Localization of genes controlling resistance to trypanosomiasis in mice.

Tsetse fly-transmitted trypanosomes (Trypanosoma spp.) cause "sleeping sickness' in man and have a serious impact on livestock-based agriculture in large areas of Africa. Multigene control of variation in susceptibility to trypanosomiasis is known to occur in mice, where the C57BI/6 (B6) strain is relatively resistant and the A/J (A) and Balb/c (B) strains are susceptible. Such resistance is also well described among several types of west African cattle. We report here the results of genome-wide scans for genes controlling this trait in the B6 mouse using crosses with two different susceptible strains. Regions on mouse chromosomes 5 and 17 were found to be important in determining resistance in both crosses while an additional region on chromosome 1 showed evidence of involvement in only one cross. We confirmed the size of the effect due to chromosome 17 in F3 intercross populations fixed for alternative parental chromosomes. The three loci are of large effect and account for most of the genetic variation in both F2 populations. We propose that they be designated Tir1, Tir2 and Tir3.

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray.

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr-smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)-salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community.

Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray.

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

Identification of mechanisms of natural resistance to African trypanosomiasis in cattle.

Natural resistance to African trypanosomiasis in certain Bos taurus cattle in West Africa, called trypanotolerance, may hold solutions for control of this economically crippling disease. Comparison of immune responses between trypanotolerant and trypanosusceptible cattle have shown some differences in antibody response, complement level and cytokine expression, but it is not known whether these differences are the cause of resistance. Two experiments were carried out to assess the contribution of the immune and haemopoietic systems to trypanotolerance. The production of haemopoietic chimaeras from trypanotolerant and susceptible twin calves and comparison of their responses after infection with singleton calves, allowed an assessment of the role of the haemopoietic system in trypanotolerance. An in vivo depletion of CD4 cells in the two breeds allowed an appraisal of the role of T and B lymphocytes in trypanotolerance. The results of the two experiments suggest that natural resistance comprises at least two mechanisms, an innate mechanism that controls parasite growth, and another, involving the haemopoietic system, that is able to limit anaemia. This supports the hypothesis that innate mechanisms in trypanotolerant cattle are more efficient in controlling disease, making them less reliant on antibody responses.

Differential in vitro and in vivo expression of MHC class II antigens in bovine lymphocytes infected by Theileria parva.

The expression of major histocompatibility complex class II (MHC II) non-polymorphic antigens detected by four monoclonal antibodies was investigated in Theileria parva-infected and non-infected cloned lymphoid cell lines, bulk cultures, and in peripheral blood mononuclear cells (PBMC) and lymph node cells (LNC) of experimentally infected calves. Compared with non-infected cell lines, both immunofluorescence microscopy and flow cytofluorometry analysis of infected lines of alpha beta T-cell, gamma delta T-cell and B-cell origin revealed high expression of MHC II MHC molecules. After T. parva infection in vitro, three alloreactive T cell clones, three interleukin-2 (IL-2)-dependent cell lines and a concanavalin A (Con A)-stimulated bulk culture all had an increase both in the proportion of MHC II+ cells and in their mean fluorescence intensity. Radioimmunoprecipitation of class II molecules biosynthesized in infected and non-infected cells revealed that they were constitutively produced in infected cells, and were a slightly larger relative mass than the MHC II molecules of uninfected cells. In a study of the serial expression of MHC II antigens in PBMC and LNC of six calves inoculated with a lethal dose of T.parva, MHC II expression by non-parasitized cells peaked at Days 7 (LNC) or 9 (PBMC) following inoculation and, subsequently, MHC II non-expressing parasitized lymphocytes progressively outnumbered MHC II-expressing parasitized cells. In two calves studied in detail, MHC II expression in PBMC and LNC generally, and in T cells particularly, increased during the course of the disease. Finally, among LNC sorted for MHC II expression at 11 and 17 days after parasite inoculation, the proportion of parasitized cells increased markedly in MHC II non-expressing populations and was reduced or increased only slightly in MHC II-expressing populations. These findings indicate that: (1) enhanced MHC II antigen expression by parasitized lymphocytes may be important in the pathogenesis of the lymphoproliferation that characterizes T. parva infection; (2) the in vivo preponderance of MHC II non-expressing over MHC II-expressing T. parva-infected cells may reflect host-mediated destruction or antigenic modulation of parasitized MHC II-expressing cells.

Genetic control of resistance to trypanosomiasis.

To map the genetic sources of trypanotolerance in mice, a linkage analysis of survival following trypanosome challenge was performed by selective genotyping in a large F2 population produced by crossing the resistant C57BL/6 and susceptible BALB/c inbred mouse lines. We report evidence of a chromosomal region of large effect, possibly comprising more than one resistance locus, on Chromosome 17; and of further loci on Chromosomes 1 and 5. Together, these genes can account for all of the difference between the mean parental phenotypes.

