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Biomedical subjects

B M Goddeeris

Publications and source records attributed to B M Goddeeris.

At least 73 records · Page 4Linked to original sources
PubMed2000

The bovine cytotoxic T cell: MHC restriction and antigen-specificity.

The analysis of the immune mechanisms operative against the bovine intracellular protozoan parasite Theileria parva resulted in the identification and characterisation of the bovine cytotoxic T lymphocyte (CTL) and its important role in protection against this parasite. By the use of polyclonal and monoclonal antibodies specific for bovine MHC antigens and lymphocyte populations and the use of MHC-haplotyped animals it was possible to demonstrate the MHC restriction and antigen-specificity of the Theileria-specific cytotoxicity. In cross-protection studies with different parasite stocks or strains, a consistent correlation could be observed between the strain specificity of the CTL and the in vivo cross-protection. Moreover, the in vitro generation of CTL lines and clones and the development of a standardised technique for analysing the frequency of parasite-specific CTL precursors in cattle immunised with Theileria parva, enabled the identification of parameters influencing the parasite strain specificity of the cytotoxic response. Not only the immunising parasite but also the bovine MHC haplotype of the respective immunised animal influenced whether the Theileria-specific CTL were strain-specific or cross-reactive between strains. These results have important implications for the identification of cross-reactive CTL epitopes of the parasite in the development of subunit vaccines for outbred animals.

Animals↗
PubMed1999

Development of immunocompetence of broiler chickens.

Subpopulations of T-cells, B-cells, macrophages and ellipsoid-associated reticular cells (EARC) could be demonstrated by immunohistochemical staining early in the development of chicken spleen. However, the typical structures of the spleen, such as the peri-arteriolar lymphoid sheath (PALS) and the ellipsoids with their surrounding ring of macrophages, were only formed around embryonic day (ED) 20. These structures and especially the B-cell compartment, i.e., the peri-ellipsoid lymphoid sheath (PELS) gradually matured during the first week posthatch. Therefore, we analysed at what age broiler chickens could generate a humoral response against the thymus-dependent antigen bovine serum albumin (BSA). Chickens were immunised in ovo (ED16 and ED18) and at 1, 7 and 12 days of age and subsequent BSA-specific immunoglobulin (Ig) M and IgG responses were measured up to 10 days postimmunisation (DPI). No major differences were observed in the relative growth rates, while hatchability was only slightly reduced. Only in chicks immunised on 12 days of age, IgM and IgG responses were high with a normal kinetic pattern. In chicks immunised on 7 days of age, responses were just detectable, but they were absent in chicks immunised in ovo and on the day of hatching (Day 1). In a subsequent experiment, 1-, 7- and 12-day-old chicks were BSA-immunised and Ig responses were measured for a longer period up to the age of 28 days. The IgG response of chicks immunised at 1 day of age was lower and occurred later (from 28 DPI) than the response of chicks immunised at 7 and 14 days of age (from 14 DPI). It was not increased by a booster immunisation on 29 days of age, in contrast to the response of chicks immunised at 7 and 14 days of age. These findings indicate that vaccination at 1 day of age does not activate the B-cell response resulting in antibody production and support the idea that the immune function of the late embryonic and neonatal chickens is not entirely developed due to the incomplete structural organisation of their secondary immune organs.

Aging↗
PubMed1999

Seroprevalence of F4+ enterotoxigenic Escherichia coli in regions with different pig farm densities.

