A field evaluation of the polymerase chain reaction procedure for the detection of bovine leukaemia virus proviral DNA in cattle.

A polymerase chain reaction (PCR) procedure that detects proviral bovine leukaemia virus (BLV) in peripheral blood mononuclear cell DNA was evaluated. Blood samples from all animals (164) in a commercial dairy herd with a 30% prevalence of BLV infection, and from 194 animals from BLV free herds were tested. The absence of any positive PCR results in animals from BLV free herds confirmed the specificity of the assay. Initial testing of the infected herd using a single amplification PCR (SA-PCR), detected BLV infection in 62 of 72 adult animals that were seropositive by the agar gel immunodiffusion (AGID) test and in one persistently seronegative cow. Infection in this cow was confirmed by sheep bioassay. Subsequent testing of the SA-PCR negative, seropositive animals using a double amplification PCR (DA-PCR) detected proviral BLV in eight of nine animals that were available for retesting. The PCR assay was also able to distinguish BLV infected calves from uninfected calves that were serologically positive because of the presence of colostral antibody. Lymphocytes from all seropositive animals were cultured for determination of BLV antigen expression. Cultures from 37 of 62 SA-PCR positive animals produced detectable quantities of viral antigens. However, antigen expression was not detected in cultures from seropositive animals that were negative in the SA-PCR. In addition, in experimental transmission tests, inoculation of more than 10(6) lymphocytes from these cows was required for sheep to become seropositive to BLV.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of bovine leukaemia virus transmission by selective culling of infected cattle on the basis of viral antigen expression in lymphocyte cultures.

The use of viral antigen expression in lymphocyte cultures to prioritize the culling of bovine leukaemia virus (BLV) infected cattle was evaluated as a means of controlling the spread of infection in heavily infected herds. Selective culling was implemented in five commercial dairy herds containing between 126 and 304 cattle with infection prevalences, based on serological testing using the agar gel immunodiffusion test, of 19.4%, 20.3%, 20.1%, 20.6% and 39%. All seropositive cattle were tested for BLV antigen expression in lymphocyte cultures, and 51% found to express detectable quantities of viral antigens. In the four herds with 19% to 21% infection prevalence, all antigen-positive animals were culled immediately. Antigen-negative animals were retained in the herds for at least 16 months. Only two new infections were recorded in these four herds after antigen-positive animals had been culled, despite the continued presence of the antigen-negative animals. In the herd with 39% infection prevalence, a rapid reduction in the incidence of infection was achieved, even though only those animals with the highest levels of antigen expression were culled initially. Experimental transmissions from seropositive cattle indicated that sheep could be infected from an antigen-positive cow with fewer than 10(3) lymphocytes, whereas more than 10(6) lymphocytes were required to transmit infection from an antigen-negative cow. Estimation of the amount of integrated BLV DNA in serial dilutions of blood from antigen-positive and antigen-negative cattle provided an explanation for the higher infectivity of antigen-positive cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

T lymphocyte responses of sheep to bovine leukaemia virus infection.

Sheep were experimentally infected with bovine leukaemia virus (BLV) by inoculation of peripheral blood lymphocytes (PBL) from BLV infected sheep. Monoclonal antibodies were used to monitor changes in lymphocyte subpopulations in the first few weeks after inoculation. The polymerase chain reaction (PCR) detected BLV DNA in PBL of infected sheep 11-15 days after inoculation, that is, before antibodies to viral structural proteins were detected at 15-39 days post-inoculation. A rise in the number of both B and T lymphocytes coincided with detection of infection by PCR. At this time, an increase in the number of circulation CD8+ lymphocytes resulted in a low CD4: CD8 ratio. It appears that in BLV infection there is a host specific cell-mediated immune response to infected lymphocytes rather than a general immune response to foreign antigens. This response, which is characterized by an increase in the number of circulating CD8+ lymphocytes, precedes seroconversion. There is considerable variation between animals in this cytotoxic T lymphocyte response.

Infectivity of bovine leukaemia virus infected cattle: an ELISA for detecting antigens expressed in in vitro cultured lymphocytes.

