Kristin A. Clothier

Genomic Comparison of Campylobacter spp. and Their Potential for Zoonotic Transmission between Birds, Primates, and Livestock

ABSTRACT Campylobacter is the leading cause of human gastroenteritis worldwide. Wild birds, including American crows, are abundant in urban, suburban, and agricultural settings and are likely zoonotic vectors of Campylobacter. Their proximity to humans and livestock increases the potential spreading of Campylobacter via crows between the environment, livestock, and humans. However, no studies have definitively demonstrated that crows are a vector for pathogenic Campylobacter. We used genomics to evaluate the zoonotic and pathogenic potential of Campylobacter from crows to other animals with 184 isolates obtained from crows, chickens, cows, sheep, goats, humans, and nonhuman primates. Whole-genome analysis uncovered two distinct clades of Campylobacter jejuni genotypes; the first contained genotypes found only in crows, while a second genotype contained “generalist” genomes that were isolated from multiple host species, including isolates implicated in human disease, primate gastroenteritis, and livestock abortion. Two major β-lactamase genes were observed frequently in these genomes (oxa-184, 55%, and oxa-61, 29%), where oxa-184 was associated only with crows and oxa-61 was associated with generalists. Mutations in gyrA, indicative of fluoroquinolone resistance, were observed in 14% of the isolates. Tetracycline resistance (tetO) was present in 22% of the isolates, yet it occurred in 91% of the abortion isolates. Virulence genes were distributed throughout the genomes; however, cdtC alleles recapitulated the crow-only and generalist clades. A specific cdtC allele was associated with abortion in livestock and was concomitant with tetO. These findings indicate that crows harboring a generalist C. jejuni genotype may act as a vector for the zoonotic transmission of Campylobacter. IMPORTANCE This study examined the link between public health and the genomic variation of Campylobacter in relation to disease in humans, primates, and livestock. Use of large-scale whole-genome sequencing enabled population-level assessment to find new genes that are linked to livestock disease. With 184 Campylobacter genomes, we assessed virulence traits, antibiotic resistance susceptibility, and the potential for zoonotic transfer to observe that there is a “generalist” genotype that may move between host species.

Draft Genome Sequences of Campylobacter jejuni Strains That Cause Abortion in Livestock

ABSTRACT Campylobacter jejuni is an intestinal bacterium that can cause abortion in livestock. This publication announces the public release of 15 Campylobacter jejuni genome sequences from isolates linked to abortion in livestock. These isolates are part of the 100K Pathogen Genome Project and are from clinical cases at the University of California (UC) Davis.

Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014

ABSTRACT Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was <1%. The prevalence in dogs (60 of 2,422) was 2.5%. Diarrhea was present in only 55% of positive dogs; however, 3.8% of the all diarrheic dogs were positive, compared with 1.8% of the nondiarrheic dogs. Salmonella-positive dogs were significantly more likely to have consumed raw food (P = 0.01), to have consumed probiotics (P = 0.002), or to have been given antibiotics (P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban (P = 0.002) or suburban (P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella-positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella-positive animals were clinically nondiarrheic.

Disseminated histoplasmosis in two juvenile raccoons (Procyon lotor) from a nonendemic region of the United States

Two 6-month-old raccoon kits, which had been rescued and fostered in preparation for return to the wild, became acutely ill and died 3 weeks before scheduled release. At necropsy, the kits had grossly enlarged livers and spleens, diffusely consolidated lungs, and generalized lymphadenopathy. Histologically, extensive infiltrates of macrophages containing yeast organisms were identified in lung, liver, kidney, spleen, lymph nodes, intestinal tissues, brain, adrenal gland, bone marrow, and thymus of both animals. Histiocytic inflammation with accompanying fibrosis was widespread, with necrotic foci evident in lungs, spleen, and intestinal sections. Fungal organisms were observed on sheep blood agar plates; however, repeated subcultures to fungal media designed to induce conidial structures for fungal identification were unsuccessful. Partial DNA sequencing of the 28S ribosomal RNA gene of the blood agar isolate identified 100% homology with Ajellomyces capsulatus (anamorphic name Histoplasma capsulatum). The kits were rescued and fostered in the San Francisco Bay area and it is likely that the exposure to H. capsulatum occurred in this area. Histoplasma sp. infection in wild mammal species is often used as an indication of spore contamination of a geographic region. Northern California is not known to be an endemic region for H. capsulatum, which is not a reportable disease in this state. The presence of severe, disseminated disease and the need for molecular identification associated with the isolate from a nonendemic region identified in the present report may indicate genetic adaptation and altered characteristics of this agent and may warrant further investigation.

Draft Genome Sequence of Multidrug-Resistant Abortive Campylobacter jejuni from Northern California

ABSTRACT Campylobacter jejuni is an enteric bacterium that can cause abortion in livestock. This is the release of a multidrug-resistant Campylobacter jejuni genome from an isolate that caused an abortion in a cow in northern California. This isolate is part of the 100K Pathogen Genome Project.