Phenotypic and functional characteristics of bovine T lymphocytes.

Monoclonal antibodies have been derived which detect the bovine equivalents of the human pan-T cell marker CD2 and the T lymphocyte subpopulation markers CD4 and CD8. We refer to the bovine analogues as BoT2, BoT4 and BoT8. Monoclonal antibodies have also been derived which detect an antigen(s) with similarities to CD3, although the precise nature of the target molecule(s) in this instance remains to be elucidated. In general there is close similarity between the tissue distributions and, where these have been determined, the molecular masses of the BoT2, BoT4, BoT8 and putative BoT3 entities and their counterparts in other species. BoT2 is expressed on a majority of peripheral blood T lymphocytes and thymocytes and BoT2+ cells are found in both thymic cortex and medulla. In contrast, the putative BoT3 marker is expressed by a minority of thymocytes which are moreover, largely restricted to medulla. Monoclonal antibodies detecting BoT2 determinants have been shown to precipitate 55 kDa molecules. Antibodies to the BoT2 and BoT3 entities have been shown to induce proliferation in peripheral blood mononuclear cells of some cattle, and to be capable of inhibition of antigen-driven proliferative responses and cytolytic function. The BoT4 and BoT8 markers are expressed in a mutually exclusive manner by bovine peripheral blood mononuclear cells but they are coexpressed on a large population of thymocytes. Monoclonal antibodies have been used to precipitate molecules of 52 and 55 kDa in the case of those detecting BoT4 and 34 and 35 kDa in the case of an antibody reactive with a BoT8 determinant. The BoT4 and BoT8 markers have been associated with specificity for, and restriction by, MHC class II and class I molecules respectively.

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species.

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Detection of antibody to Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells by immunoperoxidase techniques.

Peroxidase-labeled antibody procedures were described for detecting bovine antibodies reactive with intracellular Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells. Indirect tests were performed where the reacting bovine antibodies were localized with affinity purified rabbit-anti-bovine IgG coupled to horseradish peroxidase. A 4- to 8-fold increase in sensitivity for detecting bovine antibodies was obtained with unlabeled rabbit-anti-bovine IgG which in turn was detected with peroxidase labeled goat-anti-rabbit IgG. The T. parva infected cells used as antigen were attached to poly-l-lysine treated glass slides and all reaction steps were performed on the slides. The intracellular schizonts and cell surface staining reactions were dependent upon the status of the cells; acetone-fixed cells were required for schizont reactions and viable unfixed cells for cell surface membrane reactions. Sera from cattle stimulated in various ways with T. parva were examined by the techniques. Cattle infected by stabilate inoculation or inoculated with infected autologous lymphoblastoid cells developed relatively high levels of antibody to schizonts, but no detectable antibody to cell surface membrane antigens. This would indicate that parasite antigens do not occur on the surface of infected lymphoblasts. Cattle inoculated with infected allogeneic lymphoblasts developed low-levels of antibody to schizonts and readily demonstrable antibody to cell surface antigens. The immunoperoxidase procedures have certain advantages over immunofluorescence in that light microscopy is used; therefore, the reactions do not fade which permits a more detailed examination and provides a relatively permanent record, the preparations can be counterstained, and the reagents may be used for immunoelectron-microscopy. The procedures could provide suitable alternatives to immunofluorescence methods for East Coast fever investigations and other systems having intracellular and/or cell surface membrane antigens.

Experimentally induced toxoplasmosis in young rams: the clinical syndrome and semen secretion of toxoplasma.

Six rams were inoculated subcutaneously with 2000 toxoplasma tissue cysts. Semen samples from these rams, and from three uninfected controls, were screened for the presence of infective forms of toxoplasma. Three of the infected rams produced infective semen, each on two occasions, between 14 and 26 days post infection (pi). Five of the infected rams showed a febrile response from the fourth to 10th days pi and their indirect haemagglutination titres rose sharply from 10 days pi; the sixth ram was seropositive before infection and showed no clinical or serological response. In the experimental infection studied there were no symptoms or haematological changes of possible diagnostic value. The production of infective semen was restricted to a brief period shortly after infection although observations were continued for over 90 days. It was concluded that venereal transmission was unlikely to be significant in the spread of ovine toxoplasmosis.

Toxoplasmosis in rams: possible significance of venereal transmission.

Three rams were infected with tick-borne fever eight months after infection with toxoplasma. There was no clinical evidence of reactivation of the latent toxoplasma infection and semen samples from the rams were not infective to mice. Semen samples from 77 apparently normal rams were tested for toxoplasma infection by passage through mice; none of the samples produced patent infections in mice. Two rams with chronic toxoplasma infection were repeatedly mated with seronegative ewes. None of the ewes showed serological evidence of acquired infection and all lambed satisfactorily.

