Study On The Isolation And Identification Of Bacillus Thuringiensis From The Feces Of Animals And Its Effects On Avian Coccidiosis

Avian coccidiosis is a remarkable disease that spreads widely with a high incidence in chicken farms and is often found in chicks at 15-day-old to 50-day-old. If it were not controlled effectively, the prevalence of infection would be high up to 50%-70% with a mortality of 20%-30% or even 80% in severe cases. According to the official statistics, the disease could cost an estimated 800 million US$ considerable economic loss around the world. Current control of coccidosis is mainly based on either synthetic chemicals or antibiotics. However, a durable use of the chemical drugs in the poultry industry would easily lead to the resistant strains as well as the drug residues in the chicken and their products are harmful to human beings. Therefore, more and more researches have been focused onto non-antibiotic approaches.The crystal proteins produced by Bacillus thuringiensis (Bt) are the most widely used biological pesticides. These toxins, used by organic farmers, offering a remarkable alternative to chemical pesticides cause of nontoxic to vertebrates, generally targeting insects within a single order, are more benign to the environment, and can be genetically engineered into crops to provide constant protection. The later studies demonstrated that the spore-crystal proteins are effective to helminthes in animals.Something about that Bt targeted at avain coccidiosis had not been reported. In order to find novel Bacillus thuringiensis (Bt) strains, we investigated Bt from the feces of zool maintained animals. 253 selected colonies of the Bacillus cereus- B. thuringiensis group from 50 fecal samples obtained from 41 species of animals (33 mammals and 8 avians) were isolated. The animals were residents of the Fuzhou Zoo and the Sanming Zoo, Fujian, China. Of all these colonies, 15 were assigned to Bt on the basis of the formation of parasporal inclusions. The organism was detected in 15 (30%) samples. Fecal samples from herbivorous, omnivorous and carnivorous animals contained Bt frequencies 35.5%, 25% and 18.2%, respectively. The results suggested that a daily food intake of plant origin yielded the feces containing Bt at high levels. Using single oocyst infected techniques, three strains of Eimeria tenella, PT0705, FQ0709 and LJ0711, were obtained from classically symptomic coccidian chicken in Putian, Fuqing and Lianjiang Couties, Fujian Province. In order to compare their toxicity against chicken, healthy chicken of 14th-day and 21th-day were artificially infected with 1×104, 5×104 and 10×104 coccidian oocysts of PT0705, FQ0709 and LJ0711, respectively. The results showed that the strain PT0705 was the most toxic to tested chicken based on the clinical symptom, survival rate, weight increment and lesion grade investigations.Chick embryos inoculated with sporozoites of E. tenella were used as a model for testing the activity of Bt crystal proteins. Each embryo were inoculated by a dose of 1×104 pure sporozoites was injected with testing Bt crystal proteins before 24 hours. The varying oocyst inhibition rates of embryos brought about by Bt crystal proteins that were given at various doses were used as a criterion for determining the activity of Bt crystal proteins. The results demonstrated that BT6 was the most effective isolate among the 15 strains.According to the experimental results mentioned above, we believe that the use of chicken embryo infection with E. tenella sporozites for the determination of the activity of Bt crystal proteins is an efficacious method, which offers some advantages, such as simplicity of operation, good accuracy and only small requirement of expenses.In order to compare the efficacy of Bt crystal proteins and chemical medicine against avian coccidiosis. Chicken (n=50) were randomly grouped in control (Ⅰ), control (Ⅱ), Bt crystal proteins- treated (Ⅲ), Diclazuril-treated (Ⅳ), and Amprolium-treated (Ⅴ) group. GroupⅡ,Ⅲ,Ⅳ, andⅤwere inoculated with 10×104 sporulated E. tenella PT0705 occysts per bird. The results showed that the anticoccidia indexes (ACI) of the Bt crystal proteins,Diclazuril and Amprolium were 202.3,184.1 and 128.2 respectively. This meant that the efficacy of Bt crystal proteins against avian coccidiosis was more excellent than that of chemical Diclazuril, which has been proved to have a high efficacy against coccidiostat while Amprolium only has low efficacy against coccidiosis. The average body weight in groupⅢincreased by 458.9g, while according to t test, the value of p was less then 0.05 as compared with that in groupⅠandⅣ, and less then 0.01 as compared with that in groupⅡandⅤ. It could draw a conclusion that the Bt crystal proteins had the effect of prevention and treatment of avian coccidiosis and improved the growth of chicken.In order to understand the physiological characteristics of BT6, its morphology and biochemical reaction were studied, and cry-type genes had been identified by using PCR-RFLP identification system. The evidence showed that this isolate harbored cry1Aa, cry1Ba, cry1Ea and cry1Ia.In a word, we thought that strain BT6 was the especially effective on avian coccidiosis in all the 15 strains. The crystal proteins of strain BT6, which was toxic to E. tenella with no side effect to chicken, could be applied in avian clinical practice.