MXPA99000264A - Veterinary use of a pleuromutil derivative - Google Patents

Abstract

The present invention relates to the use of the compound of the formula I: in the therapy of veterinary diseases whose expression is improved by the increase in the supply density.

Description

VETERINARY USE OF A PLEUROMUTILINE DERIVATIVE The invention relates to pleuromutilin derivatives. It relates to the veterinary use of the compound of the formula I: 15 ie 14-0- [1- [(R) -2-amino-3-methylbutyrylamino] -2-methylpropan-2-thioacetyl] mutilin in the form of free base or of veterinarily acceptable salt, in the therapy of diseases veterinarians whose expression is improved by increasing the density of supply, briefly referred to hereinafter as 20 as "the use of the invention". The compound of the formula I, in the form of free base or of veterinarily acceptable salt (EconorR) is briefly referred to below as "the agent of the invention". Preferably it is a salt, especially in a salt form 25 hydrochloride. In that form, it is known under the generic name of valnemulin hydrochloride. It should be understood that the term "therapy" is applied for a prophylactic as well as curative treatment. The compound of the formula I in the form of the free base or in the form of a chemotherapeutically or veterinarily acceptable salt is known, for example, from European Patent Number EP 153,277 and its equivalents, for example, the Patent of the United States of North America USP No. 4,675,330, specifically as Example 12 thereof. The same is also known: an inhibitory activity against different bacteria in vi tro, in concentrations of approximately 0.008 to 25 micrograms / milliliter; an inhibitory activity in vi tro against mycoplasmas and chlamydias, in general, in concentrations of approximately 0.008 to 0.5 micrograms / milliliter; an inhibitory activity in vivo in mice, using different bacterial strains, and in hens, using strains of mycoplasma, in a dosage of approximately 12 to 50 milligrams / kilogram of body weight. an antiparasitic activity, in particular in vivo against coccidia in poultry, in dosages of 20 to 150 milligrams / kilogram of feed; and a growth-promoting activity in hens and pigs in vivo, in a dosage of 10 to 50 milligrams / kilogram of feed, thereby making the compound useful as an antibacterially active antibiotic in general, and as a veterinary agent, in particular for the chemotherapeutic treatment of coccidiosis in poultry, as well as a growth promoter in hens and pigs. Surprisingly, it has now been found that the agent of the invention is particularly effective in the therapy of veterinary diseases whose expression is enhanced by the higher density of delivery, such as enzootic pneumonia in pigs caused by infection with Mycoplasma. hyopneumoniae, dysentery of pigs caused by infection with Serpulina (formerly Treponema) hyodysenteriae, colitis of pigs (inflammation of the colon) associated with infection with Serpulina pilosicoli ileitis in pigs (porcine proliferative enteropathy, porcine intestinal adenomatosis) associated with Lawsonia intracellularis infection, chronic respiratory disease and arthritis in poultry, associated with Mycoplasma gallisepticu infection, secondary pneumonia in pigs associated with infection with Pasteurella multocida, Actinobacillus (Haemophillus) pleuropneumoniae, and / or Haemophilus parasuis, pneumonia in sheep, sheep, and cattle (Boarding Fever, Traffic Fever, Respiratory Complex of Calves, Pneumonic Bovine Pasteurellosis) associated with infection by Pasteurella haemolytica, and polyarthritis in pigs associated with Mycoplasma hyosynoviae infection. Furthermore, in an even more surprising way, it has been found that the induction of drug resistance is extremely low. Accordingly, the invention relates to veterinary use as defined in the foregoing. It also relates to the use of the agent of the invention for the manufacture of a medicament for use in the therapy of veterinary diseases., whose expression is improved by the increase in the density of supply. It also relates to a method of treating veterinary diseases whose expression is improved by increasing the density of supply, which comprises administering a therapeutically effective amount of the agent of the invention to an animal in need of such treatment. It also relates to a veterinary agent for use in the therapy of veterinary diseases whose expression is enhanced by the increase in the density of supply, which comprises the compound of formula I in the form of free base or of veterinarily acceptable salt, together with at least one veterinarily acceptable vehicle or diluent. It also relates to a process for the preparation of a medicament for use as defined above, which comprises mixing the agent of the invention, together with at least one veterinarily acceptable vehicle or diluent. The animal suffering from veterinary diseases whose expression is improved by the increase in the density of supply, for example, may not have already been treated antibacterially with the agent of the invention, or may no longer be receiving the agent of the invention for the growth promotion. A) Infection with Mycoplasma hyopneumoniae: The invention by Mycoplasma hyopneumoniae can be diagnosed in a conventional manner, for example, as described in veterinary manuals, such as Taylor, D.J. in Pig Diseases, 6th edition (1995), Publ. DJ. Taylor, Glasgow, United Kingdom on pages 164-165. The beneficial activity of the agent of the invention in this use is determined, for example, as follows: ! _. Activity vi: The test includes 10 field isolates of outbreaks of enzootic pneumonia in different herds and the type of strain "J". The isolates are cloned into a filter, identified by the disk growth inhibition test, and used in the seventh to tenth passages. The test for the minimum inhibitory concentration (MIC) is carried out in a liquid medium (Friis, N.F. et al., 32.

[1991] 425-429) in 1.8 milliliter tubes containing a double concentration of valnemulin fumarate acid and tiamulin. The mycoplasmas are inoculated in 10-fold dilutions, and the cultures are read visually to determine growth, using tubes seeded with 102 to 104 color-forming units. The initial reading is made after 2 to 4 days, and the final reading after 10 to 14 days, when no further progress has taken place in the change of color. The minimum inhibitory concentration is determined as the lowest concentration of the test compound showing inhibition of growth, compared to the control (Friis, N.F. and Szancer, J., Acta Vet. Scand. 35

[1994] 389-394). The results are shown in Table 1: Table 1 Minimum Inhibitory Concentration (micrograms / milliliter) hfu = acid fumarate All strains of hyopneumoniae tested are highly susceptible to the effect of valnemulin with values of minimum inhibitory concentration of 10 to 40 times lower than those for tiamulin, both in the initial and in the final reading, making the compound particularly interesting to be used in the treatment of clinical cases in infected herds. 2. In vitro activity: Tissue samples are obtained from the lungs of pigs from different flocks affected by enzootic pneumonia of pigs (PPE). Samples are shipped on dry ice for cultivation. M. hyopneumoniae is recovered from freshly sectioned lung tissue for direct culture on Mycoplasma Experience agar. This technique allows the distinctive colonies of M. hyopneumoniae to be recognized and easily separated, by cloning from other faster-growing mycoplasma species, such as Mycoplasma hyorhinis, often present in lung samples of enzootic pneumonia. pigs The isolates are subcultured and identified by disc growth inhibition using specific rabbit antiserum raised for M. hyopneumoniae NCTC 10110. Cultures are prepared for antibiotic stimulation in Mycoplasma Experience broth, incubated aerobically at 36 ° C.

