Introduction: Farm mink (Neovison vison) can be naturally exposed to T. canis and T. leonina pathogens on the farm. If mink were hosts, it would imply some veterinary public health as well as animal welfare issues. For this reason, the aim of the study was to determine whether mink might be definitive or paratenic hosts of these parasites. Material and Methods: Four groups of mink were infected with both parasite species using larvated eggs or feed containing mouse tissue previously infected with the parasites. Following inoculation, the infections were monitored in vivo by faecal examination for 14 weeks p.i., and then western blotting and ELISA were performed. Results: Coprology did not reveal any canine roundworm eggs, neither were nematodes found in mink intestines during post mortem examination. The specific IgG antibodies recognising excretory/secretory (ES) antigens of both parasite species were identified in mink sera. Single T. leonina tissue larvae were found in digested organs. Conclusions: Our results confirm that farm mink may contribute both T. canis and T. leonina infections. It was proved that farm mink were not their definitive hosts, and therefore mink faeces need not be considered a source of canine roundworm eggs in any soil it fertilises. Nonetheless, as farm mink may be a paratenic host for both parasite species, this may have some impact on the health and welfare of infected animals.

Introduction: Coxiella (C.) burnetii, the aetiological agent of Q fever, is able to modulate the macrophage/T-lymphocyte axis in an infected organism and impair synthesis of monokines and lymphokines. Material and Methods: The purpose of this research was to determine the levels of the cytokines that play a key role in the response to C. burnetii antigens (IL-1β, IL-2, IL-6, IL-10, IFN-γ and TNF-α) in the serum of animals originating from an infected herd prior to vaccination (day 0) and at 1, 7, and 21 days afterwards. Results: The vaccination of animals did not affect the production of IL-6, IL-1β, or IL-2. The serum levels of these cytokines were too low to measure in most of the samples. The initial levels of TNFα, IFNγ, and IL-10 were higher in seropositive than in seronegative animals, although significant differences between seropositive shedders and seropositive nonshedders appeared only in the levels of IFNγ and IL-10. Additionally, the course of the post-vaccination response concerning these two cytokines was different among seronegative nonshedders, seropositive nonshedders, and seropositive shedders. Conclusion: It seems that analysis of the IFNγ and IL-10 concentrations in animal blood serum may have some practical value in an assessment of the health status of seropositive animals and post-vaccination response.

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: In the present study apoptosis was investigated in the cornu ammonis and cerebellum of 10 dogs naturally infected with rabies virus. Diagnosis of rabies was based on the results of fluorescent antibody staining and experimental inoculation.Material and Methods: The paraffin tissue sections were stained with haematoxylin and eosin, avidin-biotin complex peroxidase (ABC-P), and terminal deoxynucleotidyl transferase biotin-dUTP nick end-labelling (TUNEL) methods.Results: Histopathological examination revealed encephalomyelitis of varying severity and the presence of Negri bodies. Dense rabies antigens were determined in the motor neurons with ABC-P method. On the other hand, Bcl-2 protein and Bax protein gave positive reaction in seven and five cases, respectively. TUNEL staining demonstrated very marked apoptotic changes in the nuclei of neurons localised deep in the substantia alba of the cerebellum. Similar changes were also determined in perivascular mononuclear cells and glia cells within the substantia alba. No apoptopic changes were found in the motor neurons of the cornu ammonis.Conclusion: The absence of apoptotic changes in the neurons was considered to be the consequence of the necrotic changes that developed in these neurons.

Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs, leading to economic losses around the world. Attenuated live vaccines with CSFV antigens have played an important role in the prevention and control of the disease. Porcine kidney 15 (PK15) cells have been widely used for the propagation of CSFV, but this cell line is not efficient or homogeneously susceptible to viral infection. To achieve a homogeneous PK15 cell line which enabled high titre replication of CSFV, we used the limiting dilution cell cloning method. We developed two cell clones, PK15-1A6 and PK15-3B1, which respectively have high- and low-permissive phenotypes to CSFV infection. The PK15-1A6, PK15-3B1, and PK15 parent cells showed different characteristics in cell proliferation rate, susceptibility to CSFV infection, and CSFV production. The mean virus titres per millilitre reflected by TCID₅₀ values in PK15-1A6, PK15-3B1, and PK15 parent cells were 106.85, 103.63, and 104.74, respectively. The PK15-1A6 cell clone is more permissive to CSFV infection than the PK15 parent cells. The screened high-permissive cells will be useful for CSFV propagation and vaccine development in vitro, and facilitate research on the pathogenicity of CSFV.

Riemerella anatipestifer (RA) infections can lead to high mortality in ducklings. Inactivated vaccines against RA are commercially available, but they fail to provide cross-protection against various serotypes. We have previously demonstrated that a subunit vaccine containing recombinant outer membrane protein A (rOmpA) antigen of serotype 2 formulated with CpG oligodeoxynucleotides (ODN) as the adjuvant was able to stimulate both humoral and cellular immunities. In the present study, thirty healthy 7-day-old Pekin ducks were randomly assigned to three equal treatment groups: rOmpA-vaccinated, rOmpA + CpG-vaccinated, and control. Vaccine was injected intramuscularly and a booster dose of the same vaccine was given two weeks after primary immunisation. The long-term antibody response and cross-serotype reaction of this vaccine were evaluated in ducks. Compared to ducks immunised with rOmpA alone, ducks immunised with rOmpA + CpG ODN had significantly (p < 0.05) increased serum antibody titre from two weeks until nine months after primary immunisation. In addition, expression of cytokines including interferon (IFN)-α, IFN-γ, interleukin (IL)-6, and IL-12 was significantly (p < 0.05) enhanced in PBMC of ducks immunised with rOmpA + CpG ODN two weeks after primary immunisation. Antibodies from ducks immunised with the rOmpA + CpG ODN vaccine could also detect RA serotypes 1 and 6 in Western blot analysis. Combination of rOmpA and CpG ODN could be a feasible strategy for developing a subunit RA vaccine with long term and broader-ranging protection.

Dengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines. Blood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors. The gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells. Recombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.

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.

Introduction: Bovine viral diarrhoea (BVD), caused by the bovine viral diarrhoea virus (BVDV), is one of the most important diseases of cattle worldwide. The purpose of the study was to determine the BVDV infection status in a dairy herd vaccinated against BVD. Before vaccination started in 2008, there had been no prior identification or the removal of the possible source of infection (persistently infected animals). It was expected that vaccination itself would enable the elimination of viral shedders on a long term basis. Material and Methods: Serological screening for antibodies against BVDV with determination for antibodies titres, BVDV antigen, and the presence of the viral genome with phylogenetic analysis of positive samples in the herd were performed, despite the lack of any clinical problems indicating possible presence of BVDV infection. Results: 19 individuals persistently infected with BVDV were identified among calves and heifers but not in adult cattle. All virus shedders were antibody negative and the genotype of isolated virus was BVDV-1b, indicating a single source of infection. The vaccine used in the herd was composed of BVDV-1a strain. In each of the tested cowsheds, antibody titres against BVDV-1b were higher than against BVDV-1a (median values). Conclusion: Despite a long-lasting vaccination programme and relatively high sequence homology of vaccinal and field strains of BVDV (83.6%), it was not possible to avoid transplacental infections of foetuses and the birth of persistently infected calves from vaccinated heifers although the protection against clinical disease was accomplished.