Purification of a leucine aminopeptidase from Eimeria falciformis.

A leucine aminopeptidase was purified from the oocysts of Eimeria falciformis using affinity chromatography and gel filtration techniques. It had a molecular weight of 45-50 kDa. Its maximal activity against leucyl-p-nitro anilide was at pH 8.6. It is a metallo-enzyme highly inhibited by bestatin.

Kinetics of specific immunoglobulin A, M and G production in the duodenal and caecal mucosa of chickens infected with Eimeria acervulina or Eimeria tenella.

The development and appearance of antibody was studied in the intestine and serum from histocompatible GB1 chickens orally infected with oocysts of Eimeria acervulina (restricted to the duodenum) or Eimeria tenella (restricted to the caeca). The local immune response was measured as the specific antibody levels in the supernatants of intestinal fragments (duodenum and caecum) maintained in culture for 16 h at 41 degrees C, 5% CO2, 95% air. Specific IgM was detected 1 week after E. acervulina infection, and the specific IgA and IgG contents of the duodenum and caecum were significantly elevated (P < 0.001) after 2 weeks. The intestinal specific IgG content was raised. E. tenella infection resulted in specific IgA only in the parasitized area during the second week post-infection (P < 0.05). Specific IgM and IgG were both detected in the duodenum and caecum, respectively, 1 and 2 weeks p.i. Production of parasite-specific immunoglobulins was always significantly higher in the parasitized than in the unparasitized areas (caeca for E. acervulina, duodenum for E. tenella). This ex vivo culture assay of intestinal fragments used to measure the mucosal immune response of intestinal areas showed a significant production of specific IgA and IgM. In addition, high levels of IgG were also measured. The role of this specific IgG in Eimeria infection remains to be determined.

A survey of Eimeria species in commercially-reared chickens in France during 1994.

In a survey of chicken coccidia in France during 1994, samples of litter were collected from 41 farms. On 31 of these farms, eimerian oocysts were abundant enough to allow monitoring of their numbers in the litter. Peak total oocyst counts on these farms ranged from 16,200 to 1,254,000/g of litter, but no coccidiosis was observed. The chickens reared without anticoccidial agents in their food (poulets biologiques) produced higher and earlier peak oocyst counts in litter than the chickens given medicated food (poulets labels). The oocysts in litter samples from 22 farms (13 poulet biologique, five poulet label, two standard broiler, one breeder and one layer) of the original 41 were identified. Six of the seven eimerian species known to parasitize chickens were found, using combinations of five methods (oocyst morphology, intestinal lesions, enzyme electrophoresis, growth in embryonating eggs and prepatent time). Multispecific infections predominated (95% of 22 farms), up to six species occurring together. Of farms where oocysts were detected, the percentages with each species were: Eimeria acervulina (100%), E. mitis (82%), E. tenella (77%), E. maxima (73%), E. praecox (45%) and E. brunetti (27%). These appear to be the first definite records of E. mitis and E. praecox for France. Although E. necatrix was not found in this survey, it had recently been detected by other workers in France, so that all seven chicken Eimeria species were known to be contemporaneous.

Changes in intestinal intra-epithelial and systemic T-cell subpopulations after an Eimeria infection in chickens: comparative study between E acervulina and E tenella.

During chicken coccidiosis, the growth of the parasite in the intestinal epithelium cells leads to the development of host immune response. Cell-mediated immune mechanisms appear to be mainly responsible for the acquired resistance to disease. The action of two species of Eimeria, with two different intestinal localizations, on T-lymphocyte subsets was followed by fluorescent antibody cell-sorter analysis, locally at the intestinal site of the parasitic development and systemically in spleen and blood. An Eimeria acervulina infection, localized in duodenum, induced a significant increase in the proportion of CD4+ (up to 15%), CD8+ (up to 12%) and TCR gamma/delta (up to 6%) in the duodenal intraepithelial leucocytes (IEL) from day 4 to day 8 Pl, and an increase in the proportion of IgM+ cells (12%) on day 8. At the same time, the proportion of CD8+ cells dropped significantly in the blood and spleen (-5 to -10%) on days 4 and 6 Pl and then increased with the proportion of CD4+ cells on day 8. An E tenella infection, localized in caecum, increased the proportion of CD4+ cells on day 8 Pl (20%) and of CD8+ cells (10%) on days 6 and 8 Pl in caecal IEL. A negative or zero effect on the proportion of TCR gamma/delta + cells was observed as well as on the IgM+ cells. At the same time, the proportion of CD4+ cells dropped in the spleen on day 8 Pl (-10%) and that of CD8+ cells dropped in the blood on day 6 (-15%). In conclusion, Eimeria infection seems to rapidly induce, locally at the site of the parasite development, a dramatic modification of the proportion of T-cell subsets in IEL, accompanied by systemic variations that are generally opposing, in the lymphocyte populations. The timing of the changes seems to follow the phases of the parasitic cycle for the Eimeria species considered.

