Effect of Eimeria acervulina infection history on the immune response and transmission in broilers.

Heterogeneity in exposure to Eimeria spp. of chickens in a flock will result in differences between individual birds in oocyst output and acquired immunity, which subsequently affects transmission of the parasite in the population. The aim of this study was to quantify effects of previous infection of broilers with Eimeria acervulina on immune responses, oocyst output and transmission. A transmission experiment was carried out with pair-wise housed broilers, that differed in infection history. This "infection history" was achieved by establishment of a primary infection by inoculation of birds with 50,000 sporulated E. acervulina oocysts at day 6 of age ("primed"); the other birds did not receive a primary infection ("naïve"). The actual transmission experiment started at day 24 of age: one bird (I) was inoculated with 50,000 sporulated oocysts and was housed together with a non-inoculated contact bird (C). Oocyst excretion and parameters describing transmission, i.e. the number of infected C birds and time passed before start of excretion of C birds, were determined from day 28 to day 50 for six pairs of four different combinations of I and C birds (I-C): naïve-naïve, naïve-primed, primed-naïve and primed-primed. Immune parameters, CD4(+), CD8(+), αßTCR(+) and γδTCR(+) T cells and macrophages in duodenum, were determined in an additional 25 non-primed, non-inoculated control birds, and in the naïve-naïve and naïve-primed groups, each group consisting of 25 pairs. Although the numbers of CD4(+) T cells and γδTCR(+) T cells increased after primary infection, none of the immunological cell types provided an indication of differences in infectivity, susceptibility or transmission between birds. Oocyst output was significantly reduced in primed I and C birds. Transmission was reduced most in the primed-primed group, but nonetheless transmission occurred in all groups. This study also showed that acquired immunity significantly reduced oocyst output after inoculation and contact-infection, but not sufficiently to prevent transmission to contact-exposed birds.

Immune responses to an Eimeria acervulina infection in different broilers lines.

The (T-cell) immune responses of two different broiler lines to a primary Eimeria acervulina infection were investigated. The lines used were a commercial fast-growing broiler line and a slow-growing type of broiler as used in organic farming. Seven-day-old broilers of both lines were infected with 5 x 10(4) oocysts of E. acervulina. The animals were weighed and a species-specific real-time PCR was used to quantify the total amount of parasites in the duodenum. In the fast-growing line, a lower parasite load was seen from day 4 onwards compared to the slow-growing line. In both lines the intestinal peak of Eimeria DNA was observed at day 5 post infection (p.i.). In the duodenum no increase in CD4(+) T-cells was found in both infected lines, but a fast increase in CD8(+) T-cells was observed in the fast-growing line. At day 3 p.i. in the slow-growing broilers an IL-18 mRNA response was observed. At day 4 p.i. strong IFN-gamma and IL-8 mRNA responses were found in both lines. No IL-4 mRNA responses were found in the duodenum. In conclusion, both lines have different growth rates and control and infected conditions. Based on the kinetics of observed phenomena a primary infection with E. acervulina in 7-day-old broilers seems to generate an early CD8alpha(+) response in fast-growing broilers compared to the slow-growing broilers. This difference in immune reaction after an E. acervulina infection could result in a different Eimeria load in the duodenum.

The effect of low-density broiler breeder diets on performance and immune status of their offspring.

