An international comparison of different laboratory tests for the diagnosis of bovine leukosis: suggestions for international standardization

A collaborative study was conducted to compare the detection limit of different laboratory tests for antibodies against bovine leukemia virus (BLV). Serum and milk samples were tested in agar gel immunodiffusion (AGID), different modifications of indirect ELISA, blocking ELISA and ELISA procedures using monoclonal antibodies to BLV gp51 or BLV p24. The detection limit of reference serum E4 diluted 2-fold in negative serum gave a median value of 1:16 in AGID, indirect ELISA, and monoclonal ELISA p24, 1:128 in monoclonal ELISA gp51, and 1:1024 in blocking ELISA. The detection limit of a 4% immunoglobulin preparation of E4 diluted in negative milk showed median values of 1:800 in indirect ELISA, 1:1000 in monoclonal ELISA, and 1:2400 in blocking ELISA. None of the ELISA procedures could detect all the positive individual milk samples diluted 1:50. The AGID test is the official reference test for detection of antibodies against BLV. Reference serum E4 diluted 1:10 in negative serum must be scored positive in the AGID test. It is suggested that an international reference serum standard be established rather than an official recommendation of a particular ELISA test.     

Demonstration of antibodies against bovine leukemia virus (BLV) by blocking ELISA using bovine polyclonal anti-BLV immunoglobulin.

A blocking enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukemia virus (BLV) is described. The test is based on the biotin-streptavidin system using unlabelled polyclonal bovine IgG against BLV as catching antibody and biotinylated bovine anti-BLV IgG as detecting antibody. The sensitivity was found to be 50-100 times higher than the agar gel immunodiffusion test, with a specificity of practically 100%. The blocking ELISA proved to be suitable for detection of antibodies against BLV in serum and milk. In 34 paired milk/serum samples, the average ratio of BLV antibody titres was 1:26. So far, more than 700,000 sera have been screened by blocking ELISA for BLV antibodies in the course of the Danish surveillance programme for BLV infection.     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.     

Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.     

Effects of bovine leukemia virus infection on production and reproduction in dairy cattle.

The purpose of this study was to determine the effects of bovine leukemia virus (BLV) infection on production, reproduction and longevity in dairy cattle. The study population was a commercial Holstein dairy herd of approximately 400 milking cows. Cattle were tested for antibodies to BLV at least annually for three years and when culled. Four groups of culled cows were compared: seronegative cows (n = 79), seropositive cows without lymphocytosis (n = 176), seropositive cows with lymphocytosis (> or = 9,000 lymphocytes/microliter) (n = 74), and seropositive cows with lymphosarcoma (n = 29). Seropositive groups of cows were bred more times and had longer calving intervals than seronegative cows. The seropositive groups had greater 305-day ME (mature equivalent) FCM (3.5% fat-corrected milk) per lactation and were older when culled than seronegative cows. However, the percent fat per lactation was greater in seronegative cows. In the last complete lactation, differences in 305-day ME FCM, days open and cull age between groups were reduced and none were significant (p > 0.05). In the cull lactation, only cows with lymphocytosis had reduced milk production relative to seronegative cows, although this difference was not significant. After adjustment for initial production and reproductive values, only seropositive nonlymphocytotic cows were culled at a significantly older age than seronegative cattle. Lymphocytotic cows were culled four months younger on average than nonlymphocytotic seropositive cows. Hence, BLV infected cows had greater milk production on average than uninfected cows. Adverse effects of BLV infection were primarily limited to lymphocytotic cows which were culled earlier and had reduced milk production in the cull lactation.     

Effects of subclinical bovine leukemia virus infection on some production parameters in a dairy farm in southern Turkey

Some production parameters of seropositive cows (age, first calving age, 305 day mature equivalent last milk yield production, lifetime mature equivalent milk yield production, lifetime total milk production, lifetime total milking period, lifetime monthly milk production, lifetime daily milk production, lifetime total days of milking, number of inseminations per pregnancy (for last pregnancy), number of calves and calving interval (for last pregnancy)) were analysed in the current study. The study population was clinically healthy Holstein cows from a commercial dairy herd in southern Turkey. Of 109 animals, 65 cows were seropositive by ELISA and the prevalence of bovine leukemia virus (BLV) infection was 59.6%. The prevalence of seropositive cows in 2nd (62.8%), 3rd (64.7%), 4th (61.5%), and 5th (66.6 %) lactations was slightly higher than that of cows in 1st (52.6%) lactations. No statistical differences were observed between BLV seronegative and seropositive cows for production and reproduction parameters analysed in this study (P > 0.05).     

