Walter M. Boyce

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens (Gallus gallus domesticus, n = 8), American crows (Corvus brachyrhynchos, n = 17), a mallard duck (Anas platyrhynchos, n = 1), and a western scrub-jay (Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR (p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences (p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

There are approximately 3,000 southern sea otters (Enhydra lutris nereis) in the nearshore environment along the California coast, US, and the species is classified as Threatened under the Endangered Species Act. We tested sera from 661 necropsied southern sea otters sampled from 1997 to 2015 to determine overall exposure to influenza A viruses (IAVs) and to identify subtype-specific antibody responses. Using an enzyme-linked immunosorbent assay (ELISA), antibodies to IAV nucleoproteins were detected in 160 (24.2%) otters, with seropositive animals found in every year except 2008. When the ELISA-positive samples were tested by virus microneutralization, antibody responses were detected to avian-origin hemagglutinin subtypes H1, H3, H4, H5, H6, H7, H9, and H11. Strong antibody responses to pandemic H1N1 (pdmH1N1) were also detected, indicating that epizootic transmission of pdmH1N1 occurred among the southern sea otter population after the emergence of this human-origin virus in 2009. We conclude ...

Waterfowl, especially ducks and geese, are primary reservoirs for influenza A viruses (IAVs) that evolve and emerge as important pathogens in domestic animals and humans. In contrast to humans, where IAVs infect the respiratory tract and cause significant morbidity and mortality, IAVs infect the gastrointestinal tract of waterfowl and cause little or no pathology and are spread by fecal-oral transmission. For this reason, we examined whether IAV infection is associated with differences in the cloacal microbiome of mallards (Anas platyrhyncos), an important host of IAVs in North America and Eurasia. We characterized bacterial community composition by sequencing the V4 region of 16S rRNA genes. IAV-positive mallards had lower species diversity, richness, and evenness than IAV-negative mallards. Operational taxonomic unit (OTU) cooccurrence patterns were also distinct depending on infection status. Network analysis showed that IAV-positive mallards had fewer significant cooccurring OTUs and exhibited fewer coassociation patterns among those OTUs than IAV-negative mallards. These results suggest that healthy mallards have a more robust and complex cloacal microbiome. By combining analytical approaches, we identified 41 bacterial OTUs, primarily representatives of Streptococcus spp., Veillonella dispar, and Rothia mucilaginosa, contributing to the observed differences. This study found that IAV-infected wild mallards exhibited strong differences in microbiome composition relative to noninfected mallards and identified a concise set of putative biomarker OTUs. Using Random Forest, a supervised machine learning method, we verified that these 41 bacterial OTUs are highly predictive of infection status. IMPORTANCE Seasonal influenza causes 3 to 5 million severe illnesses and 250,000 to 500,000 human deaths each year. While pandemic influenza viruses emerge only periodically, they can be devastating—for example, the 1918 H1N1 pandemic virus killed more than 20 million people. IAVs infect the respiratory tract and cause significant morbidity and mortality in humans. In contrast, IAVs infect the gastrointestinal tract of waterfowl, producing little pathology. Recent studies indicated that viruses can alter the microbiome at the respiratory and gastrointestinal mucosa, but there are no reports of how the microbiota of the natural host of influenza is affected by infection. Here we find that the mallard microbiome is altered during IAV infection. Our results suggest that detailed examination of humans and animals infected with IAVs may reveal individualized microbiome profiles that correspond to health and disease. Moreover, future studies should explore whether the altered microbiome facilitates maintenance and transmission of IAVs in waterfowl populations.

We used surveillance data collected in California before, concurrent with, and subsequent to an outbreak of highly pathogenic (HP) clade 2.3.4.4 influenza A viruses (IAVs) in 2014–2015 to (i) evalu...

