Escherichia coli O157:H7 is the predominant cause of diarrhea-associated hemolytic uremic syndrome (HUS) worldwide. Its cardinal virulence traits are Shiga toxins, which are encoded by stx genes, the most common of which are stx1a, stx2a, and stx2c. The toxins these genes encode differ in their in vitro and experimental phenotypes, but the human population-level impact of these differences is poorly understood. Using Shiga toxin-encoding bacteriophage insertion typing and real-time polymerase chain reaction, we genotyped isolates from 936 E. coli O157:H7 cases and verified HUS status via chart review. We compared the HUS risk between isolates with stx2a and those with stx2a and another gene and estimated additive interaction of the stx genes. Adjusted for age and symptoms, the HUS incidence of E. coli O157:H7 containing stx2a alone was 4.4% greater (95% confidence interval (CI) −0.3%, 9.1%) than when it occurred with stx1a. When stx1a and stx2a occur together, the risk of HUS was 27.1% lower (95% CI −87.8%, −2.3%) than would be expected if interaction were not present. At the population level, temporal or geographic shifts toward these genotypes should be monitored, and stx genotype may be an important consideration in clinically predicting HUS among E. coli O157:H7 cases.

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.

The sero-prevalence of antibodies against blue tongue virus (BTV) in 408 local breeds of sheep in Rajasthan state in India was investigated using standard agar gel immunodiffusion (AGID) test. Maximum seropositivities of 11.3% (13/115), 10.7% (13/121), 7.1% (11/155) and 5.9% (1/17) were recorded in the Chokla, Magra, Nali and Pugal breeds, respectively. Out of 107 goat serum samples, 6 (5.6%) were AGID positive. The performance of the standard AGID, counter current immuno-electrophoresis (CCIE) and the competitive enzyme-linked immunosorbent assay (cELISA) for the detection of serum antibody against BTV in indigenous breeds of sheep were compared. Out of 178 sheep serum samples tested, 17 (9.5%), 22 (12.3%) and 54 (30.3%) were positive for group-specific bluetongue antibodies by AGID, CCIE and cELISA, respectively. There was appreciable difference in the seroprevalence detected by AGID, CCIE and cELISA in clinically healthy and diseased sheep with regard to relative sensitivities and specificities of the tests with cELISA being highly sensitive and specific followed by CCIE and AGID test. It was concluded that these indigenous breeds of sheep may be a potential reservoir of BTV infection and cELISA should be routinely used for the detection of antibodies against BTV in these local breeds of sheep.

Subcutaneous vaccination of cattle for enterohemorrhagic E. coli O157:H7 reduces the magnitude and duration of fecal shedding but the often required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx^-, LEE⁺E. coli O157:H7 reduced recto-anal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for six weeks with 3x10⁹ CFU of a pool of three stx^-, LEE⁺E. coli O157:H7 strains (vaccine group) or three stx^-, LEE^-, non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx⁺, LEE⁺E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for four weeks. Serum antibodies against TTSP, Tir and EspA were determined by ELISA at day 0 (pre-immunization), day 61 (post-immunization, pre-challenge) and day 89 (post-challenge). Vaccine group cattle had lower numbers of WT strains at the RAJ compared to control group cattle on post-challenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP post-immunization were associated with decreased numbers of WT E. coli O157:H7 at the RAJ. IMPORTANCE The bacterium E. coli O157:H7 causes food-borne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A de-toxified E. coli O157:H7, missing genes that cause disease, was fed to cattle as an oral vaccine to create immunity to carrying pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first seven days post-challenge and shed the bacteria for a shorter duration compared to the non-vaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

According to the Humane Society, 25 to 40 percent of pet dogs in the United States are adopted from animal shelters. Shelter dogs can harbor bacterial, viral, fungal, and protozoal pathogens, posing risks to canine and human health. These bacterial pathogens may also carry antibiotic resistance genes (ARGs), serving as a reservoir for antimicrobial resistance (AMR) transmission. This study aimed to utilize whole metagenome sequencing (WMS) to screen for microbial pathogens and assess the resistome in healthy shelter dogs. Fecal samples from 58 healthy shelter dogs across 10 shelters in Kentucky, Tennessee, and Virginia were analyzed using WMS. Genomic DNA was extracted, and bioinformatics analyses were performed to identify pathogens and ARGs. The WMS detected 53 potentially zoonotic or known pathogens including thirty-eight bacterial species, two protozoa, five yeast species, one nematode, four molds, and three viruses. A total of 4560 ARGs signatures representing 182 unique genes across 14 antibiotic classes were detected. Tetracycline resistance genes were most abundant (49%), while β-lactam resistance genes showed the highest diversity with 75 unique ARGs. ARGs were predominantly detected in commensal bacteria; however, nearly half (18/38, 47.4%) of known bacterial pathogens detected in this study carried ARGs for resistance to one or more antibiotic classes. This study provides evidence that healthy shelter dogs carry a diverse range of zoonotic and antibiotic-resistant pathogens, posing a transmission risk through fecal shedding. These findings highlight the value of WMS for pathogen detection and AMR surveillance, informing therapeutic and prophylactic strategies to mitigate the transmission of pathogens among shelter dog populations and the risk associated with zoonoses.

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.