Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle.

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.

Characteristics of lymphocytes appearing in persistent lymphocytosis induced experimentally in cattle by bovine leukemia virus infection.

Properties of peripheral blood lymphocytes (PBL) appearing during persistent lymphocytosis (PL) of cattle that was induced by inoculation of a large number of bovine leukemia virus (BLV)-infected lymphocytes were studied. The PBL possessed surface immunoglobulin at a high ratio and had high syncytium-forming activity. The PBL could not be distinguished from normal PBL cytogenetically. These PBL were shown to be polyclonal cells integrating BLV-proviral DNA randomly into their cellular DNA. These properties were similar to those of PBL appearing at the stage of PL of cattle naturally infected with BLV. The BLV-proviral DNA content of PBL and transcription of viral genes increased in parallel with development of PL and decreased along with a decline of PL indicating the possibility that gene expression and activation of BLV play an important role in induction and maintenance of PL.

Bovine leucosis virus challenge infection of calves following application of BL-3 cells.

8 calves were vaccinated 3 times with 1.5.10(7) BL-3 cells. 2 and 6 weeks, respectively, after the 3rd vaccination each 4 calves were subjected to a challenge infection with 2.5.10(5) blood lymphocytes of a BLV-infected cow. For control a challenge infection was performed on 3 non-vaccinated calves. The vaccination caused in all calves the appearance of complement-dependent cytotoxic antibodies against BL-3 cells but not of antibodies against BLV-gp51. After the challenge infection there occurred in the sera of all vaccinated calves and of all non-vaccinated control animals antibodies against gp51, and BLV could be demonstrated in the cultivated blood lymphocytes of all animals. Consequently, the application of BL-3 cells had no protective effect against the BLV infection.

Evaluation of the stable fly (Stomoxys calcitrans) as a vector of enzootic bovine leukosis.

Experiments reported here were directed at 2 questions: (1) Can the stable fly (Stomoxys calcitrans) transmit enzootic bovine leukosis? (2) Could early viremia augment the probability of transmission by this insect? In one vector experiment, calves and bovine leukemia virus (BLV)-infected cows were housed with and without stable flies. The calves were monitored serologically during a 3-month postexposure period, using the agar gel immunodiffusion test. All fly-infested and fly-free calves remained BLV-seronegative. For a second vector experiment, donor calves, newly injected with blood from BLV-infected cows with high virus expression, were tethered alternately between uninoculated, weaned BLV-seronegative calves. These groups were housed with or without flies in 2 replicate trials. The inoculated calves from the first replicate seroconverted at 16 and 23 days after inoculation; the inoculated calves from the second replicate seroconverted at 11, 16, 16, and 37 days after inoculation. All uninoculated calves remained BLV-seronegative. In a manual transmission experiment, 50 unfed stable flies were allowed to complete a meal on each of 3 BLV-seronegative calves after feeding on a BLV-seropositive cow with high (42%) virus expression. One control calf was injected with blood from the cow. Seroconversion occurred in the control calf and 1 calf on which flies were given access. A scanning electron microscopic study was made of the everted and closed mouth parts of the stable fly. Given the lymphocyte count in blood from the cow used in the manual vector transmission experiment, it was calculated that 3,950 mouth part volumes would be necessary to transmit BLV.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of virus dose in experimental bovine leukemia virus infection in sheep.

Twenty-four, six month old lambs were assembled into four groups of five animals each and one group of four animals. All groups were inoculated with lymphocytes from a single donor lamb infected with bovine leukemia virus. The inoculum varied from 250 to 250,000 lymphocytes, in tenfold increments. Animals were exposed by intradermal injection in the neck region immediately anterior to the left shoulder joint. All groups were monitored at 0, 3, 7 and 12 weeks after inoculation using the following procedures: a. Syncytia induction assay for detection of bovine leukemia virus in peripheral blood lymphocytes. b. Agar gel immunodiffusion against the gp51 antigen of bovine leukemia virus for the detection of antibovine leukemia virus gp51 antibody. c. Lymphocyte stimulation test for the assessment of cell-mediated immunity using mitogen, nonfractionated bovine leukemia virus antigen, and partially purified bovine lymphoma tumor-associated antigen for the in vitro activation of lymphocytes from bovine leukemia virus-inoculated and sham-inoculated, control animals. d. Routine hematological techniques for the assessment of total leukocyte and lymphocyte counts. The median infectious dose for lymphocytes from the single bovine leukemia virus-infected donor used in this study was determined to be 2000 cells. The syncytia induction assay detected more infected individuals (13/23) at an earlier time than did the agar gel immunodiffusion assay (10/23). Using either serological or virus isolation techniques, infected animals were first detected at three weeks postinoculation in the group receiving the high-dose inoculum and at seven weeks postinoculation in groups receiving low- or medium-dose inocula.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of cattle persistently infected with bovine virus diarrhoea virus to bovine leukosis virus.

