Effect of bovine lactoferrin on a transmissible AIDS-like disease in mice.

The effect of bovine lactoferrin (bLF) was examined on an AIDS-like disease (ALD) in mice. Induction of disease was achieved by inoculation with infected cell-free plasma from diseased mice to uninfected ones. The effect of treatment with bLF was investigated when administered simultaneously with the virus, 20 days prior to infection, or 20 days after infection. Animals underwent clinical surveillance and enumeration of white blood cells (WBC) and lymphocytes, as well as fluorescent staining of CD4 and CD8 bearing cells. Simultaneous administration of bLF and virus did not affect the pattern of ALD progress along the course of the experiment. Pretreatment with bLF prior to virus inoculation abolished on day 21 the detrimental effect of viral infection that lasted for two months. An opposite outcome was observed when bLF was administered 20 days after the virus. It seems that bLF had played a preventive role for a restricted period of time. However, an adverse response was elicited when bLF was administered 20 days after viral infection.

Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model.

Bovine mastitis caused by Staphylococcus aureus (S. aureus) is a most important infection disease that affects both the quality and the quantity of milk production. Antibiotic therapies formulated for intramammary use are generally unsuccessful in eliminating existing S. aureus infections. Vaccination is a logical approach to the control of S. aureus udder infections. However, to date commercially available S. aureus vaccine have shown limited efficacy under field conditions, mainly due to the paucity of information regarding relevant antigens which will induce a broad spectrum immunization. In the present paper the attempt to develop a new vaccine designated MASTIVAC I is described. MASTIVAC I is composed of three strains of S. aureus namely: VLVL8407; ZO3984 and BS449 which were isolated from clinical and sub-clinical cases of bovine mastitis. A mouse model was used to evaluate the S. aureus specific antibody production and protection of mice against virulent S. aureus strains. The results obtained showed that this vaccine exhibits a broad spectrum of antigenic and immunogenic properties that protects mice from homologues and hetrologous S. aureus challenge.

Reactions of peripheral blood mononuclear cells (PBMC) of camels with monoclonal antibodies against ruminant leukocytes.

The particular immune system of the camel has been but little investigated. In this work circulating camel peripheral blood mononuclear cells (PBMC) were studied by flow cytometry. Monoclonal antibodies (mAbs) raised against ruminant leukocytes were used for the detection of cell surface antigens. Monoclonals to T-cell markers, CD4 (CACT138A) and CD8 (CACT80C), exhibited no reactivity towards camel PBMC in contrast to their reactivity to PBMC of other ruminant species and those of cattle in particular. A relatively high percentage (29.1+/-8.9%) of camel PBMC reacted with a non-immunoglobulin cell surface marker, B-B2, comparable to the reactivity of bovine PBMC. The B-B7 cell marker revealed 22.4+/-10.0% of reactive camel PBMC while the CD45 leukocyte common antigen was identified only on 19.4+/-3.1% of camel PBMC as compared to 74.7+/-4.9% for bovine PBMC. IgM (PIg45A) was detected on 9.1+/-1.4% of camel PBMC and on 46.6+/-19.5% of the bovine PBMC. Double fluorescent labeling with two B-cell markers and an anti-ruminant lambda light-chain mAb revealed 7-9% of cells bearing both B and lambda L-chain markers. Light chain reactivity was also assessed using an anti-goat F(ab')(2) antiserum. The values obtained, 14.3+/-5.8% for the camel and 47.8+/-2.7% for the cattle, are close to the values observed for surface IgM. These data suggest that camels, like other ruminants, possess L-chain bearing cells of the B-cell lineage. However, in the camel, Igs are different in that in addition to regular four chain Igs, about 65% of them possess two heavy chain Igs devoid of light chains. Because different sets of V(H) gene segments are used by four and two chain Igs, it is possible that there might be two lineages of B-cells each secreting a different form of antibodies.

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.

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.

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.

Effect of permeabilization on peripheral blood lymphocytes of bovine leukemia virus (BLV)-infected cattle.

Cell surface proteins serve as markers for immunophenotypic characterization of lymphocyte subsets by appropriate monoclonal antibodies and fluorescence-activated cell sorter (FACS) analysis. By the same method, internal antigens or those that are only partially expressed on the cell surface can be determined after permeabilization of the cells. Peripheral blood lymphocytes obtained from bovine leukemia virus (BLV)-infected cattle and from BLV-free cattle were permeabilized and several lymphocyte populations were examined. BoCD4, BoCD8 and three CD4 CD8-T-cell subsets retained their original frequencies after permeabilization in both groups of animals. The recognition of the B-B2 lymphocyte molecule was only partially expressed on the cell surface of intact lymphocytes and was further revealed on permeabilization. The frequency of permeabilized, but not intact, cells stained with this mAb was significantly higher for BLV-infected cattle than for BLV-free animals (P = 0.006). Reactivities of an anti-heat shock protein (Hsp) 70 were measured before and after permeabilization of PBLs. Similar increased cell frequencies were obtained for both groups of bovines. These data indicate that flow cytometry studies should be conducted on both permeabilized and intact cells for a better assessment of protein expression on the cell surface, as well as in the cytoplasm.

Levels and role of cytokines in bovine leukemia virus (BLV) infection.

Bovine leukemia virus belongs to a small subfamily of exogenous retroviruses that includes the human retroviruses HTLV-1, HTLV-II and the simian virus, STLV-1. Like other retroviruses, infection with BLV results in deregulation of the host immune system at both humoral and cellular levels. An approach which might help in the elucidation of some immune impairment phenomena is the investigation of the role that cytokines play in the pathogenesis and immune response of BLV infected animals. Here we describe our findings on IL-6 and TNF. We have found that the levels of IL-6 in the sera of BLV infected cows which show persistent lymphocytosis (BLV+ PL+) were significantly higher than those of BLV infected with no lymphocytosis (BLV+ PL-) or BLV negative cows (BLV-). The same results were obtained by measuring the spontaneous production of IL-6 in peripheral blood mononuclear cells (PBMC). Furthermore, PBMC derived from BLV+PL+ cows secrete higher levels of IL-6 and TNF alpha than those derived from BLV+PL- and BLV- ones following in vitro exposure to the BLV gp51 antigen, bacterial endotoxins (LPS) and ConA. Similar results were obtained when supernatants from stimulated adherent (monocytes, macrophages) and non-adherent cells (B and T lymphocytes) were tested. When exogenous IL-6 and TNF alpha were added to BLV infected cells in vitro, the expression of viral antigens was strongly suppressed. Thus, the possibility exists that the elevated production of IL-6 and even more than that of TNF alpha play a role as contributing factors to the latency of the clinical expression in BLV infection.

A new retroviral AIDS-like disease and/or acute leukemia induced in mice by alloimmune stimuli.

We have shown a new phenomenon demonstrating that BALB/c female mice mated to C57BL/6 males during a year (7-10 pregnancies) develop AIDS-like disease or acute leukemia after an additional immunization with fixed ConA activated paternal (C57BL/6) lymphocytes. The AIDS-like disease is sexually and vertically transmissible and easily transferable to intact BALB/c and C57BL/6 mice by filtered plasma of affected animals.

A new transmissible AIDS-like disease in mice induced by alloimmune stimuli.

The search for a suitable and reliable animal model for human AIDS that is easy to use on a large scale continues. Here we describe a new condition in mice that closely resembles human AIDS, namely, chronic lymphoproliferation with dramatic depletion of CD4-positive cells, progressive impairment of the immune responses, and Kaposi's sarcoma-like tumors or terminal B-lymphomas. The AIDS-like disease was primarily induced by mating BALB/c female mice to C57BL/6 males during a 1-year period (7-10 allogeneic pregnancies) followed by immunization with paternal lymphocytes. The disease is sexually and vertically transmissible, transferrable by cell-free plasma and is associated with autoimmune reactions to major histocompatibility complex antigens and CD4 cells. We hope that this becomes a model for studying the mechanisms of AIDS immunopathogenesis and immune-based treatment approaches.

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.

gamma delta T-lymphocytes and anti-heat shock protein reactivity in bovine leukemia virus infected cattle.

Bovine leukemia virus (BLV) induces a chronic infection in cattle that may result in persistent lymphocytosis (PL) and, sometimes, enzootic bovine leukosis. The cellular and humoral immune responses of the host following infection have been extensively investigated but little is known about the involvement of gamma delta T-cells in BLV pathogenesis. The affluence of these cells in cattle, and particularly in the peripheral blood of young ruminants, may suggest a particular role for them in defense mechanisms. In this study we have examined circulating gamma delta lymphocytes that express workshop clusters 1 (WC1) and 2 (WC2). In healthy cattle the WC1 cell count tends to decrease with age and adult cattle blood has statistically lower numbers (19.0 +/- 6.6%) than that of young animals (40.1 +/- 7.2%). However, in the blood of BLV-seropositive adult cattle and mainly in BLV+ PL+ animals the population of WC1 cells is elevated compared with uninfected animals (P < 0.007). Likewise, the WC2 cells count is increased (P < 0.01) in BLV+PL+. Furthermore, we have investigated whether BLV infection up-regulates the expression of heat shock proteins (HSP) which in turn could augment the humoral response. Anti-HSP70 activity was examined in the sera of 34 BLV-infected cattle and 40 healthy controls by ELISA. Significantly higher activities (P < 0.001) were observed in BLV-infected cattle.

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.

Foot and mouth disease virus replication in bovine skin Langerhans cells under in vitro conditions detected by RT-PCR.

The replication of foot and mouth disease virus (FMDV) was studied in isolated bovine skin Langerhans cells (LC), in keratinocytes from epidermal cell suspension, and in migrating LC obtained from cultured bovine epidermal sheets in vitro. Viral RNA replication in infected cells was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) of the negative FMDV RNA strand and by the plaque forming assay of FMDV. It was established that bovine skin LC, keratinocytes, and migratory bovine LC infected with FMDV strain 01 Geshur supported virus replication. This RT-PCR method to detect the negative strand of FMDV RNA in migratory bovine skin LC may be useful for determining FMD virus replication in tissue cells.

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.

Circulating immune complexes in bovine leukemia virus (BLV)-infected cattle.

Circulating immune complexes (ICs) were detected in the sera of bovine leukemia virus (BLV)-seropositive cattle. Immune complexes were precipitated in 2.5% polyethylene glycol (PEG) and further dissociated. Bovine leukemia virus antigens, IgG and IgM molecules were detected after solubilization in the presence of sodium dodecyl sulphate, and quantitated by enzyme-linked immunosorbent assay (ELISA) assays. Mean values of IgG and IgM in BLV-containing ICs did not significantly differ from those obtained from ICs originating from BLV-seronegative animals. However, differences were found in the composition of ICs from older BLV-positive animals as compared to those obtained from young animals. The ratio of IgG/IgM was 5.02 in animals aged 5-10 years, while this ratio was 11.66 in animals of less than 5 years of age and 10.19 in controls. This might indicate a possible increase in the contribution of IgM molecules to the structural composition of ICs in BLV-infected cattle as related to age or stage of infection.

A lactogenic-immune-deficiency-syndrome in cows: unexplained phenomenon.

The majority of adult cows in a certain dairy herd, were found to have very low levels of immunoglobulins (Igs) in their colostrum. This phenomenon was defined by us as Lactogenic-Immune-Deficiency-Syndrome (LIDS). The mean IgG levels were 44.5 and 57.2 mg ml-1 respectively (on two different occasions) as compared to that of a control group which was 103.4 mg ml-1. The levels of Igs in the colostra of heifers from the same herd were found to be higher than those of adult cows. The degree of LIDS was found to be closely related to the age of cows in the herd. The low levels of Igs in the colostra were not directly linked to their concentrations in the sera of the affected cows. The relatively low amount of IgA in the affected colostra suggests that the local production in the lymph tissue associated with the mammary glands is impaired as well. In order to investigate the etiology of the phenomenon, tests were carried out to reveal whether bovine leucosis virus (BLV) infection or immune complexes were involved in the pathogenesis of LIDS. The results were negative. The etiology of LIDS remains for the time being unknown.

Immunogenicity and allergenicity of Culicoides imicola (Diptera: Ceratopogonidae) extracts.

Summer seasonal recurrent dermatitis (SSRD) or "sweet itch" is a seasonally occurring allergic dermatitis of horses provoked by biting midges. The allergic skin reactions have been attributed to allergens present in various Culicoides species. C. imicola is the suspected etiological agent of SSRD in Israel. Whole body extracts of this midge induced hypersensitivity reactions upon injection into susceptible horses and in this study attempts were made to define components of C. imicola which have immunogenic and allergenic properties. Immunogenic potency was evaluated by raising antisera to whole body extracts of C. imicola in rabbits and examining their reactivity towards fractionated extracts. Allergenic potency was examined by reacting fractionated extracts with horse sera. Humoral reactivity of susceptible and non susceptible horses was assayed by specific IgE and IgG ELISAs. Although there are many antigenic components in whole body extracts of C.imicola capable of eliciting an immune response, no conclusive evidence was obtained indicating that allergic reactivity was associated with increased IgE levels of defined specificity.

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.