AU682354B2 - Compositions for administration to animals with coccidiosis - Google Patents

Description

WO 94/24886 PCT/FI94/00166 -1- Compositions for Administration to Animals With Coccidiosis Background of the Invention Cross Reference to Related Applications This application is a continuation-in-part of U.S. Application No.

08/053,138, filed April 29, 1993, the contents of which are hereby incorporated by reference.

Field of the Invention This invention relates to industrial diets for domestic food animals, feed additives and methods for treating animals infected with coccidia.

Related Art Coccidiosis is a common disease in domestic food animals, caused by protozoa belonging to the Eimeria family. Coccidiosis is found worldwide, and its economical impact, particularly on poultry farming, is huge. In the U.S. poultry industry alone, coccidiosis causes losses of 200-250 million dollars yearly. World-wide, coccidiosis is estimated to cause one third of all disease and mortality losses in the poultry industry (Trends in Veterinary Research and Development, part 6, Anti-coccidials, Lloyd-Evans, L.P.M.

PJB Publications Ltd., 1991).

Since the 1950s, several dietary drugs (coccidiostats) have been developed to treat coccidiosis, but with only moderate success. The most serious diawbacks associated with the use of coccidiostats have been: 1) rapidly developing resistance of the parasites; 2) adverse effects on host animals; and 3) risk for residuals or quality defects in the consumer products.

While the universal use of coccidiostats has decreased serious mortality outbreaks, subclinical effects of coccidiosis on animals markedly decrease productivity (Jeng Edgar, Highlights of Agricultural Research Alabama, Agricultural Experiment Station, v.28, p.

6 (1981)).

WO 94/24886 PCTIFI94/00166 -2- In chickens, typical clinical signs of coccidiosis include ill-thrift, rapid loss of weight, diarrhea, and dysentery. The most serious effects take place in the intestine, where coccidia invade the mucosae and cause epithelial damage, lesions and hemorrhage. Physiologically, coccidiosis causes severe disturbances in the acid-base, ionic and osmotic balance of the gut, and decreases nutrient absorption (Ruff, Georgia Coccidiosis Conference, Nov. 19- 21 (1986) pp. 169-183; Gwyther et al., Coccidia and Intestinal Coccidiomorphs, Vth International Conf., October 17-20 (1989) pp. 279-284).

Many coccidiostats in common use kill parasites by breaking down their ionic and osmotic regulation. However, these drugs are not coccidia specific; they also alter the ionic and osmotic balance of the host and, consequently, may decrease nutrient absorption in the gut (Speight, 6th European Symposium on Poultry Nutrition, World's Poultry Science Assoc., Oct. 11-15 (1987), abstract 7A). As a result, the positive effect of coccidiostats on mortality is offset by their detrimental side effects with regard to nutrient availability, growth and feed efficiency.

Betaine is an osmoprotectant. It increases the osmotic strength of cells without adversely affecting enzyme activity, and it protects enzymes from ionic or temperature inactivation (Nash et al., Aust. J. Plant Physiol. 9:47-57 (1982); Yancey et al., Science 224:1064-1069 (1982); Rudolph etal., Archives Biochem. Biophys. 245:134-143 (1986); McCue Hanson, Trends in Biotechnology 8:358-362 (1990); Papageorgiou et al., Curr. Res. In Photosynthesis 1:957-960 (1990)). While some organisms (and tissues) can accumulate betaine in high quantities under osmotic stress through osmotically induced betaine synthesis, most animals lack this capability, and are dependent upon the intake of exogenous betaine. For example, isolated salmon liver mitochondria, when exposed to osmotic stress, show increased betaine intake, but not synthesis (Bj6rk6y, Synthesis and Accumulation of glvcine betaine in Salmon (Salmo salar) and a Mussel, MSc thesis, Norwegian College of Fisheries, University of Troms6, pp. 94).

WO 94/24886 PCT/FI94/00166 -3- Although its osmoprotective properties were known prior to the present invention, betaine has mainly been studied for its ability to act as a methyl donor in transmethylation reactions (Stekol et al., J. Biol. Chem. 203:763-773 (1953)) ana for its ability to transfer methyl groups to homocysteine to produce methionine (Harper, in Review of Physiol. Chem., 120 and 351 (1973)). It was not appreciated that betaine might be useful in alleviating the undesirable side effects associated with the use of coccidiostats or that, betaine and coccidiostats might act synergistically to improve the commercial performance of domestic food animals suffering from coccidiosis.

Summary of the Invention The present invention is directed to feeds for domestic food animals which contain a combination of betaine and coccidiostat. This combination has been found to be especially effective at reducing the mortality and improving the overall commercial performance of chicks infected with coccidia. Coccidiostats used may be of either the chemical or ionophore type.

Preferred ionophore coccidiostats are salinomycin (tradename "Bio-Cox") and lasalocid (tradename Avatec). The preferred chemical coccidiostat is halofuginone hydrobromide (tradename "Stenerol"). In all cases, betaine is present in feed at a concentration of between 0.5 and 2.0 kg/ton of dry feed.

The most preferred feeds are those used for chickens. The invention is also directed to dietary additives which contain a premixed combination of betaine and coccidiostat. Such additives can either be included in the feed given to animals or administered separately from such feed.

In addition, the invention is directed to a method for reducing the mortality of animals, especially chickens, infected with coccidia by administering betaine to such animals. The betaine may either be administered alone or, more preferably, in combination with coccidiostat. The preferred ionophore coccidiostats for use in the method are salinomycin and lasalocid.

The preferred chemical coccidiostat is halofuginone hydrobromide.

I

WO 94/24886 PCT/FI94/00166 -4- Brief Description of the Figures FIGURE 1: Figure 1 shows the percent mortality for chicks grown for days on unsupplemented diets, diets supplemented with 44 ppm of Bio- CoxTM (salinomycin), and or on diets with 66 ppm Bio-Cox. The effect of each diet was examined both with and without added betaine. It can be seen that betaine addition reduced chick mortality for each of the diets studied.

FIGURE 2: Chicks grown for 45 days on unsupplemented feeds or on feeds supplemented with 44 ppm coccidiostat and 66 ppm coccidiostat were necropsied and the severity of gut lesions ranked on a scale of 0 to 4. It can be seen that, in all cases, supplementation of diets with betaine led to a reduced severity of gut lesions.

