Ovine Herpesvirus 2 Glycoprotein B Complementation Restores Infectivity to a Bovine Herpesvirus 4 gB-Null Mutant.

Ovine herpesvirus 2 (OvHV-2) and bovine herpesvirus 4 (BoHV-4) are gamma herpesviruses that belong to the genera Macavirus and Rhadinovirus, respectively. As with all herpesviruses, both OvHV-2 and BoHV-4 express glycoprotein B (gB), which plays an essential role in the infection of host cells. In that context, it has been demonstrated that a BoHV-4 gB-null mutant is unable to infect host cells. In this study, we used homologous recombination to insert OvHV-2 ORF 8, encoding gB, into the BoHV-4 gB-null mutant genome, creating a chimeric BoHV-4 virus carrying and expressing OvHV-2 gB (BoHV-4∆gB/OvHV-2-gB) that was infectious and able to replicate in vitro. We then evaluated BoHV-4∆gB/OvHV-2-gB as a potential vaccine candidate for sheep-associated malignant catarrhal fever (SA-MCF), a fatal disease of ungulates caused by OvHV-2. Using rabbits as a laboratory model for MCF, we assessed the safety, immunogenicity, and efficacy of BoHV-4∆gB/OvHV-2-gB in an immunization/challenge trial. The results showed that while BoHV-4∆gB/OvHV-2-gB was safe and induced OvHV-2 gB-specific humoral immune responses, immunization conferred only 28.5% protection upon challenge with OvHV-2. Therefore, future studies should focus on alternative strategies to express OvHV-2 proteins to develop an effective vaccine against SA-MCF.

A Vaccine Targeting Ovine Herpesvirus 2 Glycoprotein B Protects against Sheep-Associated Malignant Catarrhal Fever.

Malignant catarrhal fever (MCF) is a complex and often fatal disease of ungulates. Effective vaccines are needed to avoid MCF outbreaks and mitigate losses. This study aimed to evaluate a sheep-associated MCF (SA-MCF) vaccine candidate targeting ovine herpesvirus 2 (OvHV-2) glycoprotein B (gB). Rabbits were used as a laboratory animal model to test the safety, immunogenicity, and protective efficacy of a chimeric virus consisting of a recombinant, non-pathogenic strain of alcelaphine herpesvirus-1 encoding OvHV-2 ORF8 to express gB (AlHV-1∆ORF73/OvHV-2-ORF8). Viral-vectored immunizations were performed by using the AlHV-1∆ORF73/OvHV-2-ORF8 chimera alone or as a DNA prime (OvHV-2-ORF8)-virus boost regimen. The viral vector was inoculated by intravenous or intramuscular routes and the DNA was delivered by intradermal shots using a gene gun. The vaccine candidates were deemed safe as no clinical signs were observed following any of the immunizations. Anti-OvHV-2 gB antibodies with neutralizing activity were induced by all immunogens. At three weeks post-final immunization, all animals were challenged intranasally with a lethal dose of OvHV-2. MCF protection rates ranging from 66.7% to 71.4% were observed in vaccinated rabbits, while all mock-vaccinated animals developed the disease. The significant protective efficacy obtained with the vaccine platforms tested in this study encourages further trials in relevant livestock species, such as cattle and bison.

OvHV-2 Glycoprotein B Delivered by a Recombinant BoHV-4 Is Immunogenic and Induces Partial Protection against Sheep-Associated Malignant Catarrhal Fever in a Rabbit Model.

An efficacious vaccine for sheep-associated malignant catarrhal fever (SA-MCF) is important for the livestock industry. Research towards SA-MCF vaccine development is hindered by the absence of culture systems to propagate the causative agent, ovine herpesvirus-2 (OvHV-2), which means its genome cannot be experimentally modified to generate an attenuated vaccine strain. Alternative approaches for vaccine development are needed to deliver OvHV-2 antigens. Bovine herpesvirus 4 (BoHV-4) has been evaluated as a vaccine vector for several viral antigens with promising results. In this study, we genetically engineered BoHV-4 to express OvHV-2 glycoprotein B (gB) and evaluated its efficacy as an SA-MCF vaccine using a rabbit model. The construction of a viable recombinant virus (BoHV-4-AΔTK-OvHV-2-gB) and confirmation of OvHV-2 gB expression were performed in vitro. The immunization of rabbits with BoHV-4-AΔTK-OvHV-2-gB elicited strong humoral responses to OvHV-2 gB, including neutralizing antibodies. Following intra-nasal challenge with a lethal dose of OvHV-2, 42.9% of the OvHV-2 gB vaccinated rabbits were protected against SA-MCF, while all rabbits in the mock-vaccinated group succumbed to SA-MCF. Overall, OvHV-2 gB delivered by the recombinant BoHV-4 was immunogenic and partly protective against SA-MCF in rabbits. These are promising results towards an SA-MCF vaccine; however, improvements are needed to increase protection rates.

