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E N Meeusen

Publications and source records attributed to E N Meeusen.

35 records · Page 2Linked to original sources
PubMed1997

cDNA cloning of galectins from third stage larvae of the parasitic nematode Teladorsagia circumcincta.

A monoclonal antibody raised to a Teladorsagia circumcincta 31-33 kDa doublet antigen was used to immunoscreen a T. circumcincta cDNA expression library. Sheep antibodies eluted from the proteins expressed by two clones immunopositive with the monoclonal antibody specifically recognised the doublet antigen on Western blots of third stage larval extract, confirming that these clones coded for the antigen. Database searches revealed high levels of similarity with beta-galactoside-binding lectin-like proteins (Ga1BPs or galectins) from Caenorhabditis elegans and Onchocerca volvulus. By analogy with these sequences, both T. circumcincta cDNA clones contain the full-length protein coding region. The native doublet proteins could be preferentially extracted from homogenates of third stage larvae with lactose and could be affinity purified on an asialofetuin column, confirming the identity of these bands as galectins. Reverse transcriptase-polymerase chain reaction amplification using a primer based on the C. elegans Spliced Leader SL1 sequence showed that the corresponding T. circumcincta mRNAs are also trans-spliced at their 5' ends. While there are considerable nucleotide differences between the two clones, the majority are located in the non-coding regions. Within the coding region there are 87 nucleotide differences but only three of these result in amino acid substitutions.

Amino Acid Sequence↗
PubMed1996

Characterization of ES products involved in wound initiation by Lucilia cuprina larvae.

Excretory/ secretory (ES) products were collected up to 6 h after egg hatch and analysed by SDS-PAGE. The larvae produced a complex array of molecules, the pattern of which changed dramatically over the time of culture. When larvae were cultured on isolated sheep skin, skin degradation was found to occur immediately upon egg hatch with digestion of the available soluble skin molecules virtually complete within 6 h of culture. Proteolytic activity of the larval ES products was assessed by gelatine substrate SDS-PAGE gels and revealed numerous secreted proteases, the majority of which belong to the serine class of protease. Several of these proteases appeared to be developmentally regulated including a 28 kDa protease which was secreted only during the time of egg hatch. Metabolic labelling experiments indicated that culture conditions can influence the production of ES products with larvae cultured in the absence of sheep skin producing greater amounts and numbers of specific proteins.

Animals↗
PubMed1996

Developmentally regulated expression of a Haemonchus contortus surface antigen.

All life cycle stages of Haemonchus contortus were screened with antibody-secreting cell (ASC) probes generated from immune sheep 5 days after a challenge infection. An antigen with an approximate molecular weight of 70-90 kDa was found to be predominantly expressed in the 3rd-stage larvae, while a molecule with an approximate molecular weight of 28-31 kDa was recognised by the ASC-probes in the earlier developmental stages. Antisera generated against the purified 70-90 kDa molecule and antibodies eluted from the 70-90 kDa region in the 3rd-stage larvae were found to react with the 28-31 kDa region in the 2nd-stage larvae. These results suggest that the 70-90 kDa antigen, previously identified as a major 3rd-stage surface antigen, is synthesised as a lower molecular weight molecule in the earlier larval stages and assembled into a higher molecular weight complex through non-reducible covalent bonds.

Age Factors↗
PubMed1996

Vaccination of sheep against larvae of the sheep blowfly (Lucilia cuprina).

Four first stage larval antigens from the sheep blowfly were identified using supernatants from cultures of antibody secreting cells. These partially purified larval antigens, when added to Montanide ISA-25 containing recombinant ovine IL-1 beta (rovIL-1 beta) were used to successfully vaccinate sheep against larvae of the sheep blowfly. Significantly less strikes were recorded on vaccinated sheep compared to controls (P < 0.033) with surviving larvae from vaccinated sheep up to 85% smaller than larvae from control sheep. RovIL-1 beta was found to be an important component of the vaccine. Vaccinated sheep showed both humoral and cellular immune responses to the larval antigens. Antibody levels generally correlated directly with delayed-type hypersensitivity (DTH) responses, but neither antibody nor DTH correlated positively with protection in vaccinated sheep. Skin sections removed from individual sheep immediately after challenge revealed aggregations of CD4+, gamma delta-TCR+ and CD1+ cells located directly under the epidermis in vaccinated sheep.

Animals↗
PubMed1996

Distribution of antigen specific memory T cells in lymph nodes after immunization at peripheral or mucosal sites.

