MXPA00000614A - Use of an enzyme for the manufacture of an agent for controlling bacterial infection - Google Patents

Abstract

Provided is the use of a xylanase or a cellulase for the manufacture of an agent for the treatment and/or prophylaxis of bacterial infection in an animal caused by i(Salmonella, Campylobacter) or i(Clostriduim perfringens). It is preferred that xylanase is used in combination with wheat to form an animal feed. Such a diet is particularly effective in controlling i(Campylobacter) and i(Salmonella) in chickens. The use provided by the present invention affords an alternative to antibiotics when controlling bacterial infection in animals. This leads to considerable health, environmental and economic benefits.

Description

USE OF AN ENZYME FOR MANUFACTURING AN AGENT TO CONTROL BACTERIAL INFECTIONS DESCRIPTION OF THE INVENTION The present invention relates to the use of an enzyme for the manufacture of an agent for the treatment and / or prophylaxis of a bacterial infection. The breeding of many different types of animals is important throughout the world for the production of food for human consumption. When the animals are raised, they come into contact with a variety of bacteria that cause infections such as Campylobacter and Salmonella. In some cases these bacteria can spread directly from animals to humans (zoonoses). Accordingly, it is necessary from an economic, environmental and sanitary perspective, that such bacterial infections be prevented or eradicated in the animal before human consumption, to prevent the spread of the disease to humans. Domestic animals of particular concern, but not exclusive, regarding zoonoses, are chickens. Campylobacter and Salmonella are particularly prevalent in chickens. Bacteria are transmitted to the bird in a variety of ways, including through food, water, dirt and bugs. The bacteria REF .: 32582 initially infected the large intestine of chickens. Later, the disease progresses to the small intestine, where the infestation can cause weight loss in the bird. A particular problem with chickens is that it is almost impossible to eviscerate them in a sterile manner, which results in the bacteria that inhabit the intestines will invariably be transmitted to the consumable meat. Accordingly, the potential of a zoonosis is great, unless the meat is properly handled or cooked. The cost of human infections caused by eating chicken treated inappropriately is significant in terms of both time and lives. In accordance with this, there is currently a demand for better methods to reduce bacterial infection in animals such as. chickens, in particular those that are intended for human consumption. Several solutions have been proposed to the problem of bacterial infections. Current methods of control include the application of antibiotics, sterilization of the food and careful and controlled handling and cooking of the meat after the animal has been slaughtered. The sterilization of the food has proved ineffective in the absence of a sterile breeding environment (which is impractical), while controlled handling and cooking can not be relied on at all times. The application of antibiotics has proved unpopular in groups of consumers who want to reduce the amount of potentially harmful chemicals in food. The use of antibiotics has the additional problem that if they are not introduced into the animal in an appropriately controlled manner, strains of bacteria resistant to antibiotics are created, making infections more difficult to treat in the future. The prophylactic use of antibiotics in an animal fed, therefore, has been regulated in some countries (notably in Sweden and Finland) effectively reducing the available control methods. In fact, no single method has proven to be a barrier that completely prevents the transfer of bacteria from the animal to humans. As alternatives to the above methods, it has been proposed is Poul try Science, 1994, 73: 402-407, to introduce flora into the chickens to compete with the bacteria that cause infections. Such mucosal competitive exclusion flora (MCE) was found to reduce the level of Campylobacter jejuni infections in chickens. However, the treatment of competitive exclusion is not consistently effective, its effectiveness varies from one animal to another. Japanese Patent JP-A-81-73055 describes animal foods