FIGURE 3: Figure 3 shows the end weights of chicks grown for days on each of the three different types of diets described above. In each case, supplementation with betaine resulted in chicks with an increased end weight.

FIGURE 4: The feed conversion efficiency of chickens grown on each of the three different types of diets was determined and the results are shown in Figure 4. In each instance, betaine resulted in chicks with improved efficiencies.

FIGURE 5: Coccidia infected chicks were gown on diets containing either adequate methionine or on diets low in methionine was examined. All chicks received 66 ppm of salinomycin. The figure shows that the addition of betaine reduced the severity of gut lesions regardless of the level of methionine.

FIGURE 6: Figure 6 shows the effect of betaine addition on the mortality of coccidia-infected chicks. At a dose of 0.75 kg/ton, betaine reduced the mortality of chicks fed diets containing either a low or an adequate amount of methionine.

FIGURE 7: Figure 7 shows that betaine increases the end weight of coccidia-infected chicks in diets which are both low in methionine or which contain adequate methionine L s P- C I WO 94/24886 PCT/FI94/00166 FIGURE 8: Figure 8 shows that betaine improves feed conversion efficiency in chicks infected with coccidia regardless of whether the chicks are grown on feed low in methionine or with adequate methionine.

Detailed Description of the Preferred Embodiments A. Definitions 1. Starter diet: A "starter diet" is the diet fed to chickens during the first 21 days of life.

2. Grower diet: The term "grower diet" refers to the diet fed to chickens 21 to 40 days of age.

3. Finisher diet: The term "finisher diet" refers to the diet fed to chickens 40 to 49 days of age.

4. Betaine: "Betaine", also called "glycine betaine", is defined chemically as 1-Carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt.

Betaine is sold by Finnsugar Bioproducts under the tradename of "Betafin." 5. Feed conversion efficiency: "Feed conversion efficiency" is the ratio of the amount of weight gained by an animal divided by the amount of feed consumed by the animal. For example, a feed with an efficiency of would mean that for every kilogram of feed consumed, the animal gained kilogram.

6. Feed conversion ratio: "Feed conversion ratio" is the ratio of the amount of feed consumed by an animal divided by the amount of weight gained by the animal.

7. Corn-soy feed: "Corn-soy feed" is feed mainly comprised of yellow corn, soybean meal and soy oil.

8. Significant: As used herein the term "significant" means statistically significant. Thus, a statement that "treated chicks had significantly reduced mortality relative to untreated chicks" means that P<0.05 using standard statistical analyses.

-r I -a rl Qs~ L _e~l~ WO 94/24886 PCT/FI94/00166 -6- 9. Mortality: Mortality is defined as the number of chicks within a treatment group that die during the course of an experiment. Typically mortality is expressed as a percentage and determined by dividing the number of chicks that die by the total number of chicks at the start of the experiment and then multiplying by 100.

Feedstuffs: "Feedstuffs" are defined as those commonly used ingredients such as yellow corn or soybean meal which are combined to formulate the diet of chicks. 11. Basal diet: "Basal diet" is defined as the diet to be fed to chicks prior to supplementation with either methionine or betaine.

12. Domestic Food Animal: For the purposes of the present invention, the term "domestic food animal" is defined as any domestic animal that is consumed as a source of protein in the diet of humans or other animals.

Typical domestic animals include: bovine animals(e.g. cattle); ovine animals sheep); swine pigs); fowl chickens and turkeys); rabbit and the like.

13. Commercial Performance: As used herein, the term "commercial performance" refers to the extent to which animals raised under a specific set of conditions are commercially desirable as food animals. For the purposes of this invention, there are 4 parameters which determine commercial performance: mortality; end weight; feed conversion efficiency; and severity of gut lesions. An improvement in any one of these parameters results in an improved commercial performance provided that the remaining parameters either remain unchanged or also improve.

14. Premix: When used as a noun, "premix" refers to a composition of two or more components which have been combined for a particular use. For example, a premix of the components betaine and coccidiostat could be added to chicken feed to produce a mixture with a desired final concentration without the necessity of making further adjustments in the concentration of either betaine or coccidiostat.

a -er i rpa WO 94/24886 PCT/FI94/00166 -7lonophore coccidiostat: lonophore coccidiostats are anticoccidial agents which, by virtue of their molecular shape and structure, can act as ion transporters across biological membranes. All other anti-coccidial agents are referred to herein as "chemical coccidiostats." B. The Synergistic Action of Betaine and Coccidiostat The data presented herein establish, for the first time, that coccidiostat and betaine act together to offset the commercially detrimental effects caused by coccidia parasites. Synergism occurs whether the coccidiostat is of either the chemical or ionophore type. Example 1 describes experiments in which chicks were inoculated with coccidia and then fed diets supplemented with betaine, coccidiostat (salinomycin) or a combination of betaine and coccidiostat. Chicks raised on the supplemented diets were compared with inoculated chicks fed unsupplemented diets and with chicks not inoculated with coccidia at all. As can be seen from Table 5, chicks fed a diet containing a combination of betaine and salinomycin for 21 days had significantly higher body weights and significantly fewer gut lesions than chicks fed a diet containing an equivalent concentration of either betaine or salinomycin alone.

Although neither betaine alone nor coccidiostat alone were able to completely offset the effects of coccidia infection on feed conversion efficiency, when administered together, they produced an efficiency which was comparable to that of the noninoculated controls. Mortality in 21 day old chicks fed diets containing 44 ppm of salinomycin was significantly reduced relative to the mortality of infected chicks fed either an unsupplemented diet or a diet supplemented with betaine alone. When betaine and 44 ppm coccidiostat were used together, mortality was reduced to a level which was not significantly higher than the mortality of the noninoculated chicks.

It may also be seen from Table 5 that, at 45 days, chicks receiving feed containing both betaine and coccidiostat had a mortality significantly lower than chicks receiving feed with only one of the agents and that the mortality of the chicks administered both agents was reduced to the point where it was -l-~,~ICT-ar~eP P--l WO 94/24886 PCT/FI94/00166 -8no longer significantly higher than that of the noninoculated controls.

Similarly, the body weight and feed conversion efficiency of chicks on diets with betaine and 66 pm of coccidiostat were not significantly different from the control group, whereas chicks receiving only one of the agents had significantly lower body weights and a significantly higher feed conversion efficiency.