A commercially available solid medium (Mycoplasma Experience Ltd.) is used to isolate M. hyopneumoniae from lung tissue. A liquid medium (Mycoplasma Experience Ltd.) containing phenol red and glucose (pH of 7.6) is used for the tests of the minimum inhibitory concentration. Preparation solutions of the test compound are prepared in a concentration of 1 milligram / milliliter in deionized water, sterilized by filtration through membrane filters with a pore size of 0.2 microns (Sartorius Minisart, N), and stored at -20 ° C. To be used in the minimum inhibitory concentration tests, the supply solutions are diluted in a liquid medium until the required final concentrations are doubled. Minimum inhibitory concentration tests are performed according to the method of Tanner and Wu, Avian Diseases 36 (1992) 714-717. Active stimulus cultures are prepared either from aliquots of 1 milliliter of broth cultures stored at -70 ° C, or from cultures stored in agar at -70 ° C. The stimulus cultures are diluted to give an objective titration of 103 to 105 units of color change / milliliter. Aliquots of 0.1 milliliter of stimulus inoculum are mixed with aliquots of 0.1 milliliter of a dilution of antibiotic in microtitre cavities. Each microtiter plate contains uninoculated medium at a pH of 6.8 (M. hyopneumoniae) (endpoint control), and inoculated stimulus controls free of antibiotics. All plates are sealed with an adhesive film, and incubated aerobically at 36 ° C. Minimal inhibitory concentrations are recorded when the color change in the stimulus control cavities matches the pH of the endpoint control. The minimum inhibitory concentration is the lowest concentration that shows no color change. The results are shown in Table 2 for valnemulin, and the two reference compounds tiamulin and enrofloxacin: Table 2 In vitro sensitivity of Ten Field Isolates of M. hyopneumoniae It was found that all strains were highly susceptible to valnemulin. Most of the strains showed a very good sensitivity to enrofloxacin, but for the individual strains, the minimum inhibitory concentration of valnemulin was at least five times lower, even with those sensitive to 0.0025 micrograms / milliliter of enrofloxacin. These results confirm that field isolates are extremely sensitive to valnemulin. 3. Development of Resistance; The reference strain of Mycoplasma hyopneumoniae NCTC 10110 (obtained from the National Collection of Type Cultures, London, United Kingdom), and a fresh field isolate (MEVT G23), are grown aerobically at 36 ° C in a Mycoplasma glucose broth which contains phenol red, until an acid color change occurs. After the addition of sterile glycerol (5 percent by volume / volume), the cultures are dosed in 1 milliliter aliquots, and frozen at -70 ° C. These cultures are used to start other cultures in broth that are titrated to obtain the number of units of color change (ccu) in microtitre plates after incubation (36 ° C). The replication allows the stimulation of dilutions of antibiotic with previously determined numbers of units of color change in tests of minimum inhibitory concentration (MIC) and in the primary step in habituation studies. A commercially available medium (Mycoplasma Experience Ltd) containing glucose and phenol red (MEGB) is used at a pH of 7.6. Preparation solutions of the test compound are prepared at 1,000 micrograms / milliliter in deionized water. After swirling, the solutions are stylized by filtration through membrane filters with a pore size of 0.2 microns (Sartorius Minisart, N). For use in the minimum inhibitory concentration tests, the delivery solutions are diluted in the mycoplasma broth to double the final concentrations required. In the habituation studies, the supply solutions are dosed in aliquots of 1 milliliter, and frozen at -20 ° C. These are thawed and used at intervals of 5 days to 7 days, to prepare the ranges of drug concentrations (duplication series) in MEGB, in aliquots of 1.9 milliliters, covering the minimum inhibitory concentrations against both strains of M. hyopneumoniae. New oxytetracycline hydrochloride is prepared on each occasion. The dilution ranges are gradually changed step by step, to allow the development of resistance in the mycoplasmas.

Test Procedures: Minimal inhibitory concentration tests are performed by the method of Tanner and Wu, Avian Diseases 36 (1992) 714-717, before performing the habituation study (below), and after the tenth step, aliquots are mixed 0.1 milliliter of the dilution of the compound with 0.1 milliliter aliquots of the stimulus inoculum, containing between 103 and 105 units of color change (ccu) per milliliter, in microtitre cavities. Each microtitre plate contains the non-inoculated radicle, a medium at a pH of 6.8 (endpoint control), and inoculated stimulus controls free of drug. All plates are sealed and incubated aerobically at 36 ° C. Minimal inhibitory concentrations are recorded when the color change in the stimulus control cavities agrees with the pH control of 6.8 (orange-yellow). The minimum inhibitory concentration is the lowest concentration that shows no color change.

Habituation Study: In the primary step experiment, volumes of 1.9 milliliters of antibiotic solutions are inoculated at concentrations that cover the minimum inhibitory concentration of the strain of M. hyopneumoniae under test, with 0.1 milliliters of culture in broth containing between 103 units of color change / milliliter and 105 units of color change / milliliter. In each step experiment, a growth control consisting of the drug-free broth inoculated with Af is included. hyopneumoniae, and a control of medium not inoculated. After 7 days of incubation (36 ° C), the two highest concentrations of drug-containing broth that show an acid color change are grouped together and used to inoculate a new series of drug dilutions in mycoplasma broth (0.1 milliliters of culture in 1.9 milliliters of broth containing drug). This process is repeated every 5-7 days for up to 10 steps. When the mycoplasma strains become resistant to particular antibiotics, or to the tenth step, each strain is once again cultured in drug-containing broth, and the minimum inhibitory concentrations are determined.