Effects of low-density broiler breeder diets on offspring performance and mortality were studied using 2,100 female and 210 male Cobb 500 breeders. Breeder treatments involved 4 experimental groups and a control group with normal density diets (ND, 2,600 kcal of AME/kg during rearing and 2,800 kcal of AME/kg during laying). In treatment 2, nutrient densities were decreased by 12% (LD12) and 11% (LD11) during the rearing and laying periods, respectively, whereas in treatment 3, nutrient densities were decreased by 23% (LD23) and 21% (LD21) during the rearing and laying periods, respectively. The nutrient density in these treatments was decreased through inclusion of palm kernel meal, wheat bran, wheat gluten feed, and sunflower seed meal in the diets. Treatment 4 included diets with the same nutrient densities as in treatment 2 but included oats and sugar beet pulp (LD12(OP) and LD11(OP)). In treatment 5, the same low-density diet was given to the breeders as in treatment 2 during the rearing period, but it was followed by a normal density diet during the laying period (LD12-ND). Treatments were applied from 4 to 60 wk of age. On low-density diets, offspring showed an increased 1-d-old weight. As compared with offspring of breeders that received ND, the d 38 live weight of chickens from 29-wk-old breeders fed LD11 was improved. Mortality was reduced in offspring from 60-wk-old parent stock given low-density diets. The IgM titers in 35-d-old offspring from eggs with a lower-than-average weight were reduced when 29-wk-old broiler breeders were fed low-density diets. In offspring from eggs with a higher-than-average weight from 60-wk-old parent stock given LD11 or LD21 diets, IgM titers were higher compared with ND. It was concluded that low-density broiler breeder diets can improve offspring growth rates, reduce mortality, and reduce or increase immune responses, depending on breeder age and egg weight.

Immune responses in Eimeria acervulina infected one-day-old broilers compared to amount of Eimeria in the duodenum, measured by real-time PCR.

T-cell responses are supposed to be the major immune reactions in broilers infected with Eimeria. The nature of such T-cell responses is influenced by the species of Eimeria involved, age of the host, amount of parasites and the preceding infection history. In young chicks the intestine is still developing in length while the lymphocyte populations in the gut develop and differentiate. In chicks infected at young age the immune response may be different in quality as compared to responses in adults. We investigated the (T-cell) immune responses of young broilers to a primary Eimeria acervulina infection in relation to the number of parasites used for infection. In our experiment we infected one-day-old broilers with a low (5 x 10(2) oocysts) and a high (5 x 10(4) oocysts) dose of E. acervulina. We used a newly developed species specific real-time PCR to quantify total amount of parasites in the duodenum as the number of oocysts in faeces may not be representative for the exposure of the gut immune system. We characterized T-cell subsets in the duodenum by means of FACS-analyses, lymphocyte proliferation assays with spleen lymphocytes and the mRNA profiles of different cytokines (TGF-beta2, -4, IFN-gamma, IL-2, -6, -8 and -18) in the duodenum by means of real-time PCR. From day 5 p.i. broilers with a high dose of E. acervulina had a significantly lower body weight than the control group. No increase in CD4(+) cells, but a strong increase in CD8(+) cells was observed at days 7 and 9 p.i. in the duodenum of broilers infected with a high dose E. acervulina. IL-8 mRNA responses were observed after infection with low and with high infection doses, but no IFN-gamma and TGF-beta mRNA responses were found in the duodenum. The specific proliferative T-cell responses to a low infectious dose were not significantly different as compared to the control group. In conclusion, based on the kinetics of observed responses a primary infection with a high dose of E. acervulina in one-day-old broilers seems to generate an immune response that shows a peak at the time of oocyst excretion, whereas the immune response to a low dose is less explicit.

A DNA vaccine coding for gB and gD of pseudorabies virus (suid herpes type 1) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines.

DNA vaccines are capable of priming the immune system of neonates in the presence of maternal antibodies. However, it is still not clear whether the extent of priming and protection against challenge infections induced by a DNA vaccine in maternally immune newborns is better than that induced by conventional vaccines. To study this, we used the pseudorabies virus (PRV) infection model in the natural host, the pig. We compared the efficacy of a DNA vaccine with the efficacy of a conventional modified live vaccine (MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets. We measured the priming of the immune response and the degree of protection against challenge infection for all vaccine types. We vaccinated piglets with or without maternal immunity twice, at the age of 5 and 9 weeks, and we assessed protection by challenge infection with virulent PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines induced both humoral and cell-mediated immune responses in maternally immune animals. DNA vaccination seemed not to suffer from suppression by maternal immunity and resulted in similar or stronger immune responses in maternally immune piglets as compared in naïve piglets. In contrast, vaccination with conventional vaccines resulted in weaker immune responses in maternally immune piglets than in naïve piglets. Moreover, DNA vaccination provided better protection against challenge infection in maternally immune piglets than in naive piglets, whereas vaccination with conventional vaccines did not.

