The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries.

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

Antibiotics in 16-day-old broilers temporarily affect microbial and immune parameters in the gut.

Animal health benefits from a stable intestinal homeostasis, for which proper development and functioning of the intestinal microbiota and immune system are essential. It has been established that changes in microbial colonization in early life (the first 2 wk post hatch) impacts the functioning of the adult gut and the associated crosstalk between microbiota and intestinal mucosal cells. The aim of the present study was to study the effect of the administration of antibiotics later in life (d 15 to 20 post hatch) on microbiota and immune parameters. For this purpose, chickens received from 15 d post hatch during 5 d amoxicillin or enrofloxacin through their drinking water. Before and at 6, 16, and 27 d after start of the administration of antibiotics, the composition of the microbiota in the jejunum was determined using a 16S ribosomal RNA gene-targeted DNA microarray, the CHICKChip. At 6 d after the start of the administration of the antibiotics, the composition and diversity of the microbiota were affected significantly (P < 0.05), but this change was small and observed only temporarily since differences disappeared at 16 d after initiating treatment with amoxillin and at 27 d after starting treatment with enrofloxacin. Intestinal morphology and development were not visibly affected since there were no differences between villus/crypt ratios and numbers of PAS+ and PCNA+ cells in the duodenum and jejunum at any time point. At 16 d after the start of antibiotic administration, the number of CD4+ T-cells and CD8+ T-cells in the duodenum was lower compared to the control animals; however, this difference was not significant. At some time points, significant differences (P < 0.05) were observed among the groups to locally expressed IL-8, IL-1ß, IFN-γ, IL-2, and IL-4 mRNA. However, this effect was not long lasting, as differences that were observed at 16 d after starting the treatment had disappeared at 27 d after treatment was started. The results of this study indicate that later in the broiler's life, antibiotics only temporarily affect intestinal microbial and immune parameters.

Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12ng/assay) and ~60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4×10(4) cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

Differences in highly pathogenic avian influenza viral pathogenesis and associated early inflammatory response in chickens and ducks.

We studied the immunological responses in the lung, brain and spleen of ducks and chickens within the first 7 days after infection with H7N1 highly pathogenic avian influenza (HPAI). Infection with HPAI caused significant morbidity and mortality in chickens, while in ducks the infection was asymptomatic. The HPAI viral mRNA load was higher in all investigated tissues of chickens compared with duck tissues. In the lung, brain and spleen of HPAI-infected chickens, a high, but delayed, pro-inflammatory response of IL-6 and IL-1ß mRNA was induced, including up-regulation of IFN-ß, IFN-γ, TLR3 and MDA-5 mRNA from 1 day post infection (p.i.). Whereas in ducks already at 8 h p.i., a quicker but lower response was found for IL-6, IL-1ß and iNOS mRNA followed by a delayed activation of TLR7, RIG-I, MDA5 and IFN-γ mRNA response. Virus-infected areas in the lung of chickens co-localized with KUL-01⁺ (macrophages, dendritic cells), CD4⁺, and CD8α⁺ cells, during the first day after infection. However, only KUL-01⁺ cells co-localized with the virus after 1 day p.i. In ducks, CVI-ChNL-68.1⁺ (macrophage-like cells), CD4⁺ and CD8α⁺ cells and apoptosis co-localized with the virus within 8 h p.i. Apoptosis was detected in the brain and lung of HPAI-infected chickens after 2 days p.i. and apoptotic cells co-localized with virus-infected areas. In conclusion, excessive delayed cytokine inflammatory responses but inadequate cellular immune responses may contribute to pathogenesis in chickens, while ducks initiate a fast lower cytokine response followed by the activation of major pattern recognition receptors (TLR7, RIG-I, MDA5) and a persistent cellular response.

Differential innate responses of chickens and ducks to low-pathogenic avian influenza.

