Detection of human pathogenic bacteria in rectal DNA samples from Zalophus californianus in the Gulf of California, Mexico.

Human intrusions into undisturbed wildlife areas greatly contribute to the emergence of infectious diseases. To minimize the impacts of novel emerging infectious diseases (EIDs) on human health, a comprehensive understanding of the microbial species that reside within wildlife species is required. The Gulf of California (GoC) is an example of an undisturbed ecosystem. However, in recent decades, anthropogenic activities within the GoC have increased. Zalophus californianus has been proposed as the main sentinel species in the GoC; hence, an assessment of sea lion bacterial microbiota may reveal hidden risks for human health. We evaluated the presence of potential human pathogenic bacterial species from the gastrointestinal (GI) tracts of wild sea lions through a metabarcoding approach. To comprehensively evaluate this bacterial consortium, we considered the genetic information of six hypervariable regions of 16S rRNA. Potential human pathogenic bacteria were identified down to the species level by integrating the RDP and Pplacer classifier outputs. The combined genetic information from all analyzed regions suggests the presence of at least 44 human pathogenic bacterial species, including Shigella dysenteriae and Bacillus anthracis. Therefore, the risks of EIDs from this area should be not underestimated.

Multi-locus evaluation of gastrointestinal bacterial communities from Zalophus californianus pups in the Gulf of California, México.

Background: The gastrointestinal (GI) bacterial communities of sea lions described to date have occasionally revealed large intraspecific variability, which may originate from several factors including different methodological approaches. Indeed, GI bacterial community surveys commonly rely on the use of a single hypervariable region (HR) of 16S rRNA, which may result in misleading structural interpretations and limit comparisons among studies. Here, we considered a multi-locus analysis by targeting six HRs of 16S rRNA with the aims of (i) comprehensively assessing the GI bacterial consortium in rectal samples from Zalophus californianus pups and (ii) elucidating structural variations among the tested HRs. In addition, we evaluated which HRs may be most suitable for identifying intrinsic, structurally related microbiome characteristics, such as geographic variations or functional capabilities. Methods: We employed a Short MUltiple Regions Framework (SMURF) approach using the Ion 16S™ Metagenomic Kit. This kit provides different proprietary primers designed to target six HRs of the 16S rRNA gene. To date, the only analytical pipeline available for this kit is the Ion Reporter™ Software of Thermo Fisher Scientific. Therefore, we propose an in-house pipeline to use with open-access tools, such as QIIME2 and PICRUSt 2, in downstream bioinformatic analyses. Results: As hypothesized, distinctive bacterial community profiles were observed for each analyzed HR. A higher number of bacterial taxa were detected with the V3 and V6-V7 regions. Conversely, the V8 and V9 regions were less informative, as we detected a lower number of taxa. The synergistic information of these HRs suggests that the GI microbiota of Zalophus californianus pups is predominated by five bacterial phyla: Proteobacteria (~50%), Bacteroidetes (~20%), Firmicutes (~18%), Fusobacteria (~7%), and Epsilonbacteraeota (~4%). Notably, our results differ at times from previously reported abundance profiles, which may promote re-evaluations of the GI bacterial compositions in sea lions and other pinniped species that have been reported to date. Moreover, consistent geographic differences were observed only with the V3, V4, and V6-V7 regions. In addition, these HRs also presented higher numbers of predicted molecular pathways, although no significant functional changes were apparent. Together, our results suggests that multi-locus analysis should be encouraged in GI microbial surveys, as single-locus approaches may result in misleading structural results that hamper the identification of structurally related microbiome features.

PATHOGENIC LEPTOSPIRA SEROVARS IN FREE-LIVING SEA LIONS IN THE GULF OF CALIFORNIA AND ALONG THE BAJA CALIFORNIA COAST OF MEXICO.

The California sea lion ( Zalophus californianus ), a permanent inhabitant of the Gulf of California in Mexico, is susceptible to pathogenic Leptospira spp. infection, which can result in hepatic and renal damage and may lead to renal failure and death. During summer 2013, we used the microscopic agglutination test (MAT) to investigate the prevalence of anti-Leptospira antibodies in blood of clinically healthy sea lion pups from seven rookery islands on the Pacific Coast of Baja California (Pacific Ocean) and in the Gulf of California. We also used PCR to examine blood for Leptospira DNA. Isolation of Leptospira in liquid media was unsuccessful. We found higher antibody prevalence in sea lions from the rookery islands in the gulf than in those from the Pacific Coast. Antibodies against 11 serovars were identified in the Gulf of California population; the most frequent reactions were against serovars Bataviae (90%), Pyrogenes (86%), Wolffi (86%), Celledoni (71%), and Pomona (65%). In the Pacific Ocean population, MAT was positive against eight serovars, where Wolffi (88%), Pomona (75%), and Bataviae (70%) were the most frequent. Serum samples agglutinated with more than one Leptospira serovar. The maximum titer was 3,200. Each island had a different serology profile, and islands combined showed a distinct profile for each region. We detected pathogenic Leptospira DNA in 63% of blood samples, but we found no saprophytic Leptospira. Positive PCR results were obtained in blood samples with high and low MAT titers. Together, these two methods enhance the diagnosis and interpretation of sea lion leptospirosis. Our results may be related to human activities or the presence of other reservoirs with which sea lions interact, and they may also be related to sea lion stranding.