Genetic diversity of Enterococcus faecium isolated from Bryndza cheese.

One hundred and seventy-six Enterococcus faecium isolates from Slovak dairy product Bryndza were tested for the presence of plasmid DNA. Eighty-two isolates were positive and their plasmid DNA was isolated and digested by EcoRI and HindIII restriction endonucleases. The patterns obtained were compared with those obtained after pulsed-field gel electrophoresis of macrorestriction fragments (PFGE), (GTG)(5)-PCR and ERIC-PCR. All these molecular approaches were applied for the study of genetic variability and determination of strain relatednesses among plasmid-positive isolates of E. faecium. In general, all methods revealed a considerable genetic diversity of E. faecium isolates. Plasmid profiling and ERIC-PCR have offered a higher resolution than PFGE and (GTG)(5)-PCR.

Identification and characterization of enterococci from bryndza cheese.

AIMS: To identify enterococci isolated from sheep milk cheese--bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. METHODS AND RESULTS: Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. CONCLUSIONS: Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.

Decreased expression of L-selectin (CD62L) on lymphocytes in enzootic bovine leukaemia.

Expression of L-selectin was determined by single- and two-colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L-selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV-infected animals comprised lymphocytotic and non-lymphocytotic cows. L-selectin was expressed on 90-98% of granulocytes in all tested animals. The percentage of PBMC expressing L-selectin was lower in cattle with persistent lymphocytosis than in non-lymphocytotic or BLV-free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L-selectin was significantly decreased from 60.2 +/- 1.9% in BLV-free cattle to 43.8 +/- 3.6 and 22.5 +/- 5.7% in non-lymphocytotic and lymphocytotic cattle, respectively. B-lymphocytes stained for L-selectin exhibited about 50% reduction in L-selectin expression in BLV-infected cattle compared with BLV-free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L-selectin-positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV-free and BLV-infected cattle. However, L-selectin expression on T lymphocytes was reduced (about 50%) in BLV-infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L-selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.

Antihemolytic effect of Rooibos tea (Aspalathus linearis) on red blood cells of Japanese quails.

The antihemolytic activity of Rooibos and black tea on Japanese quail erythrocytes was studied. Peroxide and hypotonic hemolysis of the red blood cells of quails, either fed with Rooibos tea supplemented food or fed without tea, was performed. Long-term consumption of Rooibos tea did not change the erythrocyte fragility to either peroxide or hypotonia induced hemolysis. However, Rooibos and black teas decreased peroxide induced hemolysis of erythrocytes incubated with each of them, but not hemolysis induced by hypotonic NaCl solution. Stronger inhibition of hemolysis has been obtained when a boiled water extract of Rooibos tea was used for the inhibition. The degree of inhibition was comparable with the effect of ascorbic acid.

Ruminant cluster WC13.

Five monoclonal antibodies (mAbs) belonged to preliminary cluster 27; however, only two mAbs, Buf13 (3W-506) and Co-3D1D4 (3W-202), were shown to detect the same surface antigen and belong to WC13. The other three mAbs, IVA120 (3W-323), IVA197 (3W-533) and IVA198 (3W-290), detected a 45 kDa molecule that could be fibrinogen (Mateo A., Perez de la Lastra, J., Moreno, A., Dusinsky, R., Bilka, F., Simon, M., Horovska, L., Naessens, J. and Llanes, D., 1996. Biochemical characterization of antigens detected with anti-platelet monoclonal antibodies. Vet. Immunol. Immunopathol., 52: 363-370; Perez de la Lastra, J.M., Mateo, Dusinsky, R., Bilka, Simon, M., Horovska, L. and Llanes, D., 1996. Two monoclonal antibodies from the platelet panel recognize sheep plasma fibronigen. Vet. Immunol. Immunopathol., 52:).

Biochemical characterization of antigens detected with anti-platelet monoclonal antibodies.