that are intended to prevent contamination by Salmonella. It is indicated that the foods contain mannan partially decomposed in the form of polysaccharides of mannose. These are produced by the degradation of mannan by an enzyme produced by microorganisms. The resulting feed was found to be moderately effective against Salmonella in chickens, but it is not effective against Campylobacter. US-A-5,124,262 discloses a mannose isomerase enzyme used to transform fructose into mannose. The mañosa produced in this manner is thought to be useful in foods to inhibit the growth of Salmonella in chickens. In Bamboo J. 1993, pp. 29-35, it is mentioned that the xylan prepared from bamboo, inhibits the growth of various human intestinal bacteria. In particular, it is indicated that xylan is effective against Salmonella. However, the inhibition effect is reversed after a period of 24 hours. The above methods have proven to be more desirable, from an environmental and health point of view, than the administration of antibiotics. However, none has proven to be effective enough to be commercially available. International Publication WO 93/01800 describes the use of a protease for the preparation of an effective medicament against intestinal pathogens in animals. Pathogens of interest include Campylobacter. However, no mention is made of enzymes other than proteases that are useful for controlling animal pathogens. European Patent EP-A-O, 681, 787 describes the use of a carbohydrase or protease for the manufacture of an agent for the treatment of Coccidiosis. However, Coccidiosis in chickens is caused by protozoan oocysts. The document does not indicate how bacterial pathogens can be controlled in chickens or other animals. Accordingly, an object of the present invention is to provide an agent that can be used to control bacterial infections, which is more effective than currently available agents and particularly more effective than those described in the prior art mentioned above. Another objective of the present invention is to provide an agent that can be used to control bacterial infections, that is less harmful to the environment, less expensive than currently available agents and that has advantages for human health. Accordingly, the present invention provides the use of a xylanase or a cellulase for the manufacture of an agent for the treatment and / or prophylaxis of bacterial infections in animals caused by Salmonella, Campylobacter or Clostridium perfringens. A preferred cellulase is β-glucanase. Figure 1 shows the effect of three diets on colonization by Campylobacter in 12-day-old chickens. Three different levels of dilution were used in the initial stock solution of Campylobacter, to introduce the pathogen to the chickens. The results are presented as thick positive intestine averages and represent the combined results of two blocks A and B, comparing a total of 108 birds (12 per dilution for each of the three diets). Figure 2 shows the results of Figure 1, but for block A only. • Figure 3 shows the results of Figure 1, but for block B only. Figure 4 shows results similar to the Figure 1, but at three alternative dilution levels of the initial stock solution of Campylobacter. The results are presented as the logioUFC (Training Units of Colony) / ml of average counts. Figure 5 shows the results of Figure 4, but for block A only.

Figure 6 shows the results of Figure 4, but for block B only. Figure 7 shows the effect of three diets on colonization by Campylobacter of the small intestine and large intestine of 17-day-old chickens. The results are presented as the logioUFC / ml of the average accounts and represent the combined results of two blocks A and B, comprising a total of 72 birds (12 per treatment) for each of the three diets. Figure 8 shows the results of Figure 7, but for block A only. Figure 9 shows the results of the Figure 7, but for block B only. Figure 10 shows a comparison of the weight of chickens of 1, 5, 12, 19, 25 and 33 days of age (20 in total) dosed with Campylobacter jej uni and similar chickens (25 in total) that were not dosed, where all chickens were fed a wheat-based diet. Figure 11 shows results similar to those in Figure 10, but for chickens with a diet of wheat plus xylanase. Figure 12 shows results similar to those in Figure 10, but for chickens with a corn-based diet.

