Jan Naessens

Analysis of monoclonal antibodies specific for the gamma delta TcR.

gamma delta T cells in ruminants can be subdivided in two or more subpopulations on the basis of the expression of surface antigen WC1, which can exist in different isoforms. In this study, 18 monoclonal antibodies (mAbs) submitted to the Third International Workshop that were predicted to react with gamma delta TcR molecules were analysed and expression of their antigens was investigated on the different gamma delta T cell subpopulations. A set of control mAbs positive for TcR1 (86D), BoCD3 (MM1A), WC1 (B7A1, BAQ4A, CACTB32A, and BAQ89A) was included for comparative studies. Previous investigations demonstrated eight of the mAbs immunoprecipitated peptides with apparent M(r)s of 37 and/or 47 kDa, indicating they recognized determinants on the T cell receptor, TcR1. Two color flow cytometric analyses in the present study demonstrated the mAbs formed three groups; group 1, a set of mAbs that recognize TcR1 determinants expressed on all gamma delta T cells and groups 2 and 3, sets of mAbs that recognize TcR1 determinants on some gamma delta T cells: TcR1-N6 and TcR1-N7 respectively. mAbs from the latter groups define families of TcR1 molecules that express either one or both of the determinants. These antigenically distinct forms of TcR1 are expressed in equal proportion on the two gamma delta T cell populations that express one of the mutually exclusive isoforms of WC1, WC1-N3 and WC1-N4. The data indicate usage of the mAb-defined families of the gamma delta TcR is primarily restricted to the WC1+ subpopulation of gamma delta T cells. However, a small subpopulation of CD2+, WC1- gamma delta T cells expresses a form of TcR1 positive for the determinant TcR1-N6.Twenty-six monoclonal antibodies (mAbs) selected after the first round of analysis in the Third International Swine Workshop were grouped with additional mAbs from the first and second workshops and mAbs under study for further evaluation. Preparations of peripheral blood leukocytes were used in single and multicolor flow cytometric (FC) analyses. Six mAbs did not react with gammadelta T-cells. Two were negative for all tested specificities. Seven mAbs recognized molecules expressed on gammadelta T-cells that were not lineage restricted. One of these from the first workshop (2B11) yielded a pattern of labeling identical to a mAb under study (PGB73A). Ten mAbs were characterized in previous workshops and known to react with the gammadelta TCR or molecules expressed on subsets of gammadelta T-cells. One belonged to SWC4, two to SWC5, and one to SWC6. Two mAbs from the second workshop recognized a molecule or molecules expressed on subsets of gammadelta T-cells. A new mAb (PPT16) added late to the workshop following a request by the workshop chairs appeared to recognize a determinant expressed on the gammadelta TCR/CD3 molecular complex.

Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies

A panel of 10 monoclonal antibodies specific for bovine immunoglobulins M, A, G1, G2 and light chains were produced and enzyme-linked immunosorbent assays developed to measure Ig levels in body fluids and culture supernatants using this panel of MAbs. An inhibition ELISA was accurate and sensitive for MAbs of high affinity, detecting levels as low as 10 ng ml-1 of IgM using a high-affinity MAb, IL-A50 (dissociation constant = 1.3 X 10(-11) M). For MAbs of lower affinity (KD of less than 0.25 X 10(-9) M) a sandwich ELISA was more sensitive, detecting 0.1-1.0 microgram ml-1 Ig, provided a conjugate of an anti-light chain MAb was used. Using these ELISA techniques, four pairs of MAbs specific for bovine IgM, IgA, IgG1 and IgG2 respectively, were screened on sera from over 100 cattle of different breeds to determine whether any detected a polymorphic epitope. MAbs IL-A30, IL-A60, IL-A66, IL-A71, IL-A72, IL-A73 and IL-A74 were shown to recognise monomorphic determinants on their respective heavy chains. In contrast, the epitope recognised on the mu-heavy chain by MAb IL-A50, which had previously been shown to be polymorphic, was found to be allelic and inherited under the control of a single gene, probably Cu.

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.

Nomenclature and characterization of leukocyte differentiation antigens in ruminants

Abstract Three international workshops held over the past eight years have addressed the identification and nomenclature of leukocyte differentiation antigens in ruminants. This article explains the progress the workshops have made.

TNF- a mediates the development of anaemia in a murine Trypanosoma brucei rhodesiense infection, but not the anaemia associated with a murine Trypanosoma congolense infection