Cytokine responses in broiler lines that differ in susceptibility to malabsorption syndrome.

1. Based on earlier studies it was hypothesised that there is an immunological basis for the differences in susceptibility to malabsorption syndrome (MAS). A study was conducted to investigate base-line and MAS-induced cytokine levels in the intestine of broilers that differ in MAS susceptibility. 2. The transcription of cytokine mRNA in the intestine was quantified using a real-time polymerase chain reaction (PCR) method. At different time points after disease induction the intestines of broilers were investigated for expression of interleukin (IL)-2, IL-6, IL-8, IL-18 and interferon (IFN)-gamma. Age-matched non-MAS-induced chickens served as controls. 3. Control chickens from a MAS-resistant line had higher concentrations of mRNA for IL-2, IL-6, IL-18 and IFN-gamma in the small intestine while no difference between the lines was found for IL-8. After induction of MAS the relative amounts of IL-2, IL-6, IL-8 and IFN-gamma mRNA increased more in the intestines of the susceptible line than in the gut of the resistant line. 4. We suggest that differences in cytokine mRNA in the base-line situation and in MAS-induced conditions indicate a difference in immune response regulation in the two broiler lines. This difference in response could lead to the difference in susceptibility to MAS.

Immunomodulation by probiotic lactobacilli in layer- and meat-type chickens.

1. The aim of the experiments was to evaluate whether selected probiotic lactobacillus strains have different immunomodulating effects in layer- and meat-type strain chickens. 2. Humoral and cellular specific and non-specific immune responses were studied by experiments on cellular proliferation, entry and survival of Salmonella bacteria in gut and spleen leukocytes, immunoglobulin isotypes and specific immunoglobulin titres. 3. The effects of two different feeding regimes (short and continuous feeding) and doses for administration of lactobacilli were studied. 4. The lactobacillus strains that were evaluated showed modulating effects on the immune system of layer- and meat-type chickens. 5. In meat-type strain chickens the lactobacilli had a stimulating effect when the chickens were young (up to 3 weeks) and the dose was relatively high, whereas in layer-type chickens a lower effective dose and discontinuous administration was also effective. 6. Immunoprobiotic lactobacilli can have a positive effect on humoral and cellular immune responses in layer- and meat-type strain chickens, but the lactobacillus strain to be used, the age of the animals and effective dose of lactobacilli to be administered need to be optimised.

The presence of co-infections in pigs with clinical signs of PMWS in The Netherlands: a case-control study.

In this study, 60 pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) from 20 different pig herds and 180 control pigs (without clinical signs of PMWS) were examined to get more insights into the frequencies of porcine circovirus 2 infections and the presence of co-infections in pigs with and without clinical signs of PMWS in the Netherlands. Porcine circovirus type 2 was detected in 100% of the pigs with clinical signs of PMWS by virus isolation and/or PCR and in 50% of the pigs from PMWS-free herds. There was an association between the levels of infectious PCV2 and/or PCV2 DNA load and the severity of clinical signs as described for PMWS. A high variation in PCV2 antibody titres was found in the clinically affected pigs, and 27% of these pigs did not mount PCV2 antibody titres higher than 1:200. A concurrent infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) was found in at least 83% of the pigs with clinical signs of PMWS and in 35% of the pigs from PMWS-free herds. Co-infections of European- and American-type PRRSV were detected only in PMWS herds and in one control herd with a history of PMWS clinical signs.

[Vaccinations in pig health care in the Netherlands--analysis of a questionnaire among veterinarians]. / Vaccinaties in de Nederlandse varkensgezondheidszorg--analyse van een enquête onder dierenartsen.

