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1.
J Proteomics ; 109: 356-67, 2014 Sep 23.
Article in English | MEDLINE | ID: mdl-25072800

ABSTRACT

The mechanisms underlying resistance to challenge by gastrointestinal nematode parasites in sheep are complex. Using DIGE, we profiled ovine lymph proteins in lambs with host resistance (R), resilience (Ri) or susceptibility (S) to a daily trickle challenge with the nematode Trichostrongylus colubriformis. Efferent intestinal lymph was collected prior to infection (day 1) and on days 5 and 10 post-infection. Eight proteins identified by LC-MS/MS, showed differences relating to host genotype. Of these, Serpin A3-3 and Serpin A3-7 have not been reported previously in the lymph proteome. Three acute phase proteins showed significant differences relating to interactions between breeding line and parasite challenge, including complement C3ß, C3α and haptoglobin (Hp) ß. In the R lambs C3α was significantly up regulated (P<0.05) on day 10, while in the Ri lambs Hp ß was significantly down regulated (P<0.05). In the S lambs, levels of C3ß were up regulated and levels of Hp ß down regulated (both P<0.05) on day 10. Hence we demonstrate that acute phase inflammation proteins contribute to differences in the innate immune response of sheep to challenge by T. colubriformis. The findings may lead to the development of new approaches to combat nematode infestations in sheep production systems. BIOLOGICAL SIGNIFICANCE: Breeding lines of sheep with resistance (R), resilience (Ri) or susceptibility (S) to nematode infections provide an experimental model to examine the biological mechanisms underlying the ability of some sheep to expel worms and remain healthy without the use of an anthelmintic. Using proteomics we identified differences in the expression of acute phase lymph proteins in the R, Ri and S lambs. The results will assist the development of alternative control strategies to manage nematode infections in livestock.


Subject(s)
Intestinal Mucosa , Intestines , Lymph/metabolism , Proteome/metabolism , Sheep Diseases , Sheep , Trichostrongylosis , Trichostrongylus , Animals , Disease Susceptibility/metabolism , Disease Susceptibility/parasitology , Disease Susceptibility/pathology , Intestinal Mucosa/metabolism , Intestines/parasitology , Sheep/metabolism , Sheep/parasitology , Sheep Diseases/metabolism , Sheep Diseases/parasitology , Sheep Diseases/pathology , Time Factors , Trichostrongylosis/metabolism , Trichostrongylosis/pathology , Trichostrongylosis/veterinary
2.
Parasite Immunol ; 32(8): 541-8, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20626809

ABSTRACT

Infections with gastrointestinal nematode parasites are a major problem for the sheep industry in Australia and New Zealand and have been the subject of intensive research to define mechanisms of resistance. The ability to take continuous biopsy samples of infected organs and cannulate both afferent and efferent lymphatics of draining lymph nodes has been particularly useful in illuminating the kinetics of immune responses at the site of infection. Distinct localized immune responses were shown to occur within and between sheep breeds at different sensitization regimes, as well as at different developmental stages of the parasite within the host. Using localized antibodies derived from mucus and lymph nodes, two major antigens have been identified on the infective L3 stage, which may be responsible for inducing protection and have potential as vaccine targets. Recent advances in sheep genomics also offer the potential of gaining further insight into the underlying genetics of resistance to nematode infections.


Subject(s)
Intestinal Diseases, Parasitic/veterinary , Nematode Infections/veterinary , Sheep Diseases/immunology , Animals , Antigens, Helminth/immunology , Australia , Genomics , Intestinal Diseases, Parasitic/immunology , Nematode Infections/immunology , New Zealand , Sheep/genetics , Sheep/immunology , Sheep/parasitology , Sheep Diseases/parasitology
3.
Parasite Immunol ; 30(11-12): 577-84, 2008.
Article in English | MEDLINE | ID: mdl-19067838