Receptivity and susceptibility of the domestic duck (Anas platyrhynchos), the Muscovy duck (Cairina moschata), and their hybrid, the mule duck, to an experimental infection by Eimeria mulardi.

The receptivity and susceptibility of the Muscovy duck, the domestic duck, and their hybrid, the mule duck, toward an experimental infection by Eimeria mulardi were studied. To appraise their susceptibility, female ducklings were infected with a dose of 75,000 oocysts per duckling (bringing about a clinical coccidiosis). In another group, to compare the multiplication rates of the parasite in the three species of ducks, thus assessing their receptivity, an infection with 750 oocysts per duckling was realized. Eimeria mulardi develops in the three species of ducks. In each case, a significant decrease in growth in the contaminated group vs. the control group was noted, even though the Muscovy and the mule ducks were more susceptible to the contamination.

Analysis of humoral immune response in chickens after inoculation with Cryptosporidium baileyi or Cryptosporidium parvum.

White leghorn chickens aged 14 days were orally inoculated with 1 x 10(6) oocysts of Cryptosporidium baileyi or C. parvum to compare the specific immune responses. Cross-reactions were evaluated using homologous or heterologous antigens in enzyme-linked immunosorbent assay (ELISA) and Western blot to determine the occurrence of an antigenic homology between these two species. Blood, bile, whole intestine, bursa of Fabricius, and feces were collected daily from the day of inoculation (day 0) to day 22 postinoculation (PI). Eight control chickens remained negative up to day 22 PI. Chickens inoculated with C. baileyi did not express clinical symptoms but shed oocysts from days 6 to 21 PI. Chickens inoculated with C. parvum exhibited no clinical signs, no oocysts in feces, and no developmental stages of the parasite. However, a specific immune response to both antigens appeared on day 9 PI. ELISA using homologous or heterologous antigens showed that anti-C. baileyi and anti-C. parvum antibodies in serum or bile were detectable using C. baileyi or C. parvum oocysts as antigen, but the intensity of the response was significantly higher when C. baileyi was used. Cross-reactions in immunoblot analysis confirmed ELISA results, revealing a greater number of bands using C. baileyi as antigen but showing that epitopes recognized on the protein with a molecular weight of 15,000-17,000 were different.

Treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum.

Ovine or bovine colostrums with different antibody titers were tested for their ability to prevent cryptosporidiosis in five groups of neonatal lambs experimentally infected with 10(6) Cryptosporidium parvum oocysts 2 days after birth (Day 0). In a control group (Group 1), six lambs were deprived of ewe colostrum and exclusively fed with milk replacer. Two groups of six lambs were allowed to suckle their non-hyperimmunized (Group 2) or hyperimmunized (Group 3) dams throughout the experiment. Two groups of seven lambs were separated from their dams at birth before suckling and fed with non-hyperimmune (Group 4) or hyperimmune (Group 5) bovine colostrum; for 7 days they received 50 ml of colostrum completed by milk replacer twice a day, then they were fed with milk replacer exclusively. Control lambs became infected and developed clinical cryptosporidiosis with diarrhea on Days 4-9 post inoculation, oocyst shedding and mortality (2/6). In all the treated groups, the colostrum prevented mortality and clinical cryptosporidiosis. The mortality (5/7) observed in Group 5 was not due to cryptosporidiosis but anemia. In treated groups, specific antibodies were detected on Day 0 after 2 days of colostrum intake and varied little in time for IgM and IgG in spite of the parasite development, whereas they appeared later in the control group, on Day 4 for IgM, Day 11 for IgA and Day 14 for IgG. In all groups, the response which was the most consistent was the IgA response which evolved from Days 11 to 18 in association with the decline of oocyst shedding. Our results show that whatever the serum antibody titers were, the specific C. parvum circulating antibodies have no influence on the control of cryptosporidiosis. The prophylaxis or the treatment of cryptosporidiosis require high titers of specific C. parvum antibodies in the gut lumen during a sufficiently long period.