Sera of young sows from 135 closed Belgian pig breeding farms were examined for the presence of serum antibodies specific for the F4 fimbrial antigen of enterotoxigenic Escherichia coli (ETEC). Since 80% of all pig farms in Belgium are located in the provinces West-Vlaanderen (44%, approximately 18 farms/10 km2), Oost-Vlaanderen (20%, approximately 9 farms/10 km2), Antwerpen (10%, approximately 5 farms/10 km2) and Vlaams-Brabant (6%, approximately 3 farms/10 km2), the farms examined were randomly selected in these four regions. On 68% of all tested farms, sows were not vaccinated against enteric colibacillosis. In general, 65% of these non-vaccinated farms were F4-seropositive (mean optical density [OD405] >0.5) of which 38% were weakly (mean OD405 = 0.5-1.0), 21% moderately (mean OD405 = 1.0-2.0) and 6% strongly positive (mean OD405 > or =2.0). These percentages showed major differences between the four provinces. In Vlaams-Brabant, only 31% of non-vaccinated farms were seropositive, of which most were regarded as weakly positive. In Antwerpen, Oost- and West-Vlaanderen however, this percentage was clearly higher and respectively 68, 71, and 79% of non-vaccinated farms were seropositive, of which most were weakly positive. These observations indicate that F4+ ETEC is widely spread and highly prevalent on non-vaccinated pig breeding farms in Belgium. Moreover, an increasing F4-seropositivity with increasing pig farm density suggests a possible influence of the swine density on the prevalence of F4+ ETEC infections.

Animals↗
PubMed1999

Receptor-specific binding of purified F4 to isolated villi.

Different procedures for preparing and purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26 (O149:K91:F4ac LT+Sta+STb+) were performed and the purity and yield of F4ac were compared. Fimbriae were prepared by either mechanical shearing or heatshock treatment of concentrated bacterial suspensions (10(11) bacteria/ml). The mechanical shearing procedure resulted in approximately 1.7 mg fimbriae (i.e. 74.4% of the isolated protein) and 0.6 mg (25.6%) contaminating proteins per 10(12) bacteria, whereas the yield of fimbriae following heatshock treatment was lower (0.3 mg per 10(12) bacteria, i.e. 26.2%) and the relative contamination higher (1.0 mg per 10(12) bacteria, i.e. 73.8%). A further purification consisted of either anion exchange chromatography (AEC) or electro-elution from SDS-polyacrylamide gels. The electroelution procedure was performed under reducing and denaturing conditions, so that purified FaeG subunits, the major subunit of F4, were finally obtained. The binding activity of fimbriae, nonpurified as well as purified, and FaeG to F4-specific receptors on isolated intestinal villi was assessed in an inhibition adhesion assay. Native fimbriae as well as major subunits were able to bind to the receptors, and the specificity of the binding was demonstrated by blockage with F4ac-specific MAb.

Animals↗
PubMed1999

Antigenicity of cortical bone allografts in dogs and effect of ethylene oxide-sterilization.

The aims of the present study were to determine the antigenicity of cortical bone allografts and the effect of ethylene oxide-sterilization (EO-sterilization). Cortical bone allografts from one donor dog were implanted in a muscle pouch in four groups of four dogs each. The grafts were either fresh, EO-sterilized, demineralized or demineralized and EO-sterilized. The immune response against the grafts was determined by measuring the antibody response against surface antigens of donor cells and by the mixed lymphocyte reaction. Dogs receiving EO-sterilized grafts or bone matrix did not demonstrate an immune response. Only two of the four dogs with fresh cortical bone grafts showed a very weak immune response. This suggests a priming of the host by the fresh bone grafts. However, implanting skin grafts from the donor dog subdermally, in one dog of each of the groups, four months after implanting the bone grafts did not induce a secondary immune response. Macroscopic and histologic examination of the bone grafts five months after their implantation consistently revealed graft resorption (activity of osteoclasts) and vascularization of the fresh bone grafts, but not of EO-sterilized fresh grafts. For most EO-sterilized grafts, a strong inflammatory reaction was present in the tissues surrounding the graft and this was not apparent around the non-sterilized grafts. The absence of resorption and the presence of the inflammation seemed to be unwanted effects of the EO-sterilization. The EO-sterilisation did not affect osteoinduction since osteocytes were observed in the EO-sterilized demineralized grafts. Results indicate that cortical bone allografts used in the present study are very weak antigens and that the EO-sterilization procedure used has no effect on osteoinduction, but decreases bone resorption.