The results shown in Example 2 indicate that the positive effects of betaine and coccidiostat on the commercial performance of chicks infected with coccidia do not depend upon the methionine content of the diet. Thus, the anti-coccidial effect of betaine is independent of its ability to substitute for methionine. The results suggest that the protective effect of betaine is probably related to its ability to offset the detrimental effects of coccidiosis on nutrient absorption. Supporting this hypothesis is the finding that infected chicks fed diets with betaine have less severe gut lesions than chickens fed either unsupplemented diets or diets supplemented with coccidiostat alone (Table Example 3 demonstrates that the synergistic improvement in commercial performance evidenced by the combination of betaine and salinomycin is maintained both with other types of ionophore coccidiostats (lasalocid) as well as with chemical coccidiostats (halofuginone hydrobromide).

As shown in Table 13, the combination of lasalocid and betaine produced chicks with significantly higher body weights and significantly lower feed conversion efficiencies compared to chicks receiving diets supplemented with either betaine or lasalocid alone. Chicks receiving the combination of halofuginone hydrobromide and betaine evidenced a significantly reduced feed conversion efficiency. In addition, the results shown in Table 13 confirm the conclusion that the combination of salinomycin and betaine improves commercial performance. Chicks receiving salinomycin together with betaine exhibited significantly increased body weights and improved feed conversion efficiencies relative to chicks receiving either salinomycin or betaine alone.

Other coccidiostats suitable for use in combination with betaine are shown in LI ~CI _L II L WO 94/24886 PCT/FI94/00166 -9- Table 1. In all cases, the appropriate dosage of coccidiostat for administration to animals is the dosage recommended by the U.S. Food and Drug Administration (see FDA 1994 Feed Additive Compendium, U.S. Food and Drug Administration, 1994) and betaine is present at a concentration of between 0.5 and 2.0 kg/ton dry feed.

Coccidiostats that have been approved in Europe include the following (product license -numbers are shown in parentheses): amprolmix (PL 0025/4008); avatecpremix (PL0031/4011); carbigran premix (PL0006/4075); clopidol (PL 3405/4017); clopidol 250 (PL 5405/4025); coyden 25 (PL 0621/4001); cycostat 66 (PL 0095/4000); cygro premix (PL 0095/4042); deccox pure (PL 8327/4038); deccox poultry premix (PL 0012/4052); deccox sheep premix (PL 8327/4066); dinitolmide (PL 10101/4000); dinormix SR (PL 0109/4000); DOT (dinitolmide (PL 0109/4002); elancoban G200 (PL 0006/4047); monensin 200 (PL 3405/4006); lerbek (PL 327/4049); maxiban G160 (PL 0006/4078); monensin-100 poultry (PL 3405/4021); monensin-100 ruminant (PL 3405/4022); monensin-200 (PL 3405/4006); monensin 100 "ABCHEM" premix (PL 10104/4004); monteban G100 (PL 0006/4061); (PL 3405/4050); nicarbazin-250 (PL 3405/4044); nicrazin premix (PL 0025/4019); sacox 120 (PL 0086/4135); salcostat (PL 1598/4036); salcostat (DOT) premix 25% (PL 1598/4033); salgain-60 (PL 3405/4053); stenerol (PL 0086/4117) stenerol for pheasants (PL 0086/4153); and ubicox pure (PL 4188/4004). The European-approved coccidiostats may also be used to practice the claimed invention. In each case, the concentration of coccidiostat used should be equivalent to that recommended by the manufacturer and betaine should be present at a concentration of between and 2.0 kg/ton dry feed.

C. Animal Feeds Formulated to Contain Coccidiostat and Betaine 1. Feeds for Chickens The present invention is directed to feeds for chickens formulated to contain both coccidiostat and betaine. In order to prepare such feeds, a basal x I I rls r~ C~ ~'qpWr"l~l WO 94/24886 PCT/I94/00166 diet for chicks is first formulated using any of a variety of routine feedstuffs such as corn, soy, wheat and barley (see AFMA Feed lngredient Guide, published by the American Feed Manufacturer's Association, Arlington, VA., H. Patrick et al., Poultry: Feeds Nutrition, Second Edition, AVI Publishing Co. Inc., Westport, Conn., chapter 37, (1980)). All mixing and other preparation of feeds takes place using routine procedures well-known in the art (see H. Patrick et al., Poultry: Feeds Nutrition, Second Edition, AVI Publishing Co. Inc., Westport, Conn., chapters 36-38 (1980); Feed Manufacturing Technology, H. Pfost and C. Swinehart eds., American Feed Manufacturer's Association Inc., Chicago, Ill., (1970)).

The nutrient content of the basal diet may be determined using standard feedstuff analysis tables (see H. Patrick et al., Poultry: Feeds Nutrition, Second Edition: AVI Publishing Co. Inc., Westport, Conn., pp.

438-449 (1980); H. Titus et al., The Scientific Feeding of Chickens, Fifth Edition, The Interstate Publishers, Danvilie, Ill., chapter 13 (1971)).

Vitamins, minerals and other nutrients may be added to concentrations determined by turning to various available references (see Nutrient Requirements of Poultry, National Research Council, National Academy of Sciences, Washington, D.C. (1984)). These references are well-known, and the data provided is generally accepted by those skilled in the art.

Once the basal diet has been formulated, betaine and coccidiostat are added. These agents may either be added individually, or they may be added together as a premix. The final concentration of betaine in the feed should be between 0.5 and 2.0 kg/ton dry feed. The final concentration of coccidiostat will vary depending upon the particular type of coccidiostat used. The concentration recommended by the U.S. Food and Drug Administration should be suitable in all instances. Typical recommended inclusion rates for chick diets are: monensin: 100-120 ppm; salinomycin: 60 ppm; naracin: 70 ppm; and lasalocid: 90 ppm. In the case of salinomycin a final concentration of between 44 and 66 ppm has been found to be sufficient (see Example 1).

1 M I -1 WO 94/24886 PCT/FI94/00166 -11- The feed may be administered to chickens in any convenient manner.

For example, it may be formed into pellets or admir.stered as a powder. It may be given to all chicks as a preventative or it may only be given to chicks after they have been identified as being infected with coccidia.

2. Feeds for Other Domestic Food Animals Coccidiosis occurs in a number of species of domestic food animals other than the chicken. Table 1 contains coccidiostats that have been approved by the Food and Drug Administration (FDA) for the treatment of various target animals (FDA 1994 Feed Additive Compendium, U.S. Food and Drug Administration, 1994). Cattle, sheep, swine and turkeys are all known to be susceptible to infection. These species are all treated with the same coccidiostats as chickens and are all subject to the same adverse side effects.