Randomly primed PCR amplification of pooled DNA reveals polymorphism in a ruminant repetitive DNA sequence which differentiates Bos indicus and B. taurus.

By amplification of pools of DNA representative of different bovine populations with single short oligonucleotide primers of random sequence, we were able rapidly to identify markers which distinguish the two major subspecies of domestic cattle, Bos taurus and B. indicus. One of the marker polymorphisms was found to be in a novel, dispersed DNA sequence which occurs in several ruminant species. The marker will assist in the detection of crossbreeding between Zebu and B. taurus types where this threatens a potentially valuable trypanosomiasis-resistant B. taurus genetic resource in West Africa. In addition, the marker will be useful for exploration of the evolutionary relationships of the major subspecies of domestic cattle. The general approach used to identify population-specific DNA polymorphisms has potentially broad application in definition of species, breeds and populations and will be of generic value in studies of genome evolution.

Genetic basis of trypanotolerance in cattle and mice.

Under most circumstances, certain breeds of domestic ruminants show a remarkable resistance to the effects of African trypanosomiasis: they can tolerate the presence of parasites while apparently controlling levels of parasitaemia and, crucially, not showing the severe anaemia and production loss that are characteristic of infection in susceptible hosts. As discussed here by Stephen Kemp and Alan Teale, the genetic control of this phenomenon might finally be yielding to gene mapping studies. Genetic regions determining susceptibility to trypanosomiasis in mice have been identified and parallel studies are well advanced in cattle. There is growing evidence that only modest numbers of genes are involved in determining the difference between a susceptible and a resistant animal. These observations raise a new series of important questions concerning the possible exploitation of major trypanotolerance genes and the way that they might function in different genetic and physical environments.

Alloreactive T cell clones transformed by Theileria parva retain cytolytic activity and antigen specificity.

Theileria parva is a protozoan parasite which infects and transforms bovine lymphocytes. This study examined the effects of Theileria-induced transformation on phenotype and function, in terms of cytolytic potency and specificity, of class I and class II-specific alloreactive T cell clones. Alloreactive T cell clones infected with T. parva (Muguga) retained expression of the T cell differentiation antigens BoT2, BoT4, BoT8 and the mature T cell antigen recognized by monoclonal antibody IL-A27, as well as cytolytic function and antigen specificity, over a period of 3-4 months in continuous culture. These features were identical to those expressed by the uninfected parent clones. During this period, neither antigenic stimulation nor exogenous growth factors were required for the maintenance of proliferation, function or antigen specificity. Thereafter, cytolytic activity declined and was eventually lost, which may reflect degenerative changes normally associated with T cell senescence rather than result from parasitization per se.

A study of BoLA class II antigens with BoT4+ T lymphocyte clones.

It has hitherto proved difficult to phenotype cattle for class II histocompatibility antigens using standard serological techniques because of problems of reagent specificity and antigen expression on peripheral blood mononuclear cells (PBMs). We recently described the production of class II-specific alloreactive bovine T cell clones characterized by the BoT4+ phenotype. In this report we describe studies of the application of four such clones, derived from a single mixed leucocyte culture (MLC), for class II phenotyping in proliferation and cytotoxicity assay systems. Proliferation assays used irradiated PBM as stimulator cells and cytotoxicity assays used Theileria parva-infected lymphoblastoid cells as targets. Proliferation assays revealed three distinct specificities among the four clones indicating that they detected three different class II determinants. Furthermore, in a family study, the genes encoding the determinants recognized by the clones were found to be linked to the gene encoding the w10 class I A locus product on one of the w10-bearing haplotypes in our study population. Two of the clones were studied in cytolysis assays. Lack of cytolysis of one of the targets, which was derived from the PBM of an animal carrying a class II determinant detected in proliferation assay, was explained by the total lack of expression of class II antigens on the target cell line in question, as determined with 4 class II-specific monoclonal antibodies (mAb). We conclude that BoT4+ alloreactive clones provide a potentially useful and particularly discriminating way of detecting polymorphic class II antigens of cattle, especially when applied in assays of proliferative response to PBM.

A panel of bovine, ovine and caprine polymorphic microsatellites.

We report a set of six new bovine microsatellite polymorphisms based on (CA)n repeats. They are highly polymorphic and thus represent valuable markers for genome mapping. Four of the six are polymorphic in sheep and two are polymorphic in goats. One, which is polymorphic in cattle and sheep and apparently monomorphic in goats, is X-chromosome specific and has potential value in, for example, sex determination and detection of chimaerism.

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers.

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in polymerase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61% of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.