Animals↗
PubMed1999

Induction of immune responses in pigs following oral administration of purified F4 fimbriae.

An effective way of stimulating the mucosal immune system was examined in piglets, using F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). It was demonstrated that purified F4 fimbriae, as opposed to ovalbumin (OVA), are powerful oral immunogens. Indeed, oral administration of purified F4 induced antigen-specific antibody-secreting cells (ASC) in the Peyer's patches, mesenteric lymph nodes (LN), blood and lamina propria 4, 7, 9 and 11 days postimmunization, respectively, indicating a stimulation of the mucosal immune system, whereas upon oral administration of OVA, no immune response was observed. Moreover, the induced F4-specific IgA and IgG antibody responses were comparable with those obtained upon oral infection with viable E. coli and intramuscular (i.m.) F4 injection, respectively. Furthermore, a priming of the mucosal immune system is better obtained by oral infection (ASC localized in mesenteric LN) than by i.m. F4 injection (ASC localized in spleen and retropharyngeal LN) since an oral boost with purified F4 induced a secondary response in the orally infected animal (mainly IgA and IgG ASC, rapid increase of IgA antibodies) while in the i.m. primed animal a secondary (more circulating antigen-specific ASC than in the unprimed animal) as well as a primary IgM and IgA response (mainly IgM ASC, slow increase of IgA antibodies), suggesting a primary mucosal response, were seen. An oral challenge of the naive control displayed a primary response (mainly IgM ASC, slow increase of IgA and IgG antibodies). The capacity of purified F4 to activate the mucosal immune system on oral administration, is of importance for the development of oral vaccines against ETEC infections.

Adhesins, Escherichia coli↗
PubMed1999

Receptor-dependent immune responses in pigs after oral immunization with F4 fimbriae.

F4 receptor-positive (F4R+) and F4 receptor-negative (F4R-) pigs were orally vaccinated with purified F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). Serum immunoglobulin G (IgG) and IgA responses were readily detected in F4R+ animals, whereas immune responses were not detected in F4R- animals. Even after a subsequent oral infection with virulent F4(+) ETEC and a booster immunization with F4, the F4R- animals remained F4 seronegative whereas the unvaccinated F4R+ pigs exhibited clear IgA and IgG responses. These results clearly demonstrate that F4Rs are a prerequisite for an immune response following oral immunization. Furthermore, indications that oral F4 vaccination can induce mucosal protection were obtained, since the experimental ETEC infection did not induce a systemic booster response or fecal ETEC excretion in orally vaccinated F4R+ pigs, in contrast to the clear immune response and ETEC excretion of unvaccinated F4R+ animals. F4-specific IgA antibodies could be found in the feces of the vaccinated F4R+ pigs. They are secreted at the intestinal mucosal surface and appear to prevent ETEC infection. The F4R-dependent induction of a mucosal immune response can be used as a model to better understand mucosal immunization and mucosal immune responses and can contribute to the development of oral vaccines in veterinary as well as in human medicine.

Adhesins, Escherichia coli↗
PubMed1998

PCR amplification and sequence of the p33 piroplasm surface antigen gene of a Theileria species isolated from cattle in west Java, Indonesia.

Oriental theileriosis, a parasitic disease of cattle caused by protozoa of the Theileria orientalis/sergenti/buffeli group, has been reported in Indonesia but its causal agent had not yet been characterized. This study was carried out to isolate and characterize the parasite through comparison of its p33 piroplasm surface antigen gene sequence, with known p32 sequences of T. sergenti and T. buffeli isolates. A Theileria spp. isolate was collected from an Ongole cow in Jonggol, West-Java, and transferred into a splenectomized calf for antigen production. Piroplasms were extracted from erythrocytes by ammonium chloride-lysis, separated from unlysed leukocytes and parasitic DNA was phenol-extracted. Polymerase chain reaction (PCR) was carried out on genomic DNA with a pair of 20 bp primers showing consensus for the p32-35 nucleotide sequence of 7 known T. orientalis/sergenti/buffeli isolates. An 875 bp fragment was amplified, and further sequenced on both strands by the dye-labeled terminators method. It showed an 88% homology with the p33 nucleotide sequence of the Japanese T. sergenti Ikeda stock and a lesser homology with 6 other sequences of Australian T. buffeli or Japanese T. sergenti stocks. It was shown to share the presence of the Pst 1 and the absence of the HindIII restriction sites of the T. sergenti Ikeda stock and of one Australian T. buffeli stock, respectively. In conclusion, the affiliation to and the relative position of this Indonesian isolate within the T. orientalis/sergenti/buffeli group has been elucidated.