The distribution of antigen-specific memory T cells in different lymph nodes of sheep was determined using an antigen-specific in vitro proliferation assay. Lymph nodes were collected from sheep immunized simultaneously with avidin or ovalbumin in a peripheral tissue site (hind leg muscle) and keyhole limpet haemocyanin (KLH) in an intestinal tissue site (gut wall or colonic mucosa). The results showed a consistently high proliferative response in typical peripheral lymph nodes (popliteal and prescapular) and a low or negative response in gastrointestinal lymph nodes (abomasal and jejunal) while the response in other nodes was variable. The low proliferative response in the gastrointestinal lymph nodes was not due to the presence of suppressor CD8- lymphocytes and the proliferative response could not be raised to peripheral lymph nodes levels with the addition to cultures of IL-2 or mitomycin-C treated peripheral lymph node cells. The high proliferative response in the peripheral lymph nodes was not suppressed by the addition of mitomycin-C-treated gastric lymph node cells but was dramatically reduced by the addition of mAb against the IL-2-receptor or by depletion of CD4- T cells. The results suggest that antigen-specific proliferative memory T cells, which may be Th1-like memory cells, preferentially migrate to peripheral lymph nodes independent of their site of induction.

Administration, Rectal↗
PubMed1996

An in vivo mammary infusion model for tissue migration of leucocytes during inflammation.

Infusion of LPS or nematode larvae into the mammary glands of sheep induces recruitment of neutrophils or eosinophils respectively. While neutrophil recruitment required only a single infusion of LPS, repeated infusions of parasite larvae were required to induce significant eosinophil migration into the lumen of the glands. Eosinophil recruitment was accompanied by a distinct population of lymphocytes consisting mainly of activated (MHC class II and CD25+) T cells. L-selectin was expressed at reduced levels on both neutrophils and eosinophils collected from the mammary gland compared with cells present in the blood of the same sheep. In addition, VLA-4 and beta 1-integrin were down-regulated or negative in mammary eosinophils compared with strong expression in the blood while neutrophils were negative for these markers in both mammary washes and blood. Eosinophils in blood and mammary glands were negative for MHC class II, CD25 and CD4. Mast cells and lymphocyte aggregates were present in the tissue of glands chronically stimulated with parasite larvae while eosinophils were only present if the gland had been recently stimulated. These studies show that detailed in vivo analysis of leucocyte migration can be easily performed in the sheep mammary infusion model which allows non-invasive and repeated sampling of inflammatory cells before and after tissue migration.

Animals↗
PubMed1996

Fasciola hepatica: rapid switching of stage-specific antigen expression after infection.

Early developmental stages of the trematode parasite Fasciola hepatica were collected from the peritoneal cavity and liver of mice during a ten day infection period. Using one dimensional SDS-PAGE, differences in protein expression profiles were observed in stages collected on the same day post-infection in different physiological locations and also in juvenile parasites collected from the same location on different days post-infection. Four rat monoclonal antibodies were raised against the parasite using lymph nodes draining infected tissues. Three monoclonal antibodies, FY3-1, FY3-2 and FY4-7, were generated using cells from the mesenteric lymph node of recently challenged immune rats, while FY1-6 was derived from hepatic lymph node cells of a chronically infected rat. The epitope recognized by FY3-2 appeared to be carbohydrate in nature and was present on the surface of newly excysted juveniles. Immunoblots revealed that the antigens recognized by FY3-1, FY3-2 and FY4-7 were only expressed for two days after infection. In contrast, FY1-6 recognized epitopes expressed across all developmental stages screened. The rapid changes in protein and antigen expression observed during the early stages of infection may assist the parasite to evade the host immune response.

Animals↗
PubMed1994

Antibody secreting cells as specific probes for antigen identification.

Parasite specific antibody probes were prepared by harvesting lymphocytes from infected tissue and incubating them in vitro to allow the spontaneous secretion of antibodies in the culture medium by antibody secreting B cells present in the lymphocyte cultures. The culture supernatant was then used to screen Western blots of parasite antigens and resulted in the detection of antigens specific for the parasite stages present in the tissue at the time of sampling. Similar antigen recognition patterns were also observed when the cells were taken from the draining lymph nodes but the same pattern was not observed with serum taken from the same animal. The use of antibody probes obtained from in vivo induced antibody secreting B cells (ASC probes) offers a unique and universal approach to study local antibody recognition during infection.

Animals↗
PubMed1994

The immune response of sheep infected with larvae of the sheep blowfly Lucilia cuprina monitored via efferent lymph.

