| Avian coccidiosis is an intestinal disease caused by protozoan parasites of the genus Eimeria. There are seven species of Eimeria in chicken, viz. E. tenella, E. acervulina, E. maxima, E. brunetti, E. necatrix, E.praecox and E. mitis, which were mixed infection in field, so it is very important to isolate the pure strains for controlling coccidiosis. Thus, using techniques of single oocyst, three pure strains were isolated and found to be E. mitis, E. praecox and E. necatrix. Using molecular biotechnology the sequence of the rDNA-ITS regions from the the E. praecox, E. mitis, E. necatrix, E. maxima strain NT, E. maxima strain YZ, E. maxima strain SH isolated in China and E. maxima strain MG isolated from a vaccine made in USA were amplified by PCR with a pair of genus-specific primers and sequenced. Evolutionary relationships among the species and strains inferred using neighbour-joining of the aligned internal transcribed spacer sequences were well supported. The results were useful to diagnosis and molecular epidemiological survery of Eimeria spp. in poultry industry. Furthermore, this study discussed the immune interaction among E. necatrix, E.maxima and E.tenella. In order to attempt a new method of controlling coccidiosis in poultry industry, the studies had been conducted as the following.1. Isolation and Identification of E. mitis and E. praecoxUsing techniques of single oocyst, two pure strains were isolated and found to be Eimeria mitis and E. praecox. E mitis:The oocysts were spherical or subspherical; with a polar granule and without residuum; mean (14.96±1.38)μm×(13.65±1.20) μm. The sporocysts were ovoid; mean (9.23±1.20)μm×(5.29±0.53)μm; with a Stieda body and without a residuum. The incubation period was96h. Sporulation time was 15.5h. The infection was most prevalent in posterior part of intestine. E. praecox:The oocysts were ovoid or ellipsoidal; with a polar granule and without residuum; mean (19.21±2.10) μm×(16.40±1.75)μm. The sporocysts were ovoid; mean (10.62±0.90) μm×(6.85±0.75)μm; with a Stieda body and without a residuum. The incubation period was84h. Sporulation time was20h. The infection was most prevalent in anterior part of intestine. To assess the pathogenicity of two species, the four groups of30-day-old Suqin Yellow chickens were infected with20×104or60×104sporulated oocysts, respectively, and with another10birds as non-infected control group. The results showed that none of the chickens were died, and the average weight gains in the infected groups were significantly different from that of the non-infected group. The visible intestinal lesions appeared in the groups infected with E. mitis, and no gross lesions were seen in the groups infected with E. praecox.2. Isolation and Identification of E. necatrixUsing techniques of single oocyst, one strain was isolated and identified to be Eimeria necatrix:The oocysts were orbicular-ovate, with a polar granule inside but without residuum and micropyle.The size of100oocysts is (12.25-25.00) μm×(12.5-30.00) μm, with the mean size of which (16.53±3.12)μm×(18.76±3.44)μm. whose shape index is1.14. The sporocysts were ovoid, with a stieda body on the cell wall but without a residuum. The size of sporangium is (4.00-7.10)μm×(9.00-14.00) μm, with the mean size of which (5.91±0.68) μm×(10.91±0.91)μm. The size of the second-generation schizoite is (32.50-75.00) μm×(28.00-62.50) μm, with the mean size of which (50.77±11.77) μm×(44.44±9.51)μm. The size of25schizozoites is (1.50-3.00)μm×(9.50-12.50)μm, with the mean size of which (2.41±0.27) μm×(10.51±0.93) μm. The incubation period was145h. The shortest time of sporulation was19h. The oocysts were parasitic at the cecum. To assess the pathogenicity of this strain coccidium, six groups of30-day-old Suqin Yellow chickens were infected with0.5×104,1×104, and5×104sporulated oocysts respectively, and each group has10chichens with repeatability setted. Results:Death occurred in two groups infected with1×104and5×104, and the mortalities were30%to40%in the group infected with5×104oocysts,0%to10%in the group infected with1×104oocysts, respectively. The average weight gain of the groups infected with0.5×104and1×104oocysts is not significantly different from that of the without infected group. The difference is extreme significant between the average weight gain of the groups infected with5x104and that of the without infected group (P<0.05). Pathological changes appeared in the intestines of the chickens in the infected groups, the scores of pathological changes were highest in the groups infected with5×104.3. Cloning and Sequencing Analyze of the Internal Transcribed Spacer of Ribosomal DNA for E.praecox, E.mitis, E.necatrix and four Strains of E.maximaIn order to further confirm the identification of seven pure strains and research the evolutionary relationships among the species and strains, the sequence of the rDNA-ITS regions from the the E. praecox, E. mitis, E. necatrix, E. maxima strain NT, E. maxima strain YZ, E. maxima strain SH and E. maxima strain MG were amplified by PCR with a pair of genus-specific primers and sequenced. The phylogenetic relations were analyzed by compared together with sequences from GenBank. The results showed that the length of amplified fragments were1084bp for E.praecox,1037bp for E. mitis,1337bp for E. necatrix,980bp for E. maxima strain YZ,875bp for E. maxima strain SH,876bp for E. maxima strain MG,1008bp for E. maxima strain NT. The sequence similarities of four species ranged from50.8%to99.1%, the genetic distance mean0.49. Phylogenetic tree showed that each species was at the same cluster with corresponding species which sequence downloaded from GenBank. One E.maxima lineage consisted of E. maxima strain SH and E. maxima strain MG isolates, the other consisted of E. maxima strain NT and E. maxima strain YZ isolates, which exhibited96.9%and99.1%homology, respectively. The tree demonstrates the close relationship of E. mitis clone171-8from American (Gen-Bank Accession No. FJ230365) and the laboratory strain from China with strong bootstrap support for these branches, the same to E. tenella and E. necatrix.Construction of a phylogenetic tree places E. praecox on the only branch, which was put E. praecox on a par with other branches.4. Immunological Interaction between E.necatrix and E.maxima or E.tenellaIn order to examine the immunological interaction between E. necatrix and E.maxima as well as between E.necatri and E.tenella, the animal experiments were performed. The protective immunities were assessed by relative weight gain, oocyst output, oocyst reduction, the mortalities, the scores of pathological changes in the intestines and antibody levels. The results showed that protective effect in the group immunized together with E.maxima and E.necatrix were better than that of the group immunized with E.necatrix alone. It shows that E.maxima has no immune effect on E.necatrix, but synergistic effect. However, it shows that E.necatrix has some immunological effect on E.maxima. The mixed immunity group of E.tenella and E.necatrix has better protective effect than the immunity group of E.necatrix alone. It shows that E.tenella has some immunological effect on E.necatrix, but synergistic. However, the E.necatrix has no effect on the immunity of E.tenella. |
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