Results: Table 3 shows the minimum inhibitory concentrations of the strain of M. hyopneumoniae "J", and a field isolate, before and after exposure to antibiotics: Table 3 Development of In vitro Resistance in Two Strains of M. hyopneumoniae 1) Step level in vi tro. 2 Minimum inhibitory concentration after 8 steps in mycoplasma broth containing tylosin It was found that before exposure, both strains were highly susceptible to valnemulin, with minimum inhibitory concentrations of 0.0025 micrograms / milliliter for the reference strain, and 0.001 micrograms / milliliter for the field isolate. These activity levels were 50 to 100 times higher than those of tylosin and oxytetracycline. These results of minimum inhibitory concentration were used to select the dilution ranges for the drugs in the habituation study. After 10 cycles of exposure to valnemulin, the development of resistance was minimal in both strains of M. hyopneumoniae, increasing the minimum inhibitory concentrations from 0.0025 micrograms / milliliter to 0.005 micrograms / milliliter, and from 0.001 micrograms / milliliter to 0.0025 micrograms / milliliter for the reference strain and the field isolate, respectively. In contrast, a marked resistance to tylosin tartrate developed in both strains of M. hyopneumoniae. In the reference strain, the resistance was first presented within 4 to 5 steps in broth containing tylosin, and for the eighth step, the minimum inhibitory concentration had risen to > 500 micrograms / milliliter, reflecting an increase of > 2000 times in resistance to this antibiotic (Table 3). A marked resistance to tylosin was developed in the field isolate within 8 steps, raising the minimum inhibitory concentration 500 times to 62.5 micrograms / milliliter. This level of resistance persisted after 10 to 11 steps in broth containing tylosin. In both strains of M. hyopneumoniae quadruple increases in resistance to oxytetracycline were observed during 10 cycles of exposure, raising the minimum inhibitory concentrations from 0.25 micrograms / milliliter to 1 microgram / milliliter. These results show that valnemulin is considerably more active against M. hyopneumoniae than tylosin or oxytetracycline, and that it does not induce significant resistance to itself in strains of M. hyopneumoniae. 4. Prevention of experimentally induced enzootic pneumonia in vivo: An experimental model of enzootic pneumonia was used, using lung material subjected to steps, originally derived by the infection of gnotobiotic pigs with M. hyopneumoniae, to evaluate the potential of valnemulin to prevent the illness. Three trials were conducted: trials 1 and 2 were dose titration studies of the novel compound, using a standard stimulus strain of M. hyopneumoniae, and in Trial 3, the efficacy of the compound was evaluated at 200 ppm in the food against a second stimulus. Large white Landrace male pigs, 6 to 7 weeks old, from a group without infection with M. hyopneumoniae were used. The stimulus materials are pneumonic lungs containing M. hyopneumoniae given as a homogenate intranasally in 3 successive days. The minimum inhibitory concentration (MIC) of valnemulin against the strain present in the material used in Tests 1 and 2 was 0.016 micrograms / milliliter, and that for the strain isolated from the material used in Test 3 was 0.0078 micrograms / milliliter. The stimulus material was originally derived by the infection of gnotobiotic pigs with Af. hyopneumoniae, and the subsequent passage in SPF pigs (free of Specific Pathogen), and that had been stored at less than -70 ° C. Trial 1: Medication with valnemulin hydrochloride by stomach tube (giving it) once a day, to 0 (control), 2.5, 5, 7.5, or 10 milligrams / kilogram of body weight per day. Six pigs per group. Test 2: Medication with valnemulin hydrochloride in food at 0, 100, 200, 300, or 400 ppm (equivalent to 0, 5, 10, 15, and 20 milligram / kilogram / day). Six pigs per group. Test 3: Medication with valnemulin hydrochloride in food at 0 or 200 ppm (equivalent to 0 and 10 milligrams / kilogram / day). Eight pigs per group. The medication was given from the first day of the stimulus infection, until the post-mortem examination performed approximately 3 weeks after the stimulus infection. The disease was evaluated by quantifying lung lesions [Cambridge lung injury rating system (Goodwin, RFW and Whittlestone, P., J. Hyq. 69

[1971] 391-397), where the rating is an approximation of percentage of the lung affected with pneumonia], by estimating the proportion of the lung-weight of the body, and by isolating M. hyopneumoniae from the lungs.

The results are shown in Table 4 Table 4 Minimum inhibitory concentration of valnemulin hydrochloride in micrograms / milliliter against M. hyopneumoniae isolated from the stimulus material. 2) Approximate daily dose of valnemulin hydrochloride per pig.

In Trial 1, the stimulated pigs, without medication, had an average injury score of 13.2. Valnemulin reduced injuries by 71 percent and 87 percent in doses of 7.5 and 10 milligrams / kilogram, respectively, while in 2.5 and 5 milligrams / kilogram / day a little less effective in reducing injuries. The M. hyopneumoniae was re-isolated from fewer medicated pigs with 10 milligrams / kilogram, compared to medicated pigs with 2.5 milligrams / kilogram (1 against 4 pigs). In Trial 2, pigs stimulated without medication had an average injury score of 22.1. Pigs medicated with valnemulin at 200, 300, or 400 ppm, showed reductions in injuries of 46 percent, 43 percent, and 71 percent, respectively. The weights of the lungs, which could reflect the microscopic lesions, as well as the large lesions, showed a more dramatic effect, with significant reductions in levels going down to 200 ppm. No reduction was seen in the lesions of medicated pigs with 100 ppm. M. hyopneumoniae was re-isolated from all unmedicated pigs in the post-mortem examination, but was not re-isolated from medicated pigs with 400 ppm or 300 ppm of valnemulin. It was re-isolated from 3 or 4 medicated pigs with 200 ppm and 100 ppm, respectively. In Trial 3, the non-medicated pigs had an average injury score of 10.8; the medication with valnemulin at 200 ppm in the food, reduced the injury ratings by 79 percent. There was no difference in the levels of? Í. hyopneumoniae detected in the post-mortem examination between medicated and non-medicated pigs. Accordingly, valnemulin is effective for the prevention of experimentally induced enzootic pneumonia in pigs in separate experiments, using the stimulus material containing two different strains of M. hyopneumoniae. Accordingly, the agent of the invention is useful in the therapy of enzootic pneumonia in pigs caused by infection with Mycoplasma hyopneumoniae. For this use, the effective dosage, of course, will vary depending on the particular salt used, the mode of administration, the size and age of the animal, and the desired effect; for example, for prophylactic treatment, relatively low doses would be administered for a long time. However, in general, satisfactory results are obtained when the agent is administered in a daily dosage of about 5 milligrams / kilogram to about 15 milligrams / kilogram of body weight of the animal, suitably given in divided doses of 2 to 4 times a day , or in a sustained release form. For most animals, the total daily dosage is from about 100 milligrams to about 1000 milligrams, preferably from about 100 milligrams to about 500 milligrams, given once or twice a day. It can be conveniently administered as a single therapy. Preferred doses in drinking water are 0.01 to 0.05 percent by weight by volume, particularly 0.01 to 0.025 percent, and in food, from 100 to 400 ppm (grams / metric ton), particularly from 100 to 200 ppm (grams / metric ton).

