Existence of cytotoxic activity against BLV-transformed cells in lymphocytes from normal cattle and sheep

Peripheral blood lymphocytes (PBL) from normal cattle and sheep were tested for their cytotoxic activity against several target cells using a 20-hour 51Cr release assay. The following characteristics of the effector cells were observed; 1) PBL from animals showed cytotoxic activity against two sheep cell lines (FLK and SF-28) that were transformed with bovine leukemia virus. However, normal sheep and bovine cells and Molony leukemia virus-induced mouse lymphoma cell line (YAC-1) were not killed by these cells. 2) A time course study showed that the activity was first observed at 4 to 8 hours and reached a maximum at 20 to 30 hours after incubation. 3) Cytotoxic activity was observed in both adherent and nonadherent cell fractions when PBL were passed through a nylon-wool column. This indicated that the effector cells showed some degree of adherence. 4) Treatment of PBL with carrageenan did not change the cytotoxic activity against target cells, indicating that phagocytic capability is not perhaps necessary for cytotoxicity to take place. These results indicate that the effector cells participating in the cytotoxic reaction resembled natural killer cells or natural cytotoxic cells which are present in murine and human systems. However, analysis of the cell surface markers of the effector cells is yet to be done in future studies.     

Factors affecting the infectivity of lymphocytes from cattle with bovine leukosis virus.

Peripheral blood mononuclear cells were obtained from 13 bovine leukosis virus infected cattle and inoculated subcutaneously into 29 recipient adult steers to determine (a) the number of mononuclear cells (equivalent amount of blood) necessary to cause infection and (b) factors influencing infectivity of mononuclear cells from bovine leukosis virus-infected animals. A total of 55 inoculations were made. Inoculation of 1 X 10(4), 2 X 10(4) and 5 X 10(4) mononuclear cells caused seroconversion in 12%, 57% and 62% of steers, respectively. No infections occurred with 1 X 10(3) or 2 X 10(3) mononuclear cells. Cattle infected for longer than 24 months and those animals greater than three years of age were more likely to cause infection with 1 to 5 X 10(4) mononuclear cells than were cattle infected for less than 24 months or animals less than three years of age. Lymphocytes from cattle with persistent lymphocytosis caused more infections when 1 X 10(4) or 2 X 10(4) mononuclear cells were inoculated, than did lymphocytes from nonpersistent lymphocytosis cattle; however, both groups were equally infectious when 5 X 10(4) mononuclear cells were inoculated. No differences were found in infectivity of experimentally vs naturally exposed animals.     

Non-specific natural cytotoxic factor released from bovine peripheral blood lymphocytes.

Natural cytotoxicity against bovine leukemia cells (PC-3 cells) was found in bovine peripheral blood lymphocytes (PBL), and in non-adherent cells but not in adherent cells to nylon-wool column. Natural cytotoxic cells (NCC), which have natural cytotoxic activity, are found in T cell-rich fraction. When NCC were cocultured with PC-3 cells, natural cytotoxic factor (NCF) was released rapidly from NCC, and dose-response curve for NCF was almost linear induction. Cytotoxicity against PC-3 cells by NCC or NCF was increased with an increment of incubation period. Cytotoxicity against K562 cells, CL-1 cells, M1 cells or EL-4 cells by NCF was almost the same level as that against PC-3 cells, but that against those cell lines by NCC was not found. NCF activity in culture fluid from NCC cocultured with K562 cells or CL-1 cells was lower than that from NCC cocultured with PC-3 cells.     

In vitro induction of cytotoxic lymphocytes from infectious bovine rhinotracheitis virus hyperimmune cattle

Cytotoxic lymphocytes against infectious bovine rhinotracheitis virus (IBRV)-infected cells were induced by in vitro stimulation with IBRV antigen of peripheral blood leukocytes obtained from hyperimmune cattle. After one in vitro stimulation with IBRV, cytotoxicity was detected against IBRV-infected autologous target cells, but not against heterologous IBRV-infected or K562 target cells. After 4 in vitro IBRV stimulations at weekly intervals (in the presence of autologous feeder cells followed by interleukin 2 treatment at 2-day intervals), cytotoxicity was detected against autologous-, and to a lesser extent, heterologous-infected cells. Most cells in cytotoxic cultures were positive to a monoclonal antibody shown to react with bovine T lymphocytes.     

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.     

