Study On The Effects Of Bacillus Thuringiensis Spore-crystal Proteins On Ascaris Suum

Ascaris suutm, the large roundworm of swine, is an ascaridoid (ascarid) nematode of cosmopolitan distribution. It's the most common gastrointestinal worm parasite in pigs with a prevalence of 50%⁷0%. It's more common in growing pigs than in mature pigs. Although now recognized as a separate species, the large roundworm of humans, A. lumbricoides, was thought to be the same as that in pigs. As that A. suum could infect humans, it was included as a public hygienic disease by the World Health Organization (WHO) and some countries. Although the drugs of Bendazole, Ivermectin and Pyrantel etc are effective on Ascaridasis, they are very easy to reduce in animal tissues and have strong toxicities to animals and humans. Most of these drugs are inhibited or restricted to use in animals, which proclaimed by WHO and our government Especially there isn't any commercial vaccine of A. suum. A new drug without toxicity and hazard to animals and humans is exigency for veterinarians, and the drugs with these characters should have amplitude market.The crystal-forming endotoxins produced by Bacillus thuringiensis (Bt) are the most widely used biologically 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. The main goals of this study were looking for the especially good effective isolates to A. suum and how to administer it into animals. No relative report was looked up.Cultured in vitro at the conditions of 28℃, 28 days with antisepsis, ova of A. suum developed to infectious ova. Animals (mice and piglets) had been restricted to rise withthe feed without any anti-parasite drugs for 7 days. Animals should be checked and confirmed to be not infected by A. suum before inoculation. 4,000 infected ova were administered into a mouse by per os, and 6×x10⁵ ova into porcine (40kg). The test pigs had all the clinical symptom of ascaridasis, A. suum eggs per gram of feces (epg) was highly to 7100 60d after inoculation. X-ray images showed the representative signs of bronchitis, lobular pneumonia and permeated pneumonia. L2, L3 and L4 were separated, and ascaridosis model was copied successfully in this study.Mice were killed for collecting the fourth stage larva (L4) 6 days later after inoculated. All larvae were confected as suspending solution with DMEM medium to 200²50 larvae per milliliter, which were used for the tests of spore-crystal proteins larvacidal activities of spore-crystal proteins.Bt H₁4, HD₁, 007, 017, 021, 027, 032, 037 and their spore-crystal was accumulated after culture; its spore-crystal proteins were dissolved with alkaline solution. Larvacidal activities of spore-crystal proteins were determined by adding 250μ proteins solution to one cell of 24 cell microtiter plate (Falcon) containing 250μ larvae suspension. Four multiple proportion concentrations and 2 repetitions of every proteins sample were tested, and the diluents was DMEM. Control cell had 250μ larvae suspension and 250μ DMEM, which the proteins was not added in. There were no less than 50 larvae in every cell. All the 8 isolates of Bt were tested. Microtiter plates were incubated at 37℃ with 5% CO₂. The larvacidal activities were determined every 12h by counting dead and living larvae 3 times, the averages were noted down. Dead rates in control were used to calculate relative dead rate. Average relative dead ratio and half lethal dose (LD₅0) of proteins on L4 of Ascaris suum were calculated. There wasn't larva died before 24h, all larvae died after 60h in trials beside H₁4 and HD_-1 trials, and 72h in control. The results demonstrated that strain 017 was the most effective isolate. It's LD₅0 were 1.2355g/L, 0.8220g/L and 0.6488g/L respectively at 24h, 36h and 48h. H₁4 and HD₁ were not toxi...