B) Infection by Serpulina hyodysentiesiae: Serpulina hyodysenteriae infection can be diagnosed in a conventional manner, for example, as described in veterinary manuals, such as Taylor, D.J., in Pig Diseases, 6th edition (1995), Publ. DJ. Taylor, Glasgow, United Kingdom on pages 143-144. The beneficial activity of the agent of the invention in this use is determined, for example, as follows: 1. Determination of minimum inhibitory concentration: Nine field isolates of S. hyodys enter iae are included from dysentery outbreaks of pigs and the type strain, ATCC 31212, and ATCC 29796 (Serpuiin innocens , group 3 (Fellstróm, C, Res. Vet. Sci. 59

[1995] 1-4)). The identification is based on hemolysis patterns on TSA (soy triticase agar) with 5 percent bovine blood, and on the test for indole production and hydrolysis of hippurate (Rosco Diagnostic Tablets, Taastrup, DK). Bacteria are transferred from agar plates to 0.9 percent serum, and turbidity is adjusted to 1.0 on the McFarland scale before 10 microliters of each isolate are inoculated onto agar plates with double hydrochloride concentrations of valnemulin, tiamulin acid fumarate, dimetridazole, lincomycin hydrochloride, and tylosin. Growth and haemolysis are recorded after 4 days of anaerobic culture, and the minimum inhibitory concentration is determined as the lowest concentration of antibiotics where spirochetes do not grow. The results of the determinations of the minimum inhibitory concentration for S. hyodysenteriae are shown in Table 5: Table 5 Minimum inhibitory concentration (MIC) values for 10 serpulin strains. hyodyaenteriae ch - hydrochloride hfu - fumarate acid.

All strains of S. hyodysenteriae were highly susceptible to valnemulin, showing minimum inhibitory concentration values of 2 to 32 times lower than those of tiamulin. The high susceptibility of valnemulin to S. hyodysenteriae makes the compound interesting for use in the treatment of clinical cases in infected herds. 2. Determination of the minimum inhibitory concentration The minimum inhibitory concentrations are determined (MICs) against 10 strains of S. hyodysenteriae isolated from porcine faeces, by the agar dilution method. Strains of NCTC (National Collection of Type Cultures) or reference strains are used as controls in all tests of minimum inhibitory concentration. The agar medium for S. hyodysenteriae consists of BAB2 (Unipath CM271) which contains 7 percent whole defibrinated sterile sheep blood. All antimicrobial agents are tested in duplicate dilutions. Antibiotic dilutions prepared are added to an approximate volume of MH agar, previously cooled to 50 ° C, mixed, and volumes of 20 milliliters are poured. All incubations of S. Hyodysenteriae are performed in an anaerobic workstation at 37 ° C (+/- 0.5 ° C) (Don Dhitley Scientific) which provides a strict anaerobic atmosphere comprising 80 percent nitrogen, 10 percent hydrogen, and 10 percent carbon hydroxide. All other incubations are carried out at 37 ° C. All strains are incubated in an aerobic atmosphere at 37 ° C for 24 hours. The numbers of the organisms are adjusted to an optical density (OD) equivalent to 1 x 108 colony-forming units (cfu) per milliliter, using MH broth as control. The antibiotic-containing plates are inoculated in duplicate using a multi-point inoculator (Denley Instruments), which delivers a plate inoculum of approximately 1 microliter, yielding an inoculum of 104-105 colony-forming units per spot for all strains (Amon J. Antimicrob Chemother 21

[1998] 701-710; Ericsson and collaborators Acta Pathol. Microbiol. et. Tmmunol. Spand. 217

[1971] (B) Suppl., 1-90). The control plates are incubated under the same conditions as the test plates. The minimum inhibitory concentrations are read after 24 and 48 hours, the last being the definitive reading. The minimum inhibitory concentration is defined as the lowest concentration of antibiotic on which there is no growth, without taking into account whether it is a single colony or a weak veil caused by the inoculum (National Committee for Clinical Laboratory Standards

[1989], Antimicrobial Suaciftpti.hil i ty Testing, NCCLC Publications SC3, Villanova, PA, USA). The minimum inhibitory concentration (in micrograms / milliliter) against Serpulina hyodysenteriae was valnemulin (0.1), tiamulin (0.3), lincomycin (50.0), tylosin (200.0) and dimetridazole (30.0). All strains of S. hyodysenteriae were susceptible to valnemulin, which was the most active agent tested by 3 to 600 times. 3. Stimulus test: Two trials were conducted in which valnemulin fed at different inclusion levels was compared with tiamulin for the prevention of dysentery of pigs. The severity of the disease is assessed by clinical disease evidence, determined by clinical evaluations to determine body condition, stool consistency, and composition. Excretion of Serpulina hyodysenteriae in feces, lesions in post-mortem examination, growth rate, and feed conversion ratio are also determined. In the first trial, valnemulin hydrochloride fed at 20, 30, and 40 ppm is compared with tiamulin acid fumarate fed at 30 ppm and with non-medicated controls. Five groups of nine pigs (5 to 6 weeks old) conventionally bred pigs are used for each treatment group.