Six cattle persistently infected with bovine virus diarrhoea virus (BVDV) and seronegative, and two control, virus negative seropositive cattle were inoculated with lymphocytes infected with bovine leukosis virus (BLV). The two controls produced a normal immune response to BLV, developing antibodies at four and five weeks after inoculation. Two of the six cattle persistently infected with BVDV developed a strong antibody response by six weeks after inoculation with BLV. Four developed a depressed response to BLV, characterised in three by a 'hooking' reaction in the immunodiffusion test which persisted in successive bleedings but was interspersed occasionally by a weak positive reaction. In one of these animals, a series of 'hooking' reactions was followed by a number of negative results. The fourth animal remained serologically negative until 16 weeks after inoculation when a 'hooking' reaction was observed followed by a series of negative results. BLV was isolated from all the cattle persistently infected with BVDV at 42 or 58 weeks after inoculation regardless of whether the serum samples gave negative, 'hooking', weak positive or positive reactions in the immunodiffusion test. BLV was consistently isolated from the nasal secretions of a steer which was BVDV negative but seropositive. The possibility of decreased immune responsiveness to BLV in animals persistently infected with BVDV should be considered when formulating regulations governing the testing of animals for freedom from BLV.

Development of leukemia and lymphosarcoma induced by bovine leukemia virus in sheep: a hematopathological study.

The hematological and neoplastic disorders induced in sheep by experimental bovine leukemia virus (BLV) infection are described. Seventeen of 19 BLV-inoculated sheep developed a marked increase in peripheral blood lymphocytes by 36 months after the intraperitoneal injection of peripheral blood lymphocytes from a BLV-infected cow. This increase correlated with an increase in the number of circulating B lymphocytes as demonstrated by the presence of surface immunoglobulins (SIg) and a high cell proliferative response to lipopolysaccharide and was considered to be a persistent B cell lymphocytosis. Lymphosarcoma developed in five BLV-infected sheep between 19 and 38 months postinoculation and was preceded in four out of five of these cases by an elevation in peripheral blood lymphocytes which began 4 to 26 months before death due to lymphosarcoma. The majority of tumor cells in all lymphosarcoma cases were of the centroblastic type, and in two cases in which the presence of SIg was assayed, the majority of tumor cells were SIg-positive. Thus, BLV-induced lymphosarcoma in sheep seems to be a B lymphocyte-derived tumor.

Binding of a cellular protein to the gibbon ape leukemia virus enhancer.

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

Rectal transmission of bovine leukemia virus in cattle and sheep.

Bovine leukemia virus (BLV) was transmitted by rectal inoculation of BLV-infective whole blood into cattle and sheep. Two cows and 2 sheep each were given 500 ml and 50 ml of blood, respectively, by rectal infusion. Two sheep which served as positive controls each were given 1 ml of the same blood, IV. All animals became seropositive to BLV by postinoculation week 5. Although relatively large volumes of blood were used for rectal inoculation, a base line for infectivity was established for the rectal route.

A protein immunoblot test for detection of bovine leukemia virus p24 antibody in cattle and experimentally infected sheep.

A protein immunoblot test for detecting antibody to the bovine leukemia virus p24 antigen is described. The test employs a crude antigen preparation derived from concentrated cell culture fluid, and an optimised biotin-streptavidin-peroxidase amplification system for immunodetection. The test is highly specific and is more sensitive than the gp51 agar gel immunodiffusion (AGID) test for detection of BLV antibody in cattle and experimentally infected sheep. In a selected set of 30 field sera from cattle which had given equivocal results in the gp51-AGID test, 21 were positive, 4 were negative, and 5 gave an uncertain result in the p24-immunoblot test.

Transmission of bovine leukosis virus by blood inoculation.

The experimental transmission of bovine leukosis virus (BLV)-infected whole blood was studied in 2 groups of Holstein calves. The 1st group of 4 BLV-seronegative calves was given 10 microliters of whole blood by either the IM, IV, subcutaneous, or intradermal routes. All 4 calves seroconverted to BLV within 8 weeks after they were inoculated. The 2nd group, also comprising 4 calves, was given the equivalent of 1 microliter of whole blood by the described routes. These calves seroconverted to BLV by 14 weeks after they were inoculated. The results indicated that small volumes of whole blood administered by 4 different routes were effective in the spread of BLV.

Analysis of wild-derived mice for Fv-1 and Fv-2 murine leukemia virus restriction loci: a novel wild mouse Fv-1 allele responsible for lack of host range restriction.