Development of anaemia in inflammatory diseases is cytokine‐mediated. Specifically, the levels of tumour necrosis factor‐α (TNF‐α), produced by activated macrophages, are correlated with severity of disease and anaemia in infections and chronic disease. In African trypanosomiasis, anaemia develops very early in infection around the time when parasites become detectable in the blood. Since the anaemia persists after the first waves of parasitaemia when low numbers of trypanosomes are circulating in the blood, it is generally assumed that anaemia is not directly induced by a parasite factor, but might be cytokine‐mediated, as in other cases of anaemia accompanying inflammation. To clarify the role of TNF‐α in the development of anaemia, blood parameters of wild type (TNF‐α+/+), TNF‐α‐null (TNF‐α–/–) and TNF‐α‐hemizygous (TNF‐α–/+) trypanotolerant mice were compared during infections with the cattle parasite Trypanosoma congolense. No differences in PCV, erythrocyte numbers or haemoglobin were observed between TNF‐α‐deficient and wild type mice, suggesting that the decrease in erythrocytes was not mediated by TNF‐α. Erythropoetin (EPO) levels increased during infection and no significant differences in EPO levels were observed between the three mouse strains. In contrast, during an infection with the human pathogen Trypanosoma brucei rhodesiense, the number of red blood cells in TNF‐α‐deficient mice remained significantly higher than in the wild type mice. These data suggest that more than one mechanism promotes the development of anaemia associated with trypanosomiasis.

Confirmation and dissection of QTL controlling resistance to malaria in mice

We developed an F11 AIL population from an F1 cross of A/J (susceptible) and C57BL/6J (resistant) mouse strains. One thousand F11 mice were challenged with P.c. chabaudi 54X, and 340 mice selected from the phenotypic extremes for susceptibility and resistance were genotyped for microsatellite markers on Chromosomes (Chrs) 5, 8, and 17. QTL originally detected in backcross and F2 populations were confirmed on the three chromosomes within narrower genomic regions, by maximum likelihood and regression analyses. Each of the previously mapped QTL on Chrs 5 and 17 resolved into two linked QTLs. The distal and proximal QTLs on Chrs 5 and 17, respectively, map to the previously reported QTL.

An immunochemical analysis of class I (BoLA) molecules on the surface of bovine cells

As in other species, bovine class I MHC molecules have been found to be cell surface heterodimers composed of a glycosylated heavy chain of approximate relative mass 44000, associated with B2m of relative mass 12000 (Hoang-Xuan et al. 1982). To date, serological definition of the BoLA system, using alloantisera, has proceeded through two international workshops (Spooner et al. 1979, Anon 1982)which have identified 17 apparent class I alleles at a single locus, BoLA-A. To gain further insights into the nature and heterogeneity of expressed bovine MHC products, we have attempted to obtain some measure of the diversity of cell surface-expressed BoLA class I molecules by establishing a structural relationship between polymorphic and monomorphic epitopes detectable on bovine class I MHC products. This was done by immunochemical analysis of detergent lysates of radiolabeled peripheral blood lymphocytes (PBL) immunoprecipitated with bovine alloantisera, mouse mAbs, and a rabbit antiserum specific for class I HLA heavy chains not complexed to B2m. BoLA typing was performed using a panel of alloantisera and monoclonal antibodies developed in our laboratory in a standard microlymphocytotoxicity assay (Teale et al. 1983). All immunochemical studies were performed with PBL of a four-year-old Boran (Bos indicus) steer, number B641. This animal is heterozygous at the BoLA-A locus with the phenotype Awl0/KN18. The KN18 specificity is defined by two alloantisera (KMA010 and KMA018) and mAb P3. The lead serum, KMA018, has a correlation coefficient with the specificity of 0.885. A population study involving more than 1600 cattle suggested that KN18 is encoded at the BoLA-A locus (Kemp 1985). Furthermore, in an extended survey of more than 2000 cattle,

B‐1‐like cells exist in sheep. Characterization of their phenotype and behaviour

Two populations of B lymphocytes, B‐1 (CD5+ and/or CD11b+) and B‐2 (CD5− and CD11b−) cells have been described. In mice, which is the species of reference for B‐1 and B‐2 cell studies, these two subsets present different developmental schemes, phenotypes, antibody repertoires, localization and behaviours. Interestingly, in sheep, B cells rearrange their immunoglobulin (Ig) loci around the neonatal period, similarly to murine B‐1 cells. However, the phenotype of the sheep B cells has not been characterized with regards to their developmental pathway. In this report, we show that two sheep B‐cell subsets can be distinguished on the basis of CD11b expression. Relative to CD11b− B cells, the CD11b+ B cells frequently co‐express CD5, CD11c, higher levels of surface IgM (sIgM), show larger cell size and higher cell‐cycling activity, and thus present a B‐1‐like phenotype. However, unlike murine B‐1 cells, sheep B‐1 like cells mainly localize in blood, display a higher propensity to spontaneous apoptosis relative to B‐2‐like cells, and proliferate after sIgM stimulation. Our data show that despite neonatal immunoglobulin loci rearrangements, sheep B cells do not all express a B‐1‐like phenotype. However, B‐1‐and B‐2‐like cells co‐exist and present phenotypic and behavioural specificities. Nevertheless, sheep B‐1‐and B‐2‐like cells differ from the murine B‐1 and B‐2 cells in their cell behaviour. These subsets can thus not be considered as true homologues among species.

Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species.

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests. In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.

Mechanisms Controlling Anaemia in Trypanosoma congolense Infected Mice

Background Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. Methodology/Principal Findings The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. Conclusions/Significance The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection.