J. Brenner

Detrimental effect of bovine leukemia virus (BLV) on the immunological state of cattle

Bovine leukemia virus (BLV) is a retrovirus which seems to affect both the humoral and the cellular immune response. Cows affected by enzootic bovine leukemia (EBL) showed a reduction of IgM-producing cells in the spleen and lymph nodes. Experimentally infected calves had lower levels of secretory IgM and a decrease in T lymphocytes in the peripheral blood. The reduction in the amount of T cells was noticed mainly in cells bearing the CD4 markers. BLV-infected animals showed diminished responsiveness to newly encountered antigens. Cows naturally infected by BLV produced Igs with impaired structural or biological reactivity. The primary immune response was shown to be deficient in BLV-infected cows following vaccination with synthetic antigen. A marked shift in the proportion of PBL, especially of the CD5+ subset, was noticed. Peripheral blood mononuclear cells from BLV-infected cows secrete elevated levels of certain cytokines and contain increased levels of cytokine mRNA. High levels of cytokines are also found in the sera of BLV-infected cows compared to non-infected animals. A correlation was found between BLV infection and lack of spontaneous recovery from Trichophyton verrucosum infection. Moreover, some studies ascertained a significant association between the herd BLV infection status and disease incidence. The culling rate was higher and milk production lower in BLV-infected vs. BLV-free herds. It seems that BLV infection affects the immune system of a cow to such an extent that it ceases to be productive enough to be kept and, in most cases, the animal is culled before any symptoms of illness associated with persistent immunodeficiency become apparent.

Humoral and cellular responses in calves experimentally infected with Bovine Leukemia Virus (BLV)

In order to elucidate whether infection by Bovine Leukemia Virus (BLV) might induce an immunodeficient state, we inoculated sixteen calves with BLV. The calves were followed up for two years and were tested for humoral and cellular responses using various parameters, namely the appearance of antibodies to the BLV antigens, the changes in the numbers of lymphocytes involved, and the ratio between the two main populations of lymphocytes. Antibodies to the BLV antigens were of both the IgG and the IgM classes of immunoglobulins. The levels of antibodies of the IgM class were higher than those of IgG. There was a temporary decrease of reactive antibodies to the BLV antigens, to below detectable levels, during the 14-24 weeks post infection. A significant decrease in the level of plasma IgM was found in all BLV infected calves exhibiting lymphocytosis, while the level of IgG in the plasma of all experimental calves did not diverge significantly from the initial values, throughout the experiment. BLV infection was followed by lymphocytosis of B-cells in most infected calves, which persisted for the whole course of the experiment, while a decrease in the population of T-cells in peripheral blood was observed for a period of several months in all infected calves.

Cellular immune response cytokine expression during the initial stage of bovine leukemia virus (BLV) infection determines the disease progression to persistent lymphocytosis.

We have established experimental models of bovine leukemia virus (BLV) infection followed by progression to persistent lymphocytosis (PL) positive (BLV+PL+) or PL negative (BLV+PL-) stages of infection. Two out of six BLV infected animals developed PL+ 4 weeks after BLV infection. One other animal became PL+ late in the course of infection and three infected animals stayed PL-. These animals (PL-) exhibited transient lymphocytosis 3-4 weeks after infection and sustained PL- lymphocyte counts up to 24 weeks after infection. Competitive RT-PCR analysis of IFN-gamma mRNA expression revealed that peripheral blood mononuclear cells (PBMC) of animals with PL+ status developed by 4 weeks after infection had augmented IFN-gamma mRNA expression 3-4 weeks after BLV infection. However PBMC of animals that sustained a long-termed PL- lymphocyte count had elevated IFN-gamma mRNA expression 1-24 weeks after infection. Competitive RT-PCR analysis of IL-2 mRNA expression showed an increase in the levels of IL-2 mRNA in PL animals. Interleukin-10 (IL-10) mRNAs expression were elevated both in PL+ and PL- animals from 3 and 12 weeks after infection respectively. We suggest that early and extended expression of cellular response cytokines may delay the progression to PL+ in enzootic bovine leukemia.