Ducks and chickens are hosts of avian influenza virus, each with distinctive responses to infection. To understand these differences, we characterized the innate immune response to low-pathogenicity avian influenza virus H7N1 infection in chickens and ducks. Viral RNA was detected in the lungs of chickens from day 0.8 to 7, in ducks mainly at day 4. In both species, viral RNA was detected in the bursa and gut. Infection in chickens resulted in up-regulation of interferon (IFN)-α and IFN-ß mRNA, while in the ducks IFN-γ mRNA was strongly up-regulated in the lung and bursa. In chickens and ducks, all investigated pathogen recognition receptor (PRR) mRNAs were up-regulated; however, in the chicken lung Toll-like receptor (TLR)7 and melanoma differentiation-associated protein (MDA)-5 mRNA were strongly induced. TLR3, TLR7 and MDA-5 responses correlated with IFN-α and IFN-ß responses in chickens, but in ducks a correlation between IFN-α and TLR7, retinoic acid-inducible gene-I and MDA-5 was absent. We studied the responses of duck and chicken splenocytes to poly(I:C) and R848 analogues to analyse the regulation of PRRs without the interfering mechanisms of the influenza virus. This revealed IFN-α and IFN-γ responses in both species. MDA-5 was only strongly up-regulated in chicken splenocytes, in which time-related PRR responses correlated with the IFN-α and IFN-ß response. This correlation was absent in duck splenocytes. In conclusion, chickens and ducks differ in induction of MDA-5, TLR7 and IFN-α mRNA after an influenza virus infection in vivo and after in vitro stimulation with TLR antagonists.

Host response to simultaneous infections with Eimeria acervulina, maxima and tenella: a cumulation of single responses.

It is well known that broilers may be infected by different Eimeria strains at the same time and that different species infect specific parts of the gut. Cell mediated responses play a major role in the immune response in broilers after infection with Eimeria species. The cell mediated responses could be intestinal site specific and if this site specific cell mediated responses differ when other parts of the intestine are infected is unknown. To investigate this in the Eimeria infection model we analyzed the cell mediated responses to an infection with a single Eimeria species and with a mixture of different species of Eimeria such as E. acervulina, E. maxima or E. tenella in the duodenum, jejunum and caecum. The immune parameters we measured were intestinal T-cell and macrophage population dynamics as well as local cytokine mRNA expression. These parameters were related to the amount of Eimeria DNA that was measured in the intestine with an Eimeria strain specific quantitative PCR. The results showed that the strongest immune response was induced in the specific part of the intestine that was affected by each Eimeria strain. An E. acervulina infection mainly induced a duodenal CD8(+) T-cell and macrophage response as well as an increased IL-2, IL-4, IL-8, IL-10, and INF-gamma response. An E. maxima infection mainly induced a CD4(+) T-cell and macrophage response but also an increased IL-4, IL-8, and very strong INF-gamma (300-fold) expression in duodenum and jejunum. E. tenella induced a CD4(+) T-cell, macrophage response and an increase in the IL-2, IL-4, IL-8, IL-10, IL-18 and INF-gamma response in the caecum. The infection with a mixture of Eimeria species resulted in responses per intestinal segment that were similar to that observed following the single species infection. No synergistic or competitive effects were thus observed following a primary infection with a mixture of Eimeria species. In contrast, we observed an accumulation of the local effects of the single infections.

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.

Cats and goat whey associated with Toxoplasma gondii infection in pigs.

In organic livestock production systems, farm-management factors are thought to play an important role in the on-farm prevalence of Toxoplasma gondii. Serological results and the results of an HACCP analysis were combined to determine important risk factors for the prevalence of this protozoan parasite. Mathematical analysis demonstrated that feeding goat whey to pigs and the presence of a high number of cats were positively correlated to T. gondii seroprevalence in pigs. Not covering roughage and the farmers' assumption that pigs can come into contact with cat feces also showed a positive relationship. In order to decrease the risk of T. gondii infecting their pigs, farmers should limit the access and number of cats on their farms and refrain from feeding goat whey to their pigs.