The present invention encompasses feeds for these animals which contain both coccidiostat and betaine.

II Ir--'A WO 94/24886 PCT/FI94/00166 Table 1 Coccidiostats and FDA Approved Target Animals Drug Trade name Animal species Amprolium* Amprol CH, LH, TU, PH, CF Clopidol* Coyden CH, TU Decoquinate* Deccox CH, CT, GO Halofugionone hydrobromide Stenerol CH, TU Lasalocid Avatec CH, CT, SH Maduramicin ammonium* Cygro CH Monensin Coban, Rumensin CH, TU, CT,' QU, GO Narasin Monteban CH Harasin/Nicarbazin Maxiban CH Nicarbazin Nicarb CH 3-nitro-4-hydroxyphenylarsonic Roxarsone CH, TU, SW acid Robenide hydrochloride* Robenz CH Salinomycin Bio-Cox CH (SW outside USA) Zoalene* Zoanix CH, TU infrequent use CH chick, LH laying hen, TU turkey, PH pheasant, CF calf, CT cattle, GO goat, SH sheep, QU quail, SW swine A diet suitable for the particular domestic food animal being raised is formulated using standard feed tables. For example, a standard diet for cattle may be formulated using the information provided by the Merck Veterinary Manual, sixth edition, pages 1104-1132 (1986). Using the same source, standard diets can be prepared for rabbits (pages 1210-12110); sheep (1211- 1221); swine (pages 1221-1230); and poultry (pages 1188-1210). After the basal diet has been formulated, coccidiostat and 'etaine are added. The concentration of coccidiostat should be that recommended by the Food and Drug Administration (FDA 1994 Feed Additive Compendium, U.S. Food and Drug Administration, 1994). The concentration of betaine which has been I~9~Ptl~P~1K~ WO 94/24886 PCT/FI94/00166 -13found to be successful in potentiating the effects of coccidiostat in chickens 0.5-2.0 kg/ton dry weight) may be used initially in formulating feeds for other animals and adjusted up or down as experience dictates. Because of the synergistic effect of betaine, it may be possible to reduce the concentration of coccidiostat administered to animals and to still obtain commercially acceptable results. Thus, the use of betaine in combination with coccidiostat may reduce the cost of formulating an acceptable feed.

D. Dietary Additives Containing Betaine and Coccidiostat In addition to being directed to feeds containing both betaine and coccidiostat, the present invention encompasses other compositions containing these two agents which are administered to domestic food animals for the purpose of preventing the adverse effects of coccidiosis. A premix of betaine and coccidiostat may be administered either in the form of tablets, capsules or liquids. In all cases the concentration of betaine and coccidiostat should result in a dietary inclusion rate comparable to that for feeds formulated to contain these agents. The betaine/coccidiostat composition may be supplemented with additives to improve its flavor or to provide other dietary supplements or therapeutic agents needed by animals.

Compounds containing betaine and coccidiostat for parenteral administration will be formulated according to known methods for preparing pharmaceutically useful compositions in which these agents are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences (16th edition, A. Oslow, ed., Mack, Easton, PA 1980). The required dosage will depend upon the type of animal being treated and the type of coccidiostat being administered (FDA 1994 Feed Additive Compendium, U.S. Food and Drug Administration, 1994).

Additional pharmaceutical methods may be employed to control the duration of action. Controlled delivery may be accomplished by selecting appropriate macromolecules such as polyesters, polyaminoacids, r L~a WO 94/24886 PCT/FI94/00166 -14polypyrrolidone, ethylene vinylacetate, methylcellulose, carboxymethylcellulose or protamine sulfate and combining these according to wellestablished procedures in order to control release. The duration of action of coccidiostat and betaine may also be controlled by incorporating these agents into particles of polymeric materials such polyesters, polyaminoacids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.

Alternatively, it may be possible to entrap betaine and coccidiostat in microcapsules. Various materials and methods for making and using microcapsules are disclosed in Remington's Pharmaceutical Sciences, (16th edition, A. Oslow, ed., Mack, Easton, PA 1980).

E. A Method of Treating Animals Infected with Coccidia The present invention is also directed to a method of treating domestic food animals infected with coccidia by administering a combination of betaine and coccidiostat. The results shown in Tables 5 and 11 demonstrate that the administration of betaine and coccidiostat to coccidia-infected chicks significantly improves their commercial performance in terms of a diminished severity of gut lesions, reduced mortality, higher end weights and improved feed conversion efficiency.

The combination of betaine and coccidiostat, may be administered to animals either orally or parenterally as described above. Preferably betaine and coccidiostat are incorporated into the feed of animals. In the case of chickens, betaine should constitute between 0.5 and 2.0 kg/ton dry feed. If salinomycin is used as the coccidiostat for treating chickens, it should be present at an inclusion rate of between 44 and 66 ppm. Other coccidiostats which may be used include monensin (100-200 ppm); naracin (70 ppm) and lasalocid (90 ppm), as well as the other coccidiostats shown in Table 1.

Betaine may be purchased from commercial suppliers such as Finnsugar Bioproducts (sold under the tradename of "Betafin"). Suitable coccidiostats and their suppliers include the following: monensin: Elanco Products Ltd. (tradename "Elancoban" or "Romensin"); salinomycin: Hoechst I g I WO 94/24886 PCT/FI94/00166 (UK) Ltd. (tradename "Sacox"); narasin: Elanco Products Ltd. (tradename "Monteban"); and lasalocid: Roche (tradename "Avatec"). Additional coccidiostats that have been approved by the FDA are shown in Table 1. In species other than the chicken, the concentration of coccidiostat should be that recommended by the U.S. Food an Drug Administration (FDA 1994 Feed Additive Compendium, U.S. Food and Drug Administration, 1994). The initial concentration of betaine in the feed should be between 0.5 and kg/ton dry feed. This may be adjusted either up or down as experience dictates. Other procedures for administering the combination of betaine and coccidiostat to animals are described above.

All citations herein are incorporated by reference in their entirety.

Having now described the invention in general terms, the same will be further described by reference to a specific example provided herein for the purpose of explanation only and not intended to be limiting unless otherwise specified.