A panel of 18 monoclonal antibodies (mAbs) defined by the third workshop as specific for platelets, clustered in three preliminary groups: PC7, PC13 and PC27. These mAbs were further analysed by immunoprecipitation using extracts of iodinated and biotinylated peripheral blood mononucleated cells (PBMC) and platelets. We could confirm the existence of mAbs with specificities to WC9 (in PC7) and CD41/61 (in PC13). Two mAbs formed a new cluster, WC13, which may be homologous to human CD31 (in PC27). The influence of EDTA and thrombin on the expression of the different antigens on the platelet membrane was assessed by flow cytometry (FCM) analysis, as well as cross-reactivity with platelets from different species.

Two monoclonal antibodies from the platelet panel recognize sheep plasma fibrinogen.

Among the monoclonal antibodies (mAbs) submitted to the third Workshop, two mAbs, IVA120 (3W-323) and IVA198 (3W-290), could be identified to recognize the sheep fibrinogen molecule. The apparent molecular weight of the immunoprecipitated 48-60 kDa cell surface protein under reducing conditions suggested this antigen could be the fibrinogen molecule. ELISA and immunoblotting assays, performed with commercially available sheep plasma fibrinogen, confirmed that these two mAbs recognize two different epitopes present on the sheep fibrinogen molecule.

Immunohistochemical reactivity of anti-platelet monoclonal antibodies.

Ten mAbs of preliminary clusters PC13 and PC27 with specificity for bovine platelets were studied by immunohistochemistry. Cryostat sections of bovine lymph node, spleen, thymus, small intestine, liver, kidney and smears of bone marrow cells were used. Five mAbs (CAPP2, IVA30, IVA125, IL-A164 and IL-A166) assigned to cluster PC13 (CD41/CD61) stained platelets and non-lymphocytic cells of various tissues. Our data confirm the presence of two specificities in PC27: three mAbs (IVA120, IVA197 and IVA198) specific for fibrinogen strongly reacted with the endothelial and reticular tissues whereas the other two mAbs Co-3D1D4 and Buf13 (WC13) were negative.

[Association between BoLA antigens and bovine mastitis]. / Asociácia medzi BoLA antigénmi a bovinnou mastitídou.

The association between BoLA class I antigens and mastitis was studied in Bohemian Pied breed (n = 17) and its crosses--Bohemian Pied x Red Pied Holstein (n = 161), Bohemian Pied x Red Pied Holstein x Ayrshire (37). The diagnostics of mastitis was followed in the course of two years and two diagnostic parameters were included: 1. a modified California Mastitis Test (CMT) was performed once a month; 2. a bacteriological infection was examined once quarterly using biochemical and serological methods. BoLA class I antigens were determined by specific antisera in the standard microlymphocytotoxicity test. During testing the majority of cows had at least one positive reaction of CMT test. The bacterial findings were detected in 31.63% of animals. The antigen A16 was found to be significantly associated with susceptibility to mastitis in both diagnostic tests. Animals A16 positive showed the highest CMT values and repeated bacterial infections (Fig. 1). The high values observed in A2 positive animals were not significant due to the very low frequency of this allele in the population under study. There was a slight increase of CMT values and the infection frequency in animals with higher parity number (Fig. 2). However, the order of lactations did not influence the relationship of BoLA A16 and mastitis. This association was not significantly affected by the breed. The increased bacterial infection observed in the Bohemian Pied breed is likely due to relatively high incidence of A16 allele rather than to breed differences (Fig. 3).

A polymorphic monoclonal anti-BoLA class I antibody.

Cytotoxic monoclonal antibody IVA 44 was generated after the intraperitoneal immunization with peripheral blood mononuclear (PBM) cells and the boost by the intrasplenic inoculation of skin graft. The detected membrane antigen isolated by immunoprecipitation appears to be composed of two subunits characteristic for the MHC class I molecules. The antibody IVA 44 exhibited a different reactivity: it recognized the BoLA A14 (A8) specificity in animals typed in the Fifth BoLA workshop, while it reacted with all A8 positive animals including subtypes A14 and A15 in Czech and Slovak cattle. It is concluded that mAb IVA 44 might detect the broad subtype of A8 covering A14 and certain A15 split(s). The diverse A15 reactivity of this mAb in the workshop and our population could be explained by the different occurrence of A15 splits in both populations.