Veterinarians specializing in pig health care responded to a questionnaire regarding their experiences with vaccinations. The goal of the questionnaire was to gain insight into a) the reasons for vaccination and b) the factors involved in the efficacy of vaccination. The results indicated that vaccinations were typically initiated because of health problems at the farm. Veterinarians worked together with farmers and other concerned parties to initiate vaccinations. Respondents predicted that the number of vaccinations would decrease substantially with optimal farm management, but would increase if farmers were allowed (under strict conditions) to vaccinate their own pigs. The results further indicated that the nature of financial compensation predicted the rate of vaccination. Veterinarians paid according to a fixed 'fee for service' system vaccinated less frequently than did veterinarians who were paid according to an ongoing farm management contract. In conclusion, veterinarians appear to be restricted in their capacity to disseminate their experience and knowledge due to the competing needs of the farming network (animals, farmers, retailers).

Vaccine-induced T cell-mediated immunity plays a critical role in early protection against pseudorabies virus (suid herpes virus type 1) infection in pigs.

The aim of our study was to evaluate the relative importance of antibody and T cell-mediated immunity in protection against pseudorabies virus (suid herpes virus type 1) infection in pigs. We induced different levels of immune responses by using: (1) a modified live vaccine; (2) the same modified live vaccine with an oil-in-water (o/w) adjuvant; (3) an inactivated vaccine; and (4) the same inactivated vaccine with an o/w adjuvant. Subsequently, we challenged pigs with virulent pseudorabies virus (PRV). We demonstrated that best-protected pigs stood out by maintaining strong T cell-mediated immune (CMI) responses after challenge. Of the immune parameters tested, protection against virus shedding was correlated best with the magnitude of the IFN-gamma response of in vitro re-stimulated peripheral blood mononuclear cells (PBMC) with an additional role for PRV-specific IgG2 antibodies. The use of an o/w adjuvant resulted in higher antibody and CMI responses, in particular with an increased frequency of memory T helper blast cells of in vitro re-stimulated PBMC. However, this adjuvant-induced enhancement of the immune response had a limited additional effect on the efficacy of inactivated vaccines. This study suggests a major contribution of the CMI response in early protection against PRV infection and that PRV-induced IFN-gamma responses may serve as a suitable indicator for assessing the immune status of vaccinated pigs.

Excessive porcine circovirus type 2 antibody titres may trigger the development of porcine dermatitis and nephropathy syndrome: a case-control study.

In a case-control study, the role of porcine circovirus 2 (PCV2) and putative co-factors in the development of porcine dermatitis and nephropathy syndrome (PDNS) were investigated. Pigs with and without PDNS were examined for macroscopic lesions and histopathology. In addition, organs and tissues were collected at necropsy and examined for the presence of fibrinous deposits (immune complexes), CD8+ cells, and for the presence of bacterial and viral infections. Results from PDNS cases were compared with those of three control groups comprising pigs without clinical signs of PDNS and selected from; (1) the same compartment as PDNS cases, (2) another compartment but in the same PDNS herd, and (3) a control herd without any history of PDNS or post-weaning multisystemic wasting syndrome. Macroscopic and histopathological lesions found in PDNS cases were comparable to those previously documented for PDNS e.g. skin lesions and renal lesions representing glomerulonephritis associated with fibrinous deposits and to a lesser extent with interstitial nephritis. PCV2 was detected by PCR in 100% of the PDNS cases, mainly in lymph nodes and tonsils, and in 63% of the control pigs from PDNS free herds. Virus isolation did not reveal infectious PCV2 in all cases. In PDNS affected pigs the PCV2 serum antibody titres were consistently extremely high and the mean PCV2 antibody titre in PDNS pigs was significantly higher than the mean PCV2 antibody titres in pigs from all 3 control groups. Immunohistochemical investigation of kidneys from PDNS affected pigs revealed an increased accumulation of IgG1 + IgG2 and IgM, the complement factors C1q and C3, but also an increase of CD8+ cells. The amounts of IgA and the complement factor C5 in kidneys of PDNS pigs were only slightly increased as compared to control pigs. This study demonstrates that PCV2 infections can result in extremely high PCV2 antibody titres and that PCV2 is a candidate as primary agent in the development of PDNS. The causative physiological basis for PDNS may be the excessive levels of PCV2 antibodies.