Example 1 Animals: 2,464 one-day-old male and 2,464 one-day-old female broiler chicks of commercial strain (Peterson x Arbor Acres) were randomly allotted to 56 floor pens on built-up litter. Chicks were grown to 45 days of age. There were seven treatment groups, each with eight replicates. The experimental design is shown in Table 4.

Inoculation: Chicks in treatments 1 to 6 were inoculated at 14 days of age with a mixture of E. acervulina, E. maxima and E. tenella via drinking water. At 21 days of age, two males and two females from each pen were necropsied and scored for coccidiosis 4 is most severe). Treatment 7 was a non-inoculated control, but chicks received a natural contaminant via the litter.

Diets: Corn-soy diets (starter and grower) were formulated to meet or exceed the nutritional requirements set forth in Nutrient Requirements of Poultry, National Research Council, National Academy of Sciences, 1 s~ WO 94/24886 PCT/FI9400166 -16- Washington, D.C. (1984). Diet composition is shown in Table 2 and calculated analysis in Table 3. The diets were supplemented with betaine and a commercial coccidiostat, Bio-Cox, according to the experimental design (Table Diets were supplied in crumble and pellet form ad libitum. A complete record of feed consumption was maintained.

The results are disclosed in the following tables and figures. The letters after the numerical data indicate the statistical significance of the differences between the treatments, determined with analysis of variance.

Treatments marked with the same letter do not differ significantly as regards the measured parameter.

TABLE 2: DIET COMPOSITION Ingredient Starter Grower Yellow corn 60.88 65.88 Soybean meal (48) 32.15 27.26 Fat 4.01 4.06 -I I Iba WO 94/24886 WO 9424886PCT/F941 00166 -17- TABLE 3: CALCULATED ANALYSIS OF THE DIETS Constituent Starter Grower(% Crude protein 21.0 19.0 Total Lysine 1.20 1.00 Methionine 0.52 0.46 Tot Meth. Cyst 0.88 0.80 Calcium 0.9 Av. Phosphorous 0.45 0.40 ME kcal/k~g 3190 3250 MIJ/ kg, 13.3 13.6 TABLE 4: ExPERIMENTAL DESIGN Treatment Coccidiostat (ppm) Betaine Inoculation 1 0 0 Yes 2 0 0.15 Yes 3 Salinomycin 44 0 Yes 4 Salinomycin 44 0.15 Yes Salinomycin 66 0 Yes 6 Salinomycin 66 0.15 Yes 7 Salinomycin 66 0 No TABLE 5: RE-SULTS 1-21 d 1-45 d 21 d Treatment Coccidiostat Additive Body weight Feed conver- IMortality Body weight Feed conver- Mortality Lesion number PPM (kg) sion ratio sion ratio ()score I None None 0.580 a 1.433 a 16.62 a 1.948 a 1.977 a 19.05 a 3.63 a 2 None Betaine 0.592 b 1.397 b 14.06 a 1.853 a 1.954 ab 19.92 a 3.47 a 3 Salinomycin 44 None 0.604 c 1.381 bc 8.38 b 1.969 ab 1.936 abc 10.571h 2.88 b 4 Salinornycin 44 Betaine 0.617 d 1.346 cde 4.69 c 1.982 bc 1.898 cde 7.14 c 2.22 Wde Salinomycin 66 None 0.617 d 1.349 cd 4.69 c 1.981 bc 1.920 bcd 6.99 c 2.34 cd 6 Salinomycin 66 lBetaine 0.640 f 1311 def 2.70 c 2.004 cd 1.877 deC 4.32 c 1.81 e 7 Salinomycin 66 None 0.640 f 1.289 ef 2.84 c, 2.010 d 1.833 f 4.17 c 2.44 1)c inoculation) a, b, c, -means withi different letters differ significantly at (P ANOVA -,'t0001 0.004 0.0065 0.1135 0.0767 0.0088 10.0039 Overall Betaine effect NS NS NS NS NS NS NS NS NS WO 94/24886 PCT/FI94/00166 -19- Conclusions The data show that betaine addition in a commercial-type compound feed significantly reduces the severity of gut lesions and mortality, and improves the growth and feed efficiency of broiler chicks. While betaine and coccidiostat (Bio-Cox) both produced a significant effect on these parameters, there was no statistically significant interaction between betaine and the coccidiostat. Thus betaine addition resulted in further improvement in these production parameters in broilers treated with the coccidiostat.

Furthermore, the data shows that broilers treated with a lower dietary level (44 ppm) of coccidiostat than in commercial practice, and supplemented with betaine at a dietary level of 1.5 kg/ton, show similar performance to broilers treated with the commercial (66 ppm) level of the coccidiostat.

Example 2 Animals: 1,200 one-day-old male and 1,200 female broiler chicks of commercial strain (Peterson x Arbor Acres) were randomly allotted to 40 floor pens on built-up litter. Chicks were grown to 47 days of age on a basal diet containing salinomycin and supplemented with methionine, betaine, or a combination of both. The experimental design is shown in Table 8. Each of the five treatments had eight replicates.

Inoculation: Chicks were inoculated at 15 days of age with a mixture of E. acervulina, E. maxima and E. tenella via drinking water. At 21 days of age, six birds from each pen were necropsied and scored for coccidiosis at four points of the intestinal tract (upper, middle, lower, ceaca; scale: 0-4, 4 is most severe).

Diets: Corn-soy diet (starter and grower) was formulated to meet or exceed the nutritional requirements set forth in NRC 1984, except for methionine, which was deficient in diets 1, 3 and 4. Diet composition is shown in Table 6 and calculated analysis in Table 7. The diets were supplemented with betaine and a commercial coccidiostat, salinomycin, according to the experimental design (Table Diets were supplied in I 1 ~;sllss WO 94/24886 PCT/FI94/00166 crumble and pellet form ad libitum. A complete record of feed consumption was mainained.

The results are disclosed in Table 9 and in figures 5-8. The letters after the numerical data indicate the statistical significance of the differences between the treatments, determined with analysis of variance. Treatments marked with the same letter do not differ significantly as regards the measured parameter.

