Introduction: Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.Material and Methods: The VCAM-1 gene was cloned from bovine Peyer’s patches and inserted into the pGEX-4T-1 and pET-28a vectors. The recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1 were transferred into Escherichia coli BL21 and the recombinant strains were induced by isopropyl-D-thiogalactoside to produce fusion proteins tagged with polyhistidine (His) and glutathione S-transferase (GST), respectively. The expressed fusion proteins His-VCAM-1 and GST-VCAM-1 were identified by SDS-PAGE and Western blot. His-VCAM-1 protein was used as an antigen to immunise Wistar rats and polyclonal antibody serum against VCAM-1 was obtained.Results: The serum titre tested by indirect ELISA was 128,000 using GST-VCAM-1 as the well coating antigen. Western blots indicated that the antibody recognised recombinant VCAM-1 protein as well as endogenous VCAM-1. In addition, using qPCR and Western blot, VCAM-1 mRNA and protein expression levels were measured in dairy cows with subclinical mastitis. It was demonstrated that VCAM-1 levels in the mammary lymph nodes of the cows were significantly higher than those from healthy controls (P < 0.05).Conclusion: These results are to our knowledge the first report that VCAM-1 expression in the mammary lymph nodes is elevated in dairy cows with subclinical mastitis.

Introduction: Pregnancy is a physiological state in which the immune system undergoes certain changes. On the one hand, by depleting cell defence mechanisms, it favours development and maintenance of the pregnancy. At the same time cells of the immune system ensure resistance to many risk factors, including infectious agents.Material and Methods: The study was carried out on 24 Polish Konik breed mares which were divided into two equal groups. The first group (group I) included mares living in the reserve. The second group (group II) comprised mares maintained under conventional conditions in the stables. The blood samples were collected for the first time in the perinatal period, i.e. 2 weeks before parturition (trial 0), then within the first 24 h after delivery, and then on 7ᵗʰ and 21ˢᵗ day after foaling. Flow cytometric analysis of lymphocyte expressing TCD4+, TCD8+, CD2+, and MHC class II antigens was performed.Results: Before the delivery, in group I there was a significantly higher CD4:CD8 ratio compared to group II (P ≤0.05). Similarly, significantly increased CD4:CD8 ratio in group I was noted within 24 h after parturition (P ≤0.001) and it was also observed on 7ᵗʰ day (P ≤0.03) and 21ˢᵗ day after foaling (P ≤0.02). In the first 24 h after parturition, a significant decline of lymphocytes CD8+ (P ≤0.02) was noted. No significant differences in terms of lymphocytes CD2+ and CD3+ were observed. Expression of MHC-II molecules before and after the parturition was higher in group I compared to group II; however, the difference between the groups was not significant.Conclusion: The results obtained indicate that mares living in the reserve display higher activity of cell defence mechanisms.

Introduction: The purpose of this study was to determine the occurrence of avian reovirus (ARV) infections in wild birds in Poland and attempt to propagate the selected ARV strains in chicken embryo kidney (CEK) cells or chicken SPF embryos. Material and Methods: The study included 192 wild birds representing 32 species, collected between 2014 and 2016. A part of the S4 segment encoding the σNS protein of avian reoviruses (ARVs) isolated from different species of wild birds from that period was amplified. Results: The presence of ARV was demonstrated in 58 (30.2%) wild birds belonging to nine orders. The isolated strains were propagated in chicken embryos by yolk sac inoculation, and CPE was induced in the infected CEK monolayer. Agar gel precipitation showed that two ARV isolates from rock pigeon and mute swan shared a common groupspecific antigen with chicken reovirus S1133. Specific products of predicted size were found in two ARV isolates from the chicken embryo passage and 13 ARVs isolated from CEK cells. Conclusion: The study indicates the high prevalence of ARV among wild birds in Poland and its possible transmission to farmed birds.

Introduction: The prevention and control of Actinobacillus pleuropneumoniae in commercial production settings is based on serological monitoring. Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect specific antibodies against a variety of A. pleuropneumoniae antigens, including long-chain lipopolysaccharides (LPS) and the ApxIV toxin, a repeats-in-toxin (RTX) exotoxin unique to A. pleuropneumoniae and produced by all serovars. The objective of this study was to describe ApxIV antibody responses in serum and oral fluid of pigs.Material and Methods: Four groups of pigs (six pigs per group) were inoculated with A. pleuropneumoniae serovars 1, 5, 7, or 12. Weekly serum samples and daily oral fluid samples were collected from individual pigs for 56 days post inoculation (DPI) and tested by LPS and ApxIV ELISAs. The ApxIV ELISA was run in three formats to detect immunlgobulins M, G, and A (IgM, IgG and IgA) while the LPS ELISA detected only IgG.Results: All pigs inoculated with A. pleuropneumoniae serovars 1 and 7 were LPS ELISA serum antibody positive from DPI 14 to 56. A transient and weak LPS ELISA antibody response was observed in pigs inoculated with serovar 5 and a single antibody positive pig was observed in serovar 12 at ≥35 DPI. Notably, ApxIV serum and oral fluid antibody responses in pig inoculated with serovars 1 and 7 reflected the patterns observed for LPS antibody, albeit with a 14 to 21 day delay.Conclusion: This work suggests that ELISAs based on ApxIV antibody detection in oral fluid samples could be effective in population monitoring for A. pleuropneumoniae.

