Expression of defensin genes across house fly (Musca domestica) life history gives insight into immune system subfunctionalization.

Animals encounter diverse microbial communities throughout their lifetime, which exert varying selection pressures. Antimicrobial peptides (AMPs), which lyse or inhibit microbial growth, are a first line of defense against some of these microbes. Here we examine how developmental variation in microbial exposure has affected the evolution of expression and amino acid sequences of Defensins (an ancient class of AMPs) in the house fly (Musca domestica). The house fly is a well-suited model for this work because it trophically associates with varying microbial communities throughout its life history and its genome contains expanded families of AMPs, including Defensins. We identified two subsets of house fly Defensins: one expressed in larvae or pupae, and the other expressed in adults. The amino acid sequences of these two Defensin subsets form distinct monophyletic clades, and they are located in separate gene clusters in the genome. The adult-expressed Defensins evolve faster than larval/pupal Defensins, consistent with different selection pressures across developmental stages. Our results therefore suggest that varied microbial communities encountered across life history can shape the evolutionary trajectories of immune genes.

Whole genomic sequence analysis of Bacillus infantis: defining the genetic blueprint of strain NRRL B-14911, an emerging cardiopathogenic microbe.

BACKGROUND: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were reported. Therefore, to characterize its biological and medical significance, we sought to determine and analyze the complete genome sequence of Bacillus sp. NRRL B-14911. RESULTS: Based on the phylogenetic analysis of 16S ribosomal RNA (rRNA) genes, sequence analysis of the 16S-23S rDNA intergenic transcribed spacers, phenotypic microarray, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we propose that this organism belongs to the species Bacillus infantis, previously shown to be associated with sepsis in a newborn child. Analysis of the complete genome of Bacillus sp. NRRL B-14911 revealed several virulence factors including adhesins, invasins, colonization factors, siderophores and transporters. Likewise, the bacterial genome encodes a wide range of methyl transferases, transporters, enzymatic and biochemical pathways, and insertion sequence elements that are distinct from other closely related bacilli. CONCLUSIONS: The complete genome sequence of Bacillus sp. NRRL B-14911 provided in this study may facilitate genetic manipulations to assess gene functions associated with bacterial survival and virulence. Additionally, this bacterium may serve as a useful tool to establish a disease model that permits systematic analysis of autoimmune events in various susceptible rodent strains.

House Flies Are Underappreciated Yet Important Reservoirs and Vectors of Microbial Threats to Animal and Human Health.

House flies are well recognized as filth-associated organisms and public nuisances. House flies create sanitation issues when they bridge the gap between microbe-rich breeding environments and animal/human habitations. Numerous scientific surveys have demonstrated that house flies harbor bacterial pathogens that pose a threat to humans and animals. More extensive and informative surveys incorporating next-generation sequencing technologies have shown that house fly carriage of pathogens and harmful genetic elements, such as antimicrobial resistance genes, is more widespread and dangerous than previously thought. Further, there is a strong body of research confirming that flies not only harbor but also transmit viable, and presumably infectious, bacterial pathogens. Some pathogens replicate and persist in the fly, permitting prolonged shedding and dissemination. Finally, although the drivers still have yet to be firmly determined, the potential range of dissemination of flies and their associated pathogens can be extensive. Despite this evidence, the house flies' role as reservoirs, disseminators, and true, yet facultative, vectors for pathogens have been greatly underestimated and underappreciated. In this review, we present key studies that bolster the house fly's role both an important player in microbial ecology and population biology and as transmitters of microbial threats to animal and human health.

Bovine abortion associated with Nocardia farcinica.

Nocardia spp. are recognized as a cause of bovine mastitis, cutaneous or subcutaneous abscesses, pneumonia, and disseminated disease. Abortion caused by Nocardia spp. is uncommon, and only a few sporadic cases have been reported in horses, pigs, and cattle. In all previous reports, of nocardial abortion, the causative agent was identified as Nocardia asteroides. The current report describes an aborted bovine fetus that was infected with Nocardia farcinica. Placenta, abomasal fluid, lung, liver, and kidney specimens from a late-term bovine abortion were submitted to the Kansas State Veterinary Diagnostic Laboratory. The gross findings included purulent exudate in the placenta and numerous abscesses in lung. Histologically, there was necrotizing and suppurative placentitis, pyogranulomatous pneumonia, and nephritis with numerous intralesional branching and filamentous, Gram-positive bacteria. Nocardia farcinica was isolated by bacteriology, and the bacteriology result was confirmed by 2 established polymerase chain reaction protocols and by DNA sequencing.

