New Zealand Paient Spedficaiion for Paient Number 314486
New Zealand No, 314486 International No. PCT/
TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION
Priority dates 26 03 1997
Complete Specification Filed 26 03 1997
Classification (6) A01N63/02
Publication date 29 September 1999
Journal No 1444
NO DRAWINGS
NEW ZEALAND PATENTS ACT 1953
COMPLETE SPECIFICATION
Title of Invention
A biological rodenticide and method of production
Name, address and nationality of applicant(s) as in international application form.
LABORATORIOS BIOLOGICOS FARMACEUTICOS (LABIOFAM), a Cuban company of Ave Independencia, Km 16 y 1/2, Boyeros, Ciudad Habana 17200, Cuba
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] I *f V V ;Patents Form No 5 Our Ref JP208021 ;3 V nA ;PATENTS ACT 1953 Jz ^ ;NEW ZEALAND ;\ - r \c27 !l ;COMPLETE SPECIFICATION V ' / ;"•J1 ;A BIOLOGICAL RODENTICIDE AND METHOD OF PRODUCTION ;We, LABORATORIOS BIOLOGICOS FARMACEUTICOS (LABIOFAM), of Ave Independencia, Km 16 y 1/2, Boyeros, Ciudad Habana 17200, Cuba hereby declare the invention, for which We pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement ;PT05B33569 ;1 ;71 /. / 0 ft ;\j i i i w u ;5 The present invention is related to the field of ;Biocides and particularly with a process for the obtamment on an industrial scale, of a rodenticidal substance of microbiological origin, useful for harmful rodent cortrol, as well as with the substance obtained through this method ;10 ;Harmful rodents cause significant economic losses everywhere in the world They not only destroy crops, food and industrial reserves, but are also the carriers of inrectious diseases 15 Therefore, the fight against these animals constitutes a significant economic benefit and also plays an important role in the prophylaxis of the diseases that they transmit ;There are different methods for rodent control, but the most common ones are mechanical, chemical and biological ;20 methods ( microbiological methods are included within the "atter) ;Mechanical methods based ' on traps, baits, etc are successfully used in small areas However, they are less effective m larger areas 25 Chemical methods are based on the use of baits containing different types of poison Some of these proaucts have sharp action ( for example zinc phosphide, sodi 'm monofluoracetate 1080) and cause death immediately after ingestion However, they are very toxic for non-white 30 species, aue to the high concentrations of non-selective poisons that: most be used ;Anticoagulant substances are among the commonly used chemical methods The first generation of these substances appeared m the 50's and among the most used are 3-a- ;35 Benzylacetoml-4-hydroxycoumarin (warfarin) , coumatetraiyl, chlorophacetone and dephacmonone When using these compounds, death of the rodents occurs between 5 and 7 ;days after ingestion, so there is no rejection or reaction to the bait However, for a more effective control it is 40 necessary to keep large % Amounts of bait m the treatment ;5 ;10 ;15 ;20 ;25 ;30 ;35 ;repeated doses and the rodent must ingest the bait during several days to reach the lethal dose ;In the mid 60's the problem of resistance to traditional anticoagulants began to emerge This led to the development of a second generation of anticoagulants such as bromodialone and brodifacum (klerat) ;In general, the disadvantage of chemical methods is chat after their repeated use, rodents do not instinctively ingest the bait, which reduces the effectiveness of the product On the other hand, these products are hazardous for man, domestic animals and useful fauna ;Biological methods are based on the use of natural enemies of the rodents for controlling their population In general, they are based on the artificial contamination of rodents with microbes causing infectious diseases m them that lead to lethal epizootics In these methods a specific organism is used for each animal species ;For the obtamment of monopathogemc strains against murines(different culture media have been used, among which are meat peptone both, bread and brewer's yeast, mil, pea, blood, hydrolytic sera (neat flour hydrolysate) and yeast broth hydrolysate, which are used for growing the microbial inoculum that afterwards will be used m the production of the mam culture to obtain gram preparations ;The bacterial preparation obtained from cereal grains is a bacterial culture of strains pausing typhus m rodents, which are grown on different cereals, among which are whear, barley, rice, oats or rye This fermentation on a solia medium allows a larger yield m the growth of the microorganism than that which was attained by using liquid