[Use of monoclonal antibodies against human HLA II antigens for the detection of bovine B lymphocytes and macrophages]. / Vyuzitie monoklonálnych protilátok proti l'udským HLA II antigénom na detekciu bovinných B lymfocytov a makrofágov.

The crossreactivity of mouse monoclonal antibodies (MoAbs) (Tab. I) prepared against human HLA-DR and HLA-DP antigens was studied in various bovine cells: lymphocytes from lymph nodes and peripheral blood, adherent (B) and nonadherent (T) lymphocytes, monocytes, granulocytes and platelets. In the immunofluorescence test, MoAbs Bra13, Bra14, Bra20, Bra22, Bra30, Bra70, HL-38 reacted with bovine B lymphocytes and monocytes, but not with other tested cells (Tab. III, IV). These antibodies, except Bra22, were positive with B lymphocytes in the complement dependent cytotoxic test (Tab. II). The similarity of the bovine antigens and HLA-DR antigens determined by used MoAbs was also proved by immunoblotting. Monoclonal antibodies Bra38 and BraFB6 did not react with the bovine cells and separated antigens. The epitope (HLA-DR) recognized by the antibody Bra38 is probably absent in cattle. The presence of HLA-DP analogue determined by the antibody BraFB6 has not been confirmed. The crossreactive MoAbs could be used for the detection of B lymphocytes and macrophages in veterinary immunology.

Identification of medullary thymocytes by means of a monoclonal antibody.

A monoclonal antibody (IVA-1) reacting with bovine lymphoid cells differentiates the medullary and cortical zone of the thymus in cattle, pigs and sheep. The morphological picture indicates that the identified antigen most likely points out the route of maturation of the thymocytes. The antibody could be of use in studies of the development of thymocytes.

[Preparation of monoclonal antibodies against cell surface antigens in cattle]. / Príprava monoklonálnych protilátok proti povrchovým antigénom buniek hovädzieho dobytka.

The BALB/c mice were immunized three times with bovine lymphocytes and thrombocytes in vivo. Besides, dissociated spleens of the mice were immunized with bovine lymphocytes in vitro. Hybridomas producing monoclonal antibodies were formed as a result of the fusion of the spleen cells with the murine myeloma cells. On the whole, four fusions were performed after immunization in vivo and three fusions after immunization in vitro, and 70 stable hybridoma clones were obtained. Five monoclonal antibodies exhibited an identical specific reaction only with bovine thrombocytes, two antibodies reacted only with a certain limited population of bovine spleen cells. The remaining monoclonal antibodies exhibited no tissue specificity and were bound to the lymphocytes of peripheral blood, lymph nodes, thymus, and spleen, to thrombocytes, liver cells, and spermatozoa, but never to erythrocytes. As for the amount of the obtained hybridomas and specific antibodies, no significant difference was observed in the effectiveness of in vivo and in vitro immunization.

[Cytotoxic antibodies against lymphocytic antigens of cattle]. / Cytotoxické protilátky proti lymfocytárnym antigénom hovädzieho dobytka.

Typing reagents for the identification of bovine lymphocyte antigens were prepared. Sera obtained from cows killed in slaughterhouse were a good source of cytotoxic antibodies. Out of the 300 sera tested, 98 were cytotoxically active. The selected sera with a low reaction frequency were tested on a panel of 132 non-related animals. On the basis of the correlations between the sera, thirty of them could be included in four clusters. The sera within each cluster were closely associated; hence it can be assumed that a distinct specificity is represented by each of the clusters. It is suggested by correlation analysis and by the distribution of antigens in the population under study that the determined antigens fall within the same system and are genetically controlled by one or several closely associated loci.