Wild-derived mice originally obtained from Asia, Africa, North America, and Europe were typed for in vitro sensitivity to ecotropic murine leukemia viruses and for susceptibility to Friend virus-induced disease. Cell cultures established from some wild mouse populations were generally less sensitive to exogenous virus than were cell cultures from laboratory mice. Wild mice also differed from inbred strains in their in vitro sensitivity to the host range subgroups defined by restriction at the Fv-1 locus. None of the wild mice showed the Fv-1n or Fv-1b restriction patterns characteristic of most inbred strains, several mice resembled the few inbred strains carrying Fv-1nr, and most differed from laboratory mice in that they did not restrict either N- or B-tropic murine leukemia viruses. Analysis of genetic crosses of Mus spretus and Mus musculus praetextus demonstrated that the nonrestrictive phenotype is controlled by a novel allele at the Fv-1 locus, designated Fv-10. The wild mice were also tested for sensitivity to Friend virus complex-induced erythroblastosis to type for Fv-2. Only M. spretus was resistant to virus-induced splenomegaly and did not restrict replication of Friend virus helper murine leukemia virus. Genetic studies confirmed that this mouse carries the resistance allele at Fv-2.

Induction of lymphosarcoma in sheep inoculated with bovine leukaemia virus.

Five sheep were experimentally inoculated with BLV in order to study the humoral immune response in animals infected with bovine leukaemia virus (BLV). During experimental periods of 46 months, 2 sheep died with leukaemia and one sheep showed splenomegaly and proliferation of tumour cells. The other 2 sheep were clinically normal. All of the inoculated sheep developed antiviral antibodies 1 month after inoculation and BLV could be re-isolated in lymphocytes 2 to 3 months after inoculation. Antibody against glycoprotein antigen (gp51) of BLV appeared earlier than the antibody against protein antigen (p24) and antibody titres of the former were higher than those of the latter during the course of the experiment. The complement dependent antibody cytotoxicity test was performed for the detection of antibody against BLV-related cell membrane antigen with 2 different kinds of target cells; FLK cells which are foetal lamb kidney cells chronically infected with BLV and SF-28 cells which are sheep fibroblasts transformed with BLV in vitro. All 5 sheep developed cytotoxic antibodies against both types of cells. In sera from two leukaemic sheep, cytotoxic antibody titres against SF-28 cells gradually decreased 30 months after inoculation and finally became negative one to 3 months before they died of leukaemia. However, these leukaemic sheep persistently produced antibodies against gp51 and p24.

Safety and efficacy studies of live- and killed-feline leukemia virus vaccines.

The safety and the efficacy of several feline leukemia virus (FeLV) vaccines for 16-week-old kittens were determined. Vaccines were derived from an FL74 lymphoblastoid cell line that has been in continuous tissue culture passage for about 4 years. The vaccines were made from living virus, formaldehyde-inactivated whole FL74 cells, and formaldehyde-inactivated whole virus. The efficacy of each produced vaccine was determined by challenge exposure of vaccinated cats with virulent FeLV. The two formaldehyde-inactivated vaccines were found to be safe for use in kittens. Neither vaccine produce a significant feline oncornavirus-associated cell membrane antigen or virus-neutralizing antibody response, nor did they prevent infection with virulent FeLV. The inactivated whole-virus vaccine, however, did substantially decrease the proportion of kittens infected with virulent FeLV that became persistently viremic. In contrast, the whole FL74 cell vaccine did not reduce the number of infected kittens that became persistently viremic. The live-virus vaccine was found to be both safe and efficacious. About a half of the kittens vaccinated with live virus had transient bone marrow infection that lasted from 2 to 4 weeks. Viral antigen was not detected in peripheral blood, and infective virus was not shed in saliva, urine, or feces during the period that the vaccinal virus could be recovered from the bone marrow. In addition, there was no horizontal spread of vaccinal virus from vaccinated to non-vaccinated cagemates. Within several weeks, vaccinated kittens demonstrated no clinical or hematologic abnormalities and had high serum levels of feline oncornavirus-associated cell membrane antigen and virus-neutralizing antibody. Kittens vaccinated with living FeLV were resistant to infection with virulent virus.

Acute monocytic leukemia in an irradiated Beagle.

A purebred female Beagle dog that had received 2,000 R of protracted wholebody gamma-irradiation from 60Co when 14 months old had hematologic changes consistent with a myeloproliferative disorder 3 years after the termination of radiation exposure. Peripheral blood and bone marrow findings during the 7-month period before death showed progressive anemia with increased numbers of platelets; immature granulocytes, monocytes and promonocytes. A period of partial remission occurred during which time the peripheral blood was aleukemic, although there was marked thrombocytosis and abnormal erythropoiesis which was evidenced by bizarre circulating nucleated red cells, anisocytosis, poikilocytosis and Howell-Jolly bodies. The dog had a terminal crisis with marked leukocytosis, most cells in the peripheral blood being bizarre monocytes and promonocytes. Tissues obtained at necropsy showed diffuse as well as focal infiltration of the spleen, liver, lymph nodes, heart, kidney and gastrointestinal wall with immature neoplastic cells resembling monocytes and monocytic precursors. The monocytic differentiation of the invasive cell population was confirmed by morphological, cytochemical, histological, ultrastructural and in vitro cell culture studies.