Changes in lymphocyte traffic in efferent lymph from the prescapular lymph node of sheep were monitored during local primary and secondary infection with blowfly, Lucilia cuprina. During primary infections the response was characterised by an increase in the output of CD4+ T cells over CD8+ T cells for the first 48 h after wound initiation. By 72 h the output of CD8+ T cells exceeded that of CD4+ T cells. During secondary infections the increased output of CD8+ T cells was more pronounced and occurred earlier at approximately 48 h. The percentage of B lymphocytes as measured by sIg, CD45R and MHC class II expression increased at approximately 96-120 h after both primary and secondary infections, with the secondary response being greater than the primary. This increase in B cells corresponded with peak antibody titres recorded in the efferent lymph to a first instar antigen preparation as measured by ELISA. An increase in IFN-gamma and soluble IL-2 receptor was recorded after both primary and secondary infections, with the response after secondary infection being greater than that recorded after primary larval infections.

Animals↗
PubMed1994

Identification of a novel antigen from Corynebacterium pseudotuberculosis that protects sheep against caseous lymphadenitis.

A 40-kDa protein antigen from Corynebacterium pseudotuberculosis has been identified by application of a strategy that employs locally derived antibody-secreting cells (ASC). ASC probes generated by culture of ASC obtained from the lymph node draining the site of infection showed a specificity restricted to a 40-kDa antigen. Analysis of immunoblots with sequential serum samples taken from sheep during the course of experimental primary infection with C. pseudotuberculosis also revealed the 40-kDa antigen as an early immunodominant antigen. Sheep vaccinated with two 100-micrograms doses of a 40-kDa antigen preparation in aluminium hydroxide adjuvant were protected against infection with C. pseudotuberculosis, with an 82% reduction in the proportion of infected sheep and a 98% reduction in lung lesions. Sera from vaccinated sheep exhibited a strong response only to the 40-kDa antigen on immunoblots. These results strongly suggest that the 40-kDa antigen plays a major role in immunity to caseous lymphadenitis.

Animals↗
PubMed1993

Molecular cloning and characterization of a ruminant interleukin-6 cDNA.

By hybridization with a human interleukin-6 (IL-6) cDNA fragment a corresponding ruminant (ovine) cDNA was isolated from a lipopolysaccharide (LPS)-stimulated alveolar macrophage library. The nucleotide sequence of the cDNA and the predicted amino acid sequence of the protein showed significant homology to the human and murine molecules. Ovine IL-6 cDNA encodes a polypeptide of 208 amino acids that, based on analysis of human IL-6, is processed to a protein of 180 amino acids. Northern blot analysis and the 7TD1 bioassay were used to analyse regulatory aspects of IL-6 production by primary ovine fibroblasts. Both LPS and recombinant ovine IL-1 alpha were shown to induce IL-6 mRNA with peak levels occurring at 1 h post-stimulation and declining thereafter. When fibroblasts were pretreated with cyclohexamide prior to stimulation the level of induction by LPS and IL-1 alpha increased dramatically and peak levels were observed at 5 h post-stimulation. The level of secreted IL-6 increased rapidly over the first 24 h and continued to increase over the next 48 h.

Amino Acid Sequence↗
PubMed1993

Recombinant cytokines as immunological adjuvants.

This paper describes the bacterial expression and purification of bioactive recombinant ovine interleukin-2 (rovIL-2), interleukin-1 alpha (rovIL-1 alpha) and tumour necrosis factor alpha. These purified proteins had specific activities in appropriate bioassays of 1 x 10(7) 1 x 10(7) and 1 x 10(5) U/mg, respectively. Recombinant ovIL-1 alpha was assessed as an immunological adjuvant for the sheep response to the model protein avidin. When delivered either intradermally or intramuscularly in conjunction with avidin in aluminium hydroxide the rovIL-1 alpha significantly enhanced the secondary humoral response. Doses of 1, 10 or 100 micrograms per sheep enhanced the humoral response to a similar extent. Recombinant ovIL-1 beta had similar adjuvant activity in that it was demonstrated to significantly enhance the sheep humoral response to an experimental H. contortus antigen. This increase in specific antibody, however, did not correlate with enhanced protection against infection with third stage H. contortus larvae. In addition incorporation of rovIL-1 beta into the formulation was shown not to alter the isotype profile of H. contortus antigen specific antibody.

Adjuvants, Immunologic↗
PubMed1992

Systemic immunization of sheep with surface antigens from Haemonchus contortus larvae.