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Summary of the first round analyses of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The reactivity of 155 monoclonal antibodies submitted to the Third International Workshop on Swine Leukocyte Differentiation Antigens, together with 41 internal standards, was analysed by flow cytometry on 29 different pig cell targets as well as two human cell targets as a means of establishing suitable panels of monoclonal antibodies for more detailed clustering analyses by the various subsections of the workshop. Results were collected either without further gating, with gating based on FS/SS characteristics or with gating based on the co-expression of a reference antibody in two-colour flow cytometry. The CD or SWC reactivity of the internal standards had been established in previous workshops. Data sets were subsequently analysed by statistical clustering using the Leucocyte Typing Database IV software. The resulting 18 cluster groups were allocated to the appropriate second round sections of the workshop, after reviewing the overall cellular reactivity of each cluster as well as the specificity of known standards which clustered in a group.

Summary of workshop findings for porcine B-cell markers.

Based on cluster groups from the first-round analyses of the Third International Swine CD Workshop, 38 monoclonal antibodies (MAbs) including eight internal controls were analysed by flow cytometry (FCM) and immunohistochemistry (IH) in the second-round analysis of the B-cell section of this workshop. Targets in this section included peripheral blood lymphocytes and cells isolated from ileal Peyer's patches (PP), mesenteric lymph nodes (MLN) of adult animals, bone marrow cells from newborn piglets and thymus cells isolated from foetuses at day 105 of gestation. Immunohistochemistry of these 38 MAbs identified four sets, whose ligands were co-expressed with CD21, which showed a tissue distribution compatible with specificity for cells including those of the B-cell lineage. Another group of miscellaneous antibodies appeared to identify other cells, several antibodies were negative. Two-colour flow cytometry (2C-FCM) was carried out by pairing each antibody of interest with antibodies to SWC7, CD21, sIgM and a polyclonal rabbit anti-swine immunoglobulin antiserum (RaSwIg). The anti-CD21 MAb BB6-11C9 (no. 20) and IAH-CC51 (no. 19), established in previous workshops, as well as the cross-reactive anti-human CD21 B-1y4 (no. 146), clustered together in FCM analyses of the first round and showed similar cellular distribution in IH. A further cluster was formed by the standard CC55 (no. 55) and 2A10/8 (no. 102) submitted as SWC7 specific. The second SWC7 standard 2F6/8 (no. 100) clustered separately, but IH showed an identical pattern of reactivity to the other SWC7 MAb.Unfortunately, this work could not identify any other novel clusters with specificity for B-cells, as the statistical clustering of other MAbs could not be substantiated by IH or subsequent two-colour-FCM work. However, we could identify MAb with similar cellular distribution. The ligands for the cross-reactive anti-human CD40 G28.5 (no. 25) and STH224 (no. 153) were expressed on very similar targets, similarly the ligands for the MAb pair JM1H1 (no. 139) with BB6-10A10 (no. 142) and the MAb pair 3F7/11 (no. 115) with 1C2F10 (no. 187).

Long-term effects of social stress on antiviral immunity in pigs.

Mixing of unfamiliar pigs is common practice in intensive pig husbandry. Since pigs maintain a dominance hierarchy, mixing often leads to vigorous fighting. Apart from the negative impact that fighting has on welfare, there is evidence that the social stress associated with fighting suppresses immune function. In the present experiment, we investigated the impact of mixing on specific long-term immune responses and protection against challenge infection after vaccination with pseudorabies virus (PRV). Specific pathogen-free (SPF) pigs were mixed pairwise with an unfamiliar same-gender conspecific or left undisturbed with a same-gender littermate at 3 days after vaccination with PRV. Half of the pigs were females (gilts) and half were castrated males (barrows). Mixing increased agonistic behavior to the same degree in gilts and barrows. Cortisol concentrations in saliva and catecholamine excretion in urine were increased in mixed pigs, and these effects were independent of dominance status and gender. Subsequently, the effects of mixing, gender, dominance status and interactions between these factors on immune response parameters were studied. The main result was that mixed barrows showed suppressed immune responses after vaccination and increased clinical symptoms after challenge infection compared to control barrows. Mixed gilts however did not differ from control gilts. It also appeared that mixed dominants were more seriously affected than mixed subordinates were. We conclude that, in some pigs, social stress after mixing suppresses the immune response to a viral vaccine and consequently impairs protection against challenge infection.

