Biomedical subjects

B A Bokhout

Publications and source records attributed to B A Bokhout.

At least 19 recordsLinked to original sources

PubMed2000

Fasciola hepatica: an antigen fraction derived from newly excysted juveniles, containing an immunoreactive 32-kDa protein, induces strong protective immunity in rats.

Crude antigens of adult Fasciola hepatica and of newly excysted juveniles (NEJ) and a low-molecular-weight fraction of antigen from NEJs were tested for inducing protective immunity in rats. Two routes of vaccination were applied. The results showed that intraperitoneal vaccination induced significantly better protection (P <0.05) than intramuscular vaccination. Intraperitoneal vaccination with antigens from NEJs induced more effective protection: after challenge infection, rats that were so vaccinated had 92.6% (+/-2.5% SEM) fewer parasites in their liver and 57.3% (+/-13.3% SEM) fewer parasites penetrating the gut wall than control rats. Rats that were vaccinated with a low-molecular-weight fraction of antigen from NEJs were also highly protected against a challenge. F. hepatica antigens that are immunoreactive were identified on immunoblots, using sera collected from highly protected rats that had been vaccinated with NEJ antigens and also sera from cattle and rats that were experimentally infected with F. hepatica. The low-molecular-weight fraction of antigen from NEJs contained an immunodominant 32-kDa protein that was recognized by serum antibodies of vaccinated rats and immune cattle. This 32-kDa protein was not detected in partially purified antigens from adult flukes. We conclude that antigens of NEJs of F. hepatica, when injected intraperitoneally in rats, are highly protective. In particular, the 32-kDa protein contained in these antigens may be highly valuable for the development of an effective vaccine against F. hepatica.

Animals↗

PubMed1999

Protection against Fasciola hepatica in the intestine is highly correlated with eosinophil and immunoglobulin G1 responses against newly excysted juveniles.

Rats were infected with Fasciola hepatica and challenged at regular intervals up to 38 weeks using an ex vivo gut loop, a technique developed in our laboratory. The kinetics of the observed immune responses against F. hepatica in gut tissue and serum were investigated and correlated to protection. Immunohistochemical methods were used to measure the frequency of eosinophils, immunoglobulin (Ig)E-positive cells, and mucosal mast cells in the gut loop, and to determine whether the newly excysted juveniles were coated with IgG antibodies or surrounded by eosinophils, or both. Enzyme-linked immunosorbent assays and a radioimmuno assay were used to measure serum antibody reactive with newly excysted juveniles. Results showed that protection was highly correlated with the frequency of eosinophils and IgE-positive cells in the gut, but was only moderately correlated with the frequency of mucosal mast cells. Newly excysted juveniles taken from rats exhibiting high levels of protection were always coated with IgG antibodies and surrounded by eosinophils. Protection was highly correlated with titers of serum IgG1 antibodies directed against newly excysted juveniles, but was only weakly correlated with titers of serum IgA and IgE antibodies. Because protection was highly correlated with IgG1 in gut tissue and serum, and with eosinophils in gut tissue, we suggest that IgG1 and eosinophils are important in protecting rats against F. hepatica.

Animals↗

PubMed1998

A novel ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level.

We describe an ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level. An exact number of newly excysted juveniles (NEJs) was injected into a gut segment with an intact blood supply and which was still attached to a live anaesthetized rat. NEJs that penetrated the gut wall during the following 6 h were recovered from a beaker filled with medium and were counted under a microscope. This infection model was validated and enabled us to exactly quantify the infection dose whilst at the same time exactly quantifying the number of NEJs penetrating the gut wall. The mean sum of NEJs that migrated through the gut wall into the beaker (peritoneal fraction), plus NEJs that remained in the gut wall and the gut lumen was 87% of the infective dose (+/-3.6% SEM; n=18). The function of the ex vivo segments was well-preserved, as demonstrated by only minor leakage of an inert liquid marker. The ex vivo model enabled us to measure protection against F. hepatica at the gut level. In naive rats 52% (+/-2.4% SEM; n=40) of the injected NEJs penetrated the gut wall, whereas in previously infected rats only 12% (+/-1.8% SEM; n=40) were able to do so, irrespective of the infection dose. Thus, when rats were orally primed, the migration of NEJs through the gut wall was 77% less than the migration in naive rats. We conclude that the ex vivo model should be valuable in studies of the induction and expression of protective immunity against F. hepatica in the intestine, and will aid in development and optimization of vaccines.

