Roger Spooner

Summary   Theileria annulata infection (tropical theileriosis) is a disease of serious economic importance in Morocco. Epidemiological and vaccination studies have been carried out for many years in Doukkala, a region endemic for theileriosis. A vaccine based on schizont infected cells has been used extensively both experimentally and in the field. In Doukkala it was used for 4 years in the

The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4-6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.

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.

ABSTRACT Homozygous transferrins of adult cattle are made up of two strong pairs and one weak pair of protein bands on starch gel electrophoresis. Foetal transferrins have only the slower band of each pair with the fastest band of the three being much stronger than in the adult type. Before term the second band of each pair begins to develop and at the same time the fastest pair becomes weaker – attaining the adult type by term or soon after. The ai protein, which is present in early foetal life and almost disappears by 250 days of embryonic development, shows individual variation. Its relationship to fetuin is discussed.

This paper presents evidence to show that high titre antilymphocyte sera can be prepared more easily by skin grafting than from parous sera. It also shows that it is possible to analyse complex antilymphocyte sera by absorption with lymphocytes to produce working reagents. Following the 1 International BoLA Workshop the genotype of some of the animals used for skin grafting was ascertained and it was found that in each case where the resulting serum had been studied the main antiserum specificity that had been produced reacted with one of the BoLA specificities present in the skin donor.

The single-banding components of bovine transferrin from animals homozygous for the four transferrin variants found in the U.K. were isolated. Sedimentation equilibrium ultracentrifugation and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the bands of a single variant have molecular weights of 77500 and 73300 respectively. The different bands of a single variant and single bands of different variants show no evidence of size heterogeneity or of low-molecular-weight peptides being split off after reduction in 6m-guanidine hydrochloride. The two slower bands of a single variant, which both contain 2 molecules of sialic acid/molecule of protein, have the same molecular weight and amino acid composition, and give identical peptide ;maps', although differences in composition and peptide ;maps' occur between the different variants. The results support the concept that bovine transferrin is essentially a single polypeptide chain, but they do not explain dif...

Bovine peripheral blood mononuclear cells (PBMC) were labelled with monoclonal antibodies recognizing bovine MHC class II, sIgM, monocyte, T-helper and T-cytotoxic cell phenotypes. They were sorted into positive and negative populations with a fluorescence-activated cell sorter (FACS). The cell populations were infected in vitro with sporozoites of either Theileria annulata or T. parva, and the degree of infection and transformation determined. The results showed that despite the many similarities between these two parasites, they infected different cells of the immune system. T. annulata preferentially infected MHC class II-positive cells but did not infect T cells. Monocytes were infected very efficiently by T. annulata but were uninfectable with T. parva. B cells were infected much more efficiently by T. annulata than T. parva. Cell lines derived from infections with T. annulata were analysed phenotypically. Virtually all reactivity was lost for the anti-sIgM and the anti-monocyt...

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 effe...

A putative synthetic vaccine for foot-and-mouth disease (FMDV15) has proved less successful in a host species, cattle, than predicted by results in a small-animal model. Possible reasons for this include non-recognition by T cells influenced by major histocompatibility complex (MHC)-linked immune response gene control. It is now possible to type for human leucocyte antigen (HLA) DR-like bovine MHC (BoLA) class II polymorphisms with a one-dimensional isoelectric focusing (IEF) technique. Using this method 14 unrelated cattle were selected with eight different BoLA class II IEF types. After immunization with FMDV15, 13 cattle generated a T-cell response to FMDV15. However, the fine specificity and magnitude of the response was related to BoLA class II type. The non-response by one animal and low response by two other animals were associated with two of the BoLA class II types. Response to the region 149-158 was immunodominant and animals which did not respond to this region had low re...

