Evaluation of a commercial microbial enrichment kit used prior DNA extraction to improve the molecular detection of vector-borne pathogens from naturally infected dogs.

Accurate detection of vector-borne pathogens (VBPs) is extremely important as the number of reported cases in humans and animals continues to rise in the US and abroad. Validated PCR assays are currently the cornerstone of molecular diagnostics and can achieve excellent analytical sensitivity and specificity. However, the detection of pathogens at low parasitemia still presents a challenge for VBP diagnosis, especially given the very low volume of specimens tested by molecular methods. The objective of this study is to determine if a commercially available microbial enrichment kit, used prior DNA extraction, is capable of expanding the overall microbial community and increasing detectable levels of VBPs in canine blood samples through host DNA depletion. This study used EDTA-whole blood samples from dogs naturally infected with varying parasitemia levels of either Anaplasma phagocytophilum, Babesia gibsoni, or Ehrlichia ewingii. For two VBPs, EDTA-blood samples were diluted to determine the effect of microbial concentration at low parasitemia. Paired EDTA-blood samples from each dog were subjected to traditional, automated DNA extraction with or without the microbial concentrating kit (MolYsis®) prior DNA extraction. Relative amounts of pathogen DNA in paired samples were determined by real-time PCR and Next-Generation Sequencing targeting conserved regions of 16S rRNA (for bacteria) and 18S rRNA (for protozoa). Results from the three molecular methods suggest that the microbial concentrating kit did not improve the detection of VBPs, although significantly reduced the presence of host DNA. Alternative methods for VBP enrichment in clinical samples prior to molecular testing should continue to be investigated, as it may significantly improve clinical sensitivity and reduce the number of false-negative results.

The cecal microbiome of commercial broiler chickens varies significantly by season.

Next-generation DNA sequencing is rapidly becoming a powerful tool for food animal management. One valuable use of this technology is to re-examine long-standing observations of performance differences associated with animal husbandry practices to better understand how these differences may be modulated by the gastrointestinal (GI) microbiome. The influences of environmental parameters such as air temperature and relative humidity on broiler chicken performance have commonly been observed, but how the GI microbiome may respond to seasonal environmental changes remains largely unknown. The purposes of this study were therefore to: (1) characterize the cecal microflora of commercial broilers (N = 87) collected at harvest across all 4 seasons, and (2) identify any significant changes of the GI microbiome and specific taxa according to season and Campylobacter status. Finding taxa with significant positive or negative correlations with Campylobacter could be useful by identifying indicator or antagonistic taxa and could also inform inferences regarding the ecological niche of Campylobacter. Whole GI tracts were removed from commercial broilers representing 87 independent flocks between April 2013 and May 2014 in the U.S. state of Georgia. Intact ceca were separated, cultured for Campylobacter and cecal contents were frozen. The cecal microbiome was characterized using barcoded sequencing of 16S rRNA genes on the Illumina MiSeq platform. The composition of the microbiome measured at processing was generally not affected by Campylobacter status but was most significantly affected by season of grow-out. Significantly fewer bacterial genera were found in winter than spring or summer. Bacterial genera with prior evidence for both positive or negative influences on gut health outcomes were significantly less abundant in the fall. Identifying specific members of the GI microbiota that vary according to season may help develop novel interventions to improve husbandry practices and growth performance.

Assessing Cat Flea Microbiomes in Northern and Southern California by 16S rRNA Next-Generation Sequencing.

Flea-borne diseases (FBDs) impact both human and animal health worldwide. Because adult fleas are obligately hematophagous and can harbor potential pathogens, fleas act as ectoparasites of vertebrates, as well as zoonotic disease vectors. Cat fleas (Ctenocephalides felis) are important vectors of two zoonotic bacterial genera listed as priority pathogens by the National Institute of Allergy and Infectious Diseases (NIAID-USA): Bartonella spp. and Rickettsia spp., causative agents of bartonelloses and rickettsioses, respectively. In this study, we introduce the first microbiome analysis of C. felis samples from California, determining the presence and abundance of relevant pathogenic genera by characterizing the cat flea microbiome through 16S rRNA next-generation sequencing (16S-NGS). Samples from both northern (NoCal) and southern (SoCal) California were assessed to expand current knowledge regarding FBDs in the state. We identified Rickettsia and Bartonella, as well as the endosymbiont Wolbachia, as the most abundant genera, followed by less abundant taxa. In comparison to our previous study screening Californian cat fleas for rickettsiae using PCR/digestion/sequencing of the ompB gene, the 16S-NGS approach applied herein showed a 95% level of agreement in detecting Rickettsia spp. There was no overall difference in microbiome diversity between NoCal and SoCal samples. Bacterial taxa identified by 16S-NGS in this study may help to improve epidemiological investigations, pathogen surveillance efforts, and clinical diagnostics of FBDs in California and elsewhere.

