John Dustin Loy

Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle

BackgroundBovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen.FindingsReplicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection.ConclusionsReplicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle.

Moraxella bovoculi is a recently described bacterium that is associated with infectious bovine keratoconjunctivitis (IBK) or “pinkeye” in cattle. In this study, closed circularized genomes were generated for seven M. bovoculi isolates: three that originated from the eyes of clinical IBK bovine cases and four from the deep nasopharynx of asymptomatic cattle. Isolates that originated from the eyes of IBK cases profoundly differed from those that originated from the nasopharynx of asymptomatic cattle in genome structure, gene content and polymorphism diversity and consequently placed into two distinct phylogenetic groups. These results suggest that there are genetically distinct strains of M. bovoculi that may not associate with IBK.

Moraxella spp. isolated from field outbreaks of infectious bovine keratoconjunctivitis: a retrospective study of case submissions from 2010 to 2013

Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is the most costly eye disease of cattle. The principal etiologic agent of IBK is the Gram-negative bacterium Moraxella bovis. However, there have been reports of IBK outbreaks associated with Moraxella bovoculi. A retrospective study of IBK diagnostic cases submitted from July 1, 2010 through October 31, 2013 was conducted. Included in the study were 1,042 Moraxella isolates from 1,538 swabs of lacrimal secretions collected from 282 herds from 30 U.S. states. Moraxella isolates were identified to the species level and were composed of M. bovoculi (701 isolates), M. bovis (295 isolates), Moraxella ovis (5 isolates), and other Moraxella spp. (41). Minimum inhibitory concentrations required for 90% growth inhibition (MIC90) was calculated for representative isolates. The MIC90 values for both M. bovis and M. bovoculi were as follows: ampicillin and ceftiofur: ≤0.25 µg/ml; clindamycin: 2 µg/ml; danofloxacin and enrofloxacin: ≤0.12 µg/ml; florfenicol: 0.5 µg/ml; gentamicin: 1 µg/ml; neomycin: 4 µg/ml; tulathromycin: 2 µg/ml; and tylosin: 8 µg/ml. The MIC90 values for M. bovoculi included the following: chlortetracycline: ≤0.5 µg/ml; oxytetracycline: 4 µg/ml; penicillin: 0.25 µg/ml; spectinomycin: 32 µg/ml; sulfadimethoxine: >256 µg/ml; tiamulin: 1 µg/ml; and trimethoprim–sulfamethoxazole: 4 µg/ml. For M. bovis, MIC90 values included the following: chlortetracycline and oxytetracycline: 1 µg/ml; penicillin: ≤0.12 µg/ml; spectinomycin: 16 µg/ml; sulfadimethoxine: ≤256 µg/ml; tiamulin: ≤0.5 µg/ml; and trimethoprim–sulfamethoxazole: ≤2 µg/ml. The current work describes the frequency of isolation and differences in antimicrobial sensitivity observed among Moraxella isolates from case submissions.

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

BackgroundWe 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.ResultsBased 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.ConclusionsThe 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.

Rapid Typing of Mannheimia haemolytica Major Genotypes 1 and 2 Using MALDI-TOF Mass Spectrometry

Genotype 2M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate genotype 2 strains in veterinary diagnostic laboratories.

Experimental infection of conventional nursing pigs and their dams with Porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) is a newly identified virus that has been detected in swine herds of North America associated with enteric disease. The aim of this study was to demonstrate the pathogenicity, course of infection, virus kinetics, and aerosol transmission of PDCoV using 87 conventional piglets and their 9 dams, including aerosol and contact controls to emulate field conditions. Piglets 2–4 days of age and their dams were administered an oronasal PDCoV inoculum with a quantitative real-time reverse transcription (qRT)-PCR quantification cycle (Cq) value of 22 that was generated from a field sample having 100% nucleotide identity to USA/Illinois121/2014 determined by metagenomic sequencing and testing negative for other enteric disease agents using standard assays. Serial samples of blood, serum, oral fluids, nasal and fecal swabs, and tissues from sequential autopsy, conducted daily on days 1–8 and regular intervals thereafter, were collected throughout the 42-day study for qRT-PCR, histopathology, and immunohistochemistry. Diarrhea developed in all inoculated and contact control pigs, including dams, by 2 days post-inoculation (dpi) and in aerosol control pigs and dams by 3–4 dpi, with resolution occurring by 12 dpi. Mild to severe atrophic enteritis with PDCoV antigen staining was observed in the small intestine of affected piglets from 2 to 8 dpi. Mesenteric lymph node and small intestine were the primary sites of antigen detection by immunohistochemistry, and virus RNA was detected in these tissues to the end of the study. Virus RNA was detectable in piglet fecal swabs to 21 dpi, and dams to 14–35 dpi.

Evaluation of the Effect of Serum Antibody Abundance against Bovine Coronavirus on Bovine Coronavirus Shedding and Risk of Respiratory Tract Disease in Beef Calves from Birth through the First Five Weeks in a Feedlot

OBJECTIVE To evaluate the effect of serum antibody abundance against bovine coronavirus (BCV) on BCV shedding and risk of bovine respiratory disease (BRD) in beef calves from birth through the first 5 weeks in a feedlot. ANIMALS 890 natural-service crossbred beef calves from 4 research herds. PROCEDURES Serial blood samples for measurement of serum anti-BCV antibody abundance by an ELISA and nasal swab specimens for detection of BCV and other viral and bacterial BRD pathogens by real-time PCR methods were collected from all calves or subsets of calves at predetermined times from birth through the first 5 weeks after feedlot entry. Test results were compared among herds, over time, and between calves that did and did not develop BRD. The associations of various herd and calf factors with test results were also evaluated. RESULTS At the calf level, serum anti-BCV antibody abundance was not associated with BCV shedding, but BCV shedding was positively associated with BRD incidence before and after weaning. The mean serum anti-BCV antibody abundance at weaning for a group of calves was inversely related with the subsequent incidence of BRD in that group; however, the serum anti-BCV antibody abundance at weaning for individual calves was not predictive of which calves would develop BRD after feedlot entry. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that serum anti-BCV antibody abundance as determined with ELISA were not associated with BCV shedding or risk of BRD in individual beef calves from birth through the first 5 weeks after feedlot entry.