ABSTRACT

Sheep were immunized by multiple truncated infections with the gastrointestinal nematodes Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. Three infections with T. colubriformis of 14 days plus five booster doses of L3 stimulated highly effective protection against challenge (99%). Three infections of 14 days plus three booster doses with H. contortus also resulted in significant protection against challenge infection (87%), but the same procedure was not effective for T. circumcincta. Antibodies derived from gastrointestinal mucus of these immunized sheep were tested for their ability to reduce worm burden following injection of antibody-coated exsheathed larvae into the abomasum (H. contortus and T. circumcincta) or duodenum (T. colubriformis) of nematode-naïve sheep in a passive immunity test. The IgG fraction from the mucus of immunized sheep reduced worm burdens by 62%, 76% and 91% in three tests with T. colubriformis but was not effective for either of the abomasal dwelling nematodes H. contortus and T. circumcincta. Antibodies in immune mucus predominantly recognized two larval surface antigens on immunoblots of L3 extract, a high MW surface glycoprotein and the carbohydrate larval antigen (CarLA). Antibodies raised against purified T. colubriformis glycoprotein Tc-120 and CarLA were tested in the passive immunity model and it was found that only the antibody against CarLA resulted in a significant reduction of infection (87%). The protective anti-CarLA antibodies strongly recognized the surface of living T. colubriformis L3. Antibodies from abomasal mucus of sheep immunized by H. contortus and T. circumcincta infections reacted weakly with CarLA and the larval surface and did not reduce worm counts in a passive immunity test. The results provide further evidence that the larval surface carbohydrate antigen CarLA has potential as a mucosal immunogen for a strongylid nematode vaccine.


Subject(s)
Antibodies, Helminth/immunology , Immunization, Passive/methods , Immunization/methods , Sheep Diseases/prevention & control , Trichostrongyloidea/immunology , Trichostrongyloidiasis/veterinary , Animals , Antigens, Helminth/immunology , Blotting, Western , Carbohydrates/immunology , Epitopes/immunology , Immunization, Secondary , Intestinal Mucosa/immunology , Mucus/immunology , Sheep , Sheep Diseases/immunology , Trichostrongyloidiasis/immunology , Trichostrongyloidiasis/prevention & control
4.
Vet Parasitol ; 132(3-4): 217-22, 2005 Sep 30.
Article in English | MEDLINE | ID: mdl-16095841

ABSTRACT

The majority of attempts to develop commercial vaccines for veterinary helminths have focussed on identifying protein antigens, which could be formulated as protective vaccines. Notable successes have been achieved for some cestode parasites, where recombinant proteins have been developed into highly effective vaccines. Although effective protection can also be obtained using some nematode proteins in their native forms, it has not yet been possible to formulate commercially successful vaccines for other helminth parasites of veterinary significance. Increasing evidence suggests that parasite glycan moieties may provide an alternative source of vaccine antigens, and increased attention is now being given to this class of compounds. In addition to identifying candidate protective antigen(s), an increased research effort is needed to develop appropriate strategies for the formulation and delivery of helminth vaccines.


Subject(s)
Antigens, Helminth/immunology , Helminthiasis, Animal/prevention & control , Vaccination/veterinary , Vaccines , Animals , Drug Design , Polysaccharides/immunology , Vaccination/methods
5.
Vet Immunol Immunopathol ; 104(1-2): 69-80, 2005 Mar 10.
Article in English | MEDLINE | ID: mdl-15661332

ABSTRACT

Total and antigen-specific IgE responses in afferent (AIL) and efferent (EIL) intestinal lymph of sheep with a nematode resistant (R) or susceptible (S) genotype during challenge infection with the intestinal nematode parasite Trichostrongylus colubriformis were examined. Within each sheep line, lambs with a nematode naive or nematode field-primed pre-challenge status were used. Total IgE level in AIL and EIL was dependent on nematode infection and was further influenced by genotype or the immune phenotype (nematode immune mean FEC+/-SDM=77+/-179 or non-immune mean FEC+/-SDM=4016+/-4318) of the animal. During T. colubriformis challenge immune animals had higher levels of total IgE in lymph than non-immune sheep, R line sheep had higher concentrations of total IgE than S line sheep, and field-primed animals had higher total IgE levels than nematode naive animals. Concentrations of total IgE were consistently higher in AIL than EIL or serum and were higher in lymph draining the proximal than the distal jejunum demonstrating that polyclonal IgE in AIL was largely derived from the intestinal mucosa of the anatomical compartment where the nematodes reside. The consistently higher concentration of total IgE in AIL was dependent on phenotype or genotype and in S genotype sheep also on the pre-challenge status. Concentrations of nematode specific IgE were significantly higher in EIL than AIL indicating a preference for the production of IgE reacting with excretory secretory products of the infective T. colubriformis larvae in the regional lymph node.