An enzyme-linked immunosorbent assay (ELISA) using sporozoite, schizont and piroplasm antigens was developed to study the immune response of animals that had been immunised with either Theileria annulata sporozoites or schizont-infected cells and then challenged with sporozoites. The aim was to identify the most suitable antigen for a routine screening test and to compare the sensitivity of the latter with that of the indirect fluorescent antibody test (IFAT). As determined by ELISA, cattle produced antibodies to all three antigens, regardless of the method of immunisation. The schizont antigen was the least sensitive, whereas the sporozoite antigen displayed high pre-inoculation values. In contrast, the piroplasm antigen exhibited low non-specific pre-infection levels and high post-immunisation and post-challenge values according to both ELISA and IFAT. Therefore, the latter was though to be the most appropriate antigen for use in ELISA.

The results and agreements of the 1 international BoLA workshop, held in Edinburgh, Scotland in August 1978, are reported. Most of these concern the results from a comparison test of 249 alloantisera to bovine lymphocytes, the antisera being contributed by 9 laboratories. These sera were compared directly in Edinburgh on a panel of lymphocytes from 130 cattle of 21 breeds. In the microlymphocytotoxicity test used 75% of the sera reacted. Sixty eight of these sera were grouped into clusters according to their reaction patterns against the lymphocyte panel. Eleven of these clusters were clearly defined and were given workshop BoLA designations. In addition 22 sera were assigned to subgroups of the agreed clusters. There was no evidence that the method of production of the sera had any effect on their specificity. Although genetic data was not available, the phenotypes of the test panel of lymphocytes are consistent with the clusters detecting antigens controlled by multiple alleles at...

Recovery of calves from tropical theileriosis was accompanied by the disappearance of macroschizonts from lymph nodes and the appearance of cytotoxic cells in the blood and lymph nodes. Acute, fatal disease was associated with incremental parasitosis and parasitaemia and, in general, an absence of detectable cytotoxic cells in the blood or lymph nodes. After recovery from infection, calves were resistant to challenge. Challenge with sporozoites was followed sometimes by an immediate reappearance or by a later peak, or sometimes by twin peaks of cytotoxic cells but macroschizonts were not detected. Histocompatibility (BoLA) typing indicated that calves produced two sequential populations of cytotoxic cells during recovery from primary infection with Theileria annulata. The expression of lysis by the first appeared to be BoLA restricted. In contrast, both the peaks of lysis manifest after challenge appeared to be BoLA restricted. Results suggest that BoLA restricted cells are establis...

Alloreactive cytotoxic T cells (CTL) were generated in mixed lymphocyte culture against cells bearing subgroups of BoLA w6, as well as in BoLA w4, w10 and w16. Primary cultures were restimulated at weekly intervals with irradiated stimulator cells and tested in a 51Cr-release assay with target cells derived from Theileria annulata-infected cell lines. Generation of CTL was accelerated in animals that had been previously primed in vivo by skin grafting. CTL were generated that were specific for BoLA w6 subgroups and not w6. With w6.1 the specific killing was significant at the 5% level, and with w6.2, 6.3 and 6.4 it was significant at the 0.1% level. Where CTL were potentially generated against two BoLA-A locus allele products at the same time (i.e. with heterozygous stimulator cells), one of which was a w6 subgroup, there was similar CTL activity against both products when w6.4 and w16 or w6.1 and w4 were the combinations involved. In two generations against w10 and either w6.1 or w...

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.

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.

Summary   Theileria annulata infection (tropical theileriosis) is a disease of serious economic importance in Morocco. Epidemiological and vaccination studies have been carried out for many years in Doukkala, a region endemic for theileriosis. A vaccine based on schizont infected cells has been used extensively both experimentally and in the field. In Doukkala it was used for 4 years in the

"Exotic" European cattle are highly susceptible to T. annulata infection. In immunised animals, several effective anti-parasite responses can be demonstrated, such as anti-macroschizont cytotoxic T cells (CTL), and nitric oxide killing of parasites. The failure of infected animals to mount an effective primary immune response suggests that the presence of the parasite directly interferes with the development of immunity. When the activation pathways of CD4+ T cells in draining lymph nodes were examined during the course of a primary infection it was found that the development of this essential arm of the immune response was altered. Instead of interacting with antigen presenting cells in the paracortex, the majority of CD4+ T cells were rapidly activated by developing infected cells in the medulla of the node. Activation of T cells by infected cells also drastically alters the cytokines produced by the T cells. During effective immune responses, the principal cytokine involved appears to be IL-2, with only small, controlled "bursts" of IFN gamma production. However, IL-2 responsiveness is only transient in animals undergoing primary infection, while IFNg production is greatly elevated. IFN gamma does not appear to control parasitised cells, and may even aid the growth of infected macrophages--large numbers of macrophages enter the cell cycle during the peak period of IFN gamma production. Uncontrolled parasite-induced IFN gamma production is also likely to account for the local failure of antibody responses. Germinal centres in infected lymph nodes lose normal morphology, with IFN gamma sensitive zones failing to develop. A third strategy which the parasite uses to evade immune response destruction is through affecting CTL activity. CTL in infected draining lymph nodes lose expression of the adhesion molecule CD2--a molecule is essential in adherence to target cells for lysis. CD2- CTL are unable to lyse macroschizont infected cells.

A model for studying re-immunisation using skin grafting was developed as the allogeneic responses produced by T. annulata cell lines were similar to those produced by skin grafting. Appearance of schizonts and piroplasms post-immunisation was either delayed or prevented by already existing allogeneic responses. Isolation of parasite infected cell lines from lymph node biopsies and peripheral blood after cell line immunisation was also delayed or prevented by already existing allogeneic responses. Ability to isolate cell lines after immunisation correlated with protection i.e. if no parasite infected cell line of donor origin was isolated after immunisation, there was no protection. Allogeneic responses delayed or prevented the appearance of MHC I restricted parasite specific cytotoxic T lymphocytes post-immunisation. If the parasite transfer was prevented after immunisation; animals were fully susceptible to challenge. These experiments showed that allogeneic responses, generated in animals after immunisation with T. annulata schizont cell culture vaccine, can block parasite transfer and further development or enhancement of immunity against the parasite at the time of second immunisation with the same cell line. The observations are of immediate importance in endemic areas where T. annulata infected cell culture vaccines are being used. They are even more relevant in countries where animals are regularly moved between theileriosis free and endemic areas. It may not be advisable to re-immunise animals with the same cell line as that used for first vaccination.

Cattle immunised against Theileria annulata with one parasite strain have been found to be immune to re-challenge with different strains of the parasite. However, recent evidence of apparent strain specificity has been documented in cattle immunised with attenuated parasite-infected cells. In this study the strain specificity of major histocompatibility complex class I-restricted cytotoxic T-lymphocytes (CTL), a major anti-parasite effector mechanism, was examined. CTL generated following challenge with the Hissar (Indian) strain effectively lysed autologous cells infected with this strain of the parasite. However, CTL were less effective against cells infected with the Gharb (Moroccan) strain and showed virtually no reactivity against the Ankara (Turkish) strain, providing the first direct evidence for strain specificity in immune responses against T. annulata.

The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4-6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.

Two groups of animals were immunized with either 10(6) autologous or 10(6) allogeneic Theileria annulata-infected lymphoblastoid cells cultured in vitro. The development and specificity of cytotoxic cells generated in vivo were measured throughout immunization and challenge using a panel of target cells that were either Theileria-infected or uninfected blast cells of known bovine lymphocyte antigen (BoLA) specificities. After inoculation of the cell lines the two groups showed distinct differences in both their clinical responses and the target specificity of the cytotoxic cells detected. The allogeneic T. annulata cell line recipients showed a very mild clinical response, and on day 9 after inoculation a strong cytotoxic response was detected. The response appeared to be directed against the allogeneic major histocompatibility complex (MHC) antigens of the inoculated cell line in some form of graft rejection response. By day 23 the predominant cytotoxic response was directed against the recipient animals' own cells infected with the parasite. In contrast, the autologous T. annulata cell line recipients showed very severe clinical reactions, and low levels of cytotoxicity were detected. The cytotoxicity was directed against parasite-infected targets but did not appear to be MHC restricted until day 20. Both groups were immune to a heterologous sporozoite challenge that proved lethal to two susceptible control animals, and on day 10 after challenge a peak of cytotoxicity was detected which was directed against the autologous infected target cell. This would suggest that this cytotoxic response was MHC restricted and was also cross-reactive between the heterologous parasite stocks used.