Amino Acid Sequence↗
PubMed1998

Role of the humoral immune system in Salmonella enteritidis phage type four infection in chickens.

The role of avian humoral immunity in the clearance of S. enteritidis was evaluated through bursectomy. After oral inoculation of bursectomized and sham-treated chickens with S. enteritidis, faecal excretion of S. enteritidis was examined. Organs were collected weekly until six weeks post-inoculation (pi) for bacteriological enumeration. Antibody isotypes in serum and bile were quantified by ELISA. Faecal excretion of S. enteritidis was significantly lower in controls from 13 days pi. Numbers of S. enteritidis in caeca from controls were significantly decreased from three weeks pi. Numbers of S. enteritidis were significantly decreased at two weeks pi in the spleen and the liver and at six weeks pi in the liver. Antibodies to S. enteritidis peaked at two weeks pi in controls and were absent in bursectomized chickens. These findings indicate that elimination of S. enteritidis partly depends on humoral immunity. The intestinal humoral response appeared more effective than the systemic humoral response for elimination of S. enteritidis.

Animals↗
PubMed1998

Characterisation of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01.

The distribution, function and ontogeny of the mononuclear phagocyte system of the chicken were characterised using the monoclonal antibody (MAb) KUL01. KUL01 specifically recognises chicken monocytes, macrophages and interdigitating cells, as well as activated microglia cells. Its tissue distribution allowed to discriminate KUL01 from all earlier described MAb, reactive with mononuclear phagocytes. The specificity of KUL01 for mononuclear phagocytes was further confirmed in functional assays: KUL01-positive macrophages in spleen and liver actively took up colloidal carbon, while monocytes and spleen and gut macrophages contained non-specific esterase and acid phosphatase activities characteristic for antigen-processing. Further, it was demonstrated that KUL01-reactive peripheral blood monocytes express MHC-II, but not CD4. In all tissues investigated, the same morphological subtypes of macrophages were detected in chicken at similar localisations as in mammals, indicating a high degree of conservation between the mononuclear phagocyte system of the chicken and of mammals.

Animals↗
PubMed1998

CD57, a marker for B-cell activation and splenic ellipsoid-associated reticular cells of the chicken.

We have demonstrated that the ellipsoid-associated reticular cells of chicken spleen express CD57, a marker for B-cell activation. These cells are characterised by their spindle-shaped morphology, tissue distribution and the absence of certain leucocyte-specific markers. They are phagocytotic and possess high endogenous non-specific esterase activity. Previous reports failed to detect CD57 expression on ellipsoid-associated reticular cells, probably because the tissue sections were differently treated before immunohistochemistry. CD57 is also expressed by a small number of T-cells in the spleen and the caecal tonsils. This number is highly variable between individual chickens depending on the activation state of the immune system. Moreover, CD57 is expressed by bursal lymphocytes (90% or more) but not by B-cells of the peripheral blood. More interestingly, we have been able to discriminate and quantify three B-cell populations of the secondary lymphoid organs, i.e. resting B-cells, germinal centre B-cells and plasma cells, based on their expression levels of CD57 and Bu-1 (a pan B-cell marker). Thus, CD57 should be considered as a B-cell activation marker, rather than as a marker for bursal B-cells; it is also a valuable marker for the immunohistochemical study of ellipsoid-associated reticular cells of chicken spleen.

