Studies on bovine leukosis. VII. Further experience with an ELISA for the detection of antibodies to bovine leukosis virus.

An enzyme linked immunosorbent assay (ELISA) and the agar gel immunodiffusion test with bovine leukosis virus glycoprotein as antigen (AGIDT-BLV gp) were further used to test 633 bovine sera for antibodies to BLV. Both tests detected the same number of sera positive (149) or negative (464) for antibodies. Nine sera were negative in the ELISA but found to be weakly positive (2 sera) or bending the control line (7) in the AGDT-BLV gp. On the other hand 11 sera were scored negative in the AGIDT-BLV gp but were weakly positive (9 sera), positive (1), and strongly positive (1) in the ELISA. Both tests are used routinely in this Institute as they complement each other, specially if sera with low antibody titers are under investigation. It is concluded that ELISA can fully replace radioimmunoassays in the serodiagnosis of enzootic bovine leukosis.

Test protocol of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukosis virus.

In this communication the test procedure is described for an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukosis virus (BLV). Test sera are incubated in polystyrene microtiter plates sensitized with a partly-purified preparation of BLV. Bovine antibodies are detected with anti-species immunoglobulin conjugated to the enzyme horseradish peroxidase, followed by the addition of the enzyme substrate.

Horse anti-bovine lymphocyte serum. Its effect in calves.

Anti-bovine lymphocyte serum (ABLS) had been prepared in horses with calf thymocytes as antigen and its effects in calves following parenteral administration were studied. The optimal dose was found to be one ml/kg body weight. The ABLS suppressed both the T and B cell functions. The former was indicated by the disturbed response to sheep erythrocytes infections, by the decreased number of spontaneous E rosette forming lymphocytes, the prolonged survival of skin allografts and the significant inhibition of the delayed hypersensibility skin reaction (tuberculination) following administration of Mycobacterium microti. The latter was based on the disturbed response to a subcutaneous dose of tetanus toxoid. The reaction of lymphocytes of ABLS treated calves to phytohemagglutinin and poke week mitogen was also inhibited. The disturbed reactions of the T and B cells might be among others based on the strong reduction of lymphocytes in the blood circulation by ABLS (up to 10-20%).

[Transmission and immune response in bovine enzootic leukosis (author's transl)]. / Transmissie en immunologische respons bij enzoötische bovine leukose.

The causative agent of enzootic bovine leukosis (EBL) is an oncogenic RNA virus named bonvine leukosis virus (BLV). At present, instead of bovine leukemia, the name bovine leukosis is preferentially used to avoid an erroneous association with leucaemia in man. In all European Community Countries the serological diagnosis of EBL has gradually replaced hematology. A number of these serological techniques are available to-date. In the Central Veterinary Institute in Rotterdam the agar gel immunodiffusion technique, the FAT and the ELISA are used as diagnostical tools. Based on own experiments it is provisionally concluded that BLV shedding via faeces and urine does not occur. Saliva has been found infective in three out of fourteen cases (21 per cent). There is no evidence of transmission of BLV in the sperm. Prostate fluid and sperm from seven experimentally infected bulls did not contain neither BLV antigen nor antibodies to BLV. Five calves born from five cows which had been naturally served by the mentioned bulls did not show sero conversion after an observation period of one year. The authors recommend to use in AI only sperm form bulls which are negative for antibodies to BVL. Preferentially other cattle at the same farm-enterprise should be serologically tested with negative results within three months before shipment of the sperm. The humoral and cellular immunological status of leucotic cattle are examined by the application of pig erythrocytes, tetanus toxoid, Bordetella- and Aujeszky vaccine. The seroloical reactions of leucotic cattle did not differ significantly from those of the "normal' controls. By contrast the cutaneous tuberculine reaction following administration of Mycobacterium microtii was significantly more obvious in the leucotic animals as compared to the controls.

Horse anti-bovine lymphocyte serum. Its effect in calves.

Summary Anti-bovine lymphocyte serum (ABLS) had been prepared in horses with calf thymocytes as antigen and its effects in calves following parenteral administration were studied. The optimal dose was found to be one ml/kg body weight. The A BLS suppressed both the T and B cell functions. The former was indicated by the disturbed response to sheep erythrocytes injections, by the decreased number of spontaneous E rosette forming lymphocytes, the prolonged survival of skin allografts and the significant inhibition of the delayed hypersensibility skin reaction (tuberculination) following administration of Mycobacterium microti. The latter was based on the disturbed response to a subcutaneous dose of tetanus toxoid. The reaction of lymphocytes of ABLS treated calves to phytohemagglutinin and poke weed mitogen was also inhibited. The disturbed reactions of the T and B cells might be among others based on the strong reduction of lymphocytes in the blood circulation by ABLS (up to 10-20%). Suggestions with regard to further applications and studies with ABLS were given.

Studies on bovine leucosis V. A comparative study on the practical value of the agar gel immunodiffusion test, the indirect fluorescent antibody technique and the micro complement fixation test for the detection of antibodies to bovine leucosis virus.

The practical value of the indirect fluorescent antibody technique (I FAT), the micro complement fixation test (M CFT) and the agar gel immunodiffusion test (AGIDT) for the detection of antibodies to bovine leucosis virus (BLV) was investigated. For this purpose 1495 serum samples were examined. There was a remarkably good agreement between the three tests in the demonstration of antibodies to BLV especially if sera with a sufficient high concentration of antibodies ("late serum") were under investigation. These positive sera were derived only from farms which exploited one or more imported animals. A disagreement of results of the three tests was observed in 38 (2.5%) cases. This was due to: (1) difficulties in reading of the test; (2) presumably by the demonstration of different classes of antibody and (3) the fact that in the AGIDT sera could be used undiluted. This discrepancy was especially evident with sera with a low concentration of antibodies ("early or incubation sera"). A drawback for the M CFT is the anticomplementary activity found in 17% of the bovine serum samples.

Studies on bovine leukosis. VI. Enzyme linked immunosorbent assay for the detection of antibodies to bovine leukosis virus.

The results of examination of 2 784 sera in the AGIDT-gp were compared with those of the ELISA. A discrepancy was seen in 0.5% of the cases. It was concluded that the ELISA was another useful serological method added to the list of serological techniques presently available for the detection of antibodies to BLV. The advantages of the technique are discussed.

Virus-like particles in bovine sera for tissue culture.

Virus-like particles were found in nine different bovine sera for tissue culture from commercial suppliers. These particles were spherical with an overall diam. between 70 and 95 nm. After negative staining, surface projections of about 11 to 12 nm were clearly seen. One of the nine sera was positive in an Ouchterlony test with antisera against bovine viral diarrhoea virus.