TABLE 6: DIET COMPOSITION Ingredient Starter Grower Yellow corn 57.23 62.58 Soybean meal (46.5) 31.64 27.3 Meat and bone meal (48) 5.00 5.00 Fat 3.89 3.97 Salt 0.33 0.34 DL Methionine 0.024 0.041 Limestone 0.44 0.41 Def. phos 0.66 0.54 Trace mineral premix 0.25 0.25 Vitamin premix 0.05 0.05 Bacitracin MD 0.05 0.025 Chlorine chloride-60 0.087 0.084 3-nitro-20 0.025 0.025 Sand, fine washed 0.225 0.225 Finisher 68.33 21.02 5.00 3.90 0.36 0.00 0.48 0.30 0.25 0.05 0.025 0.06 0.00 0.225 as WO 94/24886 WO 9424886PCTIFI94/00166 TABLE 7: CALCULATED ANALYSIS OF THE DIETS Constituent Crude protein Total Lysine Total Methionine Tot Meth. Cyst Choline Calcium Av. Phosphorous ME kcal/kg MJ/kg Starter 22.0 1.164 0.376 0.709 0.165 0.92 0.48 3100 13.0 Grower 20.0 1.024 0.368 0.672 0.154 0.86 0.45 3170 13.3 Finisher 18.01 0.883 0.303 0.579 0.132 0.80 0.40 3240 13.5 TABLE 8: EXPERIMENTAL DESIGN Treatment Coccidiostat Added methionine Betaine Inoculation (ppm) above basal 1 Salinomycin 66 0 0 Yes 2 Salinornycin 66 0.15 0 Yes 3 Salinomycin 66 0 0.075 Yes 4 Salinomycin 66 0 0.15 Yes Salinomycin 66 0.15 0.075 Yes

I

0 %0 TABLE 9: RESULTS Tr. 1 Tr. 2 Tr. 3 Tr. 4 Tr. LM, 0 BET HM, 0 BET LM, 0.75 BET LM, 1.5 BET HM, 0.75 BET End weight, kg 2,227 a 2,243 a 2,253a 2,242 a 2,291 b w FCR 1,928 a 1,872 b 1,880h 1,869 b 1,846 c 5 FCR, weight adjusted 1,837 a 1,775 b 1,778b 1,772 b 1,730 c -I Lesion score 2,478 a 2,541 ab 2,188ab 1,563 b 1,770 ab Mortality 6,875 a 4,583 a 4,792a 6,667 a 3,125 a m n Explanations: LM low methionine, IIM high adequate methionine -I 0 BET no betaine, 0.75 BET 0.75 kg/t (1.5 lb/t) betaine, 1.5 BET 1.5 kg/t (3 lb/t) betaine FCR feed conversion ratio, morality adjusted FCR, weight adjusted FCR adjusted to 2.0 kg weight, using 0.04 decrease in FCR per 100 g live weight Lesion score sum of.lesion scores (0 to 4) at three points of the intestine Common letters after the mean values express non-significant differences between the treatments

C'-

0? WO 94/24886 PCT/FI94/00166 -23- Conclusions Betaine addition at levels 0.075-0.15% of the diet significantly improved the growth and feed efficiency of broilers. 0.15% betaine significantly reduced intestinal lesions caused by coccidial infection. The response was not dependent on the methionine content of the diet.

Example 3 Animals: 1200 one-day-old male broiler chicks of commercial strain (Peterson x Arbor Acres) were randomly allotted to 120 battery cages of 18" x 24." Chicks were grown to 21 days of age. There were ten treatment groups, each with twelve replicates. The experimental design is shown in Table Inoculation: Chicks in treatments 1, 2 and 5-10 were inoculated at 14 days of age with a mixture of E. acervulina, E. maxima and E. tenella via drinking water. At 21 days of age, 4 birds from each cage were necropsied and scored for coccidiosis 4 is most severe). Treatments 3 and 4 were non-inoculated controls.

Diets: Corn-soy diets were formulated to meet or exceed the nutritional requirements set forth in Nutrient Requirements of Poultry, National Research Council, National Academy of Sciences, Washington, D.C.

(1984). Diet composition is shown in Table 11 and calculated analysis in Table 12. The diets were supplemented with betaine and three kinds of commercial coccidiostat according to the experimental design (Table 10). A complete record on feed consumption was maintained.

The results are disclosed in the following tables and figures. The letters after the numerical data indicate the statistical significance of the differences between the treatments, determined with analysis of variance.

Treatments marked with the same letter do not differ significantly with regard to the measured parameter.

~I I WO 94/24886 WO 94/.4886PCTIFI94/00166 Table _________Experimental Design Treatment Added Coccidiostat ICoccidia No. betaine source jinoculation (lb/ton) TI None None Y T12 Betaine 3 lb/t None Y T3 None None N T4 Betaine 3 lb/t None N None Bio-Cox (60 g/t) Y T6 None Avatec (113 g/t) Y T7 None Stenerol (2.72 glt) Y T8 Betaine 3 lb/t Bio-Cox (60 g/t) Y T9 Betaine 3 lb/t Avatec: (113 g/t) Y Betaine 3 lb/t IStenerol (2.72 g/t) Y Table 11 DietComposition Ingredient Yellow corn 59,174 Soybean Meal 32.801 Fat 3700 2.379 Salt 0.199 Limestone 0.595 Def. Phos. (32-18) 1.604 Choline (CH-60%) 0.002 Trace Mineral Premix 0.050 Vitamin Premix 0.050 DL Methionine 0.145 Corn GL (ML-61 3.000 Table 12 'Calculated Analysis of the Diet Nutrient Type Asked Actual Adjusted Ingredient Type Asked Weight EQU 100.000 1 100.000 163.000 FAT 3700 MAX 5.000 PROTEIN MIN 20.000 22.823 22.823 TRACE MIN PRX IEQU 0.050 ENERGY-kcal/Ib. MIN 1425.000 1425.000 1425.000 VITAMIN PREMIX. QQU 0.050 LYSINE MIN 1.200 1.200 1.200 CORN GL ML-61 MIN 3.000 I LYSINE MAX 1.220 1.200 1.200 METI-I CY$T MIN 0.920 0.920 0.920 METHLONINF MIN 0.450 0.539 0.539 AVAIL. PHOS. MIN 0.430 0.430 0.430 SODIUM MIN 0.180 0.180 0.180 SODIUM MAX 0.220 0.180 0.180 CIIOLINE g/kg MIN 1.000 1.000 1.000 CALCIUM MIN 0.850 0.850 0.850 CALCIUM MAX 1.250 0.850 0.850 ARGININE MIN 0.04,10 1.569 1.569 COPPER mg/kg MIN 0.000 8.698 8.698 XANTHOPHYLL MIN 0.000 9.5 13 9.5 13 Table 13: Results Trt. Added betaine Coccidiostat Coccidia 1-21 d 1-21 d 1-21 d 110 (l/tn) oure noclaton Body weight Feed conversion Mortality no. (Ibto~) ouce noulaio ratio TI None None Y 1.066 e 1. 625 g 41.6 d T2 IBetaine 3 lb/t None Y 1.228 d 1.582 f 32.5 d T3 None None N 1.434 a 1.279 a 5.8 a T4 Betaine 3 lb/t None N 1 .427 ab 1.275 a 1.6 a None Bio-Cox (60 g/t) Y 1.238 d 1.399 e 15.8 bc T6 None Avatec (1 13 g/0) Y 1.250 cd 1.376 de 17.5 c T7 None Stencrol (2.72 glt) Y 1.357 ab 1.407 e 15.8 bc T8 IBetaine 3 lb~t Bio-Cox (60 g/t) Y 1.375 ab 1.344 bc 5.8 ab) T9 fletaine 3 lh/t Avatec (113 gft)__ Y 1.335 tic 1.304 at) 6.6 abc TO Betaine 3 lb/t Stenerol (2.72 g/t) Y 1.406 ab 1.348 cd 5.8 ab