Regional seroreactivity and vector-borne disease co-exposures in dogs in the United States from 2004-2010: utility of canine surveillance.

Vector-borne disease (VBD) pathogens remain an emerging health concern for animals and humans throughout the world. Surveillance studies of ticks and humans have made substantial contributions to our knowledge of VBD epidemiology trends, but long-term VBD surveillance data of dogs in the United States is limited. This seroreactivity study assessed US temporal and regional trends and co-exposures to Anaplasma, Babesia, Bartonella, Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia spp., and spotted fever group Rickettsia in dogs from 2004-2010. Dog serum samples (N=14,496) were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Disease Diagnostic Laboratory for vector-borne pathogens diagnostic testing using immunofluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) assays. These convenience samples were retrospectively reviewed and analyzed. The largest proportion of samples originated from the South (47.6%), with the highest percent of seroreactive samples observed in the Midatlantic (43.4%), compared to other US regions. The overall seroreactivity of evaluated VBD antigens were Rickettsia rickettsia (10.4%), B. burgdorferi (5.2%), Ehrlichia spp. (4.3%), Bartonella henselae (3.8%), Anaplasma spp. (1.9%), Bartonella vinsonii subsp. berkhoffii (1.5%), Babesia canis (1.1%), and D. immitis (0.8%). Significant regional and annual seroreactivity variation was observed with B. burgdorferi, Ehrlichia, and Rickettsia exposures. Seasonal seroreactivity variation was evident with Rickettsia. Seroreactivity to more than one antigen was present in 16.5% of exposed dogs. Nationally, the most prevalent co-exposure was Rickettsia with Ehrlichia spp. (5.3%), and the highest odds of co-exposure was associated with Anaplasma spp. and B. burgdorferi (odds ratio=6.6; 95% confidence interval 5.0, 8.8). Notable annual and regional seroreactivity variation was observed with certain pathogens over 7 years of study, suggesting canine surveillance studies may have value in contributing to future VBD knowledge.

Pharmacokinetics of N-acetylcysteine after oral and intravenous administration to healthy cats.

OBJECTIVE: To describe the pharmacokinetics of N-acetylcysteine (NAC) in healthy cats after oral and IV administration. ANIMALS: 6 healthy cats. PROCEDURES: In a crossover study, cats received NAC (100 mg/kg) via IV and oral routes of administration; there was a 4-week washout period between treatments. Plasma samples were obtained at 0, 5, 15, 30, and 45 minutes and 1, 2, 4, 8, 12, 24, 36, and 48 hours after administration, and NAC concentrations were quantified by use of a validated high-performance liquid chromatography-mass spectrometry protocol. Data were analyzed via compartmental and noncompartmental pharmacokinetic analysis. RESULTS: Pharmacokinetics for both routes of administration were best described by a 2-compartment model. Mean ± SD elimination half-life was 0.78 ± 0.16 hours and 1.34 ± 0.24 hours for the IV and oral routes of administration, respectively. Mean bioavailability of NAC after oral administration was 19.3 ± 4.4%. CONCLUSIONS AND CLINICAL RELEVANCE: The pharmacokinetics of NAC for this small population of healthy cats differed from values reported for humans. Assuming there would be similar pharmacokinetics in diseased cats, dose extrapolations from human medicine may result in underdosing of NAC in cats with acute disease. Despite the low bioavailability, plasma concentrations of NAC after oral administration at 100 mg/kg may be effective in the treatment of chronic diseases.

Candidatus Bartonella merieuxii, a potential new zoonotic Bartonella species in canids from Iraq.