Figure 13 shows the effect of two different diets (wheat and wheat plus xylanase) on colonization by Salmonella enteri tidis in chickens of 14 days of age. The results are presented as the logioUFC / ml of the average accounts. The tests were carried out in two blocks, A and B, comprising a total of 48 birds, 24 per diet. The advantage of using foods that contain a xylanase or a cellulase to raise animals is that the amount of antimicrobial drugs that have been routinely incorporated into your diet can be reduced or in some cases can be omitted altogether. This makes possible considerable economic savings in view of the relative cost of antibiotics. In countries where such drugs are prohibited, it represents a totally new approach to the control of bacterial diseases. When antibiotics are omitted from an animal's diet, there are several additional potential benefits. Previously it has been necessary to remove the antibiotics from the diet of the animals for some time before they are slaughtered. This ensures that the meat is relatively free of such drugs and thus is suitable for human consumption. In contrast, if antibiotics are completely omitted from the diet of an animal, as can be achieved with the present invention, then the animal can be sacrificed at any age instead of a certain time after having withdrawn the drugs. This gives the poultry farmer greater flexibility and eliminates the risk of the animals becoming infected shortly before being slaughtered. In addition, it can be guaranteed that the meat and eggs are free of antibiotics. This meat and eggs have an advantage in the market compared to products that do not have such a guarantee. Even if the enzyme added to the diet of the animals only makes it possible to reduce the level of inclusion of antibiotics, then the total cost to control bacterial infections will be reduced. Synergy or potentiation can extend the life of the antibiotic. The present invention also has benefits for human health. Its use reduces the selection by pressure of bacterial strains resistant to antibiotics, making it possible for antibiotics to be removed from the animal's food. Accordingly, more susceptible strains of antibiotics will be present in the intestine of the animal, thus ensuring a more positive result in the case that antibiotics are used in the equivalent human condition. The enzyme xylanase or cellulase to be used in food, it can be formulated as a premix together with any other enzyme that is included. The premix can be added to the raw materials before manufacturing the food, during the manufacture of the food or as a final stage once the food is ready to be used. It is possible to add the enzyme directly as liquid to a preformed feed material in the form of pellets or porridge. It is also possible to include the enzyme in the animal's diet by incorporating it in a second (different) food or in the drinking water to which the animal has access. Accordingly, it is not essential that the enzyme be incorporated into the food itself, although such incorporation forms a particularly preferred embodiment of the present invention. If the enzyme is incorporated into a food, then preferably it comprises at least 25% by weight of a cereal and more preferably at least 35% by weight of the cereal. The cereal may be one or more of the following, wheat, corn, rye, barley, oats, rice and sorghum. It is particularly preferred that the cereal be wheat. Although the cereal component of a cereal-based diet is a source of protein, it is usually necessary to include supplementary protein sources in - Il ¬ diet, such as those derived from fishmeal, meat or vegetable meal. These supplemental protein sources can constitute up to 50% by weight of the animal feed. Vegetable protein sources include at least one of the following, soybean with whole fat, rapeseed, canola, soybean meal, rapeseed meal and canola meal. If the enzyme is incorporated into a food, then it is preferably added in a relative amount of 0.0001-10 g of the enzyme per kilo of the food, preferably 0.001-1 g / kg and more preferably 0.01-0.1 g / kg. The xylanase for use in the present invention can be obtained from a fungus, such as Trichoderma, Aspergillus, Humicola,. or Neocallimastix. Alternatively, the xylanase can be obtained from a bacterium such as Bacillus, Streptomyces, Clostridium, or Ruminococcus. The present invention is particularly effective against strains of Salmonella and Campylobacter and especially Salmonella enteri tidis and Campylobacter jejuni. Another bacterium against which the invention is effective is Clostridium perfringens. Bacterial infection can be treated or prevented according to the present invention in a wide variety of animals, but the use of the present invention is particularly preferred in domestic animals and livestock. Animals that can benefit in particular from the present invention include poultry (such as chickens, turkeys, ducks and geese), ruminants (such as cattle, horses and