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.     

Enzyme-linked immunosorbent assay for the diagnosis of bovine leukosis: comparison with the agar gel immunodiffusion test approved by the Canadian Food Inspection Agency.

Four commercially available bovine leukemia virus (BLV)-ELISA kits from Europe or the United States were compared to the agar gel immunodiffusion (AGID) test officially approved by the Canadian Food Inspection Agency (CFIA). A total of 1200 cattle serum samples were used. Three ELISA kits based on the envelope glycoprotein (gp51) gave an excellent correlation with the AGID test. The kappa values were 0.998, 0.984, and 0.986 for the ELISA kits #1, #2, and #3, respectively. The ELISA kit based on the p24 core protein was found to be less sensitive than the officially approved AGID test and detected 5.13% of false negatives. Forty BLV AGID strongly positive serum samples were diluted. Based on the dilution experiment, the gp51 ELISA kits were found to be more sensitive than the AGID test kits. They were capable of detecting antibodies in samples diluted up to 1/5000 (kit #1), 1/20 800 (kit #2) and 1/4000 (kit #3), whereas the AGID kit was only capable of detecting antibodies in samples diluted up to 1/100. Based on these observations, the gp51 BLV-ELISA was recognized as an official test method for the serodiagnosis of bovine leukosis in Canada.     

Validation of a bulk tank milk antibody ELISA to detect dairy herds likely infected with bovine viral diarrhoea virus in New Zealand

AIMS: To assess the sensitivity and specificity of a bulk tank milk (BTM) antibody enzyme-linked immunosorbent assay (ELISA) to detect likely infection of a dairy herd with bovine vi- ral diarrhoea virus (BVDV). The ELISA was subsequently used to estimate the prevalence of likely infected herds in parts of the North Island of New Zealand.

METHODS: BTM samples from 724 randomly selected dairy herds in the Waikato, Bay of Plenty and Northland regions of New Zealand were tested for BVDV antibodies. From this group, 20 herds were again randomly selected from each of the quartiles of the ELISA percentage inhibition (%INH) result. From each participant herd, serum from 15 randomly selected calves aged 6–18 months and 15 cows was collected and tested using an indirect blocking ELISA for BVDV antibodies.

RESULTS: Among serum results from calves from 50 herds available for analysis, 34 (68%) herds were classified as likely non-infected (0-3 seropositive among 15 calves) and 16 (32%) as likely infected (5–15 seropositive among 15 calves). Receiver- operator characteristic (ROC) analysis identified an optimal cut-off for BTM of 80%INH associated with 81% sensitivity and 91% specificity for likely herd infection. The prevalence of BVDV antibodies in cows within herds and %INH for BVDV in bulk milk were positively correlated (p<0.01). The association between bulk milk %INH and the prevalence of BVDV antibodies in calves was stronger than the same association in cows. Based on the threshold of 80%INH, the 95% confidence interval (CI) for prevalence of likely infection in the 724 herds in the Waikato, Bay of Plenty and Northland regions of New Zealand was 12–17%. Vaccination against BVDV was not significantly associated with the likely infection status of the herd based on prevalence of BVDV antibodies among calves.

CONCLUSION: An ELISA test result for BVDV antibodies in BTM ≥80%INH can be used as a threshold to indicate the presence of likely infection with BVDV in dairy herds in New Zealand, with 81% sensitivity and 91% specificity.     

Enzyme immunoassay using mouse monoclonal anti-bovine antibodies for the detection of Brucella abortus antibodies in cow milk

Individual milk samples and artificially constructed tank milk samples from cows with naturally occurring brucellosis were examined by the enzyme-linked immunosorbent assay (ELISA) using sonicated B. abortus S-99 antigen, and mouse monoclonal anti-bovine IgM, IgA, and IgG1 conjugates. ELISA results were compared with the results of the milk ring test using either 1 ml milk (MRT-1) or 8 ml milk (MRT-8). The ELISA using mouse monoclonal anti-bovine IgG1 conjugate was sensitive and specific. In testing individual milk samples and constructed tank milk samples containing milk with low titers in the MRT-1 the ELISA was superior to the MRT-1, and MRT-8. In testing other milk samples, the ELISA was as sensitive or slightly less sensitive than the MRT-8. From a total of 5,910 milk samples collected from cows free from brucellosis, only 24 (0.4%) samples tested positive in the ELISA. All 500 tank milk samples collected from farms negative for brucellosis tested negative in the ELISA. We concluded that the ELISA is a good substitute for the MRT-1 to detect antibodies against Brucella in milk from individual cows. When tank milk is tested for antibodies against Brucella, however, both the MRT-8 and the ELISA should be used.     

Validation of a bulk tank milk antibody ELISA to detect dairy herds likely infected with bovine viral diarrhoea virus in New Zealand

AIMS: To assess the sensitivity and specificity of a bulk tank milk (BTM) antibody enzyme-linked immunosorbent assay (ELISA) to detect likely infection of a dairy herd with bovine viral diarrhoea virus (BVDV). The ELISA was subsequently used to estimate the prevalence of likely infected herds in parts of the North Island of New Zealand. METHODS: BTM samples from 724 randomly selected dairy herds in the Waikato, Bay of Plenty and Northland regions of New Zealand were tested for BVDV antibodies. From this group, 20 herds were again randomly selected from each of the quartiles of the ELISA percentage inhibition (%INH) result. From each participant herd, serum from 15 randomly selected calves aged 6-18 months and 15 cows was collected and tested using an indirect blocking ELISA for BVDV antibodies. RESULTS: Among serum results from calves from 50 herds available for analysis, 34 (68%) herds were classified as likely non-infected (0-3 seropositive among 15 calves) and 16 (32%) as likely infected (5-15 seropositive among 15 calves). Receiver-operator characteristic (ROC) analysis identified an optimal cut-off for BTM of 80%INH associated with 81% sensitivity and 91% specificity for likely herd infection. The prevalence of BVDV antibodies in cows within herds and %INH for BVDV in bulk milk were positively correlated (p<0.01). The association between bulk milk %INH and the prevalence of BVDV antibodies in calves was stronger than the same association in cows. Based on the threshold of 80%INH, the 95% confidence interval (CI) for prevalence of likely infection in the 724 herds in the Waikato, Bay of Plenty and Northland regions of New Zealand was 12-17%. Vaccination against BVDV was not significantly associated with the likely infection status of the herd based on prevalence of BVDV antibodies among calves. CONCLUSION: An ELISA test result for BVDV antibodies in BTM >/=80%INH can be used as a threshold to indicate the presence of likely infection with BVDV in dairy herds in New Zealand, with 81% sensitivity and 91% specificity.     

Anti-Neospora caninum antibodies in milk in relation to production losses in dairy cattle

A comprehensive field study was carried out with the following objectives: (a) to assess the usefulness of individual and bulk tank milk analysis for determining Neospora caninum serostatus in individual cows and herds, and (b) to study the associations between N. caninum infection status (based on milk testing), and several productive and reproductive parameters in the animals. Antibodies were detected with a commercially available ELISA test (Bio K 192/5). Analysis of paired serum and milk samples from 1134 lactating cows on 38 farms revealed that 97.6% of the ELISA results were coincident, irrespective of whether serum or milk samples were used. Moreover, multiple linear regression analysis revealed that 86.0% of the variations in ELISA values in milk were due to variations in the serum. The measurement of antibodies in bulk tank milk was a good estimator of the herd level status of N. caninum infection, and enabled detection of infection in 94.7% herds with ≥10.0% seropositive cows and/or in all herds with >4% highly seropositive cows. The odds ratio for abortion in seropositive animals was 9.1 times higher than in seronegative animals. The infection serostatus was also a significant risk factor, as the odds ratio for abortion was even higher (12.0 times) in cows categorized as highly seropositive. ELISA values for the bulk milk from 387 randomly selected herds were negatively associated with average milk production. Moreover, milk production losses mainly occurred on farms categorized as highly positive (i.e. herds with ≥20.0% seropositive cows).     