Characterization of Pajaroellobacter abortibovis, the etiologic agent of epizootic bovine abortion

Epizootic bovine abortion (EBA), first identified in the 1950s, is a major contributor of economic loss to western U.S. beef producers. The causative agent proved elusive for over fifty years until a novel Deltaproteobacteria was identified as the etiologic agent in 2005. The microbe, which has yet to be successfully cultured in vitro, has proven difficult to purify from necropsy tissues. Thus, phylogenetic characterization has been limited to analysis of the 16S ribosomal RNA (rRNA) gene (AF503916), which placed this bacterium in the order Myxococcales, suborder Sorangiineae, family Polyangiaceae and most closely related to Sorangium cellulosum. The focus of the current study was to further expand the morphologic characterization and taxonomic placement of this bacteria, named here as Pajaroellobacter abortibovis. Modified Gram staining, combined with transmission electron microscopy, provide strong evidence that the bacterium is gram negative. Flow cytometric analysis identified the presence of P. abortibovis in murine leukocytes. While attempts to sequence ten universally conserved protein-coding genes using previously published degenerative primers failed, redesigned primers based solely upon Deltaproteobacteria facilitated the partial sequencing of two genes; fusA (JQ173112) and pyrG (JQ173111). Primers designed in a similar fashion generated a partial sequence of the 23S rRNA gene (JQ173113) These sequences, combined with a revised 16S rRNA phylogenic analysis, support the placement of this bacteria as a unique genus separate from Sorangium.

Phenotypic and Genotypic Characterization of Animal-Source Salmonella Heidelberg Isolates

Salmonella enterica serotype Heidelberg (S. Heidelberg) is frequently implicated in human foodborne Salmonella infections and often produces more severe clinical disease than other serotypes. Livestock and poultry products represent a potential risk for transmission to humans. The purpose of this study was to evaluate 49 S. Heidelberg veterinary isolates for exponential growth rate (EGR), PFGE pattern, and antimicrobial resistance to evaluate these parameters as mechanisms by which S. Heidelberg emerged as a virulent foodborne pathogen. Isolates were categorized by species of origin; clinical or environmental sources; and time frame of recovery. Growth rates were determined in nutrient media using serial dilutions and colony counts; PFGE was performed according to the CDC PulseNet protocol. Minimum inhibitory concentration and susceptibility determinations were performed against antimicrobials important in human medicine. Eighteen unique PFGE patterns were detected in the isolates tested. Antimicrobial resistance was significantly greater (P < 0.05) for ten of 15 drugs in clinical over environmental isolates; for four drugs between the time frames; and for ten drugs between species of origin. The large genetic diversity present in isolates of this serotype may convey competitive advantages to this organism, while the presence of antimicrobial resistance represents a potential zoonotic risk via animal-source food products.

Suppurative otitis and ascending meningoencephalitis associated with Bacteroides tectus and Porphyromonas gulae in a captive Parma wallaby (Macropus parma) with toxoplasmosis

A 6-year-old female Parma wallaby (Macropus parma) at a zoo in California developed acute ataxia and left-sided circling. Despite intensive care, clinical signs progressed to incoordination and prostration, and the animal was euthanized. At necropsy, the left tympanic cavity was filled with homogeneous suppurative exudate that extended into the cranium expanding the meninges and neuroparenchyma in the lateral and ventral aspect of the caudal ipsilateral brainstem and medulla oblongata. Microscopically, the brainstem showed regional severe suppurative meningoencephalitis with large numbers of neutrophils, fewer macrophages, and lymphocytes admixed with fibrin, necrotic cellular debris, hemorrhage, and mineralization, with numerous intralesional Gram-negative bacilli. Bacteroides spp. and Porphyromonas spp. were isolated on anaerobic culture from the meninges, and the bacteria were further characterized by partial 16S ribosomal RNA gene sequencing as Bacteroides tectus and Porphyromonas gulae. Bacterial aerobic culture from the meninges yielded very low numbers of mixed flora and Proteus spp., which were considered contaminants. Culture of Mycoplasma spp. from middle ear and meninges was negative. Additionally, Toxoplasma gondii cysts were detected by immunohistochemistry in the heart and brain, and anti-Toxoplasma antibodies were detected in serum. The genera Bacteroides and Porphyromonas have been associated with oral disease in marsupials; but not with otitis and meningoencephalitis. The results of the present work highlight the importance of performing anaerobic cultures in the diagnostic investigation of cases of suppurative otitis and meningoencephalitis in macropods.

Frequency, serotype distribution, and antimicrobial susceptibility patterns of Salmonella in small poultry flocks in California

Backyard poultry operations are increasingly popular and commonplace in both rural and suburban locations. Although Salmonella surveillance programs are well established for large commercial poultry systems, information on smaller operations is lacking. We identified the occurrence and serotype distribution of Salmonella spp. recovered from backyard flock cases submitted to the California Animal Health and Food Safety Laboratory System (Davis, CA) in 2012–2015, and evaluated minimum inhibitory concentration for 12 antimicrobials as well as the lesions associated with Salmonella spp. in these cases. From records of 2,347 backyard flock cases with 2,627 samples, 44 samples (1.7%) were positive for Salmonella spp. DNA by PCR, and 41 (1.6%) of these samples yielded a Salmonella isolate by culture for further characterization. Seventeen different serotypes, including 3 isolates identified to the serogroup level, were identified from these isolates. Antimicrobial resistance was infrequent; however, 2 multidrug–resistant isolates were identified. Enteric or systemic lesions associated with Salmonella recovery were uncommon, with 77.3% of cases having no disease attributable to Salmonella. Recovered serotypes overlap with those seen in commercial poultry as well as in foodborne outbreaks reported by the Centers for Disease Control and Prevention in humans. Zoonotic risks via contact and food product contamination make monitoring of backyard flocks for Salmonella a critical part of flock surveillance programs, and we propose a potential sampling scheme.