A simple ELISA is described for quantifying expression of bovine leukaemia virus (BLV) antigens in short-term cultures of peripheral blood lymphocytes (PBL) isolated from infected cattle. The PBL-ELISA demonstrated that antigen expression levels in infected cattle could vary by more than 50-fold. Inoculation of sheep with dilutions of lymphocytes from two BLV-infected cattle, differentiated in the PBL-ELISA by 50 to 100-fold, suggested that antigen expression levels were correlated with infectivity. Haematological data indicated that increased antigen expression in PBL cultures was associated with an increased number of circulating B-lymphocytes, irrespective of whether or not an animal had lymphocytosis. This supported the hypothesis that BLV-infected cattle that are PBL-ELISA positive are more infectious and may present a greater risk of transmitting the disease. The applicability of the PBL-ELISA to a field situation was assessed with 98 BLV-infected cattle from three commercial dairy herds with infection prevalences of 11%, 23% and 47%. Similar percentages (49%, 50% and 52%) of PBL-ELISA positive cattle were identified among those infected cattle available for testing in the three herds. An additional 22 infected cattle from an experimental herd were tested to assess the stability of antigen expression levels over an 8 month period. Fewer (27%) of these cattle were identified as PBL-ELISA positive and antigen expression levels were generally lower than those observed in the commercial herds. Antigen expression levels in the experimental herd remained stable over the period of the study. The potential of the PBL-ELISA to assist in BLV eradication programs by identifying those seropositive cattle with the greatest potential to transmit infection is discussed.

Factors affecting the natural transmission of bovine leukaemia virus infection in Queensland dairy herds.

Natural transmission of bovine leukaemia virus (BLV) infection in south-eastern Queensland dairy herds was slow in 2 herds with a low to moderate (13 to 22%) prevalence of infection. Infection spread much more rapidly in a herd that had a higher prevalence (42%) when first tested. In a 13 month study of this herd, the cumulative incidence of infection was 24%. In one herd new infections were confined almost entirely to calves of uninfected dams. Following the end of feeding bulk milk to calves, a common practice in dairy herds, no more calves in this herd became infected. In laboratory experiments, neither prolonged housing of susceptible calves with infected cattle, consumption of drinking water contaminated with infected blood, nor inoculation of sheep with saliva from infected cattle resulted in transmission of BLV infection. Sheep were infected by subcutaneous inoculation of a suspension of purified lymphocytes from an infected heifer. The minimum infective dose was 10(3) lymphocytes, equivalent to the number of lymphocytes in approximately 0.1 microliter blood. Thus, procedures involving the transfer of a very small volume of blood from animal-to-animal have the potential to transmit infection.

Lymphocyte subpopulations in sheep with lymphosarcoma resulting from experimental infection with bovine leukaemia virus.

Sheep were experimentally infected with bovine leukaemia virus (BLV) and developed leukaemia and lymphosarcoma 30-88 weeks later. Ten sheep with lymphosarcoma were necropsied and lymphocyte subpopulations were evaluated in peripheral blood lymphocytes (PBL) and lymphocyte suspensions prepared from a range of lymph nodes, tumours and spleen. The leukaemic phase of BLV infection was characterized by an increase in the number of circulating B lymphocytes. The number of T lymphocytes was also increased with the CD8+ subpopulation proliferating at a much greater rate than the CD4+ subpopulation. In PBL the CD4:CD8 ratio fell rapidly as leukaemia developed, being 1.15 (+/- 0.18) 5-8 weeks before necropsy and 0.38 (+/- 0.09) at necropsy. During this period the number of B lymphocytes increased from 11.2 (+/- 0.7) to 379.4 (+/- 85.8) x 10(9)/L. CD4:CD8 ratios were also low in all lymph nodes and spleens of leukaemic sheep at necropsy. Most of the cells in solid tumours were B lymphocytes but a small population of T lymphocytes with a low CD4:CD8 ratio was identified.

Lymphocyte subpopulations in sheep during the early stage of experimental infection with bovine leukaemia virus.

Sheep were experimentally infected with bovine leukaemia virus (BLV) by the inoculation of PBL from leukaemic sheep. Antibodies to viral structural proteins were detected at from 2 to 6 weeks after inoculation. At seroconversion, all sheep had a marked increase in the number of circulating lymphocytes, due essentially to an increase in the number of B cells. The number of circulating B cells then decreased but remained higher than pre-infection levels. A second increase in this population preceded the development of a B cell lymphoblastic leukaemia. Generalized lymphosarcoma was diagnosed at necropsy of all sheep. Variation between individual sheep in the time from infection to the development of tumours allowed two clearly delineated groups of nine sheep to be compared. A study of changes in the B cell and T cell populations during the first 16 weeks of infection suggested that the initial response to infection influences the subsequent rate of leukaemogenesis. At seroconversion the number of circulating B cells was significantly higher in group 1 (10.16 +/- 1.51 X 10(9)/l) than in group 2 (6.47 +/- 2.76 X 10(9)/l). Group 1 sheep became leukaemic at 20-50 weeks after infection, whereas group 2 sheep did not do so until 70-95 weeks after infection.

Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis.