SHORT-TERMED EXPRESSION OF INTERLEUKIN-12 DURING EXPERIMENTAL BLV INFECTION MAY DIRECT DISEASE PROGRESSION TO PERSISTENT LYMPHOCYTOSIS

In this study an attempt was made to elucidate cellular response cytokine expression upon experimental bovine leukemia virus (BLV) infection in cattle. Progression of infection was monitored by BLV gp51 mRNA expression or DNA amplification by RT-PCR or PCR, respectively, to detect provirus infected cells. Antibodies to BLV were detected by an agar gel immuno-diffusion (AGID) test in 5 weeks and persistent lymphocytosis (PL+) was established in all four BLV-infected animals in 24 weeks after infection. At the initial stage of infection a strong cellular immune response was induced mediated by IL-12p40 mRNA expression. Short-termed IL-12p40 expression was observed in peripheral blood mononuclear cells (PBMC) in two out of four infected animals following 1-3 weeks after infection, while viral mRNA expression was observed 2 weeks following infection. Expression of genes coding for the pro-inflammatory TNFalpha, IL-1beta and cellular response cytokines IFNgamma and IL-2 was detected beginning with the second and third week after infection in all BLV-infected animals. However, IFNgamma expression significantly decreased in 12 weeks after infection in three animals while IL-10 message initially detected 3 weeks after infection increased by 12 weeks and persisted. The observed immediate short-termed cell mediated immune response characterized by IL-12p40 and IFNgamma expression followed by an early shift to an IL-10 induced humoral response, may change the cytokine balance and direct disease progression to the PL+ stage.

BLV-infected lymphocytes exhibit two patterns of expression as determined by Ig and CD5 markers.

Lymphocytes were defined by their cell surface markers, Ig and CD5 in three groups of cows naturally infected with bovine leucosis virus (BLV). Lymphocytes were enumerated and groups were designated BLV seropositive with persistent lymphocytosis (BLV + PL +), BLV seropositive without persistent lymphocytosis (BLV + PL-) and BLV negative. The competence of peripheral blood mononuclear cells (PBMC) from the tested cows to express these two markers was determined by the double staining immunofluorescence procedure. Cows which developed persistent lymphocytosis (PL) as a result of BLV infection consequently underwent massive proliferation of B lymphocytes which express both Ig and CD5 antigens. In contrast, cows which were defined as BLV positive and PL negative showed a remarkable decrease of CD5 + Ig-, CD5- Ig+ and CD5+ Ig+ cells and also in the total number of lymphocytes. We suggest that BLV infection affects bovine lymphocytes through two different pathways of expression which might be related to the genetic properties of the target cells.

Experimental infection of calves with bovine leukemia virus (BLV): an applicable model of a retroviral infection

An experimental model of chronic infection with bovine leukemia virus (BLV) was established in young calves within a relatively short time. In the sera of all infected calves, precipitating antibodies were detected within 5 weeks after infection but upon disease progression pattern of cellular profiles varied. Three calves exhibited transient lymphocytosis 3-5 weeks after infection, two became persistent lymphocytotic (PL+) by that time and one stayed non-lymphocytotic (PL-) for 11 weeks and became PL+ after 4.5 months. Eventually all infected calves became PL+ by the end of the experiment, 6-12 months after infection. Increase of total counts of peripheral blood mononuclear cells (PBMC) related to polyclonal expansion of B-cells. The latter was assessed in all infected calves where the expansion of CD5-bearing cells (B+ CD5+) correlated with increase or decrease of total PBMC counts. Other cell populations such as CD4 and CD8 were also affected. Percentages decreased by 5 weeks after experimental infection to about half their original values though actual cell numbers stayed relatively stable. The experimental model we established compared well with field cases of naturally BLV-infected cattle and thus permitted the investigation of the disease at early stages of infection.