Subject(s)
Immunoglobulin E/immunology , Intestinal Diseases, Parasitic/veterinary , Sheep Diseases/immunology , Sheep Diseases/parasitology , Trichostrongylosis/immunology , Trichostrongylosis/veterinary , Trichostrongylus/immunology , Animals , Antibodies, Helminth/analysis , Antibodies, Helminth/immunology , Antibody Specificity , Blotting, Western/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Feces/parasitology , Female , Genetic Predisposition to Disease , Intestinal Diseases, Parasitic/genetics , Intestinal Diseases, Parasitic/immunology , Intestinal Diseases, Parasitic/parasitology , Lymph/immunology , Lymph/parasitology , Male , Parasite Egg Count/veterinary , Sheep , Sheep Diseases/genetics , Trichostrongylosis/parasitology
6.
Parasite Immunol ; 25(2): 79-86, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12791103

ABSTRACT

In an accompanying paper we show that antibodies in intestinal mucus that recognize a 35-kDa antigen from the surface of the L3 stage of the sheep intestinal nematode parasite, Trichostrongylus colubriformis, are strongly associated with immune rejection of L3 in a truncated infection model of immunity in sheep. Monoclonal antibody PAB-1 was used to immunopurify this antigen from T. colubriformis L3. The antigen is resistant to digestion with a range of proteases including proteinase K and does not stain on gels or blots treated with protein-detecting reagents but does stain with carbohydrate-detecting reagents. The antigen is also resistant to degradation by the action of lipases and is not soluble in organic solvents, suggesting that lipid components are not present or not accessible. Treatment with glycosidases does not affect the antigen, indicating either that sialic acid and N-linked or O-linked sugars are not present or that they are not accessible to enzyme attack. The antigen is not destroyed by harsh alkaline degradation with up to 8 m NaOH with or without borohydride reducing agent, or by extensive hydrazinolysis. Strong acid hydrolysis with trifluoroacetic acid or boiling in hydrochloric acid for 20 min does destroy the antigen. The antigen migrates as a poorly defined high molecular weight complex on native electrophoresis gels, but is detected as a major band at 35 kDa on SDS PAGE either by carbohydrate staining or by immunoblotting with antibody from immune sheep intestinal mucus and with mAb PAB-1. Proteinase K digestion and alkaline degradation of extracts from L3 of 10 other parasitic nematode species revealed that L3 of each species contained a carbohydrate staining molecule which can be detected by mAb PAB-1 and by mucus antibody from immune sheep. Because antibodies in intestinal mucus are directed against these antigens and may be responsible for protective immunity, carbohydrate larval antigens (CarLA) could represent a new family of molecules with potential as targets for stimulating host immunity.


Subject(s)
Antigens, Helminth/chemistry , Carbohydrates/immunology , Trichostrongylus/immunology , Animals , Antigens, Helminth/analysis , Antigens, Helminth/genetics , Antigens, Helminth/metabolism , Carbohydrate Metabolism , Carbohydrates/chemistry , Host-Parasite Interactions , Larva/immunology , Molecular Weight , Sheep , Trichostrongylosis/immunology , Trichostrongylus/classification , Trichostrongylus/growth & development
7.
Parasite Immunol ; 25(1): 45-53, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12753437

ABSTRACT

Sheep that have been immunized by multiple truncated infections with the parasitic nematode Trichostrongylus colubriformis contain anti-larval activity in their intestinal mucus and high-speed mucus supernatants. This activity induces T. colubriformis L3 to clump in vitro and causes a significant reduction in larval establishment in naive sheep after infusion of larvae and mucus into the intestinal lumen via a duodenal cannula. In this report, we provide evidence that one factor contributing to the anti-larval activity of immune mucus is antibody against a 35-kDa L3-specific cuticular antigen. The anti-larval activity in mucus is > 100 kDa by membrane filtration, is heat labile and sensitive to either protease digestion or reduction with DTT. Immunoblotting showed that mucus and supernatants of ultracentrifuged mucus from immune sheep contained IgG1 and IgA antibodies that recognized predominantly a larval antigen with an estimated molecular weight of 35 kDa on SDS-PAGE. Antibodies eluted from the surface of washed larvae that had been incubated in immune mucus also reacted specifically with the 35 kDa antigen on blots of larval homogenate. Immunofluorescence and immunogold electron microscopy showed that the 35 kDa antigen is present on the epicuticle of L3 and is shed during the moult to L4. The antigen is not present in eggs, L1, L2, L4 or adult worms and is found only in extracts of sheaths and L3 before infection and up to 4 days after infection. We hypothesize that the binding of antibody to the larval surface prevents larvae from establishing at their preferred site, causing them to be eliminated from the intestine. Monoclonal antibody PAB-1 recognizes the 35 kDa T. colubriformis larval antigen and also cross-reacts with antigens of similar molecular weight on blots of L3 extracts of the parasitic nematodes Haemonchus contortus and Ostertagia circumcincta; and with a 22-kDa antigen on blots of L3 extracts from Cooperia curticei and Nematodirus spathiger. This indicates that an antigenically related surface antigen with immunizing potential is present on several nematode species and can be identified by mAb PAB-1. The 35 kDa T. colubriformis larval antigen and related molecules in other nematodes are potential novel targets for stimulating host-protective immunity against nematode infections.