Contamination of a batch of tick fever (babesiosis and anaplasmosis) vaccine with bovine leucosis virus (BLV) was detected when a herd, in the final stages of an enzootic bovine leucosis (EBL) accreditation program, developed a large number of seropositive cattle following use of tick fever vaccine. Investigations incriminated a single calf used to produce Anaplasma centrale vaccine from which 13,959 doses were distributed. The failure of this calf to give a positive agar gel immunodiffusion (AGID) test before use was not fully explained. A total of 22,627 cattle from 111 herds receiving contaminated vaccine was tested to validate claims for compensation. Results showed infection rates of 62% and 51.8% in vaccinated dairy and beef cattle, respectively, compared with 6.1% and 1.5% in non-vaccinated cattle in the same herds. The results also indicated that infection did not spread from vaccinated to non-vaccinated in-contact cattle. Heavy reliance is now placed on purchase of calves for vaccine production from EBL accredited-free herds and on transmission tests from the calves to sheep to prevent a recurrence of contamination. The need for a BLV antigen detection test, with the sensitivity of the sheep transmission test but simpler and faster to perform, is evident.

The toxicity of Castanospermum australe seeds for cattle.

Two calves given a mean of 16.1 g and 16.4 g ripe Castanospermum australe seeds/kg body weight daily for 13 and 16 days respectively developed haemorrhagic gastroenteritis. The first calf died. The second calf had mild myocardial degeneration and necrosis and mild nephrosis at necropsy. Two calves given a mean of 16.8 g unripe C. australe seeds/kg body weight daily for 18 days remained clinically normal and had mild gastritis at necropsy. The activity of alpha-glucosidase was reduced in the mononuclear cells of peripheral blood and in skeletal muscle. This was attributed to the presence of the indolizidine alkaloid, castanospermine, in the seeds. The toxin causing the gastroenteritis and other lesions is unknown.

BoLA antigens are associated with increased frequency of persistent lymphocytosis in bovine leukaemia virus infected cattle and with increased incidence of antibodies to bovine leukaemia virus.

The association between bovine major histocompatibility system (BoLA) type and persistent lymphocytosis in cattle with antibodies to bovine leukaemia virus was examined by comparing antigen frequencies in cattle with persistent lymphocytosis to controls matched for age, sex, breed and presence of antibodies to BLV. The cattle came from nine dairy herds in south-east Queensland, Australia; six herds were Australian Illawarra Shorthorn (AIS), two herds were Jersey and one herd was Friesian. Antigen W6 and Eu28R were more common in cattle with persistent lymphocytosis than in controls. Antigen W8 was less common in AIS cattle with persistent lymphocytosis. A study of 24 offspring from one sire, heterozygous for W10 and Eu28R, showed that offspring inheriting Eu28R from the sire were significantly more likely to have antibodies to BLV than offspring inheriting the opposing W10 haplotype.

Breed differences in the frequency of bovine lymphocyte antigens.

Lymphocytes from 1,564 cattle of 18 breeds and cross-bred groups in Australia were tested for major histocompatibility system class 1 antigens. Gene frequencies were calculated for the Angus, Belmont Red, Brahman, Hereford and Holstein-Friesian breeds. There were substantial differences among these breeds in antigen and gene frequency. There were striking differences among all 18 breeds in the presence or absence of certain antigens. Two antigens, CA13 and CA36, were strongly associated in Hereford cattle but occurred independently of each other in the other breeds.

Differences in the lymphoproliferative response of cattle and sheep to bovine leucosis virus infection.

Lymphoblastic leukaemia, preceded by a significantly increasing percentage of prolymphocytes in peripheral blood smears for from 12 to 68 weeks before death was a feature of sheep which developed lymphosarcoma following inoculation with the Australian strain of bovine leucosis virus (BLV). Lymphocytosis and/or the appearance of immature cells were a reliable predictor of tumour formation in sheep, but not in cattle. There was a terminal lymphoblastic leukaemia in only 43 of 84 cattle with lymphosarcoma. Differences in the morphological appearance and glycogen content of the leukaemic lymphoblasts of sheep and cattle were observed. In spite of these differences the high frequency of lymphocytosis and lymphosarcoma in experimentally infected sheep suggests that they could be a useful model for studying the pathological and immunological responses to BLV infection.

Serological and biochemical factors in bovine ephemeral fever.