Protection against bovine leukosis virus infection in sheep with the BL 20 bovine lymphoblastoid cell line

The bovine lymphoblastoid BL 20 cell line derived from a case of sporadic bovine leukosis when inoculated into sheep did not induce an antibody response directed against bovine leukosis virus (BLV) structural proteins. Sheep were inoculated twice with the BL 20 cell line and then challenged with BLV infected lymphocytes. Three out of four sheep challenged four weeks after BL 20 inoculation did not develop BLV antibodies. Of the 12 sheep challenged later, three sheep did not develop BLV antibodies. BLV was isolated from all the seropositive animals and from none of the seronegative animals.     

Experimental and natural infection with the enzootic leukosis virus of cattle

A trial was performed with heifers at the age of six to seven months. The animals were experimentally infected with the lymphocytes of a virus-productive donor. Infection was produced in all the nine cases, as demonstrated by means of the positive syncytial test. As indicated by the results of the trial, the antibodies to the enzootic bovine leucosis virus (BLV) were produced soon after experimental infection. A high sensitivity of the serum-neutralization test and the ELISA method was demonstrated in this connection: by these methods, the antibodies were identified already two to three weeks after experimental infection whereas by the immunodiffusion test they could be detected only after five weeks. Twenty-four animals were exposed to natural contact infection. Within 270 days of the trial, the disease after contact was recorded only in one heifer out of the four that were in close contact with the experimentally infected animals. In this case, as compared with experimental infection, the antibodies were produced much later--after 85 to 93 days. Leucosis was recorded in none of the remaining animals. The reasons why such a favourable result was obtained were the thorough disinfection of the stables after blood collections and the strict observance of the aseptic conditions. The results of experimental infection in three cows were identical with those obtained in young cattle. In the experimentally infected dairy cows, antibodies in milk were determined by the ELISA method. As found, in milk the antibodies to BLV appear two to three weeks later than they do in serum. The ELISA method of BLV antibody detection can be used for the identification of infected animals in herds where enzootic bovine leucosis occurs.     

A serological survey of bovine syncytial virus in Ontario: associations with bovine leukemia and immunodeficiency-like viruses, production records, and management practices.

Of the 920 cows tested, 56.7% showed antiretroviral serological reactivity. Prevalence rates (95% confidence interval) of antiretroviral antibodies among individual dairy cows in Ontario were: BIV 5.5% (4.0-7.0), BLV 25.7% (22.9-28.6), and BSV 39.6% (36.4-42.8). The following percentages of cows showed serological reactivity against the specified retroviruses: BIV 2.3%, BLV 14.0%, BSV 27.5%, BIV and BSV 1.3%, BIV and BLV 0.9%, BLV and BSV 9.9%, BIV and BLV and BSV 0.9%. These rates of sero-positivity are similar to those found in other countries. Serological test results were not adjusted for sensitivity and specificity. The prevalence rates of antibodies to the three retroviruses (BIV, BLV, and BSV) were significantly different, but no associations were observed between specific retroviral serological test results among individual cows. The prevalence rates of BIV and BSV seropositivity were constant across Ontario, whereas, there was a significant trend for the prevalence rate of BLV seropositivity to decrease going from southwestern to eastern Ontario; cows in eastern Ontario had approximately half the prevalence rate of those in southwestern Ontario. Cows that were seropositive for BSV were significantly older than BSV seronegative cows. There was no association between culling rate and BSV serology. Significant negative associations were found with winter or summer housing of calves separate from adults and summer outdoor exercise for dry cows. The use of calf hutches in the summer had a significant positive association with BSV seropositivity. Regression analyses were done to assess the association of retroviral (BIV, BLV, and BSV) seropositivity on calving interval, milk somatic cell count, and milk production. Serological test results for BIV, BLV, and BSV were entered into all models and all models were adjusted for intra-cluster (intraherd) correlation. Herd size and age were found to be important confounding variables. BIV seropositivity was not associated with any changes in production using this approach, however when considered in isolation BIV seropositivity remained associated with decreased milk production. BLV seropositivity was significantly associated with longer calving intervals and higher somatic cell counts in older cows. As well, in older cows, BSV seropositivity was significantly associated with higher milk production.     

