A novel cell surface proliferation-associated marker expressed on T cells and up-regulated on germinal center B cells.

In this study we present data on a novel cell surface antigen recognized by monoclonal antibody (mAb) VPM30, originally thought to recognize only bovine and ovine sIg+ B cells from peripheral blood. Here we show that the antigen, molecular mass 28 kDa, is not only found in B cell follicles in frozen sections, but when used on paraffin sections VPM30 specifically stains B cells in the light zone of germinal centers but not in the mantle or dark zones. In addition we show that the antigen is also expressed by 90% of T cells after activation, with kinetics of antigen expression mirroring those of proliferation. By both size and distribution, the antigen appears to be novel, corresponding to no known cluster of differentiation, and will be of great use in the study of ruminant cellular immune responses.

A novel expression vector for transfection of bovine MHC class I genes.

A vector is described for the expression of genomic or cDNA copies of bovine major histocompatibility complex (MHC) class I genes in transfected mouse Ltk- cells. Class I gene fragments are amplified by the polymerase chain reaction, using primers in conserved parts of exon 2 and the 3'-untranslated region of the gene. Amplified class I gene fragments can then be subcloned into the expression vector, pBoLA-21, which contains the necessary 5'- and 3'-sequences for correct expression. The vector was tested by subcloning and expressing genomic and cDNA clones.

Generation and characterisation of bovine antigen-specific T cell lines.

15 antigen-specific T cell lines have been generated from eight individual cattle immunised with ovalbumin. Several sources of interleukin-2 (IL-2) were used, including a supernatant from a gibbon cell line (MLA-Sup), human recombinant IL-2 (hrIL-2) and bovine recombinant IL-2 (brIL-2). These IL-2 sources were used alternately with autologous peripheral blood mononuclear cells (PBM) together with ovalbumin to generate the lines. They grew least well in MLA-Sup and best in brIL-2. FACS analysis indicated that the lines generated with the recombinant IL-2s were extremely homogeneous in that the majority of cells were BoCD4+ (bovine CD4 equivalent) and therefore of TH phenotype. The lines were antigen specific and responded to antigen only in the presence of autologous PBM and not allogeneic (MHC class I nonidentical) PBM. However, allogeneic PBM did support their proliferation to ConA. No MLR response was observed by the cell lines to allogeneic PBM. The response to antigen was inhibited by anti bovine class II mAbs but not an anti bovine class I mAb. The subpopulation of PBM which acted as antigen presenting cells for these bovine TH cell lines had typical macrophage characteristics.

Qualitative variation in the immune response to ovarian follicular fluid proteins in cattle.

In two experiments 44 heifers were immunised with inhibin preparations of partially purified ovarian follicular fluid (PPFF) of ovine, porcine and equine origin in non-ulcerative Freund's adjuvant. In the first experiment 20 cattle were immunised with ovine PPFF1. In the second, a further 24 were immunised with either a different sheep PPFF (ovine PPFF2) or PPFF from pigs or horses. Large molecules of over 30 kDa initiated a strong immune response in some animals, but none in others, although there was no obvious relationship with the MHC Class I (BoLA) phenotype. Immunisation with ovine partly purified follicular fluid affected ovulation rate in some cattle, and only these cows produced detectable antibodies to an approximately 40 kDa protein. These data show that the immunological response of cattle to a mixture of antigens is highly variable between individuals and may explain the variation in the ovarian response to immunological manipulations of ovulation rate seen in this species.

A stage-specific, parasite-induced, "window" of in vivo interferon-gamma production is associated with pathogenesis in Theileria annulata infection.