Clinical signs of ephemeral fever, which were observed in individual cattle during two successive epidemics in 1973 and 1976, were related to biochemical, cellular and serological changes in the blood. The rise in peripheral blood neutrophil counts in samples collected from 12 sentinel cattle on a daily basis before, during and after natural disease in the two epidemics to mean peaks of 9.6-12.5 X 10(9) per litre, and fall in counts of lymphocytes to a trough of 5-7 X 10(9) per litre was found to occur on the same day as the fever peak. A fall in serum calcium levels from a normal mean of 2.55 mmol/l to 2.0 mmol/l occurred on the day clinical signs were most pronounced. Serum magnesium levels were affected to only a minor degree. Plasma fibrinogen rose from a normal mean of 5.0 milligrams to a peak of 18 milligrams on the second day of disease and fell towards normal in the week after recovery. Neutralizing antibodies to bovine ephemeral fever virus were detected up to 63 days prior to clinical disease, and the rise of antibody after recovery was secondary in pattern. Serological evidence of a prior infection with an antigenically related virus, Kimberley virus, was found in these animals. In more severe clinical cases of ephemeral fever serum calcium levels were as low as 1.95 mmol/l. Treatment of cattle showing clinical signs of the disease with phenylbutazone and calcium borogluconate was favourable.

Babesia bovis: comparison of culture-derived parasites, non-living antigen and conventional vaccine in the protection of cattle against heterologous challenge.

Twenty Bos taurus cattle were vaccinated with either live commercial Babesia bovis vaccine, live parasites from in vitro culture or non-living supernatant antigen (NLSA) derived from in vitro culture and combined with the adjuvant saponin. Heterologous strain challenge 10 weeks later indicated that cattle vaccinated with live parasites from either source were strongly protected, those given 2 doses of NLSA 2 weeks apart were partially protected, and those given one dose of NLSA were poorly protected. Enzyme immunoassay detected comparable, increasing levels of specific babesial antibody in all vaccinated cattle during the 2 to 3 weeks following vaccination, after which levels in cattle given NLSA decreased. Antibody to bovine blood group factors was detected in 4 of the 10 animals given NLSA. Titres peaked after 3 to 4 weeks and then declined rapidly.

Isoimmune thrombocytopenic purpura in piglets.

A haemorrhagic diathesis due to isoimmune thrombocytopenia occurred in the fourth litter of a Landrace sow. Maternal antibodies, absorbed by piglets from the colostrum, were incompatible with platelet antigens inherited from the sire. The severity of haemorrhage varied between piglets although all were thrombocytopenic. Two deaths occurred, one at 24 h and the other at 3 weeks of age. The surviving 11 piglets were clinically and haematologically normal at 16 weeks of age.

Haematological investigation of a multiple case leucosis herd.

Adult cattle in a Queensland dairy herd with a history of deaths from lymphosarcoma were sampled regularly over a 4 year period for the identification of animals with persistent lymphocytosis (PL). Twenty-one of 94 animals that were sampled at least 6 times had PL. At the initial sampling 27% of the animals had lymphocytosis. Culling of haematologically positive animals in the first 18 months of the investigation reduced this to 5.3%, but cessation of the culling programme resulted in a gradual increase in the percentage of animals with lymphocytosis. Four deaths from lymphosarcoma occurred in adult animals, but only in the first 18 months of the investigation. Two of these animals had lymphocytosis and two lymphoblastic leukaemia. The calf of one of the latter cows developed lymphoblastic leukaemia and lymphosarcoma by the time it was 6 months of age. Although histological evidence of lymphosarcoma was lacking in a number of clinically normal animals with lymphocytosis, haematological investigation identified a group of animals within the herd that may develop lymphosarcoma.

Experimental infection of chickens with an australian strain of reticuloendotheliosis virus. I. Clinical, pathological and haematological effects.

A wide range of clinical, pathological and haematological effects were found over a 40-week period in chickens inoculated at 1-day-old with a low-passage, cell-culture preparation of an Australian strain of reticuloendotheliosis virus. Feathering defects and statistically significant depression of body weights occurred in chickens up to 8 weeks of age. Other findings in birds that died or were culled during the 40-week experimental period included mild anaemia, leucopenia, heterophilia, hypoplasia of immune system organs, inflammation in visceral and nervous system organs, and bacterial or fungal infections. These results suggested that ill-thrift and death in some chickens infected with reticuloendotheliosis virus may be due to secondary infections with microorganisms subsequent to damage of immune system organs by that virus. Lymphoreticular-cell tumours of the liver, kidney or spleen were found in two birds aged 22 and 24 weeks. These results establish reticuloendotheliosis virus as a possible cause of tumours in adult fowls. Horizontal transmission of virus was demonstrated but the only abnormalities detected in the in-contact chickens were feathering defects.

Evaluation of oral iron galactan as a method of iron supplementation for intensively housed sucking piglets.

Iron supplementation of piglets with oral galactan given as a single dose within 24 hours of birth was evaluated in a series of on farm trials. The growth rate of piglets receiving this treatment was faster than that of piglets receiving single injections of iron dextran or iron galactan at 3 days of age, or ferrous sulphate crystals orally at weekly intervals. Mean values for red cell parameters of oral iron galactan supplemented piglets at 2 to 4 weeks of age were lower than those of injected piglets but there was no clinical evidence of anaemia in any of the piglets.