The pigs are fed non-medicated food for 14 days, and then stimulated with the standard S strain. hyodysenteriae (P18A). The stimulus is orally on 2 consecutive days. The medicated food is introduced on the day after the second stimulus. In the second trial, a similar procedure is followed, except that four groups of approximately 9 pigs are used. These are fed with valnemulin hydrochloride at 5, 10, and 20 ppm, and compared with non-medicated controls. In this trial, the pigs used are from a flock raised outdoors, and are stimulated with a strain of S. hyodysenteriae isolated from an external herd and which previously proved to be capable of causing dysentery in pigs. In both trials, clinical disease evidence is evaluated daily, a clinical score is assigned, rectal samples are taken twice a week for the isolation of S. hyodysenteriae, and all pigs are weighed at regular intervals. The ingestion of food is also recorded. A postmortem examination is performed 21 days after the stimulus, and the large intestine is examined to determine the presence of lesions. Mucosal scrapings from 4 areas in the large intestine are also taken and cultured to determine S. hyodysenteriae. In the first trial, dysentery of clinical pigs was first seen in the non-medicated control group 8 days after the stimulus, and 8 of 9 pigs were affected. Clinical disease was also seen in a pig in the group fed with valnemulin at 30 ppm, and in 2 of 9 animals in the tiamulin group. The evidence of the disease was not seen in the groups treated with 20 and 40 ppm of valnemulin. An average clinical score per pig of 36 was recorded for the control group, compared to a score of 6 for the group treated with tiamulin. The differences between the ratings recorded for all the treatment groups were statistically different from the controls (p <0.001). The . Hyodysenteriae was isolated from rectal samples taken from all pigs in the control group, and also from two affected pigs in the group treated with tiamulin. S. hyodysenteriae was also isolated from 2 pigs that received valnemulin in the feed at 30 ppm, and this coincided with the clinical signs of dysentery of pigs that are subsequently seen as an incidental infection in this group. S. hyodysenteriae was not isolated from the groups of 20 and 40 ppm. There was little difference in the weight gain or feed conversion ratio of all the groups fed the medicated feed. In the post-mortem examination, the control pigs and the group treated with tiamulin that had clinical signs of disease, showed the typical lesions of dysentery of pigs, consisting of adherent mucus with necrosis of the mucosal surface of the large intestine. Lesions were also seen in a pig of the group treated with tiamulin, and in a control pig, both of which had previously not shown clinical signs of dysentery of pigs. The . hyodysenteriae was isolated from mucosal scrapings taken from all these affected animals. A pig of the group fed with valnemulin at 30 ppm also had dysentery lesions of pigs, but S was not isolated. hyodysenteriae of mucous scrapings. No lesions were observed in the post-mortem state in the groups fed with valnemulin at 20 and 40 ppm. In the second trial, dysentery of clinical pigs was first seen in unmedicated control pigs 5 days after the stimulus, and subsequently the nine pigs in the group were affected, and six of these had to be sacrificed due to the severity of the disease. disease. In the group fed with 5 ppm valnemulin, the clinical signs of pig dysentery were first observed 8 days after the stimulus, and 5 out of 10 animals were subsequently affected.

However, the affected numbers in the 5 ppm group of valnemulin were significantly lower than the controls (p <0.05). No clinical signs of the disease were seen in the groups fed with valnemulin at 10 or 20 ppm. The average clinical score of 11 for the 5 ppm group of valnemulin was significantly lower than the score of 77 for the controls (p <0.005). S was isolated. hyodysenteriae of rectal samples taken from all pigs during the trial. However, of the 5 affected animals in the 5 ppm group of valnemulin, S. hyodysenteriae was only isolated from two of these pigs. The pigs fed with valnemulin at all levels were significantly heavier than the controls at the end of the trial (5 ppm, p = 0.05, 10 and 20 ppm, p <0.05, respectively). In the post-mortem examination, the typical dysentery lesions of pigs were observed in the mucosa of the large intestine of the three remaining control pigs. In the group fed with 5 ppm valnemulin, of the five pigs that had originally shown signs of disease, only two of them had lesions on post-mortem examination, but S. hyodysenteriae was not isolated from these animals. However, S. hyodysenteriae was isolated from mucosal scrapings taken from two other animals that had typical lesions at that time, but had not shown the clinical signs of the disease. No lesions were seen in pigs fed valnemulin at 10 or 20 ppm, and S. hyodysenteriae was not isolated from those groups. Therefore, valnemulin at inclusion concentrations down to 10 ppm is effective in preventing the presentation of clinical signs of dysentery of pigs, lodging of spirochetes in faeces, and the presence of lesions on post-mortem examination. At 30 pppt, a few pigs showed the clinical signs of dysentery of pigs, but this coincided with an incidental infection that could have infected the assimilation of the antibiotic. The effects of concurrent disease upon taking the antibiotic, and the incidence of dysentery of pigs, have already been described previously (Burch, D.G.S., Vet. Rec. 110