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.

Comparison of natural resistance in seven genetic groups of meat-type chicken.

1. Several studies have shown that genetic variation exists in response to various Salmonella strains in mammals and poultry. In the current study immunocompetence traits related to natural resistance to Salmonella were measured in 7 genetic groups of meat-type chickens (in total 296 chickens involved). 2. Variables were measured of both innate (phagocytic activity) and adaptive immune responses that are important after a natural or experimental Salmonella enteritidis infection. Two traditional Old Dutch Breeds (groups 1 and 2), four commercial broiler groups (groups 3 to 6), and one experimental broiler group (group 7) were used. In two periods, birds of each group were killed for examination at ages between 14 and 35 d post hatch. 3. Significant differences between groups were found for most immune variables measured, with significant correlations between several of them. All groups produced an adequate immune response, of either the innate or the adaptive type. 4. In the current study, group 2 showed the highest overall natural resistance, though none of the groups was uniformly superior with respect to all traits measured. 5. In conclusion, for reliable measurements of general immunocompetence or resistance to Salmonella, for example, it is important to measure several aspects of the immune system.

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.

An experimental study on triclabendazole resistance of Fasciola hepatica in sheep.

The efficacy of triclabendazole in sheep experimentally infected with Fasciola hepatica was studied. Two groups of 12 lambs were infected with a susceptible (S) or a resistant (R) strain of F. hepatica. Eight weeks after infection, six lambs of each group (ST and RT) were treated with triclabendazole (10mg/kg). The other lambs were used as untreated controls (SC and RC). The parameters studied were: GLDH, gamma-GT, ELISA measuring antibodies against recombinant cathepsin-L(1) and eggs per gram faeces (epg). The lambs were slaughtered 16 weeks after infection and the number of flukes counted. The GLDH, gamma-GT levels and the OD value of the ELISA decreased as a result of the treatment in group ST. Patent infections were observed in all animals of groups SC, RT and RC. In group ST, occasionally a few eggs were found in five lambs. The percentage of flukes was 31.3 in SC and 37.6 in RC. In the treated groups ST and RT, the percentage of flukes was 0.06 and 33.6, respectively. These results corresponded to efficacies of 99.8% in the susceptible and 10.8% in the resistant strain. Since the resistant strain was isolated from a mixed cattle and sheep farm, it confirms the presence of triclabendazole resistance in the Netherlands.

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.

Nematode parasites of adult dairy cattle in the Netherlands.

Abomasa, blood samples and faecal samples for examination of nematode infections were collected from 125 dairy cows during the period November 1997-October 1998. Of these, 12 had no grazing history and were, therefore, excluded from this study. From the remaining 113, 88.5% had nematode eggs in the faeces. Larval identification of the positive cultures showed that Ostertagia spp. larvae were most frequent (97%), followed by Trichostrongylus spp. (29%), Oesophagostomum spp. (23%), Cooperia punctata (20%), Cooperia oncophora (4%), Haemonchus contortus (2%) and Bunostomum phlebotomum (1%). The geometric mean EPG was 2.4. Two cows excreted larvae of Dictyocaulus viviparus (0.1 and 0.6 LPG resp.). Worms were found in the abomasa of 108 cows (96%). In all these abomasa Ostertagia spp. was present (100%). Trichostrongylus axei was found in 47 abomasa (43.5%) and two cows (2%) were infected with Capillaria bovis. The geometric mean of the total abomasal worm counts was 1743 and of Ostertagia spp. alone 1615. Almost all male worms were Ostertagia ostertagi, only occasionally Skrjabinagia lyrata10,000) total worm burden. Ostertagia specific antibodies were highest in late summer and autumn and lowest in spring and early summer. The same pattern, although not so pronounced, was observed for the serum pepsinogen values. No clear seasonal pattern was found for the Cooperia specific antibodies. Antibodies against D. viviparus were detected in seven cows (6%).

Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle.

A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.

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.

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.

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.

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.