Animals↗

PubMed1998

Protection of Fasciola hepatica in the gut mucosa of immune rats is associated with infiltrates of eosinophils, IgG1 and IgG2a antibodies around the parasites.

We investigated the immune effector mechanisms that underlie protection against F. hepatica in the gut wall of immune rats, using (immuno)histochemistry. In the lamina propria of immune Wistar rats, four weeks after oral infection, frequencies of IgE-positive cells, eosinophils and mucosal mast cells were significantly increased, compared with naïve rats. These factors represent the traditional effector mechanisms against helminths. No significant differences were detected between the two groups in frequencies of IgM-, IgG2a-, IgG1- and IgA- positive cells, CD4- and CD8-positive cells, NK cells, macrophages, neutrophils or goblet cells. Upon challenge of immune rats with F. hepatica in an ex vivo gut segment, NEJs that migrated through the (sub)mucosa were coated with IgG1 and IgG2a antibodies and surrounded by eosinophils. No IgE or IgA antibodies were detected on the parasites. The onset of these immune effector responses, two h after challenge, was related to the expression of protection. These results suggest that NEJs are killed by an eosinophil-mediated cytotoxic response involving IgG antibodies. These antibodies were not produced in the intestine, but infiltrated the gut upon challenge. The observed immune effector responses were not restricted to the site where the primary infection is located, namely the small intestine, but were also detected in the large intestine. The presence of the protective immune mechanisms in two other rat strains demonstrates the pivotal importance of these responses, irrespective the genetic background of the host.

Animals↗

PubMed1998

Location of induction and expression of protective immunity against Fasciola hepatica at the gut level: a study using an ex vivo infection model with ligated gut segments.

In the present study, we investigated the site in the host where protective gut immunity to Fasciola hepatica is induced and expressed, following the infection route of the parasite. Expression of protection was studied in ex vivo gut segments with intact blood and lymph supply that were prepared at different locations along the entire length of the small and large intestine. Four weeks after oral infection, significant protection was detected in the duodenum, upper jejunum, midjejunum, and ileum. Protection at the gut level was expressed as early as 2 wk after oral priming and waned after 27 wk. The possibility that the gut wall plays a role in age-related protection was excluded. The effect of newly excysted juveniles (NEJ) penetrating the gut on the induction of protection was studied by recovering or killing the NEJs of the primary infection immediately after gut migration. Results showed that protection was low (13.9-19.8%). However, when gut migration was by-passed and NEJs of the primary infection were injected into the peritoneal cavity or between the liver lobes, high levels of protection at the gut level were detected (76.5-87.4%). The results indicate that protection expressed at the gut level is induced by the parasite at a young stage, during migration through the peritoneal cavity, or liver, or both and not during penetration of the gut.

Age Factors↗

PubMed1996

Parenteral vaccination of mice and piglets with F4+ Escherichia coli suppresses the enteric anti-F4 response upon oral infection.

We studied with a mouse model and in piglets the requirements to prime for a secondary, mucosal B-cell response against Escherichia coli F4 fimbriae, an important virulence factor of enterotoxigenic E. coli, the agent associated with postweaning diarrhoea in piglets. The major observation obtained with the mouse model was verified for piglets. Mice and piglets were primed orally or parenterally with purified F4ac antigen or whole bacterial cells carrying the F4ac antigen and were later orally infected with live F4ac+ E. coli bacteria. Cell suspensions of murine spleen or porcine serum were used to study the systemic B-cell response. Cell suspensions were also made of murine and porcine enteric lamina propria and were used to study the mucosal B-cell response. Enzyme-linked immunospot assays and enzyme-linked immunosorbent assays specific to E. coli F4ac antigen were used to quantify either the antibody-secreting cells or antibody titres in serum. Results showed that in mice only primary oral immunization with live bacteria induced an enteric immune response against the E. coli F4ac+ fimbriae. Oral immunization with killed bacteria induced hardly any mucosal immune response. Parenteral immunization induced a state of suppression that was reflected by the lack of an enteric immune response upon a subsequent oral infection with live bacteria. A comparable induction of suppression was observed in piglets using the same protocol. We conclude that parenteral vaccination of piglets with the E. coli F4ac antigen is ineffective to induce protective immunity at the mucosal level against postweaning diarrhoea and is possibly detrimental.