Fossil data are ambiguous regarding the evolutionary origin of contemporary desert bighorn sheep ( Ovis canadensis subspecies). To address this uncertainty, we conducted phylogeographic and population genetic analyses on bighorn sheep subspecies found in southwestern North America. We analyzed 515 base pairs of mtDNA control region sequence and 39 microsatellites in 804 individuals from 58 locations. Phylogenetic analyses revealed 2 highly divergent clades concordant with Sierra Nevada ( O. c. sierrae ) and Rocky Mountain ( O. c. canadensis ) bighorn and showed that these 2 subspecies both diverged from desert bighorn prior to or during the Illinoian glaciation (~315–94 thousand years ago [kya]). Desert bighorn comprised several more recently diverged haplogroups concordant with the putative Nelson ( O. c. nelsoni ), Mexican ( O. c. mexicana ), and Peninsular ( O. c. cremnobates ) subspecies. Corresponding estimates of effective splitting times (~17–3 kya), and haplogroup ages (~85–72 kya) placed the most likely timeframe for divergence among desert bighorn subspecies somewhere within the last glacial maximum. Median-joining haplotype network and Bayesian skyline analyses both indicated that desert bighorn collectively comprised a historically large and haplotype-diverse population, which subsequently lost much of its diversity through demographic decline. Using microsatellite data, discriminant analysis of principle components (DAPC) and Bayesian clustering analyses both indicated genetic structure concordant with the geographic distribution of 3 desert subspecies. Likewise, microsatellite and mitochondrial-based F_ST comparisons revealed significant fixation indices among the desert bighorn genetic clusters. We conclude these desert subspecies represent ancient lineages likely descended from separate Pleistocene refugial populations and should therefore be managed as distinct taxa to preserve maximal biodiversity. Los datos de fósiles sobre el origen evolutivo de las ovejas del desierto ( Ovis canadensis subespecies) contemporáneas son ambiguos. Para dilucidar esta incertidumbre, llevamos a cabo análisis filogeográficos y de genética de poblaciones entre cinco subespecies de ovejas del suroccidente de Norteamérica. Analizamos 515 pb de secuencia de la región control del ADN mitocondrial y 39 microsatélites en 804 ovejas de 58 localidades. Los análisis filogenéticos revelaron 2 clados altamente divergentes concordantes con ovejas de la Sierra Nevada ( O. c. sierrae ) y de las Montañas Rocosas ( O. c. canadensis ), y demostraron que estas dos subespecies divergieron antes o durante la glaciación de Illinois (315,000–94,000 años). Las ovejas del desierto formaron varios haplogrupos recientemente derivados concordantes con las subespecies de Nelson ( O. c. nelsoni ), México ( O. c. mexicana ) y peninsular ( O. c. cremnobates ). Las estimaciones correspondientes al tiempo de separación efectiva (17,000–3,000 años) y edades de haplogrupos (85,000–72,000 años) son los plazos más probables para las divergencias entre subespecies de ovejas del desierto dentro de la última glaciación máxima. Análisis de redes de haplotipos de unión de medias y análisis bayesianos de líneas de horizonte indicaron que las ovejas del desierto formaron una población históricamente grande y diversa en términos de haplotipos, que luego perdieron gran parte de su diversidad a través de un descenso demográfico. Utilizando datos de microsatélites los análisis DAPC y TESS indicaron agrupamiento genético concordante con la distribución geográfica actual de las tres subespecies. Asimismo, comparaciones de F_ST con datos de microsatélites y mitocondriales revelaron índices de fijación significativos entre los grupos genéticos de ovejas del desierto. Concluimos que estas subespecies de ovejas del desierto representan linajes antiguos que probablemente descienden de poblaciones de distintos refugios del Pleistoceno, y que por lo tanto deben ser manejadas como taxones distintos para preservar su biodiversidad máxima.

Ear mites (Otodectes cynotis) and ear canal tumors are highly prevalent among federally endangered Island foxes (Urocyon littoralis catalinae) living on Santa Catalina Island off the coast of Southern California. Since studies began in the 1990s, nearly all foxes examined were found to be infected with ear mites, and ceruminous gland tumors (carcinomas and adenomas) were detected in approximately half of all foxes ≥ 4 years of age. We hypothesized that reduction of ear mite infection would reduce otitis externa and ceruminous gland hyperplasia, a risk factor for tumor development. In this study, we conducted a randomized field trial to assess the impact of acaricide treatment on ear mite prevalence and intensity of infection, otitis externa, ceruminous gland hyperplasia, and mite-specific IgG and IgE antibody levels. Treatment was highly effective at eliminating mites and reducing otitis externa and ceruminous gland hyperplasia, and mite-specific IgG antibody levels were significantly lower among uninfected foxes. Ceruminous gland hyperplasia increased in the chronically infected, untreated foxes during the six month study. Our results provide compelling evidence that acaricide treatment is an effective means of reducing ear mites, and that mite removal in turn reduces ear lesions and mite-specific IgG antibody levels in Santa Catalina Island foxes. This study has advanced our understanding of the underlying pathogenesis which results in ceruminous gland tumors, and has helped inform management decisions that impact species conservation.