Claims (33)

1. A feed for domestic food animals c h a r a c t e r i z e d by comprising coccidiostat and betaine.

2. The feed of claim 1, ch a r a c t e r i z e d in that said coccidiostat is an ionophore coccidiostat.

3. The feed of claim 2, ch a r a c t e r i z ed in that said ionophore coccidiostat is selected from the group consisting of: monensin; naracin; lasalocid; and salinomycin.

4. The feed of claim 3, ch a r a c t e r i z e d in that said ionophore coccidiostat is salinomycin. The feed of claim 3, ch a r a c t e r i z e d in that said ionophore coccidiostat is lasalocid.

6. The feed of claim 2, c h a r a c t e r i z e d in that said betaine is present at a concentration of between 0.5 and 2.0 kg per ton of dry feed.

7. The feed of claim 2, ch a r a c t e r i z e d in that said domestic food animals are chickens.

8. The feed of claim 2, ch a r a c t e r i z e d in that said coccidiostat is salinomycin and said betaine is present at a concentration of between 0.5 and 2.0 kg per ton.

9. The feed of claim 8, ch a r a c t e r i z ed in that said feed has the composition shown in table 2. The feed of claim 1, c h a r a c t e r i z e d in that said coccidiostat is a chemical coccidiostat.

11. The feed of claim 10, c h a r a c t e r i z e d in that said chemical coccidiostat is selected from the group consisting of: amprolium; clopidol; decoquinate; halofuginone hydrobromide; maduramicin ammonium; nicarbazin; 3-nitro-4-hydroxyphenylarsonic acid; robenide hydrochloride; and zoalene.

12. The feed of claim 11, ch a r a c t e r i z e d in that said chemical coccidiostat is i ~NUNN WO 94/24886 PCT/FI94/00166 28 halofuginone hydrobromide.

13. The feed of claim 10, c h a r a c t e r i z e d in that said betaine is present at a concentra- tion of between 0.5 and 2.0 kg per ton of dry feed.

14. The feed of claim 10, c h a r a c t e r i z e d in that said domestic food animals are chickens. The feed of claim 10, c h a r a c t e r i z e d in that said coccidiostat is halofuginone hydrobromide and said betaine is present at a concentra- tion of between 0.5 and 2.0 kg per ton.

16. The feed of claim 15, c h a r a c t e r i z e d in that said feed has the composition shown in table 2.

17. A dietary additive c h a r a c t er i z e d by comprising a premixed composition of betaine and coccidiostat.

18. The dietary additive of claim 17, c h a r a c t e r i z e d in that said coccidiostat is an ionophore coccidiostat.

19. The dietary additive of claim 18, c h a r a c t e r i z e d in that said coccidiostat is selected from the group consisting of: monensin; naracin; lasalocid; and salinomycin. The dietary additive of claim 19, c h a r a c t e r i z e d in that said coccidiostat is salinomycin.

21. The dietary additive of claim 20, c h a r a ct e r i z e d in that said coccidiostat is lasalocid.

22. The dietary additive of claim 17, c h a r a ct e r i z ed in that said coccidiostat is a chemical coccidiostat.

23. The dietary additive of claim 22, c h a r a c t e r i z e d in that said chemical coccidiostat is selected from the group consisting of: amprolium; clopidol; decoquinate; halofuginone hydrobromide; I- WO 94/24886 PCT/FI94/00166 29 maduramicin ammonium; nicarbazin; 3-nitro-4- hydroxyphenylarsonic acid; robenide hydrochloride; and zoalene.

24. The dietary additive of claim 23, c h a r a ct e r i z ed in that said coccidiostat is halofuginone hydrobromide. A method for improving the commercial perform- ance of domestic food animals infected with coccidia, ch a racteri z ed by administering betaine and coccidiostat to said domestic food animals.

26. The method of claim 25, c h a r a c t e r i z e d in that said coccidiostat is an ionophore coccidiostat.

27. The method of claim 26, c h a r a c t e r i z e d in that said ionophore coccidiostat is selected from the group consisting of: monensin; naracin; lasalocid; and salinomycin.

28. The method of claim 27, c h a r a c t e r i z e d in that said ionophore coccidiostat is salinomycin.

29. The method of claim 26, c h a r a c t e r i z e d in that said domestic food animals are chickens. The method of claim 25, c h a r a c t e r i z e d in that said coccidiostat is a chemical coccidiostat.

31. The method of claim 30, c h a r a c t e r i z e d in that said chemical coccidiostat is selected from the group consisting of: amprolium; clopidol; decoquinate; halofuginone hydrobromide; maduramicin ammonium; nicarbazin; 3-nitro-4-hydroxyphenylarsonic acid; robenide hydrochloride; and zoalene.

32. The method of claim 31, c h a r a c t e r i z e d in that said chemical coccidiostat is halofuginone hydrobromide.

33. The method of claim 30, c h a r a c t e r i z e d in that said domestic food animals are chickens. I -1 30 gv 0 0 0 S* 98 *S *0 qJ CC 00 C CC*

34. A method for treating domestic food animals infected with coccidia, characterized by administering betaine to said domestic food animals. The method of claim 34, characterized in that said domestic food animals are chickens.