Animals↗

PubMed1995

The use of a double antibody sandwich ELISA and monoclonal antibodies for the assessment of porcine IgM, IgG and IgA concentrations.

Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) have been developed for the assessment of IgM, IgG and IgA concentrations in porcine serum. Isotype-specific monoclonal antibodies (mAb) were used for coating and detection. The DAS-ELISAs were examined for their ability to detect each isotype and for the assay variations. A computer programme was used to verify the parallelism of the slope of each serum sample with the slope of standard/reference serum, a prerequisite for reliable estimation of Ig concentrations. The DAS-ELISAs are easy to perform and highly specific, have adequate detection levels (ranging from 7 to 50 ng ml-1) and are very reproducible, as illustrated by the inter- and intra-assay variation coefficients (ranging between 4.9 and 7%). To illustrate the applicability of the ELISAs we assessed Ig concentrations in pig sera sampled from birth to young adulthood.

Aging↗

PubMed1993

[Serological study of the occurrence of L. hardjo in sheep in The Netherlands].

Sheep (3918) from 137 farms in the regions of North-, West- and Mid-Netherlands and Gelderland were serologically investigated for the presence of antibodies against Leptospira hardjo. Antibodies were detected in 3.3% of the sheep. There were large regional differences with respect to both the percentage of positive sheep and the percentage of positive flocks. All sera from sheep in Gelderland were negative. In West- en Mid-Netherlands there were 0.9 and 6.5% positive sera, respectively, and 19.4 and 32.7% positive flocks. The percentage of positive sheep per positive flocks varied from 1 tot 51.6. Serological positive sheep were from farms with and without cattle. On farms with cattle, there was no clear relationship between serologically sheep and the presence of hardjo antibodies in cattle. It thus seems plausible that sheep can be infected with hardjo independently of cattle.

Animals↗

PubMed1991

The role of the common vole (Microtus arvalis) in the epidemiology of bovine infection with Leptospira interrogans serovar hardjo.

Control of leptospirosis in cattle depends on the presence of other possible maintenance hosts, with which cattle may have contact. Twenty-seven common voles (Microtus arvalis) were trapped on a dairy farm where the cattle were infected with Leptospira interrogans serovar hardjo (hardjo). In the sera of 11 voles, titres greater than or equal to 100 against serogroup Grippotyphosa were measured with the microscopical agglutination test (MAT). From 8 of these 11 voles, which also showed interstitial lymphoplasmacellular nephritis, Leptospira interrogans serovar grippotyphosa was isolated. We found no evidence that the common vole is a maintenance host for hardjo in this biotope.

Agglutination Tests↗

PubMed1991

[Leptospirosis in swine; observations on the serological diagnosis of Leptospira interrogans serotype bratislava].

Leptospirosis is considered to be an important cause of porcine reproductive failure. In this respect recently attention is paid to the possible role of Leptospira interrogans serotype bratislava (bratislava). In the present paper the results of serotype bratislava serology conducted on three farms are presented and discussed. On two of the farms no reproductive failure was observed. On the third farm with a high percentage of return to service a longitudinal search was done. No relation could be demonstrated between bratislava titers and reproductive failure.

Animals↗

PubMed1991

Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine.

An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitive detection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction (PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows were used. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 10(7) to 10(8) Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelleted leptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation method enabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the need for dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by experimentally infected cows.