sheep), pigs, cats, dogs, rodents (such as rabbits) and fish. The present invention is particularly useful in roasting chickens. The most preferred food and enzyme combinations include wheat plus xylanase, corn plus xylanase and barley plus β-glucanase. The enzymes used in the present invention fall within a general class termed polysaccharidase. Its substrates are structural polysaccharides such as xylans and β-glucans that are an integral part of the cell wall of most terrestrial plants. These polysaccharides are not found in animal cells and are not expected to have any activity against proteins. Because these enzymes attack the polysaccharides found in the cell wall of plants, the only possible substrates for these enzymes in the gastrointestinal tract of an animal are contained in cereal-based foods. Therefore, it is speculated that the beneficial effects of xylanase or cellulase on bacterial infections will result in some way from the degradation products such as xylan or β-glucan derived from a cereal-based diet. As previously mentioned, International Publication WO 93/01800 describes the use of a protease for the preparation of an effective medicament against intestinal pathogens in animals. It is well established that such pathogens regulate their infectivity by binding to receptors on the surface of intestinal epithelial cells, through antigenic protein or glycoprotein molecules on the cell surface of the pathogen. It is suggested that the protease enzyme prevents the union of pathogenic intestinal epithelial cells by destroying these receptor / adhesion protein sites, to which the pathogen must bind to cause an infection. The protease enzymes mentioned in this reference are predicted to destroy proteins on the luminal surface of intestinal epithelial cells in a non-specific manner, but would not be expected to attack substrates other than proteins. Accordingly, the activity of the proteases described in this reference is fundamentally different from the activity of the enzyme used in the present invention. A person skilled in the art would not have predicted the utility of the present enzymes against bacterial infections based on the activity of the proteases described in International Publication WO 93/01800. Next, the present invention will be described in greater detail in accordance with the following Examples. EXAMPLES General Methodology Wheat and corn diets were prepared with the following formulations: Table 1 - Wheat Diet Table 2 - Corn Diet The animal feeds were prepared by introducing a cereal vehicle containing about 3 mg enzyme protein / kg in the wheat diet at a concentration of kg of enzyme and vehicle per ton of wheat diet. The final concentration of the enzyme protein in the feed, in this way, was about 3 mg per ton. The xylanase was obtained from Trichoderma longibrachiatum. Roast chickens were fed with the wheat diet plus xylanase since their hatching. For comparison purposes, chick blocks were fed separately with the wheat diet and with the corn diet without the addition of xylanase. A challenge model was used, in which 0.2 ml of a stock solution of C. jejuni was introduced into each animal by syringe on day 7 after the occlusion. The dilution level of the stock solution was varied, from 10 (highest challenge) to 10 (lowest challenge, similar to natural conditions). The undiluted stock solution contained approximately 10 4 CFU per 0.2 ml. The large intestine of the chickens was examined for damage caused by Campylojacter at various ages. EXAMPLE 1 Figures 1-6 show the effect of diets on colonization by Campylobacter in 12-day-old chickens. In each case, two blocks, A and B, were tested to minimize the effect of the environmental variance of the results. In each case it is clearly evident that a diet of wheat plus xylanase is effective in reducing the level of Campylobacter in the large intestine of chickens, as compared to a corn diet. Additionally, at a dilution of the stock solution of Campylobacter of 10 or lower (i.e., approximately more to natural conditions), the diet of wheat plus xylanase becomes considerably more effective than the wheat diet alone. Thus, in Figure 1, for the wheat diet plus xylanase at a dilution of the stock solution of Campylobacter 10", a record of 0. 