Standardized Escherichia coli O157:H7 Exposure Studies in Cattle Provide Evidence that Bovine Factors Do Not Drive Increased Summertime Colonization.

The increased summertime prevalence of cattle carriage of enterohemorrhagic Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is associated with the increased summertime incidence of human infection. The mechanism driving the seasonality of STEC O157 carriage among cattle is unknown. We conducted experimental challenge trials to distinguish whether factors extrinsic or intrinsic to cattle underlie the seasonality of STEC O157 colonization. Holstein steers (n = 20) exposed to ambient environmental conditions were challenged with a standardized pool of STEC O157 strains four times at 6-month intervals. The densities and durations of rectoanal junction mucosa (RAJ) colonization with STEC O157 were compared by season (winter versus summer), dose (10(9) CFU versus 10(7) CFU), and route of challenge (oral versus rectal). Following summer challenges, the RAJ STEC O157 colonization density was significantly lower (P = 0.016) and the duration was shorter (P = 0.052) than for winter challenges, a seasonal pattern opposite to that observed naturally. Colonization was unaffected by the challenge route, indicating that passage through the gastrointestinal microbiome did not significantly affect the infectious dose to the RAJ. A 2-log reduction of the challenge doses in the second-year trials was accompanied by similarly reduced RAJ colonization in both seasons (P < 0.001). These results refute the hypothesis that cattle are predisposed to STEC O157 colonization during the summer months, either due to intrinsic factors or indirectly due to gastrointestinal tract microbiome effects. Instead, the data support the hypothesis that the increased summertime STEC O157 colonization results from increased seasonal oral exposure to this pathogen.

Geographically distinct Escherichia coli O157 isolates differ by lineage, Shiga toxin genotype, and total shiga toxin production.

While the differential association of Escherichia coli O157 genotypes with animal and human hosts has recently been well documented, little is known about their distribution between countries and how this might affect regional disease rates. Here, we used a 48-plex single nucleotide polymorphism (SNP) assay to segregate 148 E. coli O157 isolates from Australia, Argentina, and the United States into 11 SNP lineages. We also investigated the relationship between SNP lineages, Shiga toxin (Stx) gene profiles, and total Stx production. E. coli O157 isolates clearly segregated into SNP lineages that were differentially associated with each country. Of the 11 SNP lineages, seven were detected among isolates from a single country, two were detected among isolates from all three countries, and another two were detected only among U.S. and Argentinean isolates. A number of Australian (30%) and Argentinean (14%) isolates were associated with novel, previously undescribed SNP lineages that were unique to each country. Isolates within SNP lineages that were strongly associated with the carriage of stx2a produced comparatively more Stx on average than did those lacking the stx2a subtype. Furthermore, the proportion of isolates in stx2a-associated SNP lineages was significantly higher in Argentina and the United States than Australia (P < 0.05). This study provides evidence for the geographic divergence of E. coli O157 and for a prominent role of stx2a in total Stx production. These results also highlight the need for more comprehensive studies of the global distribution of E. coli O157 lineages and the impacts of regionally predominant E. coli O157 lineages on the prevalence and severity of disease.

Risk factors for campylobacteriosis in two washington state counties with high numbers of dairy farms.

Campylobacteriosis is a frequently reported, food-borne, human bacterial disease that can be associated with ruminant reservoirs, although public health messages primarily focus on poultry. In Washington State, the two counties with the highest concentrations of dairy cattle also report the highest incidences of campylobacteriosis. Conditional logistic regression analysis of case-control data from both counties found living or working on a dairy farm (odds ratio [OR], 6.7 [95% confidence interval [CI], 1.7 to 26.4]) and Hispanic ethnicity (OR, 6.4 [95% CI, 3.1 to 13.1]) to have the strongest significant positive associations with campylobacteriosis. When the analysis was restricted to residents of one county, Hispanic ethnicity (OR, 9.3 [95% CI, 3.9 to 22.2]), contact with cattle (OR, 5.0 [95% CI, 1.3 to 19.5]), and pet ownership (OR, 2.6 [95% CI, 1.1 to 6.3]) were found to be independent risk factors for disease. Campylobacter jejuni isolates from human (n = 65), bovine (n = 28), and retail poultry (n = 27) sources from the same counties were compared using multilocus sequence typing. These results indicated that sequence types commonly found in human isolates were also commonly found in bovine isolates. These findings suggest that, in areas with high concentrations of dairy cattle, exposure to dairy cattle may be more important than food-borne exposure to poultry products as a risk for campylobacteriosis.

Aerobic oral and rectal bacteria of free-ranging Steller sea lion pups and juveniles (Eumetopias jubatus) in Alaska.