Sheep immunized systemically with a surface extract from third-stage H. contortus larvae showed high serum antibody reactivity against surface antigens and whole, viable larvae. After a first infection, no significant difference was found between the mean egg counts of the vaccinates and controls although most vaccinated sheep seemed to show an increased susceptibility to infection. The local abomasal response was stimulated by giving both vaccinated and control sheep a large, abbreviated infection cured after 11 days by drenching. Thereafter, a second challenge infection was given. This immunization regime resulted in seven of the nine vaccinated sheep showing clear protection against the second challenge infection.

Animals↗
PubMed1990

Genetic selection for disease resistance and traits of economic importance in animal production.

Significant advances have been made in recent years in improving animal stocks by selective breeding. However, existing selection techniques still rely on laborious and time-consuming progeny-testing programs and often depend on subjective assessment of the phenotype. New techniques in molecular genetics are being developed, aimed at the isolation and identification of DNA markers linked to genes for economically important production traits and disease resistance. When available, these markers will provide animal breeders with an objective test system to identify, at birth or even earlier, animals carrying desirable genes. This review outlines some of these new techniques and how they may be applied to the animal industries. Consideration is also given to some of the recent advances in our understanding of the immune system and of possible mechanisms of genetic control of animal disease resistance or susceptibility. The current knowledge of major histocompatibility complex (MHC) and non-MHC associated disease resistance/susceptibility in domestic animals is summarised and mechanisms which may be responsible for these associations are presented. Genes that control such factors as macrophage activation, cytokines, cytokine receptors and gamma delta-T cell receptors are also presented as potential candidates for analysis in genetic disease association studies. Ultimately, the goal will be to identify genes or DNA markers which can be used to select for or to genetically engineer disease resistance and enhanced production traits.

Animals↗
PubMedDate not supplied

Rational design of nematode vaccines; natural antigens.

Bacterial and viral vaccines have been used for several decades and have made an invaluable impact on the control of both human and animal diseases. As these vaccines are based on the induction of the "natural" protective immune response of the host, there is ample proof that this approach is effective and there is no a priori reason why a similar approach should not work for other infectious organisms, including helminth parasites. Two main stumbling blocks have so far precluded the development of effective anti-nematode vaccines: (i) the identification and isolation of the protective parasite antigens; and (ii) the induction of the appropriate protective immune effector mechanisms through vaccination. This paper will discuss the unique characteristics of nematode vaccine development and the significant progress that has been made in recent years in this area.

Animals↗
PubMedDate not supplied

Cytokines as adjuvants for ruminant vaccines.

Successful vaccination against any potential pathogen is critically dependent on inducing an appropriate immune response. The pivotal role of cytokines in the immune response to vaccination suggests that non-protective responses or responses that exacerbate disease subsequent to infectious challenge may be the result of limiting or preferential production of one or a number of these mediators. This suggests that the use of recombinant cytokines as vaccine adjuvants may offer a mechanism whereby the magnitude and phenotype of the immune response to vaccination can be specifically modified. In mice, recombinant cytokines have been used extensively as therapeutics, while studies describing vaccine adjuvant activity are more limited. Recombinant (r) interleukin (IL)-1, IL-2 and interferon (IFN) gamma have been used primarily to enhance humoral responses with enhanced protection assessed where appropriate. Cytokine adjuvant studies in ruminants have been restricted to recombinant ovine (rov) and bovine (rbov) IL-1 and IL-2. In sheep, their application has been optimised with respect to dose, route of delivery and formulation, for induction of humoral and cell mediated immunity (DTH and cytotoxicity) to the model protein antigen (Ag) avidin. The level of adjuvant activity of IL-1 in particular compares favourably to that of a variety of other traditional and new chemical adjuvants and detailed analysis has indicated no adverse local or systemic side-effects. Recent studies in our laboratory demonstrating the effectiveness of rovIL-1 as an adjuvant in single and multi-component bacterial toxoid vaccines, and studies from other laboratories demonstrating the application of rbovIL-1 as an adjuvant for the response in cattle to live attenuated viral vaccines, suggest that rIL-1 may become the adjuvant of choice for diseases where protection is mediated by high levels of circulating antibody (Ab). With respect to helminth parasites, IL-1 may prove useful as a component of vaccines based on "hidden Ags" which rely on induction of Ab. Based on analysis in mouse models of helminth infection, other cytokines such as IL-4 and IL-10 may be appropriate for vaccines based on induction of mechanisms involved in natural immunity.

Adjuvants, Immunologic↗