Inhibition of in vitro immunocyte function by sera from cattle with bovine leukosis

Most sera from leukaemic cattle inhibited phagocytic activity of normal bovine peripheral polymorphonuclear leukocytes, growth of interleukin 2-dependent bovine T cells and mitogen-induced (phytohaemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and protein A) blastogenesis of normal bovine lymphocytes. By contrast, antibody-dependent, and spontaneous cell-mediated cytotoxicity were suppressed by only a few sera. The antibody titer against bovine leukaemia virus in these sera correlated with the percent inhibition of lymphocyte blastogenesis. These leukotic sera had no direct cellular cytotoxicity and the inhibitory activity was not lost by dialysis or heat inactivation at 62°C for 30 min. However, the activity was reduced by heating at 80°C for 30 min. Neither the concanavalin A sepharose 4B effluent fraction nor 3.5% polyethyleneglycol-treated serum was found to contain significant lymphocyte-inhibitory activity. Blastogenic transformation of lymphocytes prepared from leukaemic cattle was hardly detectable; however, the mitogen responsiveness of these lymphocytes was improved by a 37°C 1-h preincubation followed by washing.     

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.     

天然植物中草药在牛羊业中应用的研究进展

我国天然植物中草药资源丰富 ,中草药含有多种微量养分和免疫因子 ,具有低毒、无残留、无副作用等特点。畜牧生产中可提高动物饲料转化效率 ,增强抵抗疾病的能力 ,缓减环境应激产生的负作用。为生产高效、安全、无公害畜产品 ,目前科学工作者针对激素、抗生素和化学合成类药物等产生抗药性和在产品中残留而危害人体健康的问题 ,研究开发利用中草药资源以替代激素、抗生素和化学合成类药物等。本文就天然植物中草药的作用及在牛羊业中的应用研究进行阐述。1　天然植物中草药的作用1 1　营养作用中草药含有动物生长发育所必需的各种养分 ,如…     

Suppression of mitogenic response of bovine lymphocytes during experimental ketosis in calves

Experimental ketosis was induced by feeding calves a diet containing 1,3 butanediol for 9 or 10 days. Blood beta-hydroxybutyrate was significantly (P less than 0.05) elevated during clinical ketosis. The mitogenic response of the lymphocytes collected during ketosis was significantly (P less than 0.01) suppressed, and the suppression persisted for 2 weeks. During ketosis, all calves fed a 1,3 butanediol diet had clinical signs of an upper respiratory tract infection. Possible relationships between the suppressed function of lymphocytes, the increased concentration of 1,3 butanediol, and the increased susceptibility of the calves to infections during ketosis were considered.     

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.     

Characterization and activation requirements of bovine lymphocytes acquiring cytotoxic activity after interleukin-2 treatment.

Interleukin-2 (IL-2) treatment of cells and generation of non-major histocompatibility complex (MHC)-restricted cytotoxic cells from peripheral blood mononuclear leukocytes (PBML) was studied. Effector-target conjugate assays demonstrated that bovine PBML bound but did not lyse K562, HL60S and HL60R cells unless activated with IL-2. The magnitude of IL-2-activated killing of tumor cells as well as the magnitude of antibody-dependent cellular cytotoxicity depended on the IL-2 concentration. A short treatment (12-18 h) of effector cells with IL-2 was sufficient for development of cytotoxic activity. Withdrawal of IL-2 from the culture resulted in a reduction of cytotoxic activity that could be restored by further addition of IL-2. Cytotoxic activity of IL-2-activated populations obtained after nylon wool or Sephadex G-10 passage, and Percoll gradient centrifugation of PBML suggests that lymphokine-activated killer (LAK) cell activity in PBML is mainly mediated by a non-adherent lymphocyte lacking markers for B-cells. Positive and negative selection experiments using cell sorting confirmed these findings and demonstrated that the cell responsible for LAK cell activity in cattle belongs to a non-monocyte, non-B, CD2+ lymphocyte population. Furthermore, cytotoxic activity could not be generated in CD2+ populations enriched for cells expressing molecules equivalent to human and murine CD4 and CD8. These findings suggest that effector cells mediating non MHC-restricted cytotoxicity in cattle prevail in a population bearing a CD2+, CD4-, CD8- phenotype and that this population depends on the continuous presence of IL-2 for optimal cytotoxic function.     

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.     

Eradication of enzootic bovine leukosis from Finland

Enzootic bovine leukosis (EBL) was recognized among Finnish cattle in 1966. Administrative decisions specifying and refining official control measures were given in 1966, 1976, 1980, and 1993. The measures' key principle always has been 'test and slaughter'. The EBL/bovine leukosis virus (BLV) infection situation was monitored at meat inspection, and hematologically between 1970 and 1977 and serologically between 1978 and 1989. Annual surveys including all dairy herds and samples from beef animals were conducted in 1990-2001. Bulk-tank milk samples represented the dairy herds in the surveys; the beef animals were sampled individually at slaughter. The maximum positive herd-level percentage in the surveys was 0.03%. EBL/BLV infection was evenly dispersed in the southern part of the country and nonexistent in the northern part. We conclude that herd-level prevalence of EBL/BLV infection never exceeded 5%. It nevertheless took 30 years to eradicate the disease and the infection. EBL was eradicated from mainland Finland in 1996 and from the island district of Ahvenanmaa in 1999. Annual monitoring of the EBL situation continues.