Bovine leukemia virus-gp51 antigen expression is associated with CD5 and IgM markers on infected lymphocytes

Cows that develop a persistent lymphocytosis (PL) as a result of bovine leukemia virus (BLV) infection develop massive proliferation of B-lymphocytes expressing both IgM and CD5 markers. The association of these two markers on peripheral blood mononuclear cells (PBMC) derived from BLV-infected cows and also expressing BLV-gp51 antigen marker on these cells was determined by three-color cytometric analysis. After in vitro cultivation of PBMC in the presence of PHA for 24 h, the mean percentages of marker-reactive cells of five PL+ cows were as follows; 43% +/- 4.5 of the PBMC expressed BLV-gp51 antigen; 90% +/- 1.6 of these cells expressed both IgM and CD5 at the same time, whereas but 7.5% +/- 1.9 expressed only IgM and 2.9% +/- 0.4 expressed only CD5. The PBMC, IgM positive cells accounted for 77.8% +/- 6.8, while both CD5 and BLV-gp51 were detected simultaneously on 52.0% +/- 2.4 of the IgM+ cells, while the CD5 marker and BLV-gp51 antigen were detected independently on 35.0% +/- 1.9 and in 9.0% +/- 3.1, respectively of the IgM+ cells. Of the CD5+ cells (equivalent to 75.5% +/- 9.0 of the PBMC), 54.7% +/- 4.7 expressed simultaneously IgM and BLV-gp51, while BLV-gp51 and IgM were expressed separately by 3.0% +/- 0.5 and 37.8% +/- 3.3, respectively. An association between the B-cell phenotype and BLV tropism might exist. It is also possible that cells bearing both IgM and CD5 markers are the main target cells for BLV infection.

Immunization of chickens with live Newcastle disease vaccine adjuvanted with oil

Chickens of various ages and breeds were vaccinated subcutaneously with Newcastle disease live-in-oil vaccines. These vaccines were prepared immediately prior to the vaccination by mixing the lyophilized live vaccine with the oil adjuvant, which was kept at room temperature. The live-in-oil vaccines were shown to be 30-50 times more effective in efficacy tests than either the same vaccines reconstituted in water or killed-in-oil vaccines. In addition to its biological advantage, the method of preparation of live-in-oil vaccine saves the expensive space of cold storage and shipment necessary for conventional killed-in-oil vaccines.

Enhanced antibody response in cattle against Leptospira hardjo by intradermal vaccination

A commercial killed Leptospira hardjo vaccine (with adjuvant) and non-adjuvanted preparation of the same vaccine were used in comparison of the effectiveness of the intradermal (i.d.) and subcutaneous (s.c.) routes for these vaccines. The tests were conducted in 50 females aged 6-14 months. After the first vaccination, both types of vaccine elicited a very poor antibody response by both routes of vaccination. However, after booster vaccination, the commercial vaccine (with adjuvant) elicited a remarkable immune response which was twice as high by i.d. compared with s.c. vaccination. No local or general adverse reactions were observed after i.d. vaccination with the adjuvanted commercial vaccine (potassium aluminium sulphate).

A possible linkage between gonadal hormones, serum and uterine levels of IgG of dairy cows

We have investigated the possible linkage between serum and uterine fluid immunoglobulin G (IgG) levels and the hormonal status of the cow. In cycling cows there was a significant (P < 0.01) drop in average (of 4 consecutive days) serum IgG levels, from 36.4 +/- 6.7 mg ml-1 during the luteal phase of the estrous cycle to 28.3 +/- 5.3 mg ml-1 during and around estrus. In prepartum cows, there was a significant drop (P < 0.01) from an average of 37.6 +/- 3.7 mg ml-1 from 5 consecutive days, i.e. 11-7 before parturition, to 28.0 +/- 5.5 mg ml-1 on the day of parturition. Total IgG in the uterine fluid ranged from 30 to 115 mg in one horn and from 24 mg ml-1 to 70 mg ml-1 in the other horn during the luteal phase, but was essentially undetectable at estrus. The drop in serum and uterine IgG occurred concomitantly with the drop in peripheral serum progesterone, from 2-3 ng ml-1 at the luteal phase, and 11-7 days before calving to less than 0.5 ng ml-1 around estrus and calving. Data suggest a possible linkage between steroid hormone and IgG levels.