Epidemics of H3N8 and H3N2 influenza A viruses (IAVs) in dogs, along with recognition of spillover infections from IAV strains typically found in humans or other animals, have emphasized the importance of efficient laboratory testing. Given the lack of active IAV surveillance or immunization requirements for dogs, cats, or horses imported into the United States, serotype prediction and whole-genome sequencing of positive specimens detected at veterinary diagnostic laboratories are also needed. The conserved sequences at the ends of the viral genome segments facilitate universal amplification of all segments of viral genomes directly from respiratory specimens. Although several methods for genomic analysis have been reported, no optimization focusing on companion animal strains has been described, to our knowledge. We compared 2 sets of published universal amplification primers using 26 IAV-positive specimens from dogs, horses, and a cat. Libraries prepared from the resulting amplicons were sequenced using Illumina chemistry, and reference-based assemblies were generated from the data produced by both methods. Although both methods produced high-quality data, coverage profiles and base calling differed between the 2 methods. The sequence data were also used to identify the subtype of the IAV strains sequenced and then compared to standard PCR assays for neuraminidase types N2 and N8.

Fecal pollution at coastal beaches requires management efforts to address public health and economic concerns. Feces-borne bacterial concentrations are influenced by different fecal sources, environmental conditions, and ecosystem reservoirs, making their public health significance convoluted. In this study, we sought to delineate the influences of these factors on enterococcal concentrations in southern Maine coastal recreational waters. Weekly water samples and water quality measurements were conducted at freshwater, estuarine, and marine beach sites from June through September 2016. The samples were analyzed for total and particle-associated enterococcal concentrations, total suspended solids, and microbial source tracking markers (PCR: Bac32, HF183, CF128, DF475, and Gull2; quantitative PCR [qPCR]: AllBac, HF183, and GFD). Water, soil, sediment, and marine sediment samples were also subjected to 16S rRNA sequencing and SourceTracker analysis to determine the influence from these environmental reservoirs on water sample microbial communities. Enterococcal and particle-associated enterococcal concentrations were elevated in freshwater, but the concentrations of suspended solids were relatively similar. Mammal fecal contamination was significantly elevated in the estuary, with human and bird fecal contaminant levels similar between sites. A partial least-squares regression model indicated particle-associated enterococcal and mammal marker concentrations had the most significant positive relationships with enterococcal concentrations across marine, estuary, and freshwater environments. Freshwater microbial communities were significantly influenced by underlying sediment, while estuarine/marine beach communities were influenced by freshwater, high tide height, and estuarine sediment. Elevated enterococcal levels were reflective of a combination of increased fecal source input, environmental sources, and environmental conditions, highlighting the need for encompassing microbial source tracking (MST) approaches for managing water quality issues. Enterococci have long been the federal standard in determining water quality at estuarine and marine environments. Although enterococci are highly abundant in the intestines of many animals, they are not exclusive to that environment and can persist and grow outside fecal tracts. This presents a management problem for areas that are largely impaired by nonpoint source contamination, as fecal sources might not be the root cause of contamination. This study employed different microbial source tracking methods for delineating the influences from fecal source input, environmental sources, and environmental conditions to determine which combination of variables are influencing enterococcal concentrations in recreational waters at a historically impaired coastal town. The results showed that fecal source input, environmental sources, and conditions all play roles in influencing enterococcal concentrations. This highlights the need to include an encompassing microbial source tracki...

Background Although high-throughput marker gene studies provide valuable insight into the diversity and relative abundance of taxa in microbial communities, they do not provide direct measures of their functional capacity. Recently, scientists have shown a general desire to predict functional profiles of microbial communities based on phylogenetic identification inferred from marker genes, and recent tools have been developed to link the two. However, to date, no large-scale examination has quantified the correlation between the marker gene based taxonomic identity and protein coding gene conservation. Here we utilize 4872 representative prokaryotic genomes from NCBI to investigate the relationship between marker gene identity and shared protein coding gene content. Results Even at 99–100% marker gene identity, genomes share on average less than 75% of their protein coding gene content. This occurs regardless of the marker gene(s) used: V4 region of the 16S rRNA, complete 16S rRNA , or single copy orthologs through a multi-locus sequence analysis. An important aspect related to this observation is the intra-organism variation of 16S copies from a single genome. Although the majority of 16S copies were found to have high sequence similarity (> 99%), several genomes contained copies that were highly diverged (< 97% identity). Conclusions This is the largest comparison between marker gene similarity and shared protein coding gene content to date. The study highlights the limitations of inferring a microbial community’s functions based on marker gene phylogeny. The data presented expands upon the results of previous studies that examined one or few bacterial species and supports the hypothesis that 16S rRNA and other marker genes cannot be directly used to fully predict the functional potential of a bacterial community. Electronic supplementary material The online version of this article (10.1186/s12864-019-5641-1) contains supplementary material, which is available to authorized users.

21Fecal pollution at coastal beaches in the Northeast, USA requires management efforts to address 22 public health and economic concerns. Concentrations of fecal-borne bacteria are influenced by 23 different fecal sources, environmental conditions, and ecosystem reservoirs, making their public 24 health significance convoluted. In this study, we sought to delineate the influences of these 25 factors on enterococci concentrations in southern Maine coastal recreational waters. Weekly 26 water samples and water quality measurements were conducted at freshwater, estuarine, and 27 marine beach sites from June through September 2016. Samples were analyzed for total and 28 particle-associated enterococci concentrations, total suspended solids, and microbial source 29 tracking markers for multiple sources. Water, soil, sediment, and marine sediment samples were 30 also subjected to 16S rRNA sequencing and SourceTracker analysis to determine the influence 31 from these environmental reservoirs on water sample microbial communities. Enterococci and 32 particle-associated enterococci concentrations were elevated in freshwater, but suspended solids 33 concentrations were relatively similar. Mammal fecal contamination was significantly elevated 34 in the estuary, with human and bird fecal contaminant levels similar between sites. A partial least 35 squares regression model indicated particle-associated enterococci and mammal marker 36 concentrations had the most significant positive relationships with enterococci concentrations 37 across marine, estuary, and freshwater environments. Freshwater microbial communities were 38 significantly influenced by underlying sediment while estuarine/marine beach communities were 39 influenced by freshwater, high tide height, and estuarine sediment. We found elevated 40 enterococci levels are reflective of a combination of increased fecal source input, environmental 41 3 sources, and environmental conditions, highlighting the need for encompassing MST approaches 42 for managing water quality issues. 43 IMPORTANCE 44Enterococci have long been the federal standard in determining water quality at estuarine and 45 marine environments. Although enterococci are highly abundant in the fecal tracts of many 46 animals they are not exclusive to that environment and can persist and grow outside of fecal 47 tracts. This presents a management problem for areas that are largely impaired by non-point 48 source contamination, as fecal sources might not be the root cause of contamination. This study 49 employed different microbial source tracking methods to delineate influences from fecal source 50 input, environmental sources, and environmental conditions to determine which combination of 51 variables are influencing enterococci concentrations in recreational waters at a historically 52 impaired coastal town. Results showed that fecal source input, environmental sources and 53 conditions all play a role in influencing enterococci concentrations. This highlights the need to 54 include an encompassing microb...