Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
1999-06-28
2001-10-16
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S093460, C424S424000, C424S489000, C514S002600, C530S350000
Reexamination Certificate
active
06303117
ABSTRACT:
The subject of the present invention are new ice granules produced from aqueous suspensions of
Bacillus thuringiensis israelensis
(BTI) or
Bacillus sphaericus
(BS) endotoxins which are used in the combatting of gnat larvae.
Gnats are dangerous transmitters of diseases, e.g. malaria, bleeding fever (dengue, DHF), encephalitis or lymphatic filiaroses. However, in the European region, above all things in the case of massive occurrence, they are a considerable plague which significantly diminishes the quality of life. Worldwide, extensive measures are made for the combatting of gnats and more than 50,000 tonnes of chemical insecticides are thereby used anually. However, besides the objective use in the reduction of gnat frequency, these chemicals cause considerable toxicological risks since they not only damage the gnats or their larvae but are also active against other organisms. Ecological risks are thereby also given since the simultaneous damaging of other insects, for example of the non-biting midges, disturbs the food chain for fish and birds. Therefore, one has sought alternatives which act more selectively against gnats or their larvae.
Besides the chemical insecticides, for many years bacterial insecticides have been the subject of intensive research work.
In 1977, Goldberg and Margalit isolated from a sample originating from gnat breeding places in the Negev desert a
Bacillus thuringiensis
strain which proved to be pathogenic towards larvae of various midges, especially mosquitoes and gnats. Today, this is classified as
Bacillus thuringiensis
var.
israelensis
and also designated as pathotype B for the differentiation from a
Bacillus thuringiensis
pathotype A which is active against
lepidoptera
. In U.S. Pat. No. 4,166,112, the use of BTI as insecticide is claimed.
Microbiological preparations based on
Bacillus thuringiensis
H-14 and Bacillus sphaericus have, in preceding years, proved to be effective in the battle against gnats. They act very selectively and kill off only the larvae of few kinds of midges, especially gnats. For other animals and humans, even close relatives of gnats, these agents are, according to present day knowledge, not dangerous. The effectiveness thereby depends upon protein crystals (endotoxins) which the bacillae produce at the end of their growth period simultaneously with the spore formation. When these endotoxins are taken up by the gnat larvae together with the feed, they are activated in the gut of the midge to give the toxin, deposit on the middle gut cells and cause these to burst by osmo-regulatory effects. Thus, the midge larvae die within a few minutes to hours.
Necessary prerequisites of the effectiveness of endotoxins are
a) a sufficiently high concentration in which they are taken up by the gnat larvae,
b) the activation to the toxin in the alkaline gut medium of the midge larvae by corresponding proteases, and
c) the presence of corresponding receptor sites on the gut cells of the midges to which these toxins bind.
That these endotoxins act so specifically appears to be due to the fact that, above all, the receptor sites are not present in the case of other animals or the proteolytic breakdown takes place in another way.
Since these microbiological feed poisons can only be used against the mosquito, gnat and midge larvae and, on the other hand, as proteins, are broken down relatively quickly by the most different micro-organisms occurring in nature, it is necessary to introduce the preparations in suitable concentration into the breeding waters after the larvae have hatched.
Hitherto, the product was mixed as powder formulation or liquid concentrate with appropriate amounts of water and sprayed as suspension over the infested waters. These forms of preparation have the great disadvantage that, in the case of distribution from the air, they deposit on branches or leaves projecting over the waters and thus do not get to the effective place. In unfavourable areas, for example primaeval forests, a loss of up to 80% can result.
Furthermore, it is known to work up the endotoxins, together with sand or maize bran, to give granulates which can then be sprinkled in solid form. Furthermore, from DE-41 33 889-C2 are known forms of composition, i.e. tablets, which, in the presence of water, evolve gas which takes care of the spreading out of the active material. In the case of application from the air, these solid compositions admittedly trickle through the branches and thus get to the effective place but are comparatively expensive and, due to the adjuvants contained in large amount, contaminate the waters.
Therefore, the task arises to find a form of composition which permits the preparations to be used in solid form without undesired adjuvants being used.
Surprisingly, this task is solved by the features characterised in the main claim and promoted by the features according to the subsidiary claims.
It is surprising that ice granules which are produced by freezing of appropriate aqueous endotoxin suspensions, which possibly also contain adjuvants in small amount, cause no damage to the endotoxins contained but rather a microbial breakdown is slowed down in the case of normal storage at −10 to −20° C.
The ice granules according to the invention are so adjusted in their composition that they are somewhat lighter than water (D=0.95 to 0.98) whereby, in the case of introduction into the water, they float on the surface. Due to slow thawing, the endotoxin is then liberated on the surface of the granulate which slowly sinks whereby the BTI crystals distribute uniformly in the whole of the body of water.
For the composition of the ice granules, a high portion of BTI is desired in order to transport as small a weight as possible in the supplying aircraft. On the other hand, the content is to be so small that a uniform distribution over the surface to be impinged is ensured.
It has proved to be worthwhile to apply about 250 g (2.5·10
9
ITU=international toxic units) of endotoxin to 1 ha water and to use therewith 5-50 l of water as dilution agent which leads to a 100% mortality in the case of mosquitoes of the genus Aedes and Culex.
For the production of the ice granules, the commercially available BTI and
Bacillus sphaericus
liquid concentrates are mixed homogeneously with water so that an end concentration of 1-200 g, preferably 5-100 g of endotoxin per l of water results. In the case of use of solid endotoxin powders, an addition of wetting agents, suspension adjuvants and/or thickeners is useful in order to ensure a uniform mixing. The granulate with a grain diameter of 2-20 mm, preferably of 3-5 mm, is produced either directly from the mixture with an ice machine or first a fluffy ice is produced with a “snow cannon” and further pressed in a pelleting machine to give granules. Grinding of comparatively large ice blocks and sieving out of a fine granulate is also possible but not preferred since the bodies thereby resulting have an irregular shape and thus, under pressure, easily grow together again. Commercially available machines with appropriate cooling devices can be used. The ice granulate is packed into thermo-sacks and then stored at −20° C. in a cold-storage depot. Appropriately cooled containers in the transport vehicles and in the supplying aircraft here also permit comparatively long transport times. The application itself takes place via conventional gyro- or simplex sprinklers. Sprinkling breadths of about 20 meters could be achieved.
A great advantage of the ice granulate lies, above all, therein that no losses of active material (BTI and
Bacillus sphaericus
) are to be observed. In the case of the application of BTI-sand granulate, about 20-30% of the active material are lost by the rubbing off in the case of application. The BTI/
Bacillus sphaericus
powder-oil mixture is removed from the sand grains as carrier materials when it is catapulted with pressure from the scattering device. One does not have this loss in the case of the application of the ice granulate since the active
Becker Norbert
Mercatoris Peter
Foley & Lardner
Lankford , Jr. Leon B.
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