The immunodominant Eimeria acervulina sporozoite antigen previously described as p160/p240 is a 19-kilodalton antigen present in several Eimeria species.

A lambda Zap II cDNA expression library, constructed from Eimeria acervulina (PAPa46 strain) sporulated oocyst stage, was screened with sera raised to E. acervulina or Eimeria tenella oocysts in order to isolate clones coding for antigens common to the two species. Most of the clones isolated were derived from the same gene. Antisera raised to a recombinant glutathione-S-transferase fusion protein 1P reacted with an antigen of 19 kDa in immunoblot of E. acervulina sporulated and unsporulated oocysts. Immunofluorescence of E. acervulina sporozoites indicated that the antigen is located in the cytoplasm. The anti-1P antisera reacted on immunoblots of E. tenella with a 19-kDa antigen and by immunofluorescence on E. tenella, Eimeria maxima and Eimeria falciformis sporozoites, indicating that the antigen is conserved in Eimeria species. DNA sequencing indicated that the sequence was almost identical to that of clone cSZ1 previously described by Jenkins et al. using E. acervulina strain #12. The 1P insert hybridized to a 1150-nt mRNA from E. acervulina PAPa46 strain and strain #12, a size consistent with the observed molecular weight of the protein.

Absence of protection against Eimeria ninakohlyakimovae after primo-infection with E ovinoidalis in new-born kids.

One group of kids (n = 23) was given 2 x 10(5) oocysts of Eimeria ovinoidalis (sheep coccidia) at birth; a second group (n = 23) was kept as an uninoculated control. Body weights, E ninakohlyakimovae oocyst output and serum coccidial antibody levels were monitored up to 77-102 d of age. No significant difference in any of these parameters was seen between the 2 groups, suggesting that no immune response to E ovinoidalis inoculation occurred. These results could be relevant to the absence of development of the endogenous stages of E ovinoidalis in kids and/or to the mode of inoculation (moderate and not repeated).

Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

Protective oral immunization of chickens against Eimeria tenella with sporozoite surface antigens.

Antigens were extracted from the surface of Eimeria tenella sporozoites with a solution containing Triton X 100 (1%), sodium dodecyl sulphate (0.5%) Na deoxycholate (1%) and EDTA (1 mM). After removal of the detergents, these surface antigen preparations conferred an immunity that protected chickens against a subsequent infection (10(4) sporulated oocysts). The best results were obtained after two 250 micrograms injections of Al(OH)3 adsorbed antigens (oocyst output per g caecal material on Day 7 post infection: 2.39 x 10(7) +/- 0.32 x 10(7) oocysts for controls and 7.37 +/- 10(6) +/- 3.19 x 10(6) oocysts for vaccinated birds) and after four gastric intubations of liposome entrapped antigens (oocysts output on Day 7 postinfection: 2.75 x 10(6) +/- 2.02 x 10(6) g-1 caecal material). These results represented respectively 70 and 88% protection indexes. Studies on the systemic and local antibody response after one or several infections of chickens with the parasite indicated at least 20 different molecules in the detergent antigens which are classified after immunoblotting according to their properties.

The effect of halofuginone lactate on experimental Cryptosporidium parvum infections in calves.

The chemoprophylactic effects of halofuginone lactate were tested against calf experimental cryptosporidiosis. Twenty 2-day-old calves, divided into four groups, were orally inoculated with 1 x 10(6) oocysts of Cryptosporidium parvum. The infected control group was unmedicated whereas the three other groups were medicated with the drug at 30, 60 and 120 micrograms kg-1 day-1, respectively, for 7 days, from Day (D) 2 to D8 post-inoculation (D 0 was inoculation day). The calves were weighed twice weekly and disease development and drug efficacy were assessed daily from D0 to D30 from consistency of feces, shedding of oocysts and mortality. Experimental C. parvum infection caused a severe clinical disease with profuse watery diarrhea, high oocyst shedding and mortality (3 out of 5) in the unmedicated group. The results clearly demonstrated the efficacy of halofuginone lactate in reducing the severity of clinical cryptosporidiosis. This efficacy was dose-dependent. The lowest dose (30 micrograms kg-1 day-1) was not able to prevent clinical disease and mortality (3 out of 5). No clinical signs were observed with the 60 and 120 micrograms kg-1 day-1 doses, but the animals shed oocysts after drug withdrawal. This shedding was more delayed the higher the dose of drug administered, but the delayed shedding had no effect on the growth of the animals.