Introduction: Infections with bovine foamy virus (BFV) were found in many countries but there is a lack of large-scale surveys on the prevalence of BFV among dairy cattle. The aim of this study was to develop and validate the recombinant Gag protein-based ELISA and to estimate the prevalence of antibodies against BFV. Material and Methods: Gag coding region from BFV was cloned into expression vector pT7Arg-STOP, which expressed a high level of recombinant Gag protein from E.coli. The ELISA was standardised, and the cut-off value and sensitivity and specificity of the test were calculated using a receiver operating characteristic and Bayesian estimation. Results: A total of 3,051 serum samples were tested by ELISA and 939 (30.8%) sera were recognised as positive. When Bayesian approach was used, the overall true BFV prevalence was 29.7% (95% CI: 25.9-33.4%). Conclusion: Expressed Gag protein of BFV has been used successfully as an antigen for ELISA. Eventually, this study provides basic information about the epidemiological status of infection with BFV in dairy cattle in Poland, which can be used for further studies on dissemination and transmission of BFV infection.

Data over a period of eleven years was analysed for Infectious Bursal Disease (IBD) virus isolated from chicken samples submit ted to the Regional Veterinary Laboratory at Bukit Tengah, Malaysia (RVLBT) for diagnosis. A total of 247 suspect IBD cases were tested by Virology Section, RVLBT between years of 2006 to 2016. IBD virus has been isolated by using Agar Gel Precipitation Test (AGPT), a bursal homogenate which has been used as an antigen against a known positive antiserum. About 27 cases (11%) from a total of 247 suspect cases in chickens were positive for the presence of IBD. The rate of IBD may be influenced by age of chickens with an increase in the possibility of IBD occurring in chicken older than 3 weeks. Apart from that, both broiler and local chickens are highly susceptible to this disease. Therefore, awareness on the existing IBD cases indicates the importance of strict management procedures, proper management programmes, vaccination and immunisation for chickens in Malaysia.

OBJECTIVE To determine titers of serum antibodies against 3 genotypes of bovine parainfluenza 3 virus (BPI3V) in unvaccinated ungulates in Alabama. ANIMALS 62 cattle, goats, and New World camelids from 5 distinct herds and 21 captured white-tailed deer. PROCEDURES Serum samples were obtained from all animals for determination of anti-BPI3V antibody titers, which were measured by virus neutralization assays that used indicator (reference) viruses from each of the 3 BPI3V genotypes (BPI3V-A, BPI3V-B, and BPI3V-C). The reference strains were recent clinical isolates from US cattle. Each sample was assayed in triplicate for each genotype. Animals with a mean antibody titer = 2 for a particular genotype were considered seronegative for that genotype. RESULTS Animals seropositive for antibodies against BPI3V were identified in 2 of 3 groups of cattle and the group of New World camelids. The geometric mean antibody titer against BPI3V-B was significantly greater than that for BPI3V-A and BPI3V-C in all 3 groups. All goats, captive white-tailed deer, and cattle in the third cattle group were seronegative for all 3 genotypes of the virus. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that BPI3V-A may no longer be the predominant genotype circulating among ungulates in Alabama. This may be clinically relevant because BPI3V is frequently involved in the pathogenesis of bovine respiratory disease complex, current vaccines contain antigens against BPI3V-A only, and the extent of cross-protection among antibodies against the various BPI3V genotypes is unknown.

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII–deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in factor VII–deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

OBJECTIVE To assess the immunogenicity of thermostable live-attenuated rabies virus (RABV) preserved by vaporization (PBV) and delivered to the duodenal mucosa of a wildlife species targeted for an oral vaccination program. ANIMALS 8 gray foxes (Urocyon cinereoargenteus). PROCEDURES Endoscopy was used to place RABV PBV (n = 3 foxes), alginate-encapsulated RABV PBV (3 foxes), or nonpreserved RABV (2 foxes) vaccine into the duodenum of foxes. Blood samples were collected weekly to monitor the immune response. Saliva samples were collected weekly and tested for virus shedding by use of a conventional reverse-transcriptase PCR assay. Foxes were euthanized 28 days after vaccine administration, and relevant tissues were collected and tested for presence of RABV. RESULTS 2 of 3 foxes that received RABV PBV and 1 of 2 foxes that received nonpreserved RABV seroconverted by day 28. None of the 3 foxes receiving alginate-encapsulated RABV PBV seroconverted. No RABV RNA was detected in saliva at any of the time points, and RABV antigen or RNA was not detected in any of the tissues obtained on day 28. None of the foxes displayed any clinical signs of rabies. CONCLUSIONS AND CLINICAL RELEVANCE Results for this study indicated that a live-attenuated RABV vaccine delivered to the duodenal mucosa can induce an immune response in gray foxes. A safe, potent, thermostable RABV vaccine that could be delivered orally to wildlife or domestic animals would enhance current rabies control and prevention efforts.

The objective of this study was to assess the efficacy of a trivalent inactivated Haemophilus parasuis serovars 4, 5, and 12 vaccine with polymeric adjuvant gel (GEL) and commercial vaccines against Glässer's disease in piglets. Commercial vaccines containing inactivated H. parasuis serovars 4 and 5 (China), inactivated H. parasuis serovars 1 and 6 (Spain), and inactivated H. parasuis serovar 5 (USA) were also evaluated. Our results demonstrated that the trivalent inactivated H. parasuis serovars 4, 5, and 12 vaccine with GEL adjuvant can provide better protection against the 3 most common pathogenic serovars circulating in China than other commercial vaccines tested. Our findings also indicated that inactivated H. parasuis serovars 1 and 6 vaccine cross-protects piglets against H. parasuis serovars 4 and 5; inactivated H. parasuis serovar 5 vaccine cross-protects piglets against H. parasuis serovar 4 challenge; but none of the commercial vaccines tested in this study protected piglets against H. parasuis serovar 12. Our results provide a basis for further identification of common protective antigens that can induce cross-protection against heterogeneous serovars.