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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 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.

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.

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.

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.

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.

The results of studies on the incidence of enzootic bovine leukaemia in the Netherlands from 1968 to 1975 inclusive are given. The serological methods used (fluorescent antibody technique, immunoperoxidase test, micro complement fixation test and gel diffusion test) were found to be of great value especially for the early diagnosis of the disease. Antibodies to bovine leukaemia virus are already detected long before the onset of persistent lymphocytosis. The results obtained so far with serological methods confirm earlier haematological findings that enzootic bovine leukaemia is non-existent in the Dutch Friesian-Holstein and Meuse-Rhine-IJssel breeds. The first cases of enzootic bovine leukaemia were observed in import cattle in 1970. The geographic distribution of farms exploiting import cattle in which the disease has been diagnosed is presented on chart 1. Methods used to control enzootic bovine leukaemia are briefly described.