A multiplex real-time PCR assay for the detection and differentiation of five bovine pinkeye pathogens.

Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is one of the most common eye diseases in cattle. Several pathogens have been associated with IBK cases, however, Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and bovine herpesvirus type 1 (BHV-1) are most frequently observed. A multiplex real-time PCR assay using two reactions was developed for the detection and differentiation of these five pathogens. Detection sensitivities of the multiplex assays were compared to singleplex reactions testing for the same targets. Correlation coefficients (R2) of >0.99, and PCR efficiencies between 92 and 106% were demonstrated in all singleplex and multiplex real-time PCR reactions. The limits of detection (LOD) of multiplex assays for Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and BHV-1 were 19, 23, 25, 24 and 26 copies per reaction, respectively. No cross amplification was observed for specificity testing of 179 IBK positive clinical samples and 55 non-target clinical samples. Percentage of clinical samples positive for Mycoplasma bovoculi, Moraxella bovoculi, Moraxella bovis, BHV-1 and Mycoplasma bovis were 88.8% (159/179), 75.9% (136/179), 60.3% (108/179), 11.7% (21/179) and 10.0% (18/179), respectively. Moraxella bovis, Moraxella bovoculi and Mycoplasma bovoculi were more prevalent than Mycoplasma bovis and BHV-1 in IBK samples collected from animals in this study population. Our data indicates that the multiplex real-time PCR panel assay is highly sensitive and highly specific for the detection and differentiation of the five major pathogens associated with bovine pinkeye.

Detection of Brucella canis infection in dogs by blood culture and bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Brucella canis was recovered from dogs that were canine brucellosis suspect by blood culture using a modified lysis method. Organism identity was established by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The instrument-provided security library identified the isolates as Brucella species. The isolates were further identified as B. canis with the help of phenotypic and genotypic characteristics. The mass spectral profiles from characterized B. canis isolates, when added to the MALDI-TOF MS standard reference library, allowed successful presumptive identification of B. canis.

Comparison of polymerase chain reaction assay, bacteriologic culture, and serologic testing in assessment of prevalence of urinary shedding of leptospires in dogs.

OBJECTIVE: To compare results of polymerase chain reaction (PCR) testing of urine samples, serologic testing, and bacteriologic culture of urine to determine prevalence of urinary shedding of leptospires in dogs. DESIGN: Serial case study. ANIMALS: 500 dogs evaluated serially without regard to health status. PROCEDURE: Urine samples were examined via PCR assay and bacteriologic culture for leptospires. Blood samples were analyzed for antibodies against serovars canicola, bratislava, pomona, icterohemorrhagiae, grippotyphosa, and hardjo. RESULTS: Titers > or = 1:100 against at least 1 serovar were detected in 104 (20.8%) dogs, and titers > or = 1:400 were detected in 41 (8.2%) dogs. High titers were detected most commonly to serovar grippotyphosa, followed by icterohemorrhagiae, canicola, pomona, bratislava, and hardjo. High titers to > 1 serovar were detected in 14 dogs. A positive PCR assay result was obtained in 41 (8.2%) dogs, only 9 of which had a titer > or = 1:100. Leptospires were not cultured from the urine of any dog. Only 4 dogs had clinical leptospirosis. Overall disease prevalence was 0.8% for the 6-month evaluation period. Compared with PCR assay, serologic testing for predicting shedding had a sensitivity of 22%, specificity of 79%, positive predictive value of 9%, and negative predictive value of 92%. CONCLUSIONS AND CLINICAL RELEVANCE: Irrespective of health status, 8.2% of dogs were shedding pathogenic leptospires. Serologic testing was a poor predictor of urinary shedding. Clinically normal dogs that shed leptospires may pose a zoonotic risk to their owners.