Characterization of two mouse myeloma X sheep lymphocyte cell lines secreting sheep antibody.

Two mouse X sheep interspecific cell hybrids were obtained by fusing mouse myeloma cell line Sp2/O. Ag14 with sheep lymphocytes obtained from a lymph node antigenically stimulated with azo-benzene arsonate-ovalbumin (ABA-ova). The interspecific cell lines were characterized using immunochemical, karyotypic and molecular DNA techniques. Both cell lines secreted sheep IgG1 antibody specific for the ABA haptenic determinant. Karyotypic analysis revealed that cell lines 4.11 and 11.9 had modal chromosome numbers of 91 and 106, respectively. Although C-banded spreads confirmed that fusion between sheep and mouse cells had occurred, it was not possible to differentiate sheep from mouse chromosomes. However, DNA hybridization techniques showed that each line contained sheep repetitive sequence DNA. It was calculated that cell line 11.9 contained 17640 copies while cell line 4.11 contained 734 copies of the previously characterized sheep satellite DNA.

A tandemly repetitive DNA sequence is present at diverse locations in the genome of Ostertagia circumcincta.

A novel repetitive DNA sequence in the sheep parasitic nematode Ostertagia circumcincta was cloned and sequenced. This 1.2-kb sequence (Oc1B) was not found in the closely related cattle parasite Ostertagia ostertagi, nor in the more distantly related sheep parasites Haemonchus contortus or Trichostronylus colubriformis. Sequences similar to Oc1B were found at various genomic locations and contained a pair of 33-bp direct repeats. Oc1B also contained a single copy of a 218-bp sequence (designated OcREP) which was present in 100 to 200 copies in the O. circumcincta genome and mostly organized in distinctive tandem arrays. The dual organizational pattern of OcREP as both a satellite-like sequence as well as interspersed as single copies amongst dissimilar sequences adds to the growing evidence for the fluidity of the parasitic nematode genome, and of eukaryotic genomes in general.

Characterization of a tandemly repetitive DNA sequence from Haemonchus contortus.

Genomic DNA from the sheep parasitic nematode Haemonchus contortus was shotgun cloned in the plasmid vector pUC18. Recombinants which gave the strongest hybridization signals to 32P-radiolabelled genomic DNA were selected as representatives of the repetitive component of the parasite DNA. One repetitive sequence which hybridized only with DNA from H. contortus and not with DNA from two other sheep nematodes (Trichostrongylus colubriformis and Ostertagia circumcincta) was further characterized by sequencing and dot blot analysis. A related repeat was found in the closely related species Haemonchus placei. Experiments to determine the genomic organization of the repeat showed that it existed in a multi-copy number tandem array. This is the first report on the characterization of repetitive DNA in sheep parasite nematodes.

Prospects for development of genetic markers for resistance to gastrointestinal parasite infection in sheep.

Selection of sheep for resistance to internal parasites represents a viable option for future parasite control. Many phenotypic measures are available for determining the level of infection in individual sheep, although no phenotypic markers are available which allow prediction of an individual's resistance status. Genetic markers are therefore the best way to incorporate parasite resistance into selection programmes. With the recent development of genetic maps, several experiments are underway to search for markers linked to parasite-resistance genes in sheep. It can be predicted confidently that markers associated with resistance will be discovered within 12 months. Markers useful as selection criteria will be available within 5 years, although considerable quantitative genetic analysis needs to be done to find the best way to utilise marker information in selection programmes. In future, methods for differential DNA analysis or mRNA expression will lead to isolation of the genes involved.

Monoclonal antibodies against sheep immunoglobulin light chain, IgM and IgA.

Monoclonal antibodies specific for sheep immunoglobulin light chain, IgM and IgA were produced by conventional cell fusion technology. Purified light chain and IgM were used to verify the specificity of anti-light chain and anti-IgM hybridoma supernatants using passive haemagglutination assays, radioimmunoassays and immunoelectrophoresis. In the absence of pure IgA, verification of monoclonal anti-IgA was, based on paired staining of intestinal lymph smears and comparing the percentage of cells stained with hybridoma supernatant with the percentage of cells stained with polyclonal anti-alpha serum.

Production and characterization of monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2.

Monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2 were produced using conventional cell fusion techniques. Monoclonal antibodies detected by preliminary screening assays were further characterized and their specificity verified by titration of ascites in radioimmunoassay or passive haemagglutination using pure sheep IgG1 or IgG2. Further evidence for the subclass specificity of the antibodies was obtained from immunoelectrophoretograms of sheep serum or purified proteins developed with monoclonal antibodies. Reaction of monoclonal antibodies with various IgG fragments showed that the determinants recognised were located on the pFc' portion of the heavy chain.

Organisation of the ovine immunoglobulin C epsilon gene locus: evidence for a deletion 5' of the gene.

A cosmid clone containing the ovine and C epsilon and C alpha immunoglobulin heavy chain genes was isolated and characterised. Restriction mapping and sequence analysis showed a high degree of similarity between the bovine and ovine C epsilon loci. Restriction fragment length polymorphism (RFLP) analysis of sheep genomic DNA revealed Mendelian inheritance of polymorphisms with identical variation in allele size for various restriction enzymes. This identical variation suggested that a deletion of approximately 100 bp existed at the 5' end of the smaller alleles.

The antibody-containing cell response in hepatic and intestinal lymph following intraperitoneal and intravenous administration of antigen in different adjuvants.

A comparison was made of the antibody-containing cell (ACC) response in hepatic and intestinal lymph of sheep following intraperitoneal injection of ovalbumin in Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) or the bland vegetable oil preparation, adjuvant 65. There was a substantial ACC response in intestinal lymph following intraperitoneal injection of ovalbumin in either FCA or FIA. Responses were essentially similar in magnitude and for both adjuvants ACC were distributed mainly among the IgGl, IgA and IgM immunoglobulin classes. In contrast, there was a negligible ACC response in intestinal lymph following injection of antigen in adjuvant 65. The output of ACC in hepatic lymph and the immunoglobulin class distribution of the ACC following intraperitoneal injection of antigen in either FCA or FIA were similar and results were specificity comprising 32% of ACC at the peak of the response. In contrast to results for intestinal lymph, injection of antigen in IgGl- and IgM-specific cells in hepatic lymph. The ACC response was much smaller in magnitude than with the Freund's adjuvants. Intravenous injection of ovalbumin in FIA or adjuvant 65 gave rise to substantial ACC responses in hepatic lymph which contrasted with the barely detectable response in intestinal lymph. Following intravenous administration the great majority of ACC in hepatic lymph were of the IgM class irrespective of adjuvant used although ACC of the IgA class made a transitory appearance.

Immune response of sheep to oral and subcutaneous administration of live aromatic-dependent mutant of Salmonella typhimurium (SL1479).

The major objective of the present study was to determine whether oral immunization with a live aromatic-dependent strain of Salmonella typhimurium (SL1479) was capable of stimulating an intestinal immune response in sheep similar to that induced by combined intraperitoneal injection followed by oral boosting. The results showed that repeated oral immunization was incapable of stimulating an anti-flagella antibody containing cell (ACC) response in the lamina propria of the intestine even though primary oral administration of 5 x 10(9) live SL1479 gave rise to an ACC response in intestinal lymph which was predominantly of the IgM isotype. ACC reached a peak 9-10 days after oral administration when ACC comprised 0.5-1% of total lymphocytes in lymph. An ACC response of similar isotope specificity also occurred in popliteal prefemoral lymph of unprimed sheep following regional subcutaneous injection of SL1479. Oral administration of SL1479 to orally primed sheep did not reinvoke an ACC response in lymph although IgG1-ACC were observed in medullary cords of mesenteric lymph nodes of sheep 6-8 days after the booster dose of SL1479. The results suggest that the protective immunity elicited by oral administration of SL1479 cannot be attributed to induction of a local intestinal antibody production.

Significance of Freund's adjuvant/antigen injection granuloma in the maintenance of serum antibody response.

An experimental system involving injections of ovalbumin (OVA) and ferritin (FER) in Freund's incomplete adjuvant (FIA) into the right and left flank skin folds of sheep was used to study the influence of the FIA/antigen depot and the draining lymph node in maintaining an antibody response. Excision of the injection granuloma and draining lymph node from one side 2-3 months after injections resulted in a profound decrease in serum antibody titres. This response was observed in all eight sheep in the experimental group. In five of eight animals in another experiment, excision of the injection sites had no appreciable effect on antigen-specific antibody titres when compared with antibody specific for antigen on the intact side of the sheep. In the remaining three animals, excision of the injection site did cause some fall in titre. Radiotracer studies revealed that about one-third of the original [125I]OVA/FIA injected was present in the granuloma 20 weeks after injection. Lymphatic cannulation approaches were used to study the responsiveness of the lymph node draining an FIA/antigen granuloma established 12 weeks earlier and showed that increments of 1-2 mg OVA in saline administered adjacent to the granuloma at 6-7 day intervals gave rise to strong anti-OVA containing cell (AOCC) responses in lymph. There were 2-6-fold increases in serum antibody titre in response to 3-5 doses of OVA or FER (1-2 mg) in saline injected adjacent to the FIA/antigen injection site (which had been administered 14-16 weeks previously). It is concluded that the release rate of antigen from a FIA/antigen depot is insufficient to sustain maximal antibody levels in blood serum.

Molecular analysis and nematode resistance association of a polymorphism at the 5' end of the sheep IgE gene.

Previous work using Southern analysis of genomic DNA detected a polymorphism at the 5' end of the sheep and cattle IgE gene. Identical length differences found between fragments following digestion with restriction enzymes indicated that the basis for the polymorphism was an insertion/deletion event. To characterise the polymorphism, the entire cattle and sheep Cvarepsilon genes were sequenced including 668bp of 5' untranslated DNA. Sequence comparison revealed a high degree of similarity between the ovine and bovine genes at both the nucleotide and amino acid level. A feature of the 5' untranslated DNA was the presence of an 87bp repeat starting at -365 upstream of the Cvarepsilon start site. PCR primers were designed to span most of the 5' untranslated sequence, including the repeat unit, and used to amplify genomic DNA from a panel of 40 sheep. Three alleles were found with frequencies of 0.7, 0.29, 0.01 which were identical to the Southern analysis results. Sequencing of the two commonest alleles revealed the basis for the polymorphism was a 36bp deletion from the 87bp repeat. Association studies in a sheep selection flock phenotypically assessed for parasite resistance found a highly significant association between one of the IgE alleles and resistance to the intestinal nematode parasite Trichostrongylus colubriformis (P=0.005). Attempts to confirm this finding in two other flocks using linkage analysis and genotype association failed to find any significant associations between the IgE polymorphism and resistance to either T. colubriformis or Haemonchus contortus.

Restriction fragment length patterns of DNA from parasitic nematodes of sheep.

High molecular weight DNA obtained from sheep parasitic nematodes Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis, was digested with various restriction endonucleases. Digestion with Eco R1 produced the most informative pattern of repeat sequence bands. H contortus adult or larval DNA produced bands of 2.7, 3.0 and 1.4 kb. O circumcincta adult or larval DNA had common 2.7 and 1.4 kb bands with adult specific bands of 2.2 and 0.9 kb and a larval specific 2.08 kb band. T colubriformis adults or larval DNA produced 2.7, 1.4 and 0.79 kb bands. These preliminary results show that restriction patterns of repeat sequence DNA may be useful for the identification of various trichostrongylid species parasitic for sheep.

A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae.

We proposed a novel statistical approach for the analysis of cDNA experiments based on mixed-model methodology combined with mixtures of distributions. Our objective was to detect genes that may be involved in conferring heritable differences in susceptibility to common infections in intensive pig production. We employed a microarray expression profiling strategy and a mixed-model approach to the analysis of the expression data. A cDNA microarray of pig with 6,420 probes from immune tissues and cells was used to compare gene expression in peripheral blood leukocytes of two pigs showing extreme performance in their response to infection with Actinobacillus pleuropneumoniae. Principal components analyses were used to identify the two most extreme-performing pigs after infection (i.e., pigs whose measured responses to infection fell at the extremes). Blood samples and expression profiles from 0 to 24 h after infection were compared using a bivariate, mixed-model approach, in which the effect gene x immunological status interaction was treated as a random effect. Bayesian model-based clustering via mixtures of normal distributions of the resulting BLUP of the random interaction was approached and resulted in a list of 307 differentially expressed genes, of which 179 were down-regulated in the susceptible pig. The majority of the differentially expressed genes were derived from a cDNA library of leukocytes of A. pleuropneumoniae-challenged pigs that were subtracted against leukocytes before the challenge. These results provide evidence that the proposed statistical approach was useful in enhancing the knowledge of the mechanisms involved in the genetics of the immune response.

Monoclonal antibody against sheep kappa light chain.

The production of a monoclonal antibody specific for sheep kappa light chain protein is described. The monoclonal antibody was designated McM11 and its specificity was verified using western blots of sheep IgG and slides of efferent lymph cells. The specificity of McM11 was confirmed by specific recognition of fusion proteins expressed by recombinant phage containing sheep kappa cDNAs. N terminal sequence of the light chain recognized by McM11 showed homology to kappa type light chains. McM11, together with McM6, a lambda specific monoclonal antibody, was shown by two color FACS analysis of sheep blood lymphocytes to recognize all sheep light chains.

Production and verification of an anti-ovine IgE monoclonal antibody.

Recombinant mouse/sheep IgE was used in the production of an anti-IgE monoclonal using conventional hybridoma techniques. The specificity of hybridomas secreting anti sheep IgE monoclonal antibodies was verified using a number of assays including competitive ELISAs, ability to induce mediator release from mast cells, and IgE binding using western blotting. Immunohistochemical staining demonstrated the binding of putative anti-IgE monoclonals to the sheep mast cell surface. The isotype of the antibody was IgG1:K.

Immunoglobulin class specificity of non-agglutinating antibody produced in cattle following Brucella abortus 45/20 vaccination.

The immunoglobulin class specificity of non-agglutinating antibody produced following vaccination with Br. abortus 45/20 vaccine was determined using the antiglobulin test. In previously unvaccinated cattle it was found that most of the non-agglutinating antibody was associated with the IgG1 immunoglobulin class. In cattle sensitised to Brucella antigens by prior vaccination with Brucella abortus strain 19 the majority of antibody was also associated with the IgG1 immunoglobulin class, but a significant amount of IgG2 antibody was also present. The results indicate that the significance of levels of IgG2 antibody would be difficult to determine in cattle repeatedly exposed to Brucella antigens.

Reverse phase passive haemagglutination and single radial immunodiffusion to detect epsilon antigen of Clostridium perfringens type D.

Two in vitro immunological assays were developed for detection of the epsilon (epsilon) antigen of Cl. perfringens type D. It was found that the reverse phase passive haemagglutination assay (RPHA) was able to detect concentrations of epsilon-antigen as low as 6 x 10-7 mg/ml whereas the single radial immunodiffusion techniques (SRID) was capable of detecting concentrations of epsilon-antigen above 0.01 mg/ml. When applied to gut contents from freshly dead infected sheep the RPHA test was found to be more sensitive than mouse toxicity assay in detecting the presence of epsilon-antigen. However, very low titres were detected in gut contents from normal sheep which meant that in a diagnostic situation interpretation of RPHA titres would be difficult. No epsilon-antigen was detected by SRID in gut contents from normal sheep or in gut contents from freshly dead infected sheep. The SRID assay could detect epsilon-antigen in gut contents from infected sheep allowed to decompose for 20 h post-mortem.