Animals↗
PubMed1997

Expression of beta 2 integrins on blood leukocytes of cows with or without bovine leukocyte adhesion deficiency.

Peripheral blood leukocytes of 11 normal cows, 7 cows heterozygous and 2 heifers homozygous for bovine leukocyte adhesion deficiency (BLAD) were analysed by flow cytometry for the intensity of their beta 2 integrin expression (LFA-1(CD11a/CD18), CR3 (CD11b/CD18) and CR4 (CD11c/CD18)). BLAD-homozygotes revealed no or a very weak expression of the beta 2 integrins and had a 10-fold and 4- to 5-fold increase in absolute number of neutrophils and monocytes, respectively, whereas the absolute number of lymphocytes remained normal. The mean fluorescence intensity (MFI) of the beta 2 integrins (CD18) in heterozygous animals was 56 to 90% of this in the normal cows (MFI between 14 and 512). The difference in the expression level was most pronounced for LFA-1 on the small cluster of lymphocytes with the highest MFI for LFA-1. Repeated analysis and phorbol myristate acetate stimulation revealed that the LFA-1 expression on this high-expressing cell population of the peripheral blood allowed a ready identification of BLAD-heterozygotes by flow cytometry.

Animals↗
PubMed1997

Induction and suppression of lymphocyte proliferation by antigen extracts of Ostertagia ostertagi.

To obtain an insight into the responses of T-cells of cattle to Ostertagia ostertagi, the responses of peripheral blood and lymph node lymphocytes to O. ostertagi antigen extracts were determined in both exposed and naive calves. The lymphocyte responses induced by O. ostertagi antigen extracts of the third (L3) and fourth (L4) larval stages, as well as adult worms, were analysed. Although peripheral blood lymphocyte responses were very low or absent, abomasal lymph node lymphocytes of exposed animals showed a strong response to the L3 antigen extract. No such response was observed in naive calves or in mesenteric lymph node cells of exposed calves. L4 and adult worm antigen extracts suppressed the proliferative responses induced by the L3 antigen extract. Whether or not this suppressive effect plays a role in the slow rate at which protective immunity develops against O. ostertagi is under further investigation.

Animals↗
PubMed1995

Parasite strain specificity of bovine cytotoxic T cell responses to Theileria parva is determined primarily by immunodominance.

The parasite strain specificity of CTL responses to Theileria parva varies among cattle immunized with the same parasite stock. We have investigated the influence of class I MHC on the strain specificity of CTL responses to T. parva in 19 cattle of defined class I phenotype immunized with either of two T. parva populations, in which protection to subsequent reciprocal challenge correlated with CTL strain specificity. In the majority of animals the response was restricted by the products of one MHC haplotype and there was a consistent bias to some haplotypes in preference to others. In 10 of 13 cattle expressing the molecularly defined MHC specificities A10 and KN104 on one haplotype, the CTL response was restricted entirely by this haplotype, thus allowing a precise analysis of the MHC restriction specificities. The MHC restriction specificity and the parasite population used for immunization both influenced the strain specificity of the response. By examining responses in identical twins immunized with different parasites or in animals before and after challenge with heterologous parasites, animals that mounted a strain-specific response to primary infection were shown to be capable of responding to Ags shared by the two parasite populations. These findings indicate that the strain specificity of CTL responses to T. parva is not determined primarily by immune response genes that define the inherent capacity to respond, but rather is a consequence of the response in individual animals being biased toward a limited number of immunodominant peptide-MHC determinants.

Animals↗
PubMed1995

Parasite strain specificity of precursor cytotoxic T cells in individual animals correlates with cross-protection in cattle challenged with Theileria parva.