Bartonellae are emerging vector-borne pathogens infecting erythrocytes and endothelial cells of various domestic and wild mammals. Blood samples were collected from domestic and wild canids in Iraq under the United States Army zoonotic disease surveillance program. Serology was performed using an indirect immunofluorescent antibody test for B. henselae, B. clarridgeiae, B. vinsonii subsp. berkhoffii and B. bovis. Overall seroprevalence was 47.4% in dogs (n = 97), 40.4% in jackals (n = 57) and 12.8% in red foxes (n = 39). Bartonella species DNA was amplified from whole blood and representative strains were sequenced. DNA of a new Bartonella species similar to but distinct from B. bovis, was amplified from 37.1% of the dogs and 12.3% of the jackals. B. vinsonii subsp. berkhoffii was also amplified from one jackal and no Bartonella DNA was amplified from foxes. Adjusting for age, the odds of dogs being Bartonella PCR positive were 11.94 times higher than for wild canids (95% CI: 4.55-31.35), suggesting their role as reservoir for this new Bartonella species. This study reports on the prevalence of Bartonella species in domestic and wild canids of Iraq and provides the first detection of Bartonella in jackals. We propose Candidatus Bartonella merieuxii for this new Bartonella species. Most of the Bartonella species identified in sick dogs are also pathogenic for humans. Therefore, seroprevalence in Iraqi dog owners and bacteremia in Iraqi people with unexplained fever or culture negative endocarditis requires further investigation as well as in United States military personnel who were stationed in Iraq. Finally, it will also be essential to test any dog brought back from Iraq to the USA for presence of Bartonella bacteremia to prevent any accidental introduction of a new Bartonella species to the New World.

Successful treatment of Bartonella henselae endocarditis in a cat.

This report describes the clinical presentation, diagnosis and treatment of a cat with vegetative valvular endocarditis temporally associated with natural infection with Bartonella henselae. Lethargy, abnormal gait and weakness were the main clinical signs that resulted in referral for diagnostic evaluation. Using a novel and sensitive culture approach, B henselae was isolated from the blood. Following antibiotic therapy there was total resolution of clinical signs, the heart murmur, the valvular lesion by echocardiography, and no Bartonella species was isolated or amplified from a post-treatment blood culture. In conjunction with previous case reports, infective endocarditis can be associated with natural B henselae infection in cats; however, early diagnosis and treatment may result in a better prognosis than previously reported.

Identification of bacteria from clinical samples using Bartonella alpha-Proteobacteria growth medium.

In an effort to overcome historical problems associated with the isolation of Bartonella species from animal and human blood samples, our laboratory developed a novel, chemically modified, insect-based, liquid culture medium (Bartonella alpha-Proteobacteria growth medium, BAPGM). In this study, we describe the isolation of non-Bartonella bacteria from aseptically obtained human blood and tissue samples that were inoculated into BAPGM pre-enrichment culture medium, and were obtained during attempts to define each individuals Bartonella infection status. After incubation for at least 7 days in liquid BAPGM, pre-enriched inoculums were sub-cultured onto a BAPGM/blood agar plate. Bacterial DNA was extracted from pooled plated colonies and amplified using conventional PCR targeting the 16S rRNA gene. Subsequently, amplicons were cloned, sequenced and compared to GenBank database sequences using the BLAST program. Regardless of the patient's Bartonella status, seventeen samples generated only one 16S rDNA sequence, representing the following genera: Arthrobacter, Bacillus, Bartonella, Dermabacter, Methylobacterium, Propionibacterium, Pseudomonas, Staphylococcus and bacteria listed as "non-cultured" in the GenBank database. Alkalibacterium, Arthrobacter, Erwinia, Kineococcus, Methylobacterium, Propionibacterium, Sphingomonas, and Staphylococcus were isolated from nine Bartonella-infected individuals. Co-isolation of Acinetobacter, Sphingomonas, Staphylococcus spp. and bacteria listed as "non-cultured" in the GenBank database was achieved for four samples in which Bartonella spp. were not detected. Despite the phylogenetic limitations of using partial 16S rRNA gene sequencing for species and strain identification, the investigational methodology described in this study may provide a complementary approach for the isolation and identification of bacteria from patient samples.