Subject(s)
Antibodies, Helminth/immunology , Antigens, Helminth/immunology , Intestinal Mucosa/immunology , Sheep Diseases/immunology , Trichostrongylosis/veterinary , Trichostrongylus/immunology , Animals , Antigens, Surface/immunology , Antigens, Surface/ultrastructure , Immunoblotting , Intestinal Mucosa/parasitology , Life Cycle Stages , Male , Parasite Egg Count , Sensitivity and Specificity , Sheep , Sheep Diseases/prevention & control , Trichostrongylosis/immunology , Trichostrongylosis/prevention & control , Trichostrongylus/classification , Trichostrongylus/growth & development
8.
Parasitol Res ; 88(5): 463-7, 2002 May.
Article in English | MEDLINE | ID: mdl-12049466

ABSTRACT

Sheep immunised by multiple truncated infections with Trichostronglyus colubriformis were highly resistant to subsequent challenge with homologous exsheathed larvae, administered via a surgically implanted duodenal cannula. The duration of immunity after truncated infections was 12-14 weeks against challenge with T. colubriformis or Cooperia curticei, but there was little cross-protection against challenge with Nematodirus spathiger. When immune sheep were given booster doses of T. colubriformis larvae before challenge with N. spathiger, there were 97% fewer N. spathiger larvae in the first 5 m of small intestine, and an overall 79% reduction of N. spathiger larvae in immunised sheep, compared with naive controls. Boosting T. colubriformis immune sheep with killed T. colubriformis larvae plus soluble T. colubriformis L3 antigen, or with soluble antigen alone, also caused significant displacement of N. spathiger challenge larvae (98% and 100% respectively), indicating a non-specific expulsion process. These results indicate that N. spathiger can be used as an indicator species in T. colubriformis immune sheep, to quantify the effects of stimulating mucosal immunity with specific antigens, which may lead to identification of the antigens required for immunisation against nematodes.


Subject(s)
Antigens, Helminth/administration & dosage , Antigens, Helminth/immunology , Sheep Diseases/immunology , Sheep Diseases/prevention & control , Strongylida Infections/veterinary , Strongylida/immunology , Animals , Immunity, Mucosal , Immunization , Injections/methods , Intestine, Small/immunology , Intestine, Small/parasitology , Larva/immunology , Sheep , Solubility , Strongylida/growth & development , Strongylida Infections/immunology , Strongylida Infections/prevention & control
9.
N Z Vet J ; 49(6): 247-51, 2001 Dec.
Article in English | MEDLINE | ID: mdl-16032199

ABSTRACT

New approaches to nematode control in sheep are urgently needed as anthelmintic drench resistance becomes ever more widespread among worm populations. Here we briefly describe and assess a number of new technologies which will become increasingly important in anti-nematode control programmes in the future. These include vaccines, immunomodulants, strategic grazing practices, the use of biological agents to destroy nematode larvae, biological anthelmintics and targeted silencing of genes regulating nematode development.

10.
Blood ; 96(3): 1180-3, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10910941

ABSTRACT

The spleen plays a major role in immune surveillance, but the impact that splenectomy exerts on the immune competence of an individual is not fully resolved. Here we show that neonatal splenectomy in sheep does not abrogate the development of a large, nonrecirculating pool of lymphocytes and that it has no effect on the acquisition of a normal blood lymphocyte profile. Splenectomy did, however, result in a significant decrease in blood residency time of recirculating lymphocytes and in an enhanced accumulation of recirculating lymphocytes in lymph nodes. Furthermore, nonrecirculating peripheral blood lymphocytes were less likely to migrate to the lung, possibly because of saturation of the marginal pool by recirculating lymphocytes. Although splenectomy has little effect on the development or distribution of lymphocyte subsets in blood and lymph, it has marked effects on the rate of recirculation of lymphocytes, which may have significant implications for peripheral immune surveillance in patients who undergo splenectomy.


Subject(s)
Lymphocytes/immunology , Spleen/immunology , Animals , Sheep , Splenectomy
11.
Vet Immunol Immunopathol ; 72(1-2): 17-20, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10614488

ABSTRACT

The sheep antibody repertoire was assessed by sequence analysis of Vlambda Vkappa and V(H) cDNA clones. In fetuses, all immunoglobulin chains were essentially in germline configuration, although limited diversity occurred at the junctional regions of Vkappa chains. Fetal B cells expressed 12 distinct Vlambda and six Vkappa gene segments. V(H) gene segments were also in germline configuration in fetuses but became extensively mutated with advancing post natal age. Two distinct types of mutations, point mutations and block mutations, were detected in V(H) transcripts and these localised differently along the gene. This raises a question as to whether a novel mutational mechanism may be involved in V(H) diversification in sheep.


Subject(s)
Antibody Diversity/genetics , Gene Expression Regulation, Developmental , Genetic Variation/genetics , Immunoglobulins/genetics , Sheep/genetics , Animals , Antibody Diversity/immunology , DNA/chemistry , Genes, Immunoglobulin/genetics , Immunoglobulins/immunology , Point Mutation , Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA , Sheep/embryology , Sheep/immunology
12.
Vet Immunol Immunopathol ; 72(1-2): 67-72, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10614494

ABSTRACT

The structure of sheep complement receptor type 2 (CR2) was characterised by cDNA cloning, protein sequencing and immunoprecipitation. The primary structure of sheep CR2 is similar to known mammalian homologues but the higher-order structure is unusual. Two distinct CR2 isoforms occur, one of which is ubiquitinated in the cytoplasmic domain, and the two molecular forms are expressed at the cell surface as non-covalently associated dimers. The percentage of sheep B-cells that express CR2 changes during development and varies between different body compartments. CR2+ and CR2 B-cell subsets also differ in the expression of other surface markers and in functional properties. Differential expression of CR2 may, therefore, delineate B-cells that arose by alternative developmental pathways, or it could be a marker for B-cells at different phases of antigen exposure.


Subject(s)
Gene Expression Regulation, Developmental , Receptors, Complement 3d/chemistry , Sheep , Animals , B-Lymphocyte Subsets/immunology , Cloning, Molecular , DNA/chemistry , Precipitin Tests/veterinary , Protein Isoforms/genetics , Protein Isoforms/immunology , Receptors, Complement 3d/genetics , Sequence Analysis, Protein
14.
Int Immunol ; 10(9): 1251-9, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9786424

ABSTRACT

The usage of lambda and kappa light chain variable region genes in peripheral fetal lamb B cells was assessed at intervals from 61 to 146 days of gestation (term = 150 days). Transcripts of 12 distinct Vlambda genes were identified, eight of which belonged to the previously described families I or II, while four segments defined a new Vlambda family, provisionally called family VI. A total of six different Vkappa gene segments were identified and grouped into four families, while two Jkappa gene segments were expressed. Southern blot analysis indicated that the sheep Igkappa locus probably contains approximately 10 Vkappa genes and suggests that the V gene cluster has been duplicated. Throughout fetal ontogeny, Vkappa transcripts contained more diverse CDR3 sequences than did Vlambda transcripts, where the V-J junctions were nearly always invariant and canonical. There was also a shift in usage of Vkappa genes and in the pattern of Vkappa-Jkappa gene recombination during fetal ontogeny.


Subject(s)
B-Lymphocytes/metabolism , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Animals , B-Lymphocytes/immunology , B-Lymphocytes/physiology , Base Sequence , DNA, Complementary/genetics , DNA, Complementary/metabolism , Embryo, Mammalian , Female , Gene Expression , Gene Rearrangement, B-Lymphocyte, Light Chain , Genes, Immunoglobulin , Genetic Variation , Humans , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/biosynthesis , Immunoglobulin lambda-Chains/genetics , Male , Molecular Sequence Data , Pregnancy , Sequence Homology, Nucleic Acid , Sheep
15.
J Immunol ; 161(1): 458-66, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9647256

ABSTRACT

Complement receptor type 2 (CR2) is a membrane-anchored glycoprotein that specifically binds C3d, as well as other ligands, and plays diverse roles in regulating immunity. Here we show that two distinct isoforms of CR2 are expressed on the surface of sheep B lymphocytes. One (CR2no 150 kDa) is structurally similar to known mammalian homologues while the other (CR2ub 190 kDa) has been modified by the covalent attachment of ubiquitin to the cytoplasmic domain and is identified for the first time. CR2no and CR2ub are expressed on the surface of sheep B cells as noncovalently associated dimers and the external topography of the two isoforms differs in some respect. The basis for these unusual higher-order structural properties may lie in the primary sequence of sheep CR2, since the transmembrane domain contains a region resembling a rare 7-amino acid dimerization motif, and two lysine residues in the cytoplasmic domain provide potential sites for posttranslational ubiquitination. The primary structures of sheep ubiquitin and C3d ligand are extensively conserved. In conjunction with the results of separate in vivo studies, these findings suggest that selective ubiquitination plays a role in modulating the higher-order structure and/or expression of CR2 during B cell development.


Subject(s)
B-Lymphocytes/metabolism , Receptors, Complement 3d/metabolism , Sheep/immunology , Ubiquitins/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , B-Lymphocytes/chemistry , B-Lymphocytes/immunology , Binding Sites, Antibody , Biopolymers/metabolism , Complement C3d/chemistry , Complement C3d/genetics , Complement C3d/isolation & purification , Cytoplasm/chemistry , Dimerization , Extracellular Space/chemistry , Humans , Isomerism , Ligands , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Peptide Mapping , Polyubiquitin , Protein Structure, Tertiary , Receptors, Complement 3d/genetics , Receptors, Complement 3d/immunology , Receptors, Complement 3d/isolation & purification
16.
Mol Immunol ; 34(8-9): 641-51, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9393967

ABSTRACT

In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.


Subject(s)
Cattle/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA/chemistry , Gene Rearrangement , Humans , Mice , Molecular Sequence Data , RNA/chemistry
17.
Immunology ; 91(1): 58-64, 1997 May.
Article in English | MEDLINE | ID: mdl-9203966

ABSTRACT

More than 80% of T cells in bovine skin localized in the superficial 0.5 mm of the dermis. Only 3% occurred within the epidermis or made contact with the stratum basale while the remainder occupied deeper dermal sites. The gamma delta-T-cell receptor (TCR) was expressed by 44% of T cells in skin and 39% and 35% expressed, respectively, the CD4 and CD8 markers. Some cells co-expressed CD8 and the gamma delta TCR. A highly diverse repertoire of gamma delta TCR was expressed in skin due mainly to the usage of multiple V delta segments and to extensive sequence variation at the junctions of both TCR gamma and TCR delta chains. However, a single receptor isotype was used. Transcripts encoding several new components of the bovine gamma delta TCR were identified, including three new V gamma segments, the C gamma 5 region and 13 new functional V delta segments. Taken together with earlier findings, these results emphasize that ruminant gamma delta T cells express exceptionally diverse antigen receptors and suggest they may have a more elaborate recognitive capacity than do their counterparts in other species.


Subject(s)
Cattle/immunology , Receptors, Antigen, T-Cell, gamma-delta/analysis , Skin/immunology , T-Lymphocyte Subsets/immunology , Amino Acid Sequence , Animals , Base Sequence , Gene Expression , Genes, Immunoglobulin/genetics , Immunity, Cellular , Immunoglobulin Constant Regions/genetics , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/genetics , Lymphocyte Count , Molecular Sequence Data
18.
Blood ; 90(12): 4865-75, 1997 Dec 15.
Article in English | MEDLINE | ID: mdl-9389703

ABSTRACT

The continual recirculation of lymphocytes between the blood, tissues, and lymph is essential for the coordination and dissemination of immune responses. We have compared the functional and phenotypic properties of lymphocytes isolated from blood and lymph, the two major migratory populations. Lymph-borne lymphocytes migrated readily into the lymphatic recirculation pathway, but greater than one third of all peripheral blood lymphocytes (PBLs) were excluded from the lymphatic circuit and showed an enhanced migration to the spleen. Phenotypic analysis showed that most non-recirculating PBLs were B cells. The migration competence of B cells correlated with the surface expression of CD21 and L-selectin; recirculating B cells expressed both of these molecules, whereas non-recirculating B cells lacked both antigens. These results establish that blood contains distinct pools of lymphocytes that differ in their recirculation competence. Clearly, blood sampling is not an efficient method to directly measure the status of the recirculating immune system, and implies important constraints and restrictions in the interpretation of experimental or clinical data that include phenotypic and quantitative analyses of blood lymphocytes.


Subject(s)
B-Lymphocytes/physiology , L-Selectin/analysis , Receptors, Complement 3d/analysis , Animals , Cell Movement , Immunophenotyping , Lymph/cytology , Lymph Nodes/cytology , Sheep
19.
Aust Vet J ; 73(3): 118, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8660216
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