Theileria annulata macroschizont-infected cell lines are successfully used as vaccines in several countries. The inoculated animals produce a strong allogeneic response against the MHC antigens of the immunizing cell line followed by an anti-parasite response. Immunity against the parasite wanes in the absence of challenge and re-immunization is sometimes recommended. However, it is not known if allogeneic responses generated by the first immunization with a T. annulata infected cell line will interfere with the boosting of immunity against the parasite at the time of re-immunization with the same cell line. Animals were primed against MHC antigens by skin grafting, followed by immunization with a T. annulata infected cell line prepared from the skin donor. A strong anti-MHC response was produced. This interfered with parasite transfer and the development of an anti-parasite immune response; the effect was more marked when a low vaccine cell dose was used. There was a negative correlation between the ease of isolating infected cells from the animals after cell line immunization, and the subsequent response to challenge. Where no cell lines could be isolated, the animals were fully susceptible to sporozoite challenge. These observations are of immediate importance in endemic areas where cell lines of T. annulata schizonts are being used as vaccines to control the disease.

Bovine mononuclear cell lines infected with the protozoan parasites Theileria annulata and T. parva have been studied with a panel of monoclonal antibodies reacting with bovine lymphocyte subpopulation markers. All infected lines are MHC class II positive, though the amount of class II antigen expressed varied between lines, and within individual lines there was variation in the proportion of positive cells from 100% with many, to less than 10%. All lines were negative for a macrophage/monocyte marker and for surface IgM. The T. parva lines tested were all positive for BoT4 or BoT8 or both, whereas T. annulata lines were uniformly negative for both of these markers. These results suggest that the two parasites preferentially infect different lymphocyte subpopulations.

Lymphoblastoid cell lines, infected and transformed in vitro by a Moroccan stock of Theileria annulata, infected and immunized susceptible taurine cattle, at cell doses of 10(8), 10(6), 10(4) and 10(2), regardless of whether the recipients were BoLA matched or mismatched to the donor cell line. The MHC relationship between the cell line and recipient did affect the severity of the clinical response to cell line immunization which may reflect differences in the specific priming of the immune response. At the highest cell doses the BoLA-mismatched recipients reacted more severely than the BoLA-matched. This study shows that, unlike the closely related parasite T. parva, there is no histocompatibility barrier to immunization using T. annulata-infected cell lines which could be achieved with as few as 10(2) allogeneic infected cells. The role of MHC compatibility between cell line and recipient in the priming of a protective immune response is discussed.

Using sera which defined the BoLA specificities at the two International BoLA workshops (Edinburgh, 1978 and Wageningen, 1980) and the European Regional workshop (Paris, 1979), 142 informative matings from 15 bulls have been studied. On the basis of this data, 11 of the 15 internationally agreed specificities and one of the regionally defined specificities behave as if controlled by alleles at a single autosomal locus. Data has not as yet been obtained for the other four internationally agreed specificities which are also believed to be at this locus. - The frequencies of 13 of the internationally agreed specificities and one of the regionally defined specificities have been studied for both sexes in one breed and for a single sex in another five breeds. The other two internationally agreed specificities are very recent and the populations have not been tested for them. The frequencies between sexes within a breed and within sexes between breeds differ significantly.

Polymorphism of expressed bovine major histocompatibility complex (MHC) class II DQB genes was investigated in a group of nine MHC-homozygous Kenya Boran cattle (Bos indicus). DQB second exon fragments were amplified by reverse transcription-polymerase chain reaction of total mononuclear cell RNA, cloned and sequenced. While a single DQB sequence was obtained from some animals, two DQB exon 2 sequences were found in others, implying expression of duplicated DQB genes. Two pairs of duplicated DQB genes were found in this group of homozygotes, and sequence analysis showed that both pairs contained distinct DQB alleles. One DQB duplication was observed in two related animals with the same MHC haplotype (F100 and F188), while the second was detected in three animals (F187, G166 and G277) that expressed the same class II DRB3 and DQB alleles but had different class I (BoLA-A) types, suggesting that this DR/DQ haplotype may be widespread in Boran cattle.