[Anticoccidial vaccines. Review and perspectives]. / Vaccins anticoccidiens. Bilan et perspectives.

Coccidioses are a major problem for the poultry industry. Currently, the diseases are controlled by drug chemoprevention, but the development of chemoresistant strains has for several years incited investigators to search for a vaccine. Several methods were investigated: oocyst administration at low and controlled doses, administration of strains which had been attenuated by physical or biochemical means, immunization with extracted or recombinant antigens. For some years to come genetically selected precocious live strains of Eimeria which are now commercially available will be useful as vaccines; but in the future, genetically engineered antigens will provide new opportunities for a successful vaccination.

Serum coloration as a criterion of the severity of experimental coccidiosis in the chicken.

Serum decoloration may be used as a criterion for the severity of coccidiosis. Direct measurement of serum coloration at 480 nm is easy to carry out and, with micromethods, it is not necessary to slaughter animals. With intestinal coccidia the sensitivity is high, and there is good correlation with the severity of infection and variability is small. With Eimeria tenella infection the criterion is less sensitive, but may be of value for some purposes.

Repeatability of ovine faecal oocyst counts in natural infections with Eimeria spp.

The repeatability of ovine faecal oocyst counts was studied in Merino of Arles and Romanov sheep over short (4 days) or longer periods (1 month). The repeatabilities over short periods ranged from 0.27 to 0.67 for total oocyst output, from 0.06 to 0.54 for Eimeria ovinoidalis and from 0.29 to 0.52 for E. parva counts. E. ovinoidalis had lower and more irregular repeatabilities than E. parva. Their repeatabilities ranged, respectively from 0.18 (E. ovinoidalis) to 0.22 (E. parva) for longer periods. The genetic share in the determination of magnitude of oocyst counts seems similar to that recorded for trichostrongylid egg counts.

Cryptosporidium parvum in calves: kinetics and immunoblot analysis of specific serum and local antibody responses (immunoglobulin A [IgA], IgG, and IgM) after natural and experimental infections.

Fecal and serum anti-Cryptosporidium parvum immunoglobulin A (IgA), IgM, and IgG were monitored by an enzyme-linked immunosorbent assay after experimental and natural infection of calves with C. parvum. Although all experimentally infected calves showed high levels of colostral antibodies in the feces, they acquired C. parvum infection. Three of five animals died. Calves which acquired natural infection showed only diarrhea. Levels of colostral coproantibodies dropped quickly. Experimental infection was followed by a rise in local anti-C. parvum IgM levels from day 5 postinfection (p.i.). IgM peaked at day 14 p.i. and then disappeared quickly. Anti-C. parvum IgA levels rose between days 7 and 14 p.i. and decreased slowly. Rising levels of coproantibodies coincided with falling oocyst output. Fecal anti-C. parvum IgG levels rose slightly during oocyst output, and IgG disappeared 3 weeks p.i. Similar kinetics were established in naturally infected calves. Although fecal anti-C. parvum IgA levels declined slowly, reinfections were established 5, 7, and 14 weeks after the primary contact. Serum anti-C. parvum IgG levels rose during maximal oocyst excretion, whereas serum anti-C. parvum IgA levels peaked later than did local IgA levels. Challenge reinfection of naturally infected calves at day 112 was not followed by clinical signs or oocyst output or by a secondary antibody response. Sequential Western immunoblotting with fecal extracts revealed up to 32 different parasite antigens. Convalescent-phase sera recognized up to 23 antigens. Fecal IgA reacted intensely with antigens with relative molecular weights (M(r)) of approximately 11,000 and 15,000. These antigens were not recognized by convalescent-phase serum IgG. Both local IgA and serum IgG also showed strong reactions with 23,000- and 44,000-M(r) antigens and with several antigens of between 66,200 and 200,000 M(r). Most bands remained detectable for at least 16 weeks p.i.

Effect of Eimeria maxima infection on development of Eimeria tenella in immunized chickens.

Chickens previously immunized against Eimeria tenella were reinfected with this species with and without infection by E. maxima. The present of E. maxima at the time of reinfection appears to decrease the immunity of the animals and enables E. tenella to develop. In this case, the development of E. maxima is not affected by the presence of E. tenella.