The tick-borne protozoan parasite Theileria annulata causes tropical theileriosis, a severe leukoproliferative disease of cattle, which naive susceptible animals fail to control. The parasite infects and transforms macrophages, developing in the local draining lymph node. IFN gamma has been shown to block parasite development in newly infected cells, and inhibits the growth of fully differentiated macroschizont stage-infected cells in vitro. However, the parasite has been found to specifically induce IFN gamma production by T cells and appears to flourish in the face of this T cell-derived response in vivo. Here we show that the production of IFN gamma in vivo is tightly controlled by the parasite. Induction of cytokine production by T cells is not initiated until the parasite has developed beyond the IFN gamma sensitive trophozoite stage. Cytokine production is kept high as infected macrophages develop, and IFN gamma appears to play an active role in maintaining the growth of these cells. Once the infection is fully established, IFN gamma is down regulated, avoiding potential inhibitory effects. Thus by controlling T cell IFN gamma production, the parasite induces a "window" of cytokine expression which promotes its own growth, but avoids potential inhibitory effects of the cytokine.

Anti-lymphocyte antibodies generated in animals immunised with Theileria annulata-infected cells.

Complement fixing antibodies were measured in sera from animals immunised with either Theileria annulata sporozoites or autologous or allogeneic schizont-infected mononuclear cells using a complement-mediated micro-cytotoxicity test. The test demonstrated the presence of anti-lymphocyte antibodies in allogeneic cell-immunised animals, which were not detectable in autologous cell- or sporozoite-immunised animals; also that these antibodies were directed to T. annulata-infected and (MHC) class I antigens. Their potential importance in repeated immunisations is discussed.

Possible association of antibody responses to human serum albumin and (T,G)-A--L with the bovine major histocompatibility complex (BoLA).

Antibody responses to human serum albumin (HSA) and (T,G)-A--L were determined in 130 young bulls in Norway and the BoLA types of the bulls were defined. Significant associations of some BoLA antigens with immune responsiveness were shown, indicating the likely existence of an immune response (Ir) region linked to the BoLA class I antigens. High response to HSA seems to be a dominant trait. BoLA w2 showed an association with low response to HSA. This may reflect the effect of a specific MHC-associated immune suppressor gene.

Studies on the major histocompatibility complex of indigenous cattle in the Ivory Coast.

The MHC (BoLA) type has been determined for cattle from three breeds in West Africa. Seventy Baoule, 50 N'Dama and 30 Zebu cattle from the centre and north of the Ivory Coast were tested. Lymphocytes from these cattle were tested in a lymphocytotoxicity test with alloantisera detecting all of the internationally recognised BoLA sera. 78 sera prepared in Edinburgh and 57 in Jouy-en-Josas were used in the study. The results showed that sera prepared in Europe detect similar specificities in West Africa. Although with some specificities the frequencies differ from those seen in Europe. The frequency of null alleles is higher than in Europe in the Ndama and Zebu animals indicating the existence of additional specificities which will require the production of alloantisera in these breeds. However in the Baoule the null allele frequency is lower even then in some European breeds. The population data in which no animals have more than two workshop specificities is consistent with a single locus control in West Africa as in Europe.

Common and stage-specific antigens of Theileria annulata.

Western blot analysis of Theileria annulata antigens was carried out using sera collected from cattle which had been immunised and challenged with either T. annulata sporozoites or schizont-infected cells. Three antigens between 71 and 73 kDa proved to be common to the three stages of parasite studied: sporozoites, schizonts and piroplasms. An antigen was found at 32 kDa which was specific to T. annulata piroplasms. Results were reproducible using sera from Morocco and the UK. At least one of the proteins at 71-73 kDa, but not that at 32 kDa were also recognised by sera from animals infected with Babesia species.

Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva.

Infection and transformation of cells of the bovine immune system by Theileria annulata and T. parva were compared. Preliminary experiments with mammary gland macrophages indicated that they were permissive to infection by T. annulata but only to a limited extent by T. parva. Further experiments involved several purified subpopulations of bovine cells including bovine monocytes, T cells and MHC class II positive and negative populations. These subpopulations were incubated with T. annulata or T. parva sporozoites in limiting dilution cultures. T. annulata preferentially infected macrophage type cells and also MHC class II positive cells, whereas the frequency of MHC class II negative cells infected by this parasite was negligible. T cells also showed a very low level of infection. In complete contrast, T. parva preferentially infected T cells and did not infect cells phenotypically defined as monocytes at all. These results suggested that class II expression was necessary for T. annulata infection and not necessary for, though not a barrier to T. parva infection. T. annulata infected cell lines all expressed class II molecules to varying degrees. Other available phenotypic markers were only expressed at very low levels or no longer expressed. The immunological significance of the different cell preferences and phenotypes of infected cell lines of T. annulata and T. parva is discussed.

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line.

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.

Factors influencing the transmission and incidence of tropical theileriosis (Theileria annulata infection of cattle) in Morocco.

A longitudinal epidemiological investigation of tropical theileriosis was carried out in an endemic region of Morocco during the 1991 disease season. This involved approximately 220 cross-bred cattle on 15 farms. Data were collected on the frequency of existing infections, the size of the vector tick population and the incidences of new infections and clinical disease, and these were then analysed using statistical models. The prevalence of subclinically infected carriers of Theileria annulata, the number of adult Hyalomma detritum, the vector, and the probability of becoming newly infected with T. annulata increased significantly with the age of cattle, although the age effect on new infections may be a result of increased tick numbers on older animals. The probability of clinical disease in newly infected cattle was not significantly influenced by age or by the number of adult ticks, but was significantly positively associated with the cattle population on the farm. The number of H. detritum nymphs counted on cattle in the autumn was related significantly to the previous adult tick count on the same animal.

The effect of dose and line on immunisation of cattle with lymphoblastoid cells infected with Theileria annulata.

Thirty-one Friesian calves in Morocco susceptible to tropical theileriosis were protected against a lethal sporozoite challenge by prior infection with lymphoblastoid cell lines infected and transformed in vitro by a Moroccan stock of Theileria annulata. The challenge infection of cryopreserved sporozoites killed all four susceptible control calves within 20 days. Four schizont-infected cell cultures at Passage 3 were inoculated at four different doses, 10(8), 10(6), 10(4) and 10(2), into pairs of calves. The recipient animals showed great variation in severity of disease symptoms, which did not show a linear correlation with the cell dose inoculated. The most severe disease symptoms were recorded, prior to challenge, in the 10(2) cell dose recipients; one animal died of acute theileriosis and another had to be treated. One of the four cell lines used was more virulent than the other three. Two years after the completion of this experiment, immunised animals have shown normal productivity traits.

Requirement for MHC class II positive accessory cells in an antigen specific bovine T cell response.

Purified populations of bovine antigen presenting cells (APCs) and T cells have been isolated from peripheral blood and characterised using various monoclonal antibodies (mAbs) for cell surface markers. Bovine APCs were found in an adherent cell fraction and were non-specific esterase positive, phagocytic and expressed bovine major histocompatibility complex (MHC) class II determinants, all of which are typical macrophage characteristics. T cells were rigorously depleted of accessory cell function before being used in an antigen presenting cell assay. The generation of T helper cells in response to the soluble antigen, ovalbumin, was entirely dependent upon a critical number of APCs. Further the proliferative response was inhibited by several mAbs to bovine MHC class II molecules. Thus the interaction between bovine APCs and helper/inducer T lymphocytes (TH/I) appears to be similar to that in other species.

Occurrence of cytotoxic antilymphocyte antibodies in sheep.

An earlier report that there is a high incidence (50 to 60 per cent) of cytotoxic antilymphocyte antibody in the sera of parous ewes is confirmed. This antibody is directed against paternally inherited antigens of the fetus. Antibody could be detected pre partum in four out of 34 primiparous ewes. However, there was no strong association between parity and the incidence of antibody. Antibody was also found in three out of 10 older rams which implies that pregnancy is not the only stimulus for antibody production.

Quantitative studies on bovine immunoglobulins, normal plasma levels of IgG2, IgG1, IgM and IgA.

Using monospecific sheep antisera, the mean plasma immunoglobulin concentrations in 82 Ayrshire milking cows were found, by the single radial diffusion method, to be: IgG2 10-91 plus or minus 4-30, IgG1 9-76 plus or minus 2-59 and IgM 2-71 plus or minus 1-22 mg/ml (plus or minus SD). IgA levels in the same group of 82 cows were determined by a simple, sensitive coated tube radioimmunoassay and had a mean value of 0-32 plus or minus 0-16 mg/ml. The frequency distributions of IgG1, IgM and IgA were approximately log normal, whereas that of IgG2 was bimodal with two populations with means of approximately 7 and 14 mg/ml. An 18-fold difference was found between high and low IgG2 levels whereas only three- to four-fold differences were recorded for IgG1 levels in the same population. Eight-fold differences were found for IgM and IgA levels. The effect of age (2-9 years) on sampling could be disregarded for IgG1, IgG, IgM and IgA levels, but cows above 6 years of age had significantly higher IgG2 levels.

In vitro infection of bovine alloreactive cytotoxic T cell lines with sporozoites of Theileria annulata and T parva.

Bovine alloreactive cytotoxic lymphocyte (CTL) lines of known target specificity were infected in vitro with sporozoites of Theileria annulata and T parva and cultured in limiting dilution. The phenotypes of the CTL lines both pre- and post infection were assessed using a panel of monoclonal antibodies specific for defined bovine lymphocyte subpopulations. The effector function of the resultant infected cell lines was determined using a Cr51 release assay and compared to the uninfected control CTL line. The results indicated that T parva sporozoites consistently infected and transformed the CTL lines very efficiently even at the lowest cell doses. In contrast the T annulata sporozoites were largely unable to infect and transform the alloreactive CTL except at the very highest cell and sporozoite doses. A factor which appeared to influence susceptibility to T annulata infection was an increased level of class II expression on the CTL line. None of the cell lines showed cytotoxic effector function after infection with either T annulata or T parva sporozoites.

Use of bovine lymphocyte antigens in diagnosis of parentage.

Lymphocyte antigens controlled by alleles at the BoLA-A locus were used to indicate which of two bulls could be eliminated as the sire in 18 cases of disputed parentage. The use of bovine lymphocyte antigens (BoLA) significantly increased the exclusion probability over that of the standard red cell and electrophoretic techniques.

Quantitative studies on bovine immunoglobulins.

The influence of serum immunoglobulins on the incidence of calf pneumonia, and the relationship between sub-clinical pneumonia, and calf serum Ig levels, is discussed. Monospecific antisera were used to measure levels of IgG1, IgG2, IgM and IgA in the sera of bull calves aged around 2 1/2 weeks. These calves were selected retrospectively according to their disease record up to six months of age. A clear association was found between low levels of IgG1, IgG2 and IgA in the "2 1/2-week" sample and subsequent susceptibility to pneumonia at around 2 1/2 months of age. Calves showing signs of pneumonia had low levels of IgG1 (45-5 per cent had less than 8 mg per ml compared with "now-pneumonic" calves which had relatively high levels (only 9.5 per cent had less than 8 mg per ml). In contrast, older calves bled at monthly intervals and found to have pneumonic lesions at slaughter had significantly evevated levels of both IgGa and IgG2.

Culling and wastage in dairy cows in East Anglia.

A culling survey in 80 Friesian dairy herds in East Anglia over four years investigated the age at, and reasons for, disposal or death. Failure to conceive was the most important reason except in very old animals. Low production was the second most common reason, culling being particularly heavy in the first two lactations. Mastitis was the major disease influencing wastage and increased with age until the sixth lactation. The percentage of culls for multiple reasons increased with age. There was a high proportion of young animals in most herds and the median lactating life of cows was about three years, equivalent to three lactations. Considerable variation in herd life demonstrated that there was substantial scope for improving herd longevity. Long living herds culled fewer animals for breeding problems in the early lactations and for mastitis in the later ones, enabling more animals to be culled as surplus and for production factors.