Bacteriologic cultures from oral, rectal, and lesion samples from free-ranging Steller sea lion (SSL, Eumetopias jubatus) pups and juveniles in Alaska (2001-2005) were examined to determine frequency of infection by a specific subset of common and pathogenic aerobic bacteria. Associations between isolated bacteria and age, sex, body condition, location, and sampling season were investigated. Salmonella spp. isolates were further evaluated to determine spatial clustering (n=48) and to identify serovars (n=13) and antimicrobial susceptibility patterns (n=11). We sampled 356 SSL pups (n=272) and juveniles (n=84), and identified 988 isolates of 13 bacterial genera of specific interest. Pasteurella spp. (43.8%), beta-hemolytic Streptococcus spp. (30.6%), and Mannheimia spp. (18.2%) were the most commonly isolated oral bacteria (n=499 isolates), whereas Escherichia coli (47.6%), beta-hemolytic E. coli (32.4%), Salmonella spp. (10.4%), and Campylobacter spp. (7.8%) were the most frequently isolated rectal bacteria (n=460 isolates). Salmonella was most commonly found in pups from western stocks and in samples collected during fall/winter seasons. A significant Salmonella cluster was detected at the Perry Island haulout. Five serovars were isolated: Enteritidis, Infantis, Newport, Reading, and Stanley. Pulsed-field gel electrophoresis provided evidence that Salmonella isolates were most likely being maintained within the SSL population in Alaska.

Genotypic-phenotypic discrepancies between antibiotic resistance characteristics of Escherichia coli isolates from calves in management settings with high and low antibiotic use.

We hypothesized that bacterial populations growing in the absence of antibiotics will accumulate more resistance gene mutations than bacterial populations growing in the presence of antibiotics. If this is so, the prevalence of dysfunctional resistance genes (resistance pseudogenes) could provide a measure of the level of antibiotic exposure present in a given environment. As a proof-of-concept test, we assayed field strains of Escherichia coli for their resistance genotypes using a resistance gene microarray and further characterized isolates that had resistance phenotype-genotype discrepancies. We found a small but significant association between the prevalence of isolates with resistance pseudogenes and the lower antibiotic use environment of a beef cow-calf operation versus a higher antibiotic use dairy calf ranch (Fisher's exact test, P = 0.044). Other significant findings include a very strong association between the dairy calf ranch isolates and phenotypes unexplained by well-known resistance genes (Fisher's exact test, P < 0.0001). Two novel resistance genes were discovered in E. coli isolates from the dairy calf ranch, one associated with resistance to aminoglycosides and one associated with resistance to trimethoprim. In addition, isolates resistant to expanded-spectrum cephalosporins but negative for bla(CMY-2) had mutations in the promoter regions of the chromosomal E. coli ampC gene consistent with reported overexpression of native AmpC beta-lactamase. Similar mutations in hospital E. coli isolates have been reported worldwide. Prevalence or rates of E. coli ampC promoter mutations may be used as a marker for high expanded-spectrum cephalosporin use environments.

Discovery of a gene conferring multiple-aminoglycoside resistance in Escherichia coli.

Bovine-origin Escherichia coli isolates were tested for resistance phenotypes using a disk diffusion assay and for resistance genotypes using a DNA microarray. An isolate with gentamicin and amikacin resistance but with no corresponding genes detected yielded a 1,056-bp DNA sequence with the closest homologues for its inferred protein sequence among a family of 16S rRNA methyltransferase enzymes. These enzymes confer high-level aminoglycoside resistance and have only recently been described in Gram-negative bacteria.

Multilocus variable-number tandem-repeat method for typing Salmonella enterica serovar Newport.

In recent years, the proportion of Salmonella enterica infections represented by S. enterica serovar Newport has increased markedly among humans and animals. Multilocus variable-number tandem-repeat analysis (MLVA) has proven to be useful in discriminating other highly clonal Salmonella serovars. Here, we report on the development of a highly discriminatory MLVA for Salmonella serovar Newport.

Antimicrobial resistance in Salmonella enterica serovar Dublin isolates from beef and dairy sources.

Salmonella enterica serovar Dublin (S. Dublin) is a cattle-adapted Salmonella serovar, so if antimicrobial resistance in S. Dublin arises as a result of antimicrobial use this most likely occurs within the cattle reservoir without impact from antimicrobial use in humans. We tested the antimicrobial resistance of bovine-origin S. Dublin isolates from 1986 through 2004 using a standard disk diffusion method. High proportions of isolates throughout the time period were resistant to one or more antimicrobials, and a marked increase in resistance to ceftazidime occurred between 2000 and 2004. Dairy-origin isolates were more likely to be resistant to several antibiotics than were isolates from beef operations where exposure to antimicrobials is likely to be less frequent. Plasmid analysis of a subset of isolates also supported the hypothesis that antimicrobial resistance traits in the cattle-adapted serovar Dublin were acquired within the bovine host environment.

Multidrug-resistant Salmonella typhimurium, Pacific Northwest, United States.

We compared human and bovine isolates of Salmonella enterica using antimicrobial-drug resistance profiles and pulsed-field gel electrophoresis. From 2000 through 2006, we observed an increase in a novel multidrug-resistant clone of S. Typhimurium with no recognized phage type. This clone may represent an emerging epidemic strain in the Pacific Northwest.