Patent Number: 5262399
Issue Date: 19931116
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BACKGROUND OF THE INVENTION
Flukes belong to subclass Digenea, class Trematoda in the phylum Platyhelminthes. These digeneans all have an intermediate host and are found exclusively in vertebrates, including man. Families which have members of considerable veterinary importance are Fasciolidae, Dicrocoeliidae, Paramphistomatidae, and Schistosomatidae.
The adult trematode flukes occur primarily in the bile ducts, alimentary tract, and vascular system. Most flukes are flattened dorso-ventrally, have a blind alimentary tract, suckers for attachment, and are hermaphroditic. Depending on the predilection site, the eggs pass out of the final host, usually in faeces or urine, and the larval stages develop in a molluscan intermediate host. For a few species, a second intermediate host is involved, but the mollusc is essential for all members of the group. They are worldwide in their distribution.
Several clinical syndromes may be associated with liver fluke (Fasciola sp.) infection, depending on the numbers and stage of development of the parasite and on the presence or absence of certain bacteria (Clostridium novyi). Acute fluke disease occurs during invasion of the liver by recently ingested metacercariae. In heavy invasions, the trauma inflicted by the maritas tunneling about in the liver and consequent inflammatory reaction result in highly fatal clinical illness characterized by abdominal pain with a disinclination to move. Sheep can die very quickly due to focal liver necrosis and extensive subcutaneous hemorrhage.
Subacute and chronic fluke disease is associated with the presence of adult trematodes in the bile ducts and characterized by the classical clinical signs of liver fluke infection. There is gradual loss of condition, progressive weakness, anemia, and hypoproteinemia with development of edematous subcutaneous swellings, especially in the intermandibular space and over the abdomen. There is considerable economic waste in cattle livers condemned as unfit for human consumption; destructive migrations in the livers of sheep and goats virtually preclude small ruminant production in endemic areas.
Light infections of flukes may not elicit clinical effects, but the parasites can have a significant effect on production due to an impairment of appetite and to their effect on post-absorptive metabolism of protein, carbohydrates, and minerals.
Dicrocoelium dendriticum, a fluke in the family Dicrocoeliidae, of economic importance in sheep, cattle, and pigs, has a life cycle adapted to a sequence of hosts that frequent dry habitats. Adults are parasites of the bile ducts of their hosts and have life cycles utilizing snails and ants as their intermediate hosts. Although clinical illness is absent in young animals, these trematodes are long-lived and the pathological changes in the liver increase in severity and extent with the duration of the infection. Ergo, in older sheep, D. dendriticum infection causes progressive hepatic cirrhosis manifested clinically as cachexia, lowered wool production, decreased lactation, and premature aging. Essentially, it makes sheep husbandry unprofitable by curtailing the reproductive life of the ewe flock. Platynosomum fastosum, another member of this family, is a parasite of the bile and pancreatic ducts of cats, occurring in the southern U.S. and the Caribbean; infection is acquired by eating lizards containing metacercariae.
Control of fascioliasis may be approached in two ways: by reducing populations of the intermediate snail host or by using anthelmintics.
Theoretically, aquatic snails can be controlled by draining swamps or by broadcasting molluscicides on the snail-infested waters, but the continued existence of flukes where they have always been indicates that snail control measures are impracticable in many cases. Areas connected by streams with other snail-infested regions are generally not amenable to snail control measures.
Anthelmintic medication in the U.S. currently consists primarily of albendazole, which is available in only a few states where Fasciola hepatica poses a serious problem. Special dispensation is required to treat sheep with albendazole elsewhere in the U.S. Other effective flukicides--diamphenethide, nitroxynil, oxyclozanide, rafoxanide, and triclabendazole--are not available in the U.S.
Regular use of chemicals to control unwanted organisms can select for drug resistant strains. This has occurred in many species of economically important pests. The development of drug resistance necessitates a continuing search for new control agents having different modes of action.
The bacterium Bacillus thuringiensis (B.t.) produces δ-endotoxin polypeptides that have been shown to have activity against a number of insect species. These toxins are deposited as crystalline inclusions within the organism. Many strains of B.t. produce crystalline inclusions with no demonstrated toxicit to any insect tested.
A small number of research articles have been published about the effects of δ-endotoxins from B. thuringiensis species on the viability of nematode eggs. Bottjer, Bone, and Gill (Experimental Parasitology 60:239-244, 1985) have reported that B.t. kurstaki and B.t. israelensis were toxic in vitro to eggs of the nematode Trichostrongylus colubriformis. In addition, 28 other B.t. strains were tested with widely variable toxicities. Ignoffo and Dropkin (Ignoffo, C. M. and Dropkin, V. H.  J. Kans. Entomol. Soc. 50:394-398) have reported that the thermostable toxin from Bacillus thuringiensis (beta exotoxin) was active against certain nematodes. Beta exotoxin is a generalized cytotoxic agent with little or no specificity. Also, H. Ciordia and W. E. Bizzell (Jour. of Parasitology 47:41 [abstract]1961) gave a preliminary report on the effects of B. thuringiensis on some cattle nematodes.
At the present time there is a need to have more effective means to control liver flukes that cause considerable damage to susceptible hosts. Advantageously, such effective means would employ biological agents.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns isolates and genes of Bacillus thuringiensis that are active against flukes. Specifically, the compounds of the subject invention have been shown to be highly active against the liver fluke Fasciola hepatica.
the B.t. isolates used according to the subject invention can be grown and the δ-endotoxin that is produced recovered by standard procedures and as further described herein. The recovered toxin or the B.t. isolates themselves can be formulated using standard procedures associated with the use of flukicidal products.
Among the B.t. isolates which can be used according to the subject invention are B.t. PS17, B.t. PS33F2, B.t. PS63B, B.t. PS69D1, B.t. PS80JJ1, B.t. PS158D5, B.t. PS167P, B.t. PS169E, B.t. PS177F1, B.t. PS177G, B.t. PS204G4, and B.t. PS204G6.
The subject invention further concerns the use of genes cloned from Bacillus thuringiensis isolates. Specifically exemplified are four genes cloned from the B.t. isolate designated B.t. PS17. The genes designated PS17d, PS17b, PS17a and PS17e, encode Bacillus thuringiensis δ-endotoxins which are active against flukes. The genes can be transferred to suitable hosts via a recombinant DNA vector. The transformed hosts which express the flukicidal toxins can be used in methods to control flukes. Also, the toxins expressed by these microbes can be recovered and used in fluke control procedures.
BRIEF DESCRIPTION OF THE DRAWING
Seq. ID No. 1 discloses the DNA of PS17a.
Seq. ID No. 2 discloses the amino sequence of the toxin encoded by PS17a.
Seq. ID No. 3 discloses the DNA of PS17b.
Seq. ID No. 4 discloses the amino acid sequence of the toxin encoded by PS17b.
Seq. ID No. 5 is the aminoterminal nucleotide sequence of isolate PS33F2.
Seq. ID No. 6 is the internal nucleotide sequence of isolate PS33F2.
DETAILED DISCLOSURE OF THE INVENTION
Isolates which can be used according to the subject invention have been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1915 North University Street, Peoria, Ill. 61604, USA. The accession numbers are as follows:
The toxin genes used according to the subject invention can be obtained, for example, from the B. thuringiensis isolate designated PS17. As shown above, a subculture of B.t. PS17 and the E. coli host harboring the toxin genes of the invention have been deposited.
All of the above-listed cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
As described herein, B.t. isolates of the invention have activity against liver flukes. It is expected that these isolates would be active against other flukes as disclosed herein.
The B.t. toxins of the invention can be administered orally in a unit dosage from such as a capsule, bolus or tablet, or as a liquid drench when used against flukes in mammals. The drench is normally a solution, suspension or dispersion of the active ingredient, usually in water, together with a suspending agent such as bentonite and a wetting agent or like excipient. Generally, the drenches also contain an antifoaming agent. Drench formulations generally contain from about 0.001 to 0.5% by weight of the active compound. Preferred drench formulations may contain from 0.01 to 0.1% by weight, the capsules and boluses comprise the active ingredient admixed with a carrier vehicle such as starch, talc, magnesium stearate, or dicalcium phosphate.
Where it is desired to administer the toxin compounds in a dry, solid unit dosage form, capsules, boluses or tablets containing the desired amount of active compound usually are employed. These dosage forms are prepared by intimately and uniformly mixing the active ingredient with suitable finely divide diluents, fillers, disintegrating agents and/or binders such as starch, lactose, talc, magnesium stearate, vegetable gums and the like. Such unit dosage formulations may be varied widely with respect to their total weight and content of the antiparasitic agent, depending upon the factors such as the type of host animal to be treated, the severity and type of infection and the weight of the host.
When the active compound is to be administered via an animal feedstuff, it is intimately dispersed in the feed or used as a top dressing or in the form of pellets which may then be added to the finished feed or, optionally, fed separately. Alternatively, the antiparasitic compounds may be administered to animals parenterally, for example, by intraluminal, intramuscular, intratracheal, or subcutaneous injection, in which event the active ingredient is dissolved or dispersed in a liquid carrier vehicle. For parenteral administration, the active material is suitably admixed with an acceptable vehicle, preferably of the vegetable oil variety, such as peanut oil, cotton seed oil and the like. Other parenteral vehicles, such as organic preparations using solketal, glycerol, formal and aqueous parenteral formulations, are also used. The active compound or compounds are dissolved or suspended in the parenteral formulation for administration; such formulations generally contain from 0.005 to 5% by weight of the active compound.
When the toxins are administered as a component of the feed of the animals, or dissolved or suspended in the drinking water, compositions are provided in which the active compound or compounds are intimately dispersed in an inert carrier or diluent. By inert carrier is meant one that will not react with the antiparasitic agent and one that may be administered safely to animals. Preferably, a carrier for feed administration is one that is, or may be, an ingredient of the animal ration.
Suitable compositions include feed premixes or supplements in which the active ingredient is present in relatively large amounts and which are suitable for direct feeding to the animal or for addition to the feed either directly or after an intermediate dilution or blending step. Typical carriers or diluents suitable for such compositions include, for example, distiller's dried grains, corn meal, citrus meal, fermentation residues, ground oyster shells, wheat shorts, molasses solubles, corn cob meal, edible beam mill feed, soya grits, crushed limestone and the like.
In addition to having activity against flukes within the digestive tract of mammals, spores from B.t. isolates of the invention will pass through the animals' digestive tract, germinate and multiply in the feces, and thereby provide additional control of fluke larva which hatch and multiply therein.
The gene(s) from the B.t. isolates of the subject invention can be introduced into microbes capable of occupying, surviving in, and proliferating in the phytosphere of plants according to the procedure of European Patent Application 0 200 344. Upon ingestion of such a plant by an animal hosting a fluke, the fluke-active toxin becomes available in the animal host to control the fluke infestation.
The toxin genes from the isolates of the subject invention can be introduced into a wide variety of microbial hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the toxin. With suitable hosts, e.g., Pseudomonas, the microbes can be applied to the situs of flukes where they will proliferate and be ingested by the flukes. The result is a control of the flukes. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin produced in the cell. The treated cell then can be applied to the environment of target pest(s). The resulting product retains the toxicity of the B.t. toxin.
Where the B.t. toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used. Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the toxin from environmental degradation and inactivation.
A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae. Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.
A wide variety of ways are known and available for introducing the B.t. genes expressing the toxin into the microorganism host under conditions which allow for stable maintenance and expression of the gene. The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present. The transformants then can be tested for flukicidal activity.
Suitable host cells, where the toxin-containing cells will be treated to prolong the activity of the toxin in the cell when the treated cell is applied to the environment of target pest(s), may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxin is unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. Illustrative prokaryotes, both Gram-negative and -positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene into the host, availability of expression systems, efficiency of expression, stability of the flukicide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a fluke toxin microcapsule include protective qualities for the toxin, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
Host organisms of particular interest include yeast, such as Rhodotorula sp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.; phylloplane organisms such as Pseudomonas sp., Erwinia sp. and Flavobacterium sp.; or such other organisms as Escherichia, Lactobacillus sp., Bacillus sp., and the like. Specific organisms include Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.
The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.
Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability in protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Bouin's fixative and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like.
The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of inactivation should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of inactivation or killing retains at least a substantial portion of the bio-availability or bioactivity of the toxin.
The cellular host containing the B.t. fluke toxin gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
The B.t. cells may be formulated in a variety of ways. They may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.
The fluke toxin concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The toxin will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the flukicide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 102 to about 104 cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the flukes, e.g., plants, soil or water, by spraying, dusting, sprinkling, or the like.
It is well known in the art that the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid. Thus, the genetic code can be depicted as follows:
The above shows that the novel amino acid sequence of the B.t. toxins can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the protein. Accordingly, the subject invention includes such equivalent nucleotide sequences. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E. T. and Kezdy, F. J.  Science 223:249-255). Thus, the subject invention includes mutants of the amino acid sequence depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is substantially retained. Further, the invention also includes mutants of organisms hosting all or part of a toxin encoding a gene of the invention. Such microbial mutants can be made by techniques well known to persons skilled in the art. For example, UV irradiation can be used to prepare mutants of host organisms. Likewise, such mutants may include asporogenous host cells which also can be prepared by procedures well known in the art.
The methods and compositions of the subject invention can be used to control liver flukes, which can parasitize vertebrates. Specifically, the invention can be used to control flukes in humans, livestock, domestic pets, and other animals. As used herein, the term "livestock" can include, for example, sheep, cattle, pigs, and goats. The methods and compositions of the subject invention may be used to control immature and adult flukes. The methods of control include, but are not limited to, pasture treatment (for vertebrate and intermediate hosts); liposomes or other carriers for delivering the toxins to the liver or bile ducts; and treatment of free-living forms of the flukes in the gastrointestinal tracts of vertebrate hosts. The flukicidal B.t. toxins described herein may be used alone, or in rotation or combination with other flukicides such as, for example, albendazole.
Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Culturing B.t. Isolates
A subculture of B.t. isolate can be used to inoculate the following medium, a peptone, glucose, salts medium:
The salts solution and CaCl2 solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.
The above procedure can be readily scaled up to large fermentors by procedures well known in the art.
The B.t. spores and crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.
Purification of Protein and Amino Acid Sequencing
The B.t. isolates PS33F2, PS63B, PS52A1, and PS69D1 were cultured as described in Example 1. The parasporal inclusion bodies were partially purified by sodium bromide (28-38%) isopycnic gradient centrifugation (Pfannenstiel, M. A., E. J. Ross, V. C. Kramer, and K. W. Nickerson  FEMS Microbiol. Lett. 21:39). Toxic proteins were bound to PVDF membranes (Millipore, Bedford, Mass.) by western blotting techniques (Towbin, H., T. Staehlelin, and K. Gordon  Proc. Natl. Acad. Sci. USA 76:4350) and the N-terminal amino acid sequences were determined by the standard Edman reaction with an automated gas-phase sequenator (Hunkapiller, M. W., R. M. Hewick, W. L. Dreyer, and L. E. Hood  Meth. Enzymol. 91:399). The sequences obtained were:
In addition, internal amino acid sequence data were derived for PS63B. The toxin protein was partially digested with Staphylococcus aureus V8 protease (Sigma Chem. Co., St. Louis, Mo.) essentially as described (Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli  J. Biol. Chem. 252:1102). The digested material was blotted onto PVDF membrane and a ca. 28 kDa limit peptide was selected for N-terminal sequencing as described above. The sequence obtained was:
From these sequence data oligonucleotide probes were designed by utilizing a codon frequency table assembled from available sequence data of other B.t. toxin genes. The probes were synthesized on an Applied Biosystems, Inc. DNA synthesis machine.
Protein purification and subsequent amino acid analysis of the N-terminal peptides listed above has led to the deduction of several oligonucleotide probes for the isolation of toxin genes from B.t. isolates. RFLP analysis of restricted total cellular DNA using radiolabeled oligonucleotide probes has elucidated different genes or gene fragments. The following table reviews the data.
Polymerase Chain Reaction (PCR) and Partial Nucleotide Sequence of a δ-Endotoxin Gene from PS33F2
The δ-endotoxin gene identified in PS33F2 was further characterized by the polymerase chain reaction (PCR) using a DNA Thermal Cycles, a GeneAmp DNA amplification kit (Perkin-Elmer Corp., Norwalk, Conn.), and standard reactions as specified by the manufacturer. The forward primer was: The reverse primer was:
The aminoterminal and internal nucleotide sequences are shown in Seq. ID No. 5 and Seq. ID. No. 6.
The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. These procedures are all described in Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Thus, it is within the skill of those in the genetic engineering art to extract DNA from microbial cells, perform restriction enzyme digestions, electrophorese DNA fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells, prepare plasmid DNA, electrophorese proteins, and sequence DNA.
Purification and N-Terminal Sequencing of B.t. Isolate PS17
One Bacillus thuringiensis (B.t.) isolate which can be used as the source of flukicidal toxin protein according to the subject invention is identified as B.t. strain PS17. The culture can be grown using standard media and fermentation techniques well known in the art. The toxin protein inclusions were harvested by standard sedimentation centrifugation. The recovered protein inclusions were partially purified by sodium bromide (28-38%) isopycnic gradient centrifugation (Pfannenstiel, M. A., E. J. Ross, V. C. Kramer, and K. W. Nickerson  FEMS Microbiol. Lett. 21:39). Thereafter the individual toxin proteins were resolved by solubilizing the crystalline protein complex in an alkali buffer and fractionating the individual proteins by DEAE-sepharose CL-6B (Sigma Chem. Co., St. Louis, Mo.) chromatography by step-wise increments of increasing concentrations of an NaCl-containing buffer (Reichenberg, D., in Ion Exchangers in Organic and Biochemistry [C. Calmon and T. R. E. Kressman, eds.], Interscience, New York, 1957). Fractions containing the toxin protein were bound to PVDF membrane (Millipore, Bedford, Mass.) by western blotting techniques (Towbin, H., T. Staehelin, and K. Gordon  Proc. Natl. Acad. Sci. USA 76:4350) and the N-terminal amino acids were determined by the standard Edman reaction with an automated gas-phase sequenator (Hunkapiller, M. W., R. M. Hewick, W. L. Dreyer, and L. E. Hood  Meth. Enzymol. 91:399). From these sequence data an oligonucleotide probe can be designed by utilizing a codon frequency table assembled from available nucleotide sequence data of other B.t. toxin genes. A probe was synthesized on an Applied Biosystems, Inc. DNA synthesis machine.
The above procedure can be readily scaled up to large fermentors by procedures well known in the art.
The B.t. spores and/or crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.
Cloning of Four Toxin Genes from B.t. PS17 and Transformation into Escherichia coli
Total cellular DNA was prepared by growing the cells B.t. PS17 to a low optical density (OD600 =1.0) and recovering the cells by centrifugation. The cells were protoplasted in TES buffer (30 mM Tris-Cl, 10 mM EDTA, 50 mM NaCl, pH=8.0) containing 20% sucrose and 50 mg/ml lysozyme. The protoplasts were lysed by addition of SDS to a final concentration of 4%. The cellular material was precipitated overnight at 4° C. in 100 mM (final concentration) neutral potassium chloride. The supernate was extracted twice with phenol/chloroform (1:1). The DNA was precipitated with ethanol and purified by isopycnic banding on a cesium chloride-ethidium bromide gradient.
Total cellular DNA from PS17 was digested with EcoRI and separated by electrophoresis on a 0.8% (w/v) Agarose-TAE (50 mM Tris-HCl, 20 mM NaOAc, 2.5 mM EDTA, pH=8.0) buffered gel. A Southern blot of the gel was hybridized with a [32 P]-radiolabeled oligonucleotide probe derived from the N-Terminal amino acid sequence of purified 130 kDa protein from PS17. The sequence of the oligonucleotide synthesized is (GCAATTTTAAATGAATTATATCC). Results showed that the hybridizing EcoRI fragments of PS17 are 5.0 kb, 4.5 kb, 2.7 kb and 1.8 kb in size, presumptively identifying at least four toxin genes, PS17d, PS17b, PS17a and PS17e, respectively.
A library was constructed from PS17 total cellular DNA partially digested with Sau3A and size fractionated by electrophoresis. The 9 to 23 kb region of the gel was excised and the DNA was electroeluted and then concentrated using an Elutip.TM. ion exchange column (Schleicher and Schuel, Keene, N.H.). The isolated Sau3A fragments were ligated into LambdaGEM-11.TM.(PROMEGA). The packaged phage were plated on KW251 E. coli cells (PROMEGA) at a high titer and screened using the above radiolabeled synthetic oligonucleotide as a nucleic acid hybridization probe. Hybridizing plaques were purified and rescreened at a lower plaque density. Single isolated purified plaques that hybridized with the probe were used to infect KW251 E. coli cells in liquid culture for preparation of phage for DNA isolation. DNA was isolated by standard procedures.
Recovered recombinant phage DNA was digested with EcoRI and separated by electrophoresis on a 0.8% agarose-TAE gel. The gel was Southern blotted and hybridized with the oligonucleotide probe to characterize the toxin genes isolated from the lambda library. Two patterns were present, clones containing the 4.5 kb (PS17b) or the 2.7 kb (PS17a) EcoRI fragments. Preparative amounts of phage DNA were digested with SalI (to release the inserted DNA from lambda arms) and separated by electrophoresis on a 0.6% agarose-TAE gel. The large fragments, electroeluted and concentrated as described above, were ligated to SalI-digested and dephosphorylated pBClac. The ligation mix was introduced by transformation into NM522 competent E. coli cells and plated on LB agar containing ampicillin, isopropyl-(Beta)-D-thiogalactoside (IPTG) and 5-Bromo-4-Chloro-3-indolyl-(Beta)-D-galactoside (XGAL). White colonies, with putative insertions in the (Beta)-galactosidase gene of pBClac, were subjected to standard rapid plasmid purification procedures to isolate the desired plasmids. The selected plasmid containing the 2.7 kb EcoRI fragment was named pMYC1627 and the plasmid containing the 4.5 kb EcoRI fragment was called pMYC1628.
The toxin genes were sequenced by the standard Sanger dideoxy chain termination method using the synthetic oligonucleotide probe, disclosed above, and by "walking" with primers made to the sequence of the new toxin genes.
The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. These procedures are all described in Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
The restriction enzymes disclosed herein can be purchased from Bethesda Research Laboratories, Gaithersburg, Md., or New England Biolabs, Beverly, Mass. The enzymes are used according to the instructions provided by the supplier.
The PS17 toxin genes were subcloned into the shuttle vector pHT3101 (Lereclus, D. et al.  FEMS Microbiol. Lett. 60:211-218) using standard methods for expression in B.t. Briefly, SalI fragments containing the PS17b and a toxin genes were isolated from pMYC1628 and pMYC1627, respectively, by preparative agarose gel electrophoresis, electroelution, and concentrated, as described above. These concentrated fragments were ligated into SalI-cleaved and phosphatased pHT3101. The ligation mixtures were used separately to transform frozen, competent E. coli NM522. Plasmids from each respective recombinant E. coli strain were prepared by alkaline lysis and analyzed by agarose gel electrophoresis. The resulting subclones, pMYC2311 and pMYC2309, harbored the PS17a and b toxin genes, respectively. These plasmids were transformed into the acrystalliferous B.t. strain, HD-1 cryB (Purdue University, Lafayette, Ind.), by standard electroporation techniques (Instruction Manual, Biorad, Richmond, Calif.).
Recombinant B.t. strains HD-1 cryB [pMYC2311] and [pMYC2309] were grown to sporulation and the proteins purified by NaBr gradient centrifugation as described above for the wild-type B.t. proteins.
Activity Against Fasciola hepatica
Toxins produced by wild type B.t. isolates and recombinant microbes expressing B.t. proteins were tested for their flukicidal activity.
In vitro culture (37° C., 5% CO2) was according to modification of the method of Ibarra and Jenkins (Z. Parasitenkd. 70:655-661, 1984). Culture media consisted of RPMI (7.5 pH) with 50% v/v rabbit serum and 2% v/v rabbit RBC.
In the experiments described below, the effects of various substances on liver flukes were tested and compared. As used herein, Compound PS17 refers to toxin recovered and purified from cultures of B.t. PS17 as described above. Compound PS17a refers to toxin recovered and purified from cultures of the recombinant microbe which has been transformed with the B.t. gene designated B.t. PS17a. Compound C, is a formulation blank used as a negative control. ABZ refers to the drug Albendazole, which can be obtained from SmithKline Beecham, Lincoln, Nebr. Compounds PS17, PS17a, C, D, and E were added directly to media at 100 ppm; ABZ was dissolved in 100 μl absolute ethanol prior to addition to media.
Criteria for efficacy
Flukes were examined hourly for 3-8 hours and then once or twice daily for effects of drug treatment as evidenced by death, motility disturbances, or morphologic changes as compared to untreated control flukes, using an inverted microscope at 40×.
Three-week-old Fasciola hepatica removed from the livers of experimentally infected rabbits were cultured in vitro for 5 days in 1.6 cm Linbro culture plate wells (2 ml media; 4-6 flukes/well). After 5 days incubation, flukes were transferred to media containing Compound PS17a (100 ppm; 5 flukes), Compound PS17 (100 ppm; 6 flukes), or to untreated control media (4 flukes). All flukes were dead in Compound PS17a by 18 hours. In Compound PS17 only one fluke was alive (sluggish) at 24 hours compared to three live flukes in the media control (Table 1). All flukes were fixed and stained for morphologic study; no apparent microscopic changes were observed except those attributable to analysis of dead flukes. During the 5-day pre-culture period, flukes remained active and were observed ingesting RBC's from the media, with an apparently active digestive function.
Four mature flukes recovered from a naturally infected calf were separately cultured in vitro in 25 cm2 tissue culture flasks in 5 ml media for 24 hours and then transferred to similar flasks containing Compound PS17a (100 ppm), Compound PS17 (100 ppm), ABZ (10 ppm) or untreated media (Table 2). Flukes in Compound PS17a died between 10 and 18 hours, in Compound PS17 between 18 and 20 hours, and in ABZ by 64 hours. The media control fluke died in 72 hours in association with contamination of media as evidenced by turbidity and yellow media coloration.
These flukes were cultured in RPMI 49%+Rabbit serum 49%+Rabbit RBC 2% for 5 days prior to trial initiation. The experiment terminated after 24 hours, and all flukes were fixed for morphological study. Behavior of flukes prior to mortality included less activity and oral suckers directed to the surface of the media. At death, the body is contracted, then relaxed prior to decomposition.
An additional experiment was done to test the efficacy of Compounds PS17 and PS17a against F. hepatica as compared to albendazole (ABZ), a drug with known efficacy (positive control) and an untreated media control. Tests were done against 1) mature flukes recovered from naturally infected calves and 2) immature 5-week-old rabbit origin flukes.
Five-week-old immature F. hepatica were recovered from experimentally infected rabbit livers and placed in culture media overnight (1.6 cm Linbro culture plates, 2-3 flukes per well) in triplicate. Flukes were then transferred to similar Linbro plates containing 2 ml of Compound PS17a (100 ppm), Compound PS17 (100 ppm), albendazole (ABZ, 10 ppm), or untreated media (Table 3). In Compound PS17a, 7 of 7 flukes had died by 20 hours; sluggish movement was noted at 8-hours-post-exposure. In Compound PS17, mortality (2 of 7 flukes) and sluggish movement of remaining flukes was noted at 20 hours; at 31 hours 5 of 7 flukes were dead; at 48 hours all flukes were dead. In the ABZ positive control, 1 of 7 flukes was dead at 20 hours, 4 had died by 31 hours, and remaining flukes died by 60 hours. In untreated control media, 2 of 7 flukes had died at 31 hours, 3 at 48 hours, 4 at 55 hours, 5 at 60 hours, and 7 of 7 were dead by 72 hours. (NOTE: Superficial layers of some wells became turbid at 35 hours post-exposure; all flukes were washed in RPMI and transferred to wells containing fresh media at original drug concentrations).
Mature flukes recovered from 3 calf livers that had been condemned due to fascioliasis (Roucher's Meat Packing, Plaquemine, La.) were washed and held in sterile saline (for 2-3 hours) and four flukes were placed in each of two 25 ml tissue culture flasks containing Compound PS17a (100 ppm); Compound PS17 (100 ppm); ABZ (10 ppm) or untreated control media (Table 4). All flukes were dead in Compound PS17a by 10-11 hours and in Compound PS17 by 20 hours. In ABZ, flukes died by 48-54 hours. In control media, 2 flukes died by 48 hours; all flukes died by 66 hours. (NOTE: Contamination in flasks was noted at 31-48 hours).
Flukes were recovered from bile ducts of condemned calf livers and washed in sterile RPMI for 2-3 hours in groups of 10-20 flukes. Only one fluke was cultured in each well of 6-well Linbro tissue cultured plates (10 ml size), with each well containing 4 ml treated or untreated media. A formulation blank (Compound C) having no known flukicidal activity was also tested. Additional controls were added to identify sources of potential contamination, including a media control with 2% RBC and a media control without 2% RBC. The ABZ concentration was increased to 25 ppm. Control wells containing drug or media only were also included to control for contamination source.
This experiment included 12 replicates (Table 5); six of these replicates had corresponding wells with treated or untreated media only. For Compound PS17a, all 12 flukes died by 12 hours; weak movement was observed as early as 10 hours. All Compound PS17 flukes were dead by the 19 or 24 hour observation. Compound C fluke mortality was observed at 36-58 hours. For the media with 2% RBC and media without 2% RBC controls, one fluke was dead at 36 hours, two at 58 hours, and three at 72 hours; one fluke survived to 115 hours in media with 2% RBC.
For 8 of the 12 replicates and two of the 6 corresponding media controls, the media was electively changed in both fluke containing and non-fluke containing wells at 24 hours post-exposure. This extra handling procedure may have resulted in death of all flukes by 58 hours in 6 of the 8 replicates, apparently due to contamination as evidenced by turbidity and yellow color of media. Two fluke replicates in which media was changed in 24 hours did not become contaminated. None of the six media control (no fluke) replicates became contaminated during 115 hours of incubation.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
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