Lymphocyte subset distribution in apparently normal and single intradermal test-positive water buffaloes analyzed by flow cytometry.

Monoclonal antibodies (mAbs) against bovine lymphocyte cell surface antigens namely, MHC Class I, MHC class II (DP, DQ and DR), CD3, CD4, CD8, gamma delta TCR, WC1N1 and WC1N2, were tested for their reactivity on apparently normal buffalo mononuclear cells prepared from spleen, lymph nodes and peripheral blood. All the mAbs cross-reacted with the buffalo mononuclear cells. The mean (+/-SD) CD4:CD8 cell ratio in the peripheral blood of apparently normal buffaloes was 1.08+/-0.049 while in the spleen and lymph nodes it was 0.90+/-0.080 and 1.81+/-0.430, respectively. The lymphocyte subsets in the buffaloes positive for tuberculosis by the single intra dermal (SID) test was found to be altered; the CD4 cells were reduced while the CD8 and gamma delta cells were increased. The mean CD4:CD8 ratio in the SID positive buffaloes was 0.36+/-0.010.

Comparison of hemagglutination inhibition test and ELISA in quantification of antibodies to egg drop syndrome virus.

A single-serum dilution ELISA for egg drop syndrome (EDS) virus-specific antibodies was developed. In testing 425 chicken sera it was found to have a 93.6% sensitivity and 98.7% specificity relative to a hemagglutination inhibition (HI) test. The correlation coefficient for ELISA and HI titers was 0.793. The ELISA was efficacious in quantification of both vaccinal and infection antibodies and could routinely be used for screening large numbers of field sera.

Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay or polymerase chain reaction.

Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS) virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein, presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDS virus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared with the other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum against EDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome. Following experimental infection of oestrogen-treated chickens with EDS virus, cloacal swabs, oviduct, uterus and spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly and after a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by AC-ELISA or PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommended due to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommend that PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged in embryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.

Demonstration of alternative and classical complement pathway activity in colostrum from buffalo (Bubalus bubalis).

Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50 degrees C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg(2+) in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH50 units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81-6.77 CH50/ml.