OBJECTIVE To determine the effect of a commercially available multivalent killed virus vaccine on serum neutralizing (SN) and colostrum neutralizing (CN) antibodies against bovine herpesvirus (BHV) type 1 and bovine viral diarrhea virus (BVDV) types 1 and 2 in pregnant dairy cattle. ANIMALS 49 Holstein dairy cattle. PROCEDURES 25 cattle were vaccinated (IM injection) at least 60 days prior to calving (ie, at the end of the lactation period or according to the expected calving date for heifers) and again 5 weeks later. The remaining 24 cattle were not vaccinated (control group). Titers of SN antibodies were measured at the 5-week time point. Titers of SN and CN antibodies were measured at parturition. RESULTS 5 weeks after initial vaccination, titers of SN antibodies against BHV-1 and BVDV types 1 and 2 were 1:512, 1:128, and 1:2,048, respectively, in vaccinates and 1:64, 1:128, and 1:64, respectively, in unvaccinated controls. Equivalent SN antibody titers at parturition were 1:256, 1:64, and 1:512, respectively, in vaccinates and 1:128, 1:128, and 1:64, respectively, in controls. Median titers of CN antibodies against BHV-1 and BVDV types 1 and 2 were 1:1,280, 1:10,240, and 1:20,480, respectively, in vaccinates and 1:80, 1:1,280, and 1:2,560, respectively, in controls. CONCLUSIONS AND CLINICAL RELEVANCE Titers of antibodies against viral respiratory pathogens were significantly enhanced in both serum (BHV-1 and BVDV type 2) and colostrum (BHV-1 and BVDV types 1 and 2) in cattle receiving a killed virus vaccine (with no adverse reactions) before parturition. To maximize protection of bovine neonates, this method of vaccination should be considered.

Three canine parainfluenza viruses type 5 (CPIV-5) were isolated from lung tissues of 3 Korean dogs with mild pneumonia between 2008 and 2009. The isolates were fully sequenced and compared with published reference sequences. The size of the genome was 15 246 nucleotides long and no remarkable differences were found when compared with previously published reference sequences. In phylogenetic analysis based on the F and P genes, parainfluenza virus 5 (PIV-5) strains were divided into at least 3 subgroups. Three CPIV-5 strains were clustered with CPIV-5 T1, H22 and 78524 strains. All PIV-5 strains were independent of the host species, geographical distribution, and the isolated period.

OBJECTIVE To investigate the association of bovine respiratory disease (BRD) or vaccination with serologic response in calves. ANIMALS 94 Holstein calves. PROCEDURES To assess the association between BRD and antibody titers, 38 calves < 3 months old that were treated for BRD were matched with 38 untreated calves. To investigate the effect of vaccination on antibody titers, 24 calves were randomly assigned to be vaccinated against bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus types 1 and 2, bovine herpesvirus type 1 (BHV1), and parainfluenza virus type 3 at 2 weeks of age (n = 6), 5 weeks of age (6), and both 2 and 5 weeks of age (6) or were assigned to be unvaccinated controls (6). Blood samples were obtained at I, 2, 5, and 12 weeks for determination of serum neutralization antibody titers against the vaccine viruses, bovine coronavirus, and Mannheimia haemolytica. Antibody rates of decay were calculated. RESULTS Calves with initial antibody titers against BRSV < 1:64 that were treated for BRD had a slower rate of anti-BRSV antibody decay than did similar calves that were not treated for BRD. Calves with high initial antibody titers against BRSV and BHV1 had lower odds of BRD than did calves with low initial antibody titers against those 2 pathogens. Vaccination at 2 or 5 weeks of age had no effect on the rate of antibody decay. CONCLUSIONS AND CLINICAL RELEVANCE Clinical BRD and the serologic response of dairy calves were associated with initial antibody titers against BRSV and BHV1. Serologic or clinical responses to viral exposure may differ in calves with low passive immunity.

OBJECTIVE To compare antibody responses of horses naturally infected with West Nile virus (WNV) and those vaccinated against WNV, to identify whether vaccination interferes with the ability to diagnose WNV infection, and to determine the duration of antibody responses after vaccination. SAMPLE Sera from horses naturally infected with WNV (n = 10) and adult WNV-naïve horses before and after vaccination with a live canarypox virus–vectored vaccine (7) or a killed virus vaccine (8). PROCEDURES An established WNV IgM capture ELISA was used to measure IgM responses. Newly developed capture ELISAs were used to measure responses of 8 other WNV-specific immunoglobulin isotypes. A serum neutralization assay was used to determine anti-WNV antibody titers. RESULTS WNV-specific IgM responses were typically detected in the sera of WNV-infected horses but not in sera of horses vaccinated against WNV. Natural infection with and vaccination against WNV induced an immunoglobulin response that was primarily composed of IgG1. West Nile virus–specific IgG1 was detected in the sera of most horses 14 days after vaccination. Serum anti-WNV IgG1 and neutralizing antibody responses induced by the killed-virus vaccines were higher and lasted longer than did those induced by the live canarypox virus–vectored vaccine. CONCLUSIONS AND CLINICAL RELEVANCE On the basis of these findings, we recommend that horses be vaccinated against WNV annually near the beginning of mosquito season, that both IgM and IgG1 responses against WNV be measured to distinguish between natural infection and vaccination, and that a WNV IgG1 ELISA be used to monitor anti-WNV antibodies titers in vaccinated horses.