[1982] 244-246). The analysis of the weight gain data showed no differences between the treated groups, indicating that the antibiotic did not affect the flavor of the food. At 5 ppm, valnemulin did not completely prevent clinical disease or spirochete lodging, but there were significant reductions in clinical scores and spirochete lodging when compared to controls. In this trial, tiamulin fed at 30 ppm did not prevent the dysentery of pigs, but reduced the incidence of the disease when compared to the control group, with a reduction in clinical scores. However, in this trial, the food medication with valnemulin at 10 ppm is clearly more effective than tiamulin at 30 ppm to prevent dysentery of pigs. 4. Another stimulus test: A group of 60 conventionally bred pigs (3.5 to 4 weeks old) were fed non-medicated feed for 14 days, and then stimulated orally with the standard strain of S. hyodysenteriae (P18A) . When the clinical signs of the disease are evident, the pigs are assigned to six treatment groups of 8 pigs per group, and they are fed with food containing antibiotics for 6 days, and then with non-medicated food for another 14 days. Valnemulin hydrochloride is given with food at 50, 75, 100, and 150 ppm, and compared with the tiamulin acid fumarate given with the food at 100 ppm, and with non-medicated controls. Pigs with a range of clinical signs of disease are included in each treatment group. After being assigned to the treatment groups, the clinical disease evidence is evaluated daily, a clinical score is assigned, rectal samples are taken twice a week for the isolation of S. hyodysenteriae, and all pigs are weighed at intervals regular The ingestion of food is also recorded. A post-mortem examination is performed 24 days after the stimulus, and the large intestine of each pig is examined to determine the presence of lesions. Mucous scrapings are also taken from 4 areas in the large intestine, and are cultured to determine S. hyodys in erythrocytes. After the stimulation and before the allocation, clinical dysentery of clinical pigs was observed 7 days after the infection, and the first two treatment groups (control and tiamulin) were formed 8 days after the stimulation. The other groups were formed 12, 15, 20, and 28 days after the stimulus in the following order: valnemulin at 75, 50, 100 and 150 ppm. Each group contained pigs that had blood and / or mucus in their feces, as well as animals that only had light clinical signs of the disease, that is, soft stools. On day 4 of the trial, all pigs in the non-medicated control group were showing severe clinical signs of the disease, and were sacrificed. During the treatment period, in the tiamulin group, a total of three animals died or required euthanasia due to the severe dysentery of pigs. All pigs treated with valnemulin survived until the end of the trial. In the groups fed with valnemulin at all levels, the clinical signs of the disease disappeared quickly, and the clinical disease was not appreciated by day 5 of the trial. By day 8, this improvement was also seen in the surviving pigs fed tiamulin. The average clinical ratings for the valnemulin groups were 4 to 10, compared to an average score of 30 recorded for the tiamulin group. The statistical analysis of the clinical scores for days 3 to 10 showed that there was a significant difference between all the groups fed with valnemulin and the group treated with tiamulin (p <; 0.001). All the pigs fed with valnemulin continued to increase their weight during the treatment period, and all had a greater daily live weight gain (DLWG) than the tiamulin group (range of 0.4 to 0.9, compared to 0.1). The feed conversion ratios (FCR) were much higher in the group treated with tiamulin (6.4), compared to the valnemulin groups (range 1.9 to 2.5) during the treatment period. S was isolated. hyodysenteriae of the majority of the pigs of each treatment group at the time of allocation. Five days after the assignment, S could not be detected. hyodysenteriae in rectal samples taken from pigs of any of the treatment groups. In the subsequent treatment period, a pig from the group fed with valnemulin at 50 ppm showed clinical signs of the disease 4 days after the withdrawal of the medicated feed, and a pig in the group treated with tiamulin also had dysentery of pigs on the last day of the trial. However, S was not isolated. hyodysenteriae of the feces of these pigs. In this post-treatment period, there was little observable difference in daily gain of live weight or in the feed conversion ratios between all treatment groups. In the post-mortem examination at the end of the trial, dysentery lesions of pigs were not observed in any pig fed with valnemulin at 75, 100 or 150 ppm. In the group fed valnemulin at 50 ppm, one pig had clinical dysentery of pigs and another pig of this group had some redness in the large intestine at this time. S was also isolated. hyodysenteriae of the mucosal scrapes of these two pigs, and of 3 other animals of this group that did not have dysentery lesions of pigs. A pig from the group fed with valnemulin at 75 ppm also produced S. hyodysenteriae in the mucosal scrapings, although there was no evidence of clinical dysentery of pigs in this pig. Therefore, valnemulin at 50, 75, 100 and 150 ppm produced a reduction in the number of days that were needed to recover from clinical dysentery of pigs when compared with tiamulin. Also, the animals did not die or require euthanasia in these groups treated with valnemulin. There was a significant difference (p <0.001) in the clinical ratings for the groups treated with valnemulin compared to the tiamulin group. The accommodation of S. hyodysenteriae in the stool was also prevented by all levels of valnemulin after the withdrawal of the medicated feed. However, S. hyodysenteriae was isolated from mucosal scrapings of pigs that received valnemulin at 50 and 75 ppm. It has been shown that tiamulin at 35 and 40 ppm, removes S. hyodysenteriae from feces, but does not prevent the recovery of mucosal scrapings (Taylor, D.J. Vet.Rec.106

[1980] 526-528). Tiamulin at 100 ppm has been used successfully under experimental conditions (Taylor, D.J., Proc. 7th IPVS Consress Mexico

[1982] 47) to treat dysentery of pigs in animals that were not affected so severely as to be inapetent. In this trial, tiamulin at 100 ppm reduced mortality, compared to non-medicated controls, but did not prevent some deaths, and this may have been due to the severity of the disease induced by that experimental stimulus. In this trial, therefore, valnemulin in the feed at 50, 75, 100 and 150 ppm, successfully treated experimentally produced dysentery of pigs, with the prevention of mortality and the elimination of clinical signs. Excretion of S. hyodysenteriae during treatment was prevented, with a recurrence after cessation of treatment in only one animal treated with 50 ppm. Valnemulin at 50 and 75 ppm did not completely eliminate S. hyodysenteriae from all pigs, but at 100 ppm it was highly effective. Accordingly, the agent of the invention is useful in the therapy of dysentery of pigs caused by the infection by Serpulina hyodysenteriae. For this use, the effective dosage, of course, will vary depending on the particular salt used, the mode of administration, the size and age of the animal, and the desired effect; for example, for a prophylactic treatment, relatively low doses would be administered for a long time. However, satisfactory results are generally obtained when the agent is administered in a daily dosage of about 1 milligram / kilogram to about 5 milligrams / kilogram of body weight of the animal, suitably provided in divided doses of 2 to 4 times a day, or to taste in food or water, or in some form of sustained release. For most animals, the total daily dosage is from about 10 milligrams to about 400 milligrams, for example, from about 10 milligrams to about 200 milligrams for prevention, or from about 20 milligrams to about 400 milligrams for treatment, given to taste in food or water, or once or twice a day. Preferred doses in drinking water are from 0.001 to 0.05 weight percent by volume, particularly from 0.001 to 0.005 weight percent, and in food, from 20 to 100 grams / metric ton, particularly from 20 to 75 grams / ton metrics.

C. Colitis of pigs associated with Serpulina piloaicoli infection: Colitis can be diagnosed in a conventional manner, for example as described in veterinary manuals, such as Taylor, D.J., in Pig Diseases. 6th edition (1995), Publ. DJ. Taylor, Glasgow, United Kingdom, on pages 148-149. The beneficial activity of the agent of the invention in this use is determined, for example, as follows: 1. Minimum Inhibitory Concentration Values: Nine field isolates of WBHS from dysentery outbreaks of pigs, from herds with and without diarrhea, and ATCC 29796 (Serpuiin innocens, group 3) (Fellstrom, C, Ras, Vet. Sci.

[1995] 1-4). The identification is based on the pattern of hemolysis on TSA (soy triticase agar) with 5 percent bovine blood, and on the test for the production of indole and hydrolysis of hippurate (Rosco Diagnostic Tablets, Taastrup, DK). Of the nine weakly beta-emolitic spirochetes, one was assigned to group 2, four to group 3, and five to group 4 (Fellstrom, ibid). Bacteria were transferred from agar plates to 0.9 percent serum, and the turbidity was adjusted to 1.0 on the McFarland scale before inoculating 10 microliters of each isolate onto agar plates with concentrations of twice the following antimicrobials: valnemulin hydrochloride, tiamulin acid fumarate, dimetridazole, lincomycin hydrochloride, and tylosin. Growth and haemolysis are recorded after 4 days of anaerobic culture, and the minimum inhibitory concentration is determined as the lowest concentration of antibiotics where spirochetes do not grow. The results of the determinations of the minimum inhibitory concentration for WBHS are shown in Table 6. In general, the WBHS were susceptible to the five antimicrobials. There were no differences in susceptibility between the three WBHS groups for any of the antimicrobials tested. The values of minimum inhibitory concentration obtained for valnemulin are the lowest value for 9 of 10 strains, while they are much higher for the great majority of strains with the four reference compounds. The high susceptibility of valnemulin to both WBHS makes it interesting to be used in the treatment of clinical cases of infected herds.