In this study, we examined the prevalence, pathology, and epidemiology of tumors in free-ranging island foxes occurring on three islands in the California Channel Islands, USA. We found a remarkably high prevalence of ceruminous gland tumors in endangered foxes (Urocyon littoralis catalinae) occurring on Santa Catalina Island (SCA)—48.9% of the dead foxes examined from 2001–2008 had tumors in their ears, and tumors were found in 52.2% of randomly-selected mature (≥ 4 years) foxes captured in 2007–2008, representing one of the highest prevalences of tumors ever documented in a wildlife population. In contrast, no tumors were detected in foxes from San Nicolas Island or San Clemente Island, although ear mites (Otodectes cynotis), a predisposing factor for ceruminous gland tumors in dogs and cats, were highly prevalent on all three islands. On SCA, otitis externa secondary to ear mite infection was highly correlated with ceruminous gland hyperplasia (CGH), and tumors were significantly associated with the severity of CGH, ceruminous gland dysplasia, and age group (older foxes). We propose a conceptual model for the formation of ceruminous gland tumors in foxes on SCA that is based on persistent, ubiquitous infection with ear mites, and an innate, over exuberant inflammatory and hyperplastic response of SCA foxes to these mites. Foxes on SCA are now opportunistically treated with acaricides in an attempt to reduce mite infections and the morbidity and mortality associated with this highly prevalent tumor.

Wide-ranging large carnivores pose myriad challenges for conservation, especially in highly fragmented landscapes. Over a 13-year period, we combined monitoring of radio collared pumas (Puma concolor) with complementary multi-generational genetic analyses to inform puma conservation in southern California, USA. Our goals were to generate survivorship estimates, determine causes of mortality, identify barriers to movement, and determine the genetic and demographic challenges to puma persistence among >20,000,000 people and extensive urban, suburban, and exurban development. Despite protection from hunting, annual survival for radio collared pumas was surprisingly low (55.8%), and humans caused the majority of puma deaths. The most common sources of mortality were vehicle collisions (28% of deaths), and mortalities resulting from depredation permits issued after pumas killed domestic animals (17% of deaths). Other human-caused mortalities included illegal shootings, public safety removals, and human-caused wildfire. An interstate highway (I-15) bisecting this study area, and associated development, have created a nearly impermeable barrier to puma movements, resulting in severe genetic restriction and demographic isolation of the small puma population (n ~ 17–27 adults) in the Santa Ana Mountains west of I-15. Highways that bisect habitat or divide remaining “conserved” habitat, and associated ongoing development, threaten to further subdivide this already fragmented puma population and increase threats to survival. This study highlights the importance of combining demographic and genetic analyses, and illustrates that in the absence of effective measures to reduce mortality and enhance safe movement across highways, translocation of pumas, such as was done with the endangered Florida panther (P. c. coryi), may ultimately be necessary to prevent further genetic decline and ensure persistence of the Santa Ana Mountains population.

The genome sequence of Fervidicella metallireducens strain AeB^T, a curved, heterotrophic, thermoanaerobic, and iron-reducing bacterium isolated from a gray microbial mat colonizing the free-flowing waters of a Great Artesian Basin (GAB) bore well located in outback Queensland, Australia, is reported here. The analysis of the 2.9-Mb sequence indicates that the attributes of the genome are consistent with its physiological and phenotypic traits.

We report the complete genome sequence of a reassortant H14N2 avian influenza virus isolated in 2011 from a northern shoveler in California. This introduced Eurasian subtype acquired seven segments from North American viruses and circulated in the Pacific Flyway 1 year after its detection in the Mississippi Flyway.