Interferon (IFN)-γ has been shown to be associated with immunity to Marek’s disease virus (MDV). The overall objective of this study was to investigate the causal relationship between IFN-g and vaccine-conferred immunity against MDV in chickens. To this end, 3 small interfering RNAs (siRNAs) targeting chicken IFN-g, which had previously been shown to reduce IFN-γ expression in vitro, and a control siRNA were selected to generate recombinant avian adeno-associated virus (rAAAV) expressing short-hairpin small interfering RNAs (shRNAs). An MDV challenge trial was then conducted: chickens were vaccinated with herpesvirus of turkey (HVT), administered the rAAAV expressing shRNA, and then challenged with MDV. Tumors were observed in 4 out of 10 birds that were vaccinated with HVT and challenged but did not receive any rAAAV, 5 out of 9 birds that were administered the rAAAV containing IFN-γ shRNA, and 2 out of 10 birds that were administered a control enhanced green fluorescent protein siRNA. There was no significant difference in MDV genome load in the feather follicle epithelium of the birds that were cotreated with the vaccine and the rAAAV compared with the vaccinated MDV-infected birds. These results suggest that AAAV-based vectors can be used for the delivery of shRNA into chicken cells. However, administration of the rAAAV expressing shRNA targeting chicken IFN-γ did not seem to fully abrogate vaccine-induced protection.

Porcine reproductive and respiratory syndrome (PRRS) has tremendous impact on the pork industry in North America. The molecular diagnosis of infection with PRRS virus (PRRSV) is hampered by its considerable strain diversity. In this study, 43 previously published or newly developed primers for probe-free real-time reverse transcription polymerase chain reaction (RT-PCR) were evaluated on their sensitivity, specificity, reproducibility, and repeatability, using a diverse panel of 36 PRRSV strains as well as other arteriviruses and unrelated porcine viruses. Three primer pairs had excellent diagnostic and analytical sensitivity on par with a probe-based reference assay, absolute specificity to virus genotype and species, as well as over 95% reproducibility and repeatability across a wide dynamic range.

OBJECTIVE To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. SAMPLE Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. PROCEDURES Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter. Cells that stably integrated the TAg were selected and expanded to passage 25. For each passage, differentiation and fusion properties of the cells were determined and immunocytochemical analyses were performed to evaluate expression of TAg and other muscle-specific proteins. Optimum conditions that led to cell differentiation into myotubes were also determined. RESULTS Compared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33°C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37°C or when incubated with doxycycline at 33°C. Expression of TAg was not detected when cells were incubated with doxycycline at 37°C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that production of an immortalized clonal equine skeletal muscle cell line was possible. A clonal equine skeletal muscle cell line will be a valuable in vitro tool for use in equine physiology and disease research.

The objective of this study was to identify and partially characterize 3 equid herpesviruses that were isolated postmortem from zebras in Ontario, Canada in 1989, 2002, and 2007. These 3 virus isolates were characterized by plaque morphology, restriction fragment length polymorphism (RFLP) of their genomic deoxyribonucleic acid (DNA), real-time polymerase chain reaction (PCR) assay, and sequence analyses of the full length of the glycoprotein G (gG) gene (ORF70) and a portion of the DNA polymerase gene (ORF30). The isolates were also compared to 3 reference strains of equid herpesvirus 1 (EHV-1). Using rabbit kidney cells, the plaques for the isolates from the zebras were found to be much larger in size than the EHV-1 reference strains. The RFLP patterns of the zebra viruses differed among each other and from those of the EHV-1 reference strains. Real-time PCR and sequence analysis of a portion of the DNA polymerase gene determined that the herpesvirus isolates from the zebras contained a G at nucleotide 2254 and a corresponding N at amino acid position 752, which suggested that they could be neuropathogenic EHV-1 strains. However, subsequent phylogenetic analysis of the gG gene suggested that they were EHV-9 and not EHV-1.

Pseudorabies has been controlled efficiently in China for many years by vaccination. However, it suddenly broke out in many pig farms in 2012–2013 in southern China. In this study, a systematic investigation that included virus isolation, genetic and pathological studies, and immunogenicity analysis was carried out with the aim of understanding the pathogenetic and antigenic features of novel isolates of pseudorabies virus (PRV). Of 38 tissue samples collected from pigs with clinical signs of pseudorabies on 13 farms in 4 provinces in southern China in 2012–2013, 29 showed wild-type PRV infection by polymerase chain reaction. Sequence analysis of 5 isolates from the 4 provinces showed that they belonged to a relatively independent cluster that shared 2 insertions of a single amino acid in the gE gene and 1 insertion of 7 amino acids in the gC gene. In experiments, isolate ZJ01 caused death in 100% of pigs that were either 14 or 80 days old. The serum antibodies to the commercial PRV vaccines had significantly lower neutralizing activity against the ZJ01 isolate than against the vaccine strains. The antigenic relatedness between ZJ01 and the vaccine strains was 0.378 to 0.455. These findings indicated that a novel, highly virulent PRV strain with antigenic variance had spread widely in southern China.