5 positive large intestines. The equivalent records for diets lacking xylanase were approximately 1.5 and 2.5. EXAMPLE 2 Figures 7-9 demonstrate the effectiveness of diets on colonization by Campylobacter of the small intestine and large intestine of 17-day-old chickens in two blocks. The effect of wheat diet plus xylanase to reduce colonization by Campylobacter of the large intestine of the chickens is evident as already demonstrated in Example 1. However, this reduction is even more marked with respect to the small intestine. Consistent with this, in Figure 7 the logioUFC of the average small intestine count measured for chickens with the wheat and xylanase diet was less than 4. The equivalent scores for diets that did not contain xylanase were found to be of about 6, ie, 100 times greater. EXAMPLE 3 Figures 10-12 illustrate a comparison of the effect of different diets on the weight of chickens of 1, 5, 12, 19, 25 and 33 days of age. Figure 10 shows the results for the wheat-based diet, Figure 11 the results for the wheat-based diet plus xylanase and Figure 12 the results for the corn-based diet. The weight of the chickens in each case was reduced when dosed with Campylobacter. However, the chickens to which Campylobacter was introduced increased faster in weight when they were in the diet of wheat plus xylanase, with respect to any of the other diets. EXAMPLE 4 Figure 13 demonstrates the effectiveness of the wheat and wheat plus xylanase diets on colonization by Salmonella enteri tidis of the small intestine of 14-day-old chickens, in two blocks, A and B. The methodology in these experiments was identical to that used for the Campylobacter experiments described above, except that the undiluted stock solution of Salmonella enteri tidis contained approximately 10 CFU per 0.2 ml. The effect of wheat diet plus xylanase to reduce colonization by Salmonella of the small intestine of chickens is clearly evident. So that, in block B, chickens with wheat diet were found to have logioUFC / ml counts of approximately 7. However, chickens from block B with wheat diet plus xylanase, were found to have much lower logioUFC scores / ml of approximately 4 (1000 times lower). The above Examples clearly demonstrate a reduction of bacterial infection in the intestine due to the inclusion of xylanase in the diet. Similar results have been observed when a cellulase such as a β-glucanase is used. This indicates that the use provided by the present invention significantly reduces the ability of certain bacteria to colonize the large intestine, which, in turn, prevents the migration of bacteria to the small intestine. In accordance with this, since they present a reduced infection level, the growth rate of the animals increases, producing economic benefits. The reduction of the contamination index also has obvious benefits for the health of human beings and the replacement of antibiotics by such diets has clear environmental benefits. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Claims (18)

1. CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. The use of a xylanase for the manufacture of an agent for the treatment and / or prophylaxis of bacterial infections in an animal caused by Salmonella, Campylobacter or Clostridium perfringens.
2. 2. The use according to claim 1, characterized in that the agent is in the form of an animal feed.
3. 3. Use in accordance with the claim 2, characterized in that the agent comprises at least 25% by weight of a cereal.
4. 4. Use in accordance with the claim 3, characterized in that the cereal is wheat, corn, rye, barley, oats, rice and / or sorghum.
5. 5. The use according to any of the preceding claims, characterized in that the xylanase is obtained from a fungus.
6. 6. The use according to claim 5, characterized in that the fungus is Trichoderma, Aspergillus, Humicola or Neocallimastrix.
7. The use according to any of the preceding claims, characterized in that the xylanase is obtained from a bacterium.
8. 8. The use according to claim 7, characterized in that the bacterium is Bacillus, Streptomyces, Clostridium or Ruminococcus.
9. 9. The use of a cellulase for the manufacture of an animal feed comprising at least 25% by weight of a cereal that is one or more of the following wheat, corn, rye, barley, oats, rice and sorghum, for the treatment and / or prophylaxis of bacterial infections in an animal, caused by Salmonella, Campylobacter or Clostridium perfringens.
10. 10. The use according to claim 9, characterized in that the cellulase is a β-glucanase.
11. 11. The use according to any of the preceding claims, characterized in that the cereal is wheat.
12. 12. The use according to any of the preceding claims, characterized in that the agent further comprises a supplementary source of protein.
13. 13. The use according to claim 12, characterized in that the supplementary source of protein is fishmeal, meat meal or a vegetable protein.
14. The use according to claim 13, characterized in that the vegetable protein is soybean with whole fat, rapeseed, canola, soybean meal, rapeseed meal and / or canola flour.
15. 15. The use according to any of claims 2 to 4, 9 or 10, characterized in that the animal feed comprises 0.0001 to 10 g / kg of the enzyme.
16. 16. Use in accordance with the claim 15, characterized in that the animal feed comprises 0.001 to 1 g / kg of the enzyme.
17. 17. Use in accordance with the claim 16, characterized in that the animal feed comprises 0.01 to 0.1 g / kg of the enzyme.
18. 18. The use according to any of the preceding claims, characterized in that the bacterium causing the infection is Salmonella enteri tidis, Campylobacter jejuni and / or Clostridium perfringens.