Table 6 Minimum inhibitory concentration (MIC) values for 10 weakly beta-hemolytic spirochetes 15 The figure is the number of beta-hemolytic spirochetes determined with that value of minimum inhibitory concentration, ch = hydrochloride; hfu = acid fumarate.

Accordingly, the agent of the invention is useful in the therapy of colitis of swine associated with infection by Serpulina pilosicoli. For this use, the effective dosage, of course, will vary depending on the particular salt employed, the mode of administration, the size and age of the animal, and the desired effect; for example, for a prophylactic treatment, relatively low doses would be administered for a long time. However, generally satisfactory results are obtained when the agent is administered in a daily dosage of about 1 milligram / kilogram to about 5 milligrams / kilogram of body weight of the animal, suitably given in divided doses of 2 to 4 times a day, or to taste in food or water, or in a sustained release form. For most animals, the total daily dosage is from about 10 milligrams to about 400 milligrams, for example, from about 10 milligrams to about 200 milligrams for prevention, or from about 20 milligrams to about 400 milligrams for treatment, given to taste in food or water, or once or twice a day. Preferred doses in drinking water are from 0.001 to 0.05 weight percent by volume, particularly from 0.001 to 0.005 weight percent, and in food from 20 to 100 grams / metric ton, particularly from 20 to 75 grams / metric ton .

D) Ileitis in pigs associated with L wsoais intracellularis infection: Lawsonia intracellularis infection can be diagnosed in a conventional manner, for example, as described in veterinary manuals such as Taylor, DJ, in Pig Diseases, 6th edition (1995) ), Publ. DJ. Taylor, Glasgow, United Kingdom, on pages 154-157. The beneficial activity of the agent of the invention in this use is determined, for example, as follows: 1. Activity in vi tro: Generally the method of Lawson, G.H.K. and collaborators, J_. Clin, Microbe! . H (1993) 1136-1142, with some modifications (the standard Kirby-Bauer disc methods for intracellular bacteria are not applicable): Cells: Rat enterocyte cell cultures monolayers IEC-18 (ATCC CRL 1589) are established , and are maintained by standard cell culture methods. 1-day-old monolayers of dividing cells (a confluence of approximately 30 percent) are prepared for infection on day 0, on glass coverslips, in small culture flasks (Traes). Inoculums: Lots of three strains of Lawsonia intracellularis are used as inocula. These are derived from afflicted pigs with proliferative enteropathy, proliferative hemorrhagic enteropathy (PHE), and forms of porcine intestinal adenomatosis (PIA), prepared from pig intestines, and passed to cell cultures as described in the reference of Lawson earlier. The two strains of proliferative hemorrhagic enteropathy were partially tested for pathogenicity in pigs, as described in McOrist S. et al., Infect. Immun. 61 (1993) 4286-4292. The inocula batches are collected after several cell steps, and stored frozen at -70 ° C in 1 milliliter flasks. Preliminary trials established the appropriate dilutions of thawed flasks that would result in easily recognizable cellular infections in 5-day trials. Antibiotics are prepared as lOOx supply solutions for each use. The activity of each batch of antibiotic is confirmed by standard Kirby-Bauer disc methods with a laboratory strain of Escherichia coli. Infections: On day 0, one vial of each strain is thawed, diluted 1/8 to 1/32 with culture medium, to be used in 4 groups 1 to 4. For group 4 (continuous treatment) this inoculum is incubated initially for 1 hour at 37 ° C in a culture medium containing the antibiotic in different concentrations, before being added to the cells. For group 3 (extracellular activity), this inoculum is prepared in a culture medium containing the antibiotic in different concentrations, added immediately before the time of addition to the cells. For groups 1 (control) and 2 (intracellular activity), inocula are prepared in a culture medium free of antibiotics, and added to the cells. The culture flasks (Traes) with cells and added inoculum (+/- antibiotics) in a total of 0.5 milliliters of medium, are placed in steel jars, evacuated to an atmosphere of 40 percent (8 percent of 02) , they are left for 2 minutes, they are filled with hydrogen, and finally with 10 percent C02. The trays are then removed from these jars, and placed in an established incubator to provide a humidified atmosphere of 8 percent of 02, 8.8 percent of C02, and the rest of nitrogen, at 37 ° C. On days 1 and 2, all the bottles are removed and re-fed with new medium, containing antibiotics, groups 2 and 4, or without, groups 1 and 3, and put back in the incubator. On day 5, all coverslips are removed, and stained to determine Lawsonia intracellularis, by indirect immunoperoxidase dye that incorporates a specific monoclonal antibody. The number of cells on each coverslip is used, very infected with Lawsonia intracellularis (>; 30 per cell, highly infected cell = HIC) as the main infection measure. The number of highly infected cell sites and general cellular observations are also recorded. The highly infected cells are compared in the control (group 1), in the intracellularly active antibiotic assay (group 2), in the extracellularly active antibiotic assay (group 3), and in the continuous treatment test (group 4), in trials in triplicate, for up to three strains each. Some tests are repeated for different concentrations of ass. The percentage proportions are calculated after the derivation of highly infected cells from the relevant controls for the test group. The highly infected cells of average control are the total number of highly infected cells in the control traces, divided by the number of infected control trays. The proportion of percentage of Traes test is then calculated by dividing its value of highly infected cells between highly infected cells of average control, multiplied by 100 to give a percentage. That is to say, the test traces where the antibiotics had no effect, would have a percentage ratio of around 100, and where the antibiotics completely inhibit the growth, the proportion would be 0. The results obtained with hydrochloride Valnemulin are as shown in Table 7, which shows the percentage of cells that remain uninfected after treatment: Table 7 Proportions of percentage of Lawßonia intracellularis infection of cell cultures with added valnemulin ch = hydrochloride.