culture media ;The purpose of the present invention is to provide a method for manufacturing an effective microbiological rodenticide to be used in the control of harmful rodents or to at least provide the public with a useful choice By this method a high titer of the disease germ that is used can be obtained, which assures the high effectiveness of the finished product, as well as a reduction of the time necessary for its manufacture This application also provides a rodenticidal compound obtained using the ;3 ;T A / /OA ;0 1 H 4 0 o method, which has a high pathogenicity and virulence against murines, causing lethal epizooties ;The present invention comprises obtaining a preparation with biocidal activity, with three main components ;1 Strain of Salmonella enteritidis ;2 Potentiating solution ;3. Substratum for fermentation, composed of whole rice ;These three elements constituting the preparation are integrated by means of though two confluent operations ;(a) Preparing the sterile rice base substrate containing the potentiating substance and adjusted pH ;Obtaining the Salmonella enteritidis inoculum and inoculating it m (a) ;The invention provides a method for obtaining a microbiological rodenticidal compound which uses grains of cereals as bait and, as active principles, a monopathogenic microorganism and an anticoagulant comprising the steps of ;(a) obtaining a highly concentrated inoculum of the Salmonella enteritidis enteric subspecies subgroup 1 group D through fermentation in a liquid nutrient medium, ;i ;(b) mixing bait with a mixed solution comprising sodium hydroxide and warfarin sodic salt (3- -benzylacetonil-4-hydroxycoumarin) which acts as a potentiating substance, and ;(c) inoculating the bait with the inoculum from (a) ;The invention also provides a rodenticidal compound obtained using the method according to any one of claims 1-5 which includes about 62 5% of a cereal with a humidity content between 50 and 56% which is used as a bait, about 12 5% of an inoculum of Salmonella enteritidis enteric subspecies subgroup 1 group D diluted until about 109 CFU are obtained in the final compound, and about 25% of the mixed solution containing a warfarin sodic salt as potentiating agent ;In general, the process begins by selecting and washing the rice that is going to be used as substrate The first step is to separate the impurities contained m the selected rice Rice with a 13% humidity content is first washed with hot water at 80°C during a period of nearly 20 minutes, and the water used for the washing is drained Later on th-ist t o ;°r- anH N: ;/ ;substrate is subject to a sterilization process at 121°C',and a pressure of 1 2 bar for a period of time between 35 apx.\ 45 ° ;minutes Immediately afterwards, the substrate is coo lad. and dried until it reaches a humidity content between 2 0 ana 36%' ;and a temperature of 37°C, and then is blendea with a rni-^ed^^ ;solution which contains 0 9$ soaium hydroxiae and 0 ^ ;4 ;(followed by 4a) ;34 /. r o e> I If O0 ;warfarin sodic salt (3-a~Benzyl-acetonil-4-hydroxycoumarin) which is used as a potentiator of the lethal action of tne preparation and added for 15 minutes while shaking ;At this time an adjustment of the pH of the mixture is carried out, it should be between 7,4 and 8 ;Immediately afterwards, the substrate thus obtained is inoculated with a strain of Salmonella enteritidis enteric subspecies subgroup 1, group D, which is deposited m available collections of these microorganisms This inoculum is carried out m such a way that the final concentration of the microorganism in the mixture, which originally is 10B Colony Forming Units (CFU) per mL, after a brief 4-hour r \ ;31 4486 ;fermentation period in solid state, becomes 109 CFU per mL ;For obtaining the inoculum of the previous step we start with a preculture of the microorqanism, which is transferred to a 100 L fermentator containing nutrient broth in a ratio 5 from 1 to 1 5% of the volume to be fermented, adjusting the fermentation conditions at a temperature of 37°C, a pH between 7 4 and 7 6, shaking between 400 and 600 r p m and aeration between 0 1 and 1 0 v v m The fermentation time ranges from 3 to 5 hours and an inoculum with titer between 10 1 and 2 x 1010 colony forming units (CFU) is finally obtained ;We finally obtain a batch of a rodenticidal product, ready to be packed and stored, a temperature between 5 and 9°C, m these conditions the stability of the product is 15 preserved for a long period of time ;The above-mentioned product is a rodenticidal compound containing grains of cereal that are used as a bait (such as wheat, rice, rye, oats, millet, etc ) it also contains a substance used as potentiator, composed of a mixed solution ;20 of sodium hydroxide wxth 3-a-benzylacetonil-4-hydroxycoumarm and an inoculum of a strain of Salmonella enteritidis enteric subspecies subgroup 1 group D with a final titre between 10s and 109 CFU ;The material thus obtained is later packed and stored 25 The biological tests of the finished product showed 100% ;mortality rate, with death taking place from 4 to 12 days after ingestion by the murine family, therefore showing a significant increase of its biological properties with regard to effp iveness, which is due mainly to the virulence and 30 pathoc .mcity of the resulting product. ;Said product has, as its mam features, its high ef fectiveness, as well as its stability for one year when frozen, 6 months if stored between 5 and 15°C, and 30 days if stored at room temperature 35 Since the invention has to do Vvith a process that can be scaled-up to industrial level for the of a rodenticidal compound, where the adequate operations and paraiaeters have been defined to obtain batches of the product , ;with a proper 'quality and stability, it can be mechanized'^ ;/ ;I! *
using well-known facilities with state-of-the-art technology which allows the optimization of said scale-up at industrial level EXAMPLES
EXAMPLE 1 OBTEINMENT OF THE INOCULUM
A microdrop of a deep-frozen culture of a strain of Salmonella enteritidis enteric subspecies subgroup 1 group D is added to an enriched liquid medium, which is incubated for 4 hours at 37°C m a sifter at 180 r p m From this incubation a preculture with a titre of 1010 CFU is obtained, which is afterwards transferred to a fermentator containing the liquid medium of nutrient broth, whose composition is
Bacteriological peptone - 5 0 g/L
Nutrient extract - 10 g/L
Yeast extracr - 2 0 g/L
Elna - 5 0 g/L
EXAMPLE 2 MECHANIZATION OF THE PREPARATION OF THE RODENTICIDAL COMPOUND
The rice used as bait and substrate for the growth of the pathogenic microorganism can be supplied m bulk or clean m bags
After the impurities are sepaiated from the rice, it is carried to the tanks for storing clean rice, cach with a capacity of 17 M T
If rice is supplied clean m bags, these are carried on wheelbarrows to the freight elevator, which takes them straight to the receiving mill-hoppers where the rice m bulk from the silos will also get there, using elevators and conveyer belts
The dosifying mill-hoppers with a capacity of 650 kg of rice, control and guarantee the weight of the rice mass necessary for the process, every 1 and 5 hours alternatively per batch
Rice with a 13% humidity content is unloaded from the dosifying mill-hoppers into a container where the whole processing of the different components that constitute the rodenticidal'compound will take place
314486
Immediately afterwards deirineralized water is injected and heated at 80°C until covering the entire volume of the rice for 35 minutes Simultaneously with injection of hot water a mixing mechanism is activated
The action of this mechanism makes of possible to wash the rice for 20 minutes Later on the water that was used for the washing is drained
A vacuum is applied again for 5 minutes and 225 L of hot water at 80°C are injected During this time the mechanism of the mixer is kept working At the same time, vapor is injected with pressure regulated to 1 2 bar and temperature of 121°C, directly to the chamber of said container When these parameters are reached inside the container, a sterilization process takes place for about 45 minutes
Later on the rice is cooied and dried until reaching a humidity between 27 and 28% and a temperature of 37°C
Immediately afterwards, a mixed solution containing 0 S>% sodium hydroxide and 0 1% Warfarin Sodic Salt is injected into the container Once the 282 L of said mixed solution have been injected, this operation continues for 60 minutes
The cooling process of the rice mass using mixed solution continues until a temperature of 37°C is reached Later on, an adjustment of the pH of the mixture is carried out until a value between 7 4 and 8 is reached
Immediately afterwards, 141 L of the diluted inoculum are injected and mixed with the rice mass previously treated with the mixed solution, until the homogenization of the final mixture At the end of this step, samples of the product are taken for quality analysis
Later on, the product contained m the container is carried to the mill-hopper of the filling machine The entire above-mentioned cycle must be fulfilled in a 6-hour period, making it possible to obtain 1125 kg of the rodenticidal compound
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