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease.

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.

Oral immunisation of naive and primed animals with transgenic potato tubers expressing LT-B.

The efficacy of edible vaccines produced in potato tubers was examined in mice. Transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation. The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B). A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum. Introduction of this gene under control of the tuber-specific patatin promoter in potato plants resulted in the production of functional, i.e. Gm1-binding, recLT-B pentamers in tubers. Selected tubers containing about 13 microg of recLT-B per gram fresh weight were used for immunisation. Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B. The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice. Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant. Naive and primed mice were fed 5 g of tubers ( approximately 65 microg of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract ( approximately 2 microg of recLT-B). In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres. In primed animals, however, oral immunisation resulted in significant anti-LT IgA antibody responses in serum and faeces. Intragastric intubation of tuber extract revealed higher responses than feeding of tubers. These results indicate clearly that functional recLT-B can be produced in potato tubers, that this recombinant protein is immunogenic and that oral administration thereof elicits both systemic and local IgA responses in parentally primed, but not naive, animals.

Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains.

Different Lactobacillus strains are frequently used in consumer food products. In addition, recombinant lactobacilli which contain novel expression vectors can now be used in immunotherapeutic applications such as oral vaccination strategies and in T cell tolerance induction approaches for autoimmune disease. Both for food and clinical applications of lactobacilli, proper selection of wild type strains is crucial. For that purpose, eight different common Lactobacillus strains were analysed with respect to mucosal induction of pro- and anti-inflammatory cytokines, IgA-producing plasma cells in the gut, as well as systemic antibody responses against a parenterally administered antigen. Immunohistochemical analysis of cytokine-producing cells in the gut villi showed no significant induction of the cytokines IL-1alpha, IFN-gamma, IL-4 or IL-10 after oral administration of wild type Lactobacillus strains. In contrast, oral administration of L. reuteri and L. brevis induced expression of the proinflammatory/Th1 cytokines TNF-alpha, IL-2 and/or IL-1beta. Oral administration of these two strains and L. fermentum also significantly enhanced the IgG response against parenterally administered haptenated chicken gamma globulin (TNP-CGG). The five other strains did not show this adjuvanticity. L. reuteri induced relatively high levels of IgG2a compared to L. murines, a nonadjuving Lactobacillus strain. These findings imply that different Lactobacillus strains induce distinct mucosal cytokine profiles and possess differential intrinsic adjuvanticity. This suggests that rational Lactobacillus strain selection provides a strategy to influence cytokine expression and thereby influence immune responses.

Development of the natural response of immunoglobulin secreting cells in the pig as a function of organ, age and housing.

We analysed the development of the natural immunoglobulin-secreting cell (Ig-SC) response in systemic- and mucosal-lymphoid tissues of specified pathogen free pigs between 1 and 40 weeks of age. As antigen exposure may influence the development of the Ig-SC repertoire we also compared the frequencies of Ig-SC in various lymphoid tissues of 40 weeks old specified pathogen free pigs and conventional pigs. A procedure to isolate lamina propria cells from porcine intestine was adapted for this study. The frequencies of IgM-, IgG-, and IgA-secreting (spot forming) cells were determined with a reversed enzyme linked immunospot assay, which was also adapted for detection of Ig-SC in pigs. The Ig-SC frequencies were calculated as percentage of the mononuclear leukocytes isolated from the various organs. The observations till 40 weeks of age were as follows: Splenic IgM-SC predominated at all ages and reached a plateau of 0.1-0.2% of the mononuclear leukocytes already at 4 weeks of age. The IgM-SC of mesenteric lymph node (MLN) predominated up till 12 weeks of age and reached an optimum of 0.15% reached at 4 weeks of age. The frequencies of IgG-SC of spleen and MLN had dips around 4 weeks of age and increased thereafter till 40 weeks of age (spleen 0.025%, MLN 0.05% at 40 weeks of age). The frequencies of IgA-SC were low in the spleen (< or =0.003%) and moderate in the MLN (0.01-0.02%) at all ages tested. In peripheral lymph node (PLN) and bone marrow (BM), the frequencies of IgM-SC (0.03-0.05%) were much lower than in the spleen. The IgG-SC frequencies of BM and MLN also had dips around 4 weeks of age and increased thereafter. The IgG-SC frequency of BM reached a plateau at 12 weeks of age (0.15%) and for PLN the highest frequency was observed at 40 weeks of age (0.05%). The frequencies of IgA-SC were low in BM and PLN (<0.003%). High frequencies of IgA-SC were observed in mucosa associated tissue like Peyer's patches (PP) and intestinal lamina propria (till 20% of the mononuclear leukocytes in intestinal lamina propria of 12-40 weeks of age). IgM and IgA are both important isotypes in mucosal lymphoid organs in the pig. The shift from IgM to IgAas predominant, mucosal isotype was first observed in duodenum and jejunum (12 weeks) and later in ileum (40 weeks). The influence of ageing on the frequency of Ig-SC in PP was only observed in jejunal PP. whereas in ileal PP the frequencies of Ig-SC did not vary over time. We combined our data about the frequencies of IgM-, IgG-, and IgA-SC in various organs with data obtained by others about the distribution of lymphocytes over porcine lymphoid organs at about 12 weeks of age. Based on these calculations we concluded that the small intestine, with more than 80% of all Ig-SC, is fair most the major site of Ig production in the pig. We also concluded that the small intestine is the major site of IgA and IgM production cells in the pig. Although IgA becomes predominant along the intestine, the results demonstrated that in the pig IgM is more a mucosal isotype compared with other species. With 40% of all IgG-SC the porcine BM appeared to be the major site of IgG production. Unexpected results were obtained for IgG-SC in the systemic lymphoid organs. In these organs the frequencies of IgG-SC dropped firstly from 1 to 4 weeks of age and steadily increased thereafter till 40 weeks of age. This observation is discussed in relation to the possibility that systemic IgG-SC at one week of age were passively acquired from maternal colostrum. The influence of housing/antigenic load at 40 weeks of age was mainly expressed by an increase (2-8x) of the frequency of IgG-SC in spleen, PLN, BM, and intestinal lamina propria, whereas the typical mucosal IgA-SC frequencies in the lamina propria were hardly affected.

Instruments for oral disease-intervention strategies: recombinant Lactobacillus casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis.

Lactobacillus strains possess properties that make them attractive candidates as vehicles for oral administration of therapeutics. In this report we describe the construction and analysis of recombinant Lactobacillus casei applicable in oral vaccination against an infectious disease (tetanus) and in oral tolerance induction for intervention in an autoimmune disease, multiple sclerosis. Recombinant L. casei which express surface-anchored tetanus toxin fragment C (TTFC) were generated. Quantitative analysis by flow cytometry demonstrated a high level of cell wall-bound expression of TTFC and immunogenicity was demonstrated by parenteral immunization with whole cell extracts of the recombinants. A series of expression vectors was constructed to secrete human myelin basic protein (hMBP) or hMBP as a fusion protein with beta-glucuronidase from Escherichia coli. These heterologous products produced by L. casei were detected in the growth medium and parenteral immunization with this medium evoked antibodies against hMBP, confirming that secretion indeed had occurred. Based on the different localization of the heterologous proteins, lactobacilli expressing surface-anchored TTFC are ideally suited for the induction of antibody responses, whereas lactobacilli that secrete myelin proteins can be used for the induction of peripheral T-cell tolerance. In conclusion, the specific technology described here allows the construction of a wide array of safe live recombinant lactobacilli which may prove to be useful in oral intervention strategies for the prevention of infectious diseases or treatment of autoimmune diseases.