These results indicate that the minimum concentration of valnemulin hydrochloride to cause a significant inhibition of the growth of Lawsonia intracellularis (growth <1 percent) is < 2 micrograms / milliliter. 2. Stimulation study: 35 weaned pigs were stimulated on the same day with a virulent inoculum of Lawsonia intracellularis (strain LR189 / 5/83), an isolate of the causative agent of porcine proliferative enteropathy, obtained by culturing it in the enterocyte cell line. rat IEC-18 (ATCC CRL 1589) as described in Lawson et al., J. Clin. Microbiol. 31 (1993) 1136-1142. Seven control pigs are dosed with a buffer solution. Seven of the 21 stimulated pigs are left untreated. These seven pigs had reduced weight gains, and all developed proliferative enteropathy lesions, detected in sections of the intestines taken at autopsy 3 weeks after the stimulus. Six of these seven pigs had very visible lesions, 3 had mild to moderate diarrhea two weeks after the stimulus. To test a "prevention" dosing strategy, two other groups of stimulated pigs were dosed orally with valnemulin hydrochloride in the doses of 25 ppm and 75 ppm, respectively (ie 1.25 milligrams / kilogram, and 3.7 milligrams / kilogram) by means of a premix given two days before the stimulus, continuously until euthanasia. To test a treatment strategy, two other groups of stimulated pigs are dosed orally with valnemulin at 25 ppm and 75 ppm, by means of a premix given seven days after the stimulus, continuing until euthanasia.

No lesions of proliferative enteropathy were seen in the sections of the intestines taken at autopsy in 3 of 7 pigs in the prevention group, and in 5 of 7 pigs in the treatment group. The control pigs remained normal. Therefore, valnemulin prevented or treated the disease in a large proportion of the stimulated pigs, even in the low actual doses of the medication received. Accordingly, the agent of the invention is useful in the therapy of ileitis in pigs, associated with Lawsonia intracellularis infection. For this use, the effective dosage, of course, will vary depending on the particular salt employed, the mode of administration, the size and age of the animal, and the desired effect; for example, for a prophylactic treatment, relatively low doses would be administered for a long time. However, satisfactory results are generally obtained when the agent is administered in a daily dosage of about 1.5 milligrams / ki or about 6.5 milligrams / kilogram of body weight of the animal, suitably given to taste in water or food, or in divided doses 2 to 4 times a day, or in a sustained release form. For most animals, the total daily dosage is from about 10 milligrams to about 1000 milligrams, preferably from about 15 milligrams to about 500 milligrams, provided once or twice a day. The preferred doses in drinking water are from 0. 001 to 0.05 percent by weight by volume, particularly from 0. 001 to 0.005 percent, and in food, from 20 to 400 grams / metric ton, particularly from 20 to 200 grams / metric ton.

E) Chronic respiratory disease and arthritis in poultry, associated with infection by Mycoplasma galliseptic? M: The infection can be diagnosed in a conventional manner, for example, for infection with Mycoplasma gallisepticum, as described in veterinary manuals such as Diseases of Poultry, 8th edition (1984), in Hofstad et al., Iowa State University Press, pages 196-198. The beneficial activity of the agent of the invention in this use is determined, for example, as follows: 1_. In vitro activity: Minimum inhibitory concentration The antimicrobial effect of valnemulin hydrochloride is determined by means of a standard serial microdilution technique, in comparison with tylosin acid fumarate (soluble Tylan) and tiamulin. 12.8 milligrams of the test compound are dissolved in 100 milliliters of distilled water, to obtain a supply solution, which is stored at -20 ° C until it is used. The reference strains of Mycoplasma gallisepticum used are X95, S6 Holland, S6 Bench (England), MS-16 (Japan), MK-7 (Japan), and 1226, and different fresh field isolates. The sensitivity to the antibiotic of the strains is examined using a modified microcalde dilution method (Stipkovits et al., Vet Microbio! .. 15

[1987] 65-70). The test medium (100 microliters) is added to all the wells of the microtiter plates, using duplicate dilutions of antibiotics. The inoculum (100 microliters) contains 10 colony forming units / milliliter. The plates are sealed with sealing film, and incubated at 37 ° C for a period of 3 days. The lowest concentration of the antibiotic that completely prevents the change of color of the medium in the reading of the third day, is the minimum inhibitory concentration (MIC). The cavities without change of color are verified to determine the viability of the mycoplasmas, coating them on an agar medium. All strains of Mycoplasma are propagated in 10 milliliters of medium B (Ernf, H. and Stipkovits, L., Acta Vet. Scand.

[1973] 436-449). After incubation at 37 ° C until the first stage of trunk growth, aliquots of 1 milliliter of the culture are dosed into tubes as seed cultures, and stored at -20 ° C. The Mycoplasma strains that are fermented in glucose are tested in a Bg modified medium supplemented with 1 percent glucose (pH 7.8), the strains that hydrolyze arginine in a Ba medium (Ernf, H. and Stipkovits, L., supra 450-463) containing 1 percent arginine (pH 7.3), and negative strains for glucose and arginine in a B medium that includes 1 percent triphenyl tetrazolium chloride. The values of average minimum inhibitory concentration are calculated for different groups of strains and antibiotics, taking into account the dilutions. The minimum inhibitory concentration values of the reference strains are shown in Table 8, and those of Mycoplasma isolates in Table 9: Table 8 Minimum inhibitory concentration values of reference strains of Mycoplaama gallisepticum (micrograms / milliliter) ch = hfu hydrochloride = acid fumarate, Table 9 Minimum inhibitory concentration values of mycoplasma isolates of chicken origin (microgram / milliliter) The results obtained with the reference mycoplasma strains indicate that the three compounds show more or less the same range of activity. However, the minimum field inhibitory concentration values of birds reflect a pronounced difference between the test substances: although a uniformly high sensitivity is exhibited against valnemulin (0.03 to 0.06 micrograms / milliliter), sensitivity is reduced somewhat against tiamulin (0.06 to 1 microgram / milliliter), and with tylosin, even resistant strains (0.06 to 32 micrograms / milliliter) are obvious. 2. Stimulation studies: 2.1 Prevention Groups of young male chickens are infected with a virulent strain of Mycoplasma gallisepticum (MG), and each group is medicated with one of the following: valnemulin (concentrations of 0.00312, 0.00625, 0.0125 and 0.025 percent) , tiamulin (0.00625, 0.0125, and 0.025 percent), and tylosin (0.05 percent). The infection is carried out on day 2 by the injection of 0.1 milliliter of Mycoplasma gallisepticum culture containing approximately 106 colony-forming units of viable organisms in each lung (Jordan, F.T.W., The Veterinary Record 127

[1990] 502), and