The effect of Bacillus larvicidal toxins On mammals By Nadia Adam Mohammed Ahmed B.Sc. of Veterinary Sciences University of Bahr-Elgazal ٢٠٠٠ Thesis submitted for the partial fulfillment of the requirements For the degree of Master of Science (M.Sc.) in microbiology Supervisor Professor: Mohammed Sulieman El-sanosi Department of microbiology Faculty of Veterinary Medicine University of Khartoum ٢٠٠٦ May To My lovely parents, My husband (M/Elmurtada) and my children (Reem&Rwan) To My brothers and sisters, And To Those who suffer from malaria, I dedicate this work. Nadia I would like to express my sincere thanks to my supervisor Prof. Elsanosi for his valuable supervision and encouragement. I also like to extend my thanks to Prof.Ahamed.A.Gameel for his great help in histopathological investigation of this study. Thanks also extend to all staff and technicians of the Department of Microbiology for their valuable advices and assistance. Iam also greatfull to my colleague and all my friends Special thanks to my family for their patience and continuous encouragement during this work. Abstract Mosquito control is the key for hopeful and sustainable eradication of different socio-economic as well as fatal viral, bacterial and parasitological diseases in the tropical and subtropical countries. In addition the safety of any pesticide should be assured before application. In the present study the susceptibility of arabiensis mosquito larvae to the toxin of two endo-spores forming bacilli strain was examined. Both Bacilli spp B. ٪١٠٠ thuringiensis and B. sphaericus were found to be highly toxic and fatal with was found to be tolerant to the (٤_Instar) ٤_mortality rate. However, larval stage toxin. Moreover the safety of these two toxins against different mammalian animal species was recorded during oral administration in goats, rabbits and mice, while the toxicity due to intravenous route was assessed in rabbit only. The toxins were found to be completely safe when administrated orally to goats, rabbits and mice used in this experiment, while B. sphaericus cause no signs of toxication in rabbits even when injected intravenously. However, the laboratory animal showed significant clinical, pathological and histopathological changes. ﺍﻟﺨـﻼﺼـﺔ ﺘﻌﺘﺒﺭ ﻤﻜﺎﻓﺤﺔ ﺍﻟﺒﺎﻋﻭﺽ ﺍﻟﻤﻔﺘﺎﺡ ﺍﻟﺭﺌﻴﺴﻲ ﻻﺴﺘﺌﺼﺎل ﺍﻟﻜﺜﻴﺭ ﻤﻥ ﺍﻷﻤﺭﺍﺽ ﺫﺍﺕ ﺍﻷﺜـﺭ ﺍﻻﻗﺘﺼﺎﺩﻱ ﻭﺍﻻﺠﺘﻤﺎﻋﻲ ﻭﻏﻴﺭﻫﺎ ﻤﻥ ﺍﻷﻤﺭﺍﺽ ﺍﻟﻔﻴﺭﻭﺴﻴﺔ ﻭ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﻭﺍﻟﻁﻔﻴﻠﻴﺔ ﺍﻟﻘﺎﺘﻠـﺔ ﻓـﻲ ﺍﻟﻤﻨﺎﻁﻕ ﺍﻟﻤﺩﺍﺭﻴﺔ ﻭﺘﺤﺕ ﺍﻟﻤﺩﺍﺭﻴﺔ ﺒﻤﺎ ﻓﻴﻬﺎ ﺍﻟﺴﻭﺩﺍﻥ. ﺃﻀﻑ ﺇﻟﻰ ﺫﻟﻙ ﺃﻥ ﺴﻼﻤﺔ ﺃﻱ ﻤﻥ ﺍﻟﻤﺒﻴﺩﺍﺕ ﺍﻟﺤ ﺸﺭﻴﺔ ﻴﺠﺏ ﺃﻥ ﺘﺅﻜﺩ ﻗﺒـل ﺍﺴـﺘﺨﺩﺍﻤﻬﺎ ﻋ ﻤ ﻠ ﻴ ﺎﹰ. ﺼﻤﻡ ﻫﺫﺍ ﺍﻟﺒﺤﺙ ﻟﺩﺭﺍﺴﺔ ﻗﺎﺒﻠﻴﺔ ﻴﺭﻗﺎﺕ ﺍﻟﺒﺎﻋﻭﺽ ﻟﻨﻭﻋﻴﻥ ﻤـﻥ ﺍﻟـﺴﻤﻭﻡ ﺍﻟﺒﻜﺘﻴﺭﻴـﺔ ﻟﻨـﻭﻋﻴﻥ ﻤﺨﺘﻠﻔﻴﻥ ﻤﻥ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﻜﻭﻨﺔ ﻟﻸﺒﻭﺍﻍ . ﻭﻤﻥ ﺜﻡ ﺩﺭﺍﺴﺔ ﺴﻼﻤﺔ ( ﻋﺩﻡ ﺴﻤﻴﺔ ) ﻫﺫﻴﻥ ﺍﻟﻨﻭﻋﻴﻥ ﻤﻥ ﺍﻟﺴﻤﻭﻡ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﺒﺎﻟﻨﺴﺒﺔ ﻷﻨﻭﺍﻉ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺍﻟﺜﺩﻴﻴﺎ ﺕ ( ﺍﻟﻤﺎﻋﺯ – ﺍﻷﺭﺍﻨـﺏ – ﺍﻟﻔﺌـﺭﺍﻥ ) ﺇﻥ ﻫﺫﻴﻥ ﺍﻟﻨﻭﻋﻴﻥ ﻤﻥ ﺍﻟﺴﻤﻭﻡ ﺍﻟﻘﺎﺘﻠﺔ ﻟﻠﻴﺭﻗﺎﺕ ( B. (B.thuringiensis, Sphaericus) ﻭﺠﺩﺕ ﻓﻌﺎﻟﺔ ﺒ ﺩ ﺀ ﺍﹰ ﻤﻥ ﺍﻟﻨﺴﺒﺔ (١٠٠%) ﻓﻲ ﻗﺘل ﺍﻟﻴﺭﻗﺎﺕ ﻤﺎ ﻋﺩﺍ ﺍﻟﻁﻭﺭ ﺍﻟﺭﺍﺒﻊ ﻤﻥ ﺍﻟﻴﺭﻗـﺎﺕ ( L٤ ) ﺍﻟﺘﻲ ﺘﻘﺎﻭﻡ ﻫﺫﻩ ﺍﻟﺴﻤﻴﺎﺕ ﻭﻤﻥ ﻨﺎﺤﻴﺔ ﺃﺨﺭﻯ ﻓﻘﺩ ﻭﺠﺩﺕ ﻫﺫﻩ ﺍﻟﻤﺴﺘﺨﻠﺼﺎﺕ ﻏ ﻴﺭ ﺴـﺎﻤﺔ ﻟﻬـﺫﻩ ﺍﻟﺤﻴﻭﺍﻨﺎﺕ ( ﺍﻟﻤﺎﻋﺯ – ﺍﻷﺭﺍﻨﺏ – ﺍﻟﻔﺌﺭﺍﻥ ) ﻋﻨﺩﻤﺎ ﻴﺘﻡ ﺘﻨﺎﻭﻟﻬﺎ ﻋﻥ ﻁﺭﻴﻕ ﺍﻟﻔـﻡ ( ﺍﻟـﺸﺭﺍﺏ ) ﻏﻴﺭ ﺃﻥ ﺃﺤﺩ ﻫﺫﻩ ﺍﻟﻤﺴﺘﺨﻠﺼﺎﺕ (B.thuringiensis) ﺃﺒﺩﺕ ﺴﻤﻴﺔ ﻗﺎﺘﻠﺔ ﻟﻸﺭﺍﻨﺏ ﻋﻨـﺩﻤﺎ ﻴـﺘﻡ ﺤﻘﻨﻬﺎ ﻋﻥ ﻁﺭﻴﻕ ﺍﻟﻭﺭﻴﺩ. List of Content Dedication I Acknowledgement II Abstract III Arabic abstract IV List of contents V List of tables IX List of figures X List of plates XI ١ Introduction Chapter one: Literature review ٢ Mosquitoes .١٫١ ٢ Classification .١٫١٫١ ٢ Mosquito life cycle .١٫١٫٢ ٤ Malaria .١٫٢ ٤ Larvicides .١٫٣ ٥ Classification of Bacillus Species .١٫٣٫١ ٦ Morphological Characteristics .١٫٣٫٢ ٦ Physio-chemical characteristics .١٫٣٫٣ ٦ Bacillus species overview .١٫٣٫٤ ٨ Bacillus thuringiensis .١٫٣٫٤٫١ ١٢ Bacillus sphaericus .١٫٣٫٤٫٢ ١٣ Effect of the Bacillus on mammals .١٫٣٫٤٫٣ Chapter Two: Material and methods ١٥ Collection of samples and area of collection .٢٫١ ١٥ Transportation of samples .٢٫٢٫١ ١٥ Isolation of bacteria from mosquito larvae .٢٫٢٫٢ ١٥ Dissection procedure .٢٫٢٫٣ ١٥ Culture media .٢٫٣ ١٦ Solid media .٢٫٣٫١ ١٦ Nutrient Agar .٢٫٣٫١٫١ ١٦ Blood Agar .٢٫٣٫١٫٢ ١٧ MacConkey’s Agar .٢٫٣٫١٫٣ ١٧ Starch Agar .٢٫٣٫١٫٤ ١٧ Simmon’s citrate agar .٢٫٣٫١٫٥ ١٨ Urea Agar Medium .٢٫٣٫١٫٦ ١٩ Lecitho-Vitellin (LV) agar .٢٫٣٫١٫٧ ١٩ (Casein agar (Milk agar .٢٫٣٫١٫٨ ٢٠ Aesculin agar .٢٫٣٫١٫٩ ٢٠ (Ammonium salt sugar (ASS .٢٫٣٫١٫١٠ ٢١ Semi-solid media .٢٫٣٫٢ ٢١ Motility media .٢٫٣٫٢٫١ ٢١ Huge and Leifson’s (O/F) medium .٢٫٣٫٢٫٢ ٢١ Nutrient gelatin .٢٫٣٫٢٫٣ ٢٢ Liquid media .٢٫٣٫٣ ٢٢ Nutrient broth .٢٫٣٫٣٫١ ٢٢ (NaCl ٪١٠) Nutrient broth .٢٫٣٫٣٫٢ ٢٢ Peptone water .٢٫٣٫٣٫٣ ٢٣ Peptone water sugars .٢٫٣٫٣٫٤ ٢٣ (Glucose phosphate broth (for M.R. and V.P. test .٢٫٣٫٣٫٦ ٢٤ Reagents .٢٫٤ ٢٥ Indicators .٢٫٥ ٢٥ Solutions .٢٫٦ ٢٥ (٪٠٫٨٥) Normal Saline .٢٫٦٫١ ٢٥ Method of Sterilization and Disinfection .٢٫٧ ٢٦ Sterilization .٢٫٧٫١ ٢٦ Dry Heat .٢٫٧٫١٫١ ٢٦ Hot air oven .٢٫٧٫١٫١٫١ ٢٦ Flaming .٢٫٧٫١٫١٫٢ ٢٦ Moist Heat .٢٫٧٫١٫٢ ٢٦ Autoclaving .٢٫٧٫١٫٢٫١ ٢٦ Cultural methods .٢٫٨ ٢٦ Primary Isolation .٢٫٨٫١ ٢٦ Incubation of cultures .٢٫٨٫٢ ٢٦ Purification of isolates .٢٫٨٫٣ ٢٧ Detection of pigment production .٢٫٨٫٤ ٢٧ Identification of isolates .٢٫٨٫٥ ٢٧ Cultural characteristics .٢٫٨٫٥٫١ ٢٧ Primary test .٢٫٨٫٥٫٢ ٢٧ Gram staining and microscopy ٢٫٨٫٥٫٢٫١ ٢٨ Spore stain .٢٫٨٫٥٫٢٫٢ ٢٨ Secondary Tests .٢٫٨٫٥٫٣ ٢٨ Catalse Test .٢٫٨٫٥٫٣٫١ ٢٨ Oxidase Test .٢٫٨٫٥٫٣٫٢ ٢٨ Motility Test .٢٫٨٫٥٫٣٫٣ ٢٩ Oxidation Fermentation of Glucose (O-F) test .٢٫٨٫٥٫٣٫٤ ٢٩ Sugar Fermentation .٢٫٨٫٥٫٣٫٥ ٢٩ Urease Test .٢٫٨٫٥٫٣٫٦ ٢٩ Citrate Utilization Test .٢٫٨٫٥٫٣٫٧ ٢٩ Indole Test .٢٫٨٫٥٫٣٫٨ ٣٠ Methyl Red Reaction .٢٫٨٫٥٫٣٫٩ ٣٠ Voges-Proskauer (V-P) test .٢٫٨٫٥٫٣٫١٠ ٣٠ Asculin Hydrolysis .٢٫٨٫٥٫٣٫١١ ٣٠ Starch Hydrolysis .٢٫٨٫٥٫٣٫١٢ ٣٠ Gelatin Hydrolysis or Liquefactions .٢٫٨٫٥٫٣٫١٣ ٣١ Nitrate Reduction .٢٫٨٫٥٫٣٫١٤ ٣١ Digestion of Casein .٢٫٨٫٥٫٣٫١٥ ٣١ Lecitho-Vitellin Reaction .٢٫٨٫٥٫٣٫١٦ ٣١ Ammonium salt sugar test .٢٫٨٫٥٫٣٫١٧ ٣١ Growth in medium with increased NaCl concentration .٢٫٨٫٥٫٣٫١٨ ٣١ Preservation of culture .٢٫٨٫٥٫٤ ٣٢ Preparation of the supernatant Bacillus metabolites .٢٫٨٫٥٫٥ ٣٢ Preparation of the filtrated Bacillus metabolites .٢٫٨٫٥٫٦ ٣٢ Preparation of the concentrated Bacillus metabolites .٢٫٨٫٥٫٧ ٣٢ Pathogenicity of Bacillus species to mosquito larvae .٢٫٨٫٥٫٨ ٣٢ Screening test of the larvicidal .٢٫٨٫٥٫٩ ٣٢ Effect of concentrate metabolites on different animal species .٢٫٩ ٣٣ Mice .٢٫٩٫١ ٣٣ Rabbit .٢٫٩٫٢ ٣٣ Goat .٢٫٩٫٣ ٣٣ Histopathological techniques .٢٫١٠ Chapter Three: Results ٣٧ Isolation of Bacteria .٣٫١ ٣٨ Biochemical properties of Bacillus spp .٣٫٢ ٤٠ The larvicidal activity of the Bacillus spp .٣٫٣ ٤٢ Some visual observations in the experimental larvae intoxication .٣٫٣٫١ Effect of the concentrated Bacillus metabolites on mammals .٣٫٤ ٤٢ ٤٢ The oral route .٣٫٤٫١ ٤٤ The effect of the toxin by the weight of the intestine of mice .٣٫٤٫١٫١ The intravenous route .٣٫٤٫٢ ٤٤ ٥٢ Discussion ٥٥ Conclusion ٥٦ Recommendation ٥٧ References List of tables A list of bacteria which Isolated from the mid-gut of An.Arabiensis larvae .٣٫١ ٣٧ ٣٩ Biochemical properties of the isolated Bacillus spp .٣٫٢ ٤١ The larvicidal activity of Bacillus spp .٣٫٣ ٤٣ Effect of concentrated Bacillus metabolites on mammals .٣٫٤ ٤٤ The effect of the toxins in the experimented mice .٣٫٥ Death percentage of Mosquito larvae due to different concentrations of .٣٫٦ ٤٩ two Bacillus toxins at different days List of figures ٥٠ .larvae given toxin T ٥ Death percentage of .٣٫١ ٥١ .larvae given toxin S ٥ Death percentage of .٣٫٢ List of plates An affected liver of rabbit intravenously inoculated with .٣٫١ ٤٦ B. thuringiensis toxin Alveolar haemorrhages with mononuclear cells infiltration .٣٫٢ ٤٦ In infected rabbit lung with the toxin ٤٧ Cardial myocytes atrophy due to B. thuringiensis toxin .٣٫٣ Congested kidney with mononuclear cells infiltration .٣٫٤ ٤٧ In rabbit infected with the toxin The rabbit brain lesions due to (I/V) injection of .٣٫٥ ٤٨ B. thuringiensis toxin Skeletal muscle of rabbit inoculated with the toxin (I/V) showing .٣٫٦ ٤٨ haemorrhages, degeneration And Zinker necrosis INTRODUCTION Introduction From the stand point of risk to mammalian’s health and well being, mosquitoes represent the most important group of insects. They are vector of some of the most distressing diseases, like malaria, yellow fever and filariasis. Malaria is one of the diseases that spread by some species of the anophline mosquitoes. With the essential need of determining the appropriate control program, the vector- control by synthetic insecticides used have reflected certain drawbacks on the environmental hazard, development of resistance, destruction of natural enemies of the vectors, toxicity to humans, domestic and wild animals and the disruption in the natural balance and food chain.