Class I major histocompatibility complex-restricted parasite-specific cytotoxic T lymphocytes (CTL) are known to be a major component of the bovine immune response to the protozoan parasite Theileria parva, but formal proof for their role in protection of cattle against infection with T. parva has been lacking. Animals immunized with one stock of T. parva show variations in the degree of protection against heterologous challenge and also in the parasite strain specificity of their CTL responses. The present study investigated the relationship of strain specificity of CTL responses and cross-protection in an effort to verify the role of CTL in protection. The parasite strain specificity of the CTL responses generated in 23 cattle immunized with either of two immunologically distinct parasite populations was examined, and the susceptibility of individual cattle to challenge with the heterologous parasite population was determined. The frequency of stock-specific or cross-reactive CTL precursor cells (CTLp) in individual animals was measured by a limiting-dilution microassay. A proportion of animals immunized with either parasite exhibited cross-reactive CTLp, whereas CTLp detected in the remaining animals were specific for the homologous parasite. On challenge with the heterologous stock, those animals with cross-reactive CTLp were solidly protected while those with strain-specific CTLp showed moderate to severe reactions, although many of them recovered. The finding of a close association between strain specificity of the CTL response and protection against challenge provides strong evidence that CTL are important in mediating immunity.

Animals↗
PubMed1994

Adoptive transfer of immunity to Theileria parva in the CD8+ fraction of responding efferent lymph.

Evidence that class I major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are involved in immunity to malaria has highlighted the potential importance of these cells in protection against intracellular parasites. Parasite-specific CTL are a prominent feature of the immune response of cattle to Theileria parva, a related apicomplexan parasite. The relationship between the appearance of these cells in the blood of immune cattle under challenge and the clearance of infection suggests that they are involved in the control of infection, but direct evidence is lacking that CTL can mediate protection. We have made a quantitative kinetic study of CTL responses in lymph originating from infected lymph nodes in a number of immune cattle under challenge with T. parva. Direct killing activity and the frequency of CTL precursors (CTLp) within responding cell populations were evaluated. A substantial increase in the proportion of CD8+ CTL was observed between days 8 and 11 after challenge. Frequencies of CTLp as high as 1:32 were observed and activity was essentially confined to the large blasting cell fraction. The analogous response in peripheral blood was of lower magnitude and delayed by 1-2 days. The high frequency of CTLp in efferent lymph permitted the adoptive transfer of this activity between immune and naive monozygotic twin calves. In separate experiments, naive calves lethally infected with T. parva were protected by inoculation of up to 10(10) responding CD8+ T cells derived from their immune twins. Elimination of CD8+ T cells within the inoculum abrogated this effect. These findings provide direct evidence that CD8+ T cells can control T. parva infections in immune cattle.

Animals↗
PubMed1992

Standardization of a technique for analysing the frequency of parasite-specific cytotoxic T lymphocyte precursors in cattle immunized with Theileria parva.

A limiting dilution microculture system was optimized to quantify the frequency of Theileria parva-specific cytotoxic T lymphocyte precursors (CTLp) in peripheral blood mononuclear cells (PBMC) from immune cattle. Optimal results were obtained with responder cell input levels ranging from 2 x 10(4)/well to 6.25 x 10(2)/well, along with 1-5 x 10(3)/well stimulator cells in standard supplemented RPMI 1640 medium containing 2.5-5% T cell growth factors. Thirty-six microtitre wells were established at each responder input level. Cultures were incubated for 7 days at 38 degrees C, at the end of which time individual wells were screened for cytotoxic activity in a 4-h 111indium oxine-release assay. Analysis of the cytotoxicity data, by a computer-programmed maximum likelihood estimation method indicated that they conformed to the Poisson model of single-hit kinetics. Estimates of frequencies ranged from 1:3600 to 1:5275 CTLp in PBMC of eight cattle between 1 and 24 months after immunization with T. parva. By contrast, no CTLp were detected in six naïve animals analysed to a responder cell input of 10(5)/well. Split-well analysis of individual microwells showed that the CTL clones generated under limiting dilution conditions displayed exquisite specificity for parasitized cells, were genetically restricted and in some animals were parasite strain-specific.

Animals↗