Effects of seropositivity for bovine leukemia virus, bovine viral diarrhoea virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum on culling in dairy cattle in four Canadian provinces

The purpose of this research was to determine the effects of seropositivity for exposure to bovine leukemia virus (BLV), bovine viral diarrhoea virus (BVDV), Mycobacterium avium subspecies paratuberculosis (MAP) and Neospora caninum (NC) on overall and reason-specific culling in Canadian dairy cattle. Serum samples from, approximately, 30 randomly selected cows from 134 herds were tested for antibodies against BLV, MAP and NC using commercially available ELISA test kits, while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhoea virus (BVDV). For analyzing the time (in days) to culling of cows after the blood testing, a two-step approach was utilized, non-parametric (Kaplan-Meier survival graphs) visualization and then semi-parametric survival modelling (Cox proportional hazards model), while controlling for confounding variables and adjusting for within herd clustering. For all reasons of culling, MAP-seropositive cows had a 1.38 (1.05-1.81, 95% C.I.) times increased hazard of culling compared to MAP-seronegative cows. Seropositivity for the other pathogens was not associated with an increased risk of overall culling. Among cows that were culled because of either decreased reproductive efficiency or decreased milk production or mastitis, MAP-seropositive cows were associated with 1.55 (1.12-2.15, 95% C.I.) times increased hazard compared to MAP-seronegative cows. Among cows that were culled because of reproductive inefficiency, NC-seropositive cows had a 1.43 (1.15-1.79, 95% C.I.) times greater hazard than NC-seronegative cows. Among cows that were culled because of decreased milk production, cows in BVDV-seropositive herds had a 1.86 (1.28-2.70, 95% C.I.) times increased hazard compared to cows in BVDV-seronegative herds. BLV-seropositive cows did not have an increased risk of reason-specific culling as compared to BLV-seronegative cows. No significant interaction on culling among seropositivity for the pathogens was detected, but only a limited number of cows tested seropositive for multiple pathogens. Results from our research will help in better understanding the economic impacts of these pathogens and justification for their control.     

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200.Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.     

Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R² = 0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively.In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n = 253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.     

Determination of antibodies to Streptococcus agalactiae in the milk of dairy cows using the ELISA test

A sensitive 4-layers ELISA test for determination of antibodies against the pathogens of Streptococcus agalactiae in cows' milk was used for diagnosis of mastitis, with the aim to broaden these methods. Antigen was linked on the solid phase in the form of the whole bacteria, and milk was tested, diluted in the ratio of 1:10. Antigen bound-specific antibodies were labelled with pig antibodies against bovine immunoglobulins and in the next layer with rabbit antibody conjugated with peroxidases against pig immunoglobulins. After test visualisation and reading on the photometre, the results were given in the positivity per cent as a 100-multiple of the proportion of absorbance of the unknown sample and the positive control after subtraction of the negative control. Milk was examined in 36 dairy cows from three various breeding herds by that method. The samples were parallelly examined bacteriologically and cytologically. In the milk of dairy cows with positive S. agalactiae finding, the main level of antibodies expressed a positivity per cent, was 15.0%, while in bacteriologically negative animals it was only 6.2%. The dairy cows were divided into 8 groups, characterizing various stages of mastitis, according to the results of the individual treatments.     

The fat content of milk from dairy cattle infected with bovine leukosis virus

Two groups of cows infected with the bovine leukosis virus (BLV) were kept on two different diets and the fat content of their milk was assayed. The results were compared with those obtained from two comparable groups of BLV-free cows. The cows in each group were of similar ages, those in the groups on the poorer diet being 1–4 months post partum, while those on the richer diet were 5–7 months post partum. The mean percentage of fat in the milk from uninfected cows on the poorer diet was 2.94 while that from the similar infected cattle was 3.06. Uninfected cows on the richer diet produced milk containing 3.39% fat, while those that were infected produced milk containing 3.30% fat. No statistical differences in milk fat production were observed between the BLV seropositive and seronegative cows.