Animals↗

PubMed1991

Manipulation of intestinal immune responses against ovalbumin by cholera toxin and its B subunit in mice.

We studied the effect of mucosal presentation of ovalbumin (OVA) conjugated to cholera toxin (CT) or cholera toxin B subunit (CTB) on the intestinal immune responses against OVA. Mice were primed intraperitoneally (i.p.) with OVA in a water-in-oil emulsion and boosted intraduodenally (i.d.) with OVA conjugated to CT or CTB in various molar ratios. Responses were evaluated by testing intestinal secretions for OVA-specific antibodies and by quantifying the OVA-specific antibody secreting cells (ASC) in the lamina propria of the small intestine. OVA-CT conjugates were tested in a molar ratio ranging from 1.8:1 to 4500:1. OVA-CTB conjugates were tested in a molar ratio ranging from 0.25:1 to 500:1. The optimum intestinal immune response was reached at a molar ratio of 1.8:1 for OVA-CT and 5:1 for OVA-CTB. The binding capacity of OVA-CTB, but not of OVA-CT, to GM1 ganglioside corresponded with the capacity to enhance the intestinal immune response. The effect of conjugating CTB or CT to OVA on the immune response against OVA was more striking when mice were not only boosted i.d., but also primed i.d. Both OVA-CT and OVA-CTB induced detectable immune responses, whereas free OVA did not. Therefore, the carrier effect of CT or CTB is essential to trigger a mucosal immune response against OVA when presented mucosally only. We conclude that enhancing antigen uptake greatly facilitates mucosal immune responses.

Adjuvants, Immunologic↗

PubMed1990

Evaluation of an ELISA for the diagnosis of experimentally induced and naturally occurring Leptospira hardjo infections in cattle.

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Leptospira interrogans serovar hardjo (hardjo) infection in cattle was compared with the microscopic agglutination test (MAT). Glutardialdehyde was used in the ELISA to couple sonicated hardjo antigen to the microtiter plate. Mouse monoclonal anti-bovine IgG1 coupled to peroxidase was used as conjugate. Sera from calves experimentally inoculated with hardjo reacted positively in the MAT as early as 10 days after inoculation; these sera did not react positively in the ELISA until 25 days after the first inoculation. Positive and negative field sera from 704 adult cattle on 90 farms were examined by the MAT and the ELISA; a 90% correlation between the two tests was demonstrated. Eighty-six sera from calves inoculated with four Leptospira serogroups other than hardjo and 227 field sera from adult cattle with naturally occurring leptospirosis other than hardjo were examined by the ELISA. Fewer than 1% of these heterologous sera reacted with hardjo antigen in the ELISA. We concluded that the ELISA described in this report is an advantageous alternative to the MAT for diagnosing leptospirosis.

Agglutination Tests↗

PubMed1989

[Leptospirosis in cattle; milker's fever in cattle farmers].

In this paper the symptomatology, epidemiology, and diagnosis of Leptospira interrogans serovar hardjo infections in cattle are reviewed. The possibilities on monitoring and control of this disease in both foreign countries and the Netherlands are discussed. Special attention is paid to the zoonotic aspects of the infection (dairy fever).

Animals↗

PubMed1989

[The occurrence of hardjo-positive dairy cows in the Northern Netherlands. A prospective serological survey].

More than 5000 lactating cows from 82 farms, in the northern part of the Netherlands, were serologically examined for antibodies to Leptospira hardjo. Hardjo-positive cows were found on 52% of the farms with a mean incidence of 23% positive animals. Considerable differences were observed in the percentages hardjo-positive cows per region (7-30%), as well as in the percentages positive cows per positive farm (22-46%). With the exception of one region (14%) the percentages of hardjo-positive farms per region hardly differed (average 58%). In addition to the animal survey 488 sera of men working and/or living on cattle-farms were also examined. Twenty-six persons were hardjo-positive. Most of these persons (circa 90%) milked regularly. All seropositive persons were found on farms with hardjo-positive cattle. Results from an inquiry on circa 200 farms indicated, that hardjo-infections probably occur with a higher frequency on larger farms. Finally the results from this survey confirm the observation in the literature, that symptoms of hardjo-infections are more frequently found in the autumn.

