Group B2 was the most abundant phylotype in all E. coli isolates (n = 37), with groups A, B1, and D also identified. Integrons were not observed in E. coli (n = 21) isolated from wild sea lions, but were identified in E. coli from captive animals (n = 16), from which class I integrases were detected in eight isolates. Sequencing of gene cassette arrays identified genes conferring resistance to streptomycin-spectinomycin (aadA1) and trimethoprim (dfrA17, dfrB4). Class II integrases were not detected in the E. coli isolates. The frequent detection in captive sea lions of E. coli with resistance genes commonly identified in human clinical cases suggests that conditions experienced in captivity may contribute to establishment. Identification of antibiotic resistance in the microbiota of Australian sea lions provides crucial information for disease management. Our data will inform conservation management strategies and provide a mechanism to monitor microorganism dissemination to sensitive pinniped populations.9 page(s

Colony location and captivity influence the gut microbial community composition of the Australian sea lion (Neophoca cinerea)

Gut microbiota play an important role in maintenance of mammalian metabolism and immune system regulation, and disturbances to this community can have adverse impacts on animal health. To better understand the composition of gut microbiota in marine mammals, fecal bacterial communities of the Australian sea lion (Neophoca cinerea), an endangered pinniped with localized distribution, were examined. A comparison of samples from individuals across 11 wild colonies in South and Western Australia and three Australian captive populations showed five dominant bacterial phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. The phylum Firmicutes was dominant in both wild (76.4%±4.73%) and captive animals (61.4%±10.8%), while Proteobacteria contributed more to captive (29.3%±11.5%) than to wild (10.6%±3.43%) fecal communities. Qualitative differences were observed between fecal communities from wild and captive animals based on principal-coordinate analysis. SIMPER (similarity percentage procedure) analyses indicated that operational taxonomic units (OTU) from the bacterial families Clostridiaceae and Ruminococcaceae were more abundant in wild than in captive animals and contributed most to the average dissimilarity between groups (SIMPER contributions of 19.1% and 10.9%, respectively). Differences in the biological environment, the foraging site fidelity, and anthropogenic impacts may provide various opportunities for unique microbial establishment in Australian sea lions. As anthropogenic disturbances to marine mammals are likely to increase, understanding the potential for such disturbances to impact microbial community compositions and subsequently affect animal health will be beneficial for management of these vulnerable species.10 page(s

Characterization of MHC class IA in the endangered southern corroboree frog

Southern corroboree frogs (Pseudophryne corroboree) have declined to near extinction in the wild after the emergence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in southeastern Australia in the 1980s. A major captive breeding and reintroduction program is underway to preserve this iconic species, but improving resistance to B. dendrobatidis would help the wild population to be self-sustaining. Using 3 'and 5' rapid amplification of complementary DNA ends (RACE), we characterized the major histocompatibility complex (MHC) class IA locus in this species. We then used sequences generated from RACE to design primers to amplify the peptide-binding region (PBR) of this functional genetic marker. Finally, we analysed the diversity, phylogeny, and selection patterns of PBR sequences from four P. corroboree populations and compared this with other amphibian species. We found moderately high MHC class IA genetic diversity in this species and evidence of strong positive and purifying selection at sites that are associated with putative PBR pockets in other species, indicating that this gene region may be under selection for resistance to Bd. Future studies should focus on identifying alleles associated with Bd resistance in P. corroboree by performing a Bd laboratory challenge study to confirm the functional importance of our genetic findings and explore their use in artificial selection or genetic engineering to increase resistance to chytridiomycosis