36. The method of claim 34, characterized in that betaine is incorporated into the feed of said domestic food animals.

37. The method of claim 36, characterized in 10 that said betaine is present at a concentration of between and 2.0 kg per to- of dry feed.

38. A method according to claim 34, wherein the coccidiosis is subclinical or clinical coccidiosis.

39. A method according to claims 34 or 38, 15 wherein the coccidiosis is a result of vaccination of said animal with a coccidiosis vaccine. A method according to claims 34 or 38, wherein the coccidiosis is a result of an environmental infection.

41. A method according to any one of claims 34 and 38 to 40, wherein a coccidiostat is administered in addition to betaine. DATED this 23rd day of April 1996 CULTOR LTD By Their Patent Attorneys: GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia staffnaIkeepJRETYPES/6570594 23.4.96 WO 94/24886 WO 9424886PCT/FJ94/00166 1/4 Effect of betaine on the growthL- and dfee d conversion of coccidia-challenged chicks Mortality ElControl UN Betaine No Bio-Cox Blo-Cox (44) Bio-Cox (66) 1-45 days FIGURE I Lesion score Scale 0 to 4 4 19 Control F~ WEBetaine No Bio-Cox Bio-Cox (44) Days 1-45 Bia-Cox (66) FIGURE 2 S~UBSTITUTE SHEET WO 94/24886 WO 9424886PCTIF194/00166 2/4 Effect of betaine on the growth and feed conversion of coccid ia-challenged chicks End weight 0 Control K Betaine 1.98 1.96 1.94 I 1.92 1.91 1.J 1-45 days No Blo-Cox Blo-Cox (44) Bio-Cox (66) FIGURE 3 Feed conversion 8 Control M Betaine 1.95 1.9 1.85 Days 1-45 No Bio-Cox BIo-Cox (44) Blo-Cox (66) FIGURE 4 SUBSTITUTE SHEET -1 WO 94/24886 PCTIFI14/00166 3/4 Effect of betaime on the gut lesions and mortality of broilers fed a methionine deficient and a methionine adequate diet. Lesion score El No BETAINE 9 0.75 kg/t BETAINE K 1.5 kglt BETAINE F 01L Low METHIONINE Adeq. METHIONINE FIGURE Mortality I E No BETAINE 0.75 kgA EAN a 1.5 kgtt BETAINE Low METHIONINE Adeq. METHIONINE FIGURE 6 SUBSTITUTE SHEET WO 94/24886 PTF9/06 PCT/FI94100166 4/4 Effect of betaine on the growth and feed conversion of broilers fed a methionine deficient and a methionine adequate diet. End weight kg 2.3 S~ No BETAINE kg/t BETAINE 228 W 1.5 k9lt BETAINE 2-26 2.24 1 2.2 2.18 2.16 Low METHIONINE Adeq. METHIONINE FIGURE 7 Feed conversion, adjusted to 2.00_ kg El No BETAINE M 0.75 kg/t BETAINE M 1.5 kg/t BETAINE 1.84 1.82 1.8 1.78 1.76 I 1.74 1.72 1.7 Adeq. METHIONINE Low METHIONINE FIGURE 8 SUBSTITUTE SHEET INTERNATIONAL SEARCH REPORT International application No. PCT/FI 94/00166 A. CLASSIFICATION OF SUBJECT MATTER According to Internatlonaf Patent classification (IPC) or to both I nationalI classification and IPC Minimum documentation searched (classification system followed by classification symbols) A23K, A61K Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched SE,DK,FI,NO classes as above Electronic data base consulted during the international search (name of data base .Ard, where practicable, search terms used) CA, WPI, CABA, FSTA, IFIPAT_____ C. DOCUMENTS CONSIDERED TO BE, RELEVANT Category* Citation of document, with, indication, where appropriate, of the relevant passages Relevant tr, claim No. A EP, Al, 0182177 (AMERICAN CYANAMID COMPANY), 1-24 28 May 1986 (28.05.86) A STN International, file FSTA, FSTA accession 1-24 no. 90:2222, Gilka, J. et al: 11 ***Amino*** composition of meat, fatty acid composition of fat and content of some chemical elements in the tissues of male lambs fed ***monensin*** or asalocid***. Meat Science, 1989, 25 273-280 1.1Further documents are listed in the continuation of Box C. [EJ See patent family annex. Special categories of cited documents: 'F later document published after the international filing date or priority ocuentdefnin th geera stte f ~date and not in conflict with the application but cited to undertand t ocmn b eiigte eea ae of thea reiwicviantonidee the principle or theory undecrlying the invention erlier document but published on or after the international filing date document of particular relevance: the claimed invention cannot be document which may throw doubts on priority claim(s) or which is csdewen noe ocannt btaen lonsiere oivlea ne cited to establisth the publication date of another citation or other se~we h ouetI ae ln spca reao (as spece) documnent of p.-Acular relevance: the claimed invention cannot be document referring to an oral disclosur, use, exhibition or other considered to iuvolve an inventive step when the document is Mean combined with one or more other such documents, such combination document published prior to the internatiooal filing date but later than being obvious to a person skilled in the art the pnionty date clained 'W document member o f the same patent family Date of the actual completion of the international search Date of mailing of the international search report 0 3 -08- 1994 Jul-y 1994 Name and mailing address of the ISA/ Authorized officer Swedish Patent Office Box 5055, S-102 42 STOCKHOLM Eva Johansson Facsimile No. 46 8 666 02 86 Telephone No. +46 8 782 25 00 Form PCT/ISA/210 (second sheet) (Juily 1992) INTERNATIONAL SEARCH REPORT International application No. PCT/FI 94/00166 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This international search report has not been established in respect ofcertain claims underArticle 17(2)(a) for the following reasons: 1. W Claims Nos.: 25-37 because they relate td subject matter not required to be searched by this Authority, namely: See PCT Rule 39.1(iv): Methods for treatment of the human or animal body by surgery or therapy, as well as diagnostic methods. 2. Claims Nos.: tecause they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: 3. f Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Box II Observations where unity of invention Is lacking (Continuation of Item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: 1. O As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. O As allsearchable claims couldbe searched withouteffortjustifyingan additional fee, this Authority did not invitepayment of any additional fee. 3. O As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nc.: 4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is Srestricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest II The additional search fees were accompanied by the applicant's protest. D No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (continuation of first sheet (July 1992) C I F -IP-