Agglutination Tests↗

PubMed1989

Background (spontaneous) immunoglobulin production in the murine small intestine before and after weaning.

The ontogeny of the murine intestinal B-cell compartment before and after weaning was studied by quantitative analysis of immunoglobulin-secreting cells (Ig-SC) in the small intestine (SI). Before weaning, few Ig-SC were detected in the SI, whereas spleen and bone marrow already contained many Ig-SC. The number of Ig-SC in the SI started to increase immediately after weaning. Comparing early-weaned mice with non-weaned mice of the same age clearly demonstrated that weaning brought on the development of Ig-SC in the SI. The influence of a gut flora on the number of Ig-SC in the SI was examined by comparing the number of Ig-SC in the SI of conventionally housed, specific pathogen free (SPF) and germ-free mice. A bacterial flora was apparently needed for the normal development of Ig-SC in the SI. Comparing mice containing an aerobic Gram-negative bacterial flora with mice containing only an anaerobic Gram-positive bacterial flora demonstrated that the type of bacterial flora is relatively unimportant. No evidence was found that circulating maternal antibodies suppressed the development of the "spontaneous" intestinal and systemic B cell response. The results show that bacterial colonization of the intestine plays a pivotal role in the development of the Ig-SC compartment in the SI.

Animals↗

PubMed1989

Evaluation of a delayed-type hypersensitivity test for the diagnosis of Brucella abortus infection in cattle.

The delayed-type hypersensitivity (DTH) test was used to diagnose brucellosis in two cows experimentally induced with brucellosis, and 176 dairy cows from a farm suspected of brucellosis. DTH test results were compared with results of the milk ring test, the serum agglutination test, the complement fixation test and the Coombs test. Cows positive in the DTH test and in one of the other tests were examined bacteriologically. In experimentally infected animals the DTH test was positive 10 days after infection, 1-4 weeks before serologic tests indicated brucellosis. Although the DTH test was positive during the whole experiment, on the one occasion when serologic titres were high, it was negative. Of the 176 dairy cows, 45 were positive in one or more serologic tests. In twelve cows (29%) the diagnosis was inconclusive because they were positive in only one of the serologic tests. In these cases the DTH test confirmed the infection. Three cows with high serologic response tested negative in the DTH test. B. abortus was isolated from 13 of 15 cows examined. We conclude that when serologic results are ambiguous, the DTH test is a useful additional technique for diagnosing brucellosis.

Animals↗

PubMed1989

Quantification of antigen-specific antibody-secreting cells in the small intestine and other lymphoid organs of mice after oral booster immunization.

The intestinal immune response of mice against ovalbumin (OVA) was quantified by isolating lymphoid cells from the small intestine (SI) and testing them for antigen-specific immunoglobulin (Ig) secretion. The isolation procedure for functionally active lymphoid cells from the SI, originally developed to quantify the number of 'background' Ig-secreting cells in the SI, proved to be a useful method for evaluating antigen-specific intestinal immune responses quantitatively. The method was able to detect antigen-specific antibody-secreting cells (ASC) in the SI even when these cells occurred at a minimum frequency of only 0.006%. When mice were primed intraperitoneally (i.p.) with polymerized OVA and given an oral OVA booster immunization, OVA-specific ASC appeared in the SI from Day 3 after booster. After i.p. priming and an i.p. booster these cells could not be detected in the SI. The OVA-specific IgA-ASC responses in various organs after oral booster immunization were compared. From Day 5 after booster, when the response peaked, most OVA-specific IgA-ASC occurred in the SI. This suggested that these cells are mainly responsible for the OVA-specific antibodies demonstrated by ELISA in intestinal secretions from Day 6 after oral booster immunization. It is concluded that the quantitative method used in this study detects antigen-specific ASC in the SI with great sensitivity and could be used to evaluate immunization regimes aimed at inducing intestinal mucosal immune responses.

Animals↗