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.

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.

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.

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.

IgM rheumatoid factor in cattle immunized against babesiosis.

Polyclonal bovine IgM-rheumatoid factors (IgM-RFs) were examined in sera of cattle immunized against babesiosis. An enzyme-linked immunosorbent assay (ELISA) was used enabling rapid screening of serum samples. Results obtained indicate a rise of serum IgM-RF levels with age in healthy bovines. However when animals of similar age and pertaining to the same herd were examined, levels of serum IgM-RF exhibited a wide distribution range. Mean values in 60 sera of 2 yrs old clinically healthy heifers originating from a single herd were of 452.60 +/- 201.26 e.u. at 1 in 1,000 serum dilution and of 202.37 +/- 137.86 e.u. at 1 in 4,000 serum dilution. In a herd where repeated vaccination of dams against babesiosis was carried out 3 to 6 weeks before delivery either with live Babesia bovis parasites or soluble antigens of in vitro grown organisms, no significant differences in mean IgM-RF values were found between revaccinated animals and a control group. Nor did the mean values of serum IgM-RF of calves born to the respective groups of dams exhibit significant differences. It thus appears that immunization of healthy cattle against this parasite does not affect mean serum IgM-RF levels.

Immunoglobulin M (IgM) indirect enzyme-linked immunosorbent assay and the involvement of IgM-rheumatoid factor in the serodiagnosis of BHV-1 infection.

An IgM enzyme-linked immunosorbent assay (IgM-ELISA) for the detection of antibodies to bovine herpesvirus-1 (BHV-1) was developed. Its applicability was examined by serological studies in two calves experimentally infected with virulent BHV-1 over a period of 60 days. IgM antibodies were detected by ELISA on day 6 after infection, and there was an increase in IgG antibodies on day 9. Serum neutralizing (SN) antibodies were detected only on day 13, confirming the higher sensitivity of the ELISAs. A similar study of four calves treated with a commercial inactivated virus vaccine indicated no detectable IgM-ELISA response, and late SN and IgG-ELISA reactivity. Thus IgM-ELISA appears to be of value in assessing recent infection, whereas IgG-ELISA and SN cannot distinguish between infection and vaccination. The possible limitations imposed on the specific IgM-ELISA by the presence of IgM rheumatoid factor (IgM-RF) in bovine serum were examined. IgM-RF levels were determined in bovines of various ages. Elevated values of IgM-RF were found in the sera of older animals; their occurrence may lead to false IgM-positive diagnosis (16%) of BHV-1 infection. This was examined in 113 serum specimens from suspected cases of BHV-1 infection and in 32 bulls at an insemination center. Pretreatment of serum samples with an antibovine IgG serum eliminated false positivity of the IgM-ELISA. It is concluded that IgM-ELISA should be of particular value in the diagnosis of recent infection with BHV-1, mainly in calves.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale.

An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivity was obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishable in both tests from those produced following vaccination with A. centrale.

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.

Identification and partial purification of soluble antigens from culture-grown Besnoitia besnoiti endozoites.

Soluble antigens from culture-grown Besnoitia besnoiti endozoites were identified following their partial purification by affinity chromatography. A specific eluate obtained after affinity chromatography on a column to which antibodies from serum of a naturally infected cow were bound exhibited seven polypeptide bands on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Five bands were observed in the eluate from an immunoadsorbent to which antibodies from an experimentally infected calf were coupled. Eluted antigens were reactive in the enzyme-linked immunosorbent assay (ELISA). Reactivity of electrophoretically resolved antigens with sera of endozoite-inoculated cattle and sera from field cases of besnoitiosis were studied using the Western immunoblotting technique.

Soluble antigens from culture-grown Besnoitia besnoiti endozoites.

Soluble antigens from Besnoitia besnoiti cell culture-grown endozoites, obtained either by hypotonic lysis or by freeze-thawing and ultrasonication (FTS) of the organisms, were detected by the agar gel immunodiffusion method. Each antigenic preparation yielded 1-4 precipitin lines when reacted with the corresponding rabbit hyperimmune serum, while no reaction was observed with Besnoitia-positive sera from naturally infected cattle. Soluble exoantigens released by viable Besnoitia endozoites into the supernatant of infected cell cultures formed two precipitin lines with rabbit anti-FTS hyperimmune serum and appeared as positively charged protein in immunoelectrophoresis. The precipitin lines observed were parasite specific since no reaction occurred with either lysates of normal Vero cells or with supernatants from non-infected cell cultures.

ELISA test for the serodiagnosis of Akabane virus infection in cattle.

A simple ELISA test has been developed for the detection of IgG and IgM antibodies to Akabane virus in bovine serum. The test is specific and its sensitivity higher than the serum neutralisation assay. Detection of IgM antibodies can serve as a rapid method of diagnosing primary infection with Akabane virus. The superiority of ELISA resides mainly in the rapidity of performance and that it can be performed with inactivated reagents at high dilutions of serum samples. Thus it might enable the surveillance of spread of infection in zones prone to be affected by insect-borne viral diseases.

Reactivity of IgM-rheumatoid factors of human, feline and bovine sera.

The reactions of IgM-rheumatoid factors (IgM-RFs) from human, feline and bovine sera were examined with their homologous monomeric IgGs and with IgGs of other species. Cross-reactivities with IgGs of other species were observed in each separate system. By the use of an enzyme-linked immunosorbent assay (ELISA), measurable differences in reactivities were obtained. The results suggest that IgM--RFs of human and feline origin recognize a similar determinant on the Fc region of IgG. A different moiety on the latter molecule appears to be involved in the reactivity of bovine IgM-RF.

Humoral immune response of asymptomatic cats naturally infected with feline leukemia virus.

The humoral immune response of cats that were naturally infected with the feline leukemia virus (FeLV) was examined after antigenic stimulation with the synthetic antigen poly(L-Tyr, L-Glu)-poly(DL-Ala)-poly(L-Lys). The primary humoral antibody response in FeLV-infected cats was both delayed and greatly reduced, compared with that seen in uninfected control cats. A similar discordance was observed after secondary stimulation with the antigen, in the FeLV-infected cats had both a delayed response and a reduced response, compared with uninfected cats. The levels of total immunoglobulins of the immunoglobulin G and immunoglobulin M classes in the sera of FeLV-infected cats were significantly higher (two- and threefold, respectively) than were those of the uninfected control animals. The presence of an impaired humoral immune response to newly presented antigens in the presence of elevated immunoglobulin levels has been thoroughly documented in the case of people with the acquired immunodeficiency syndrome. This further emphasizes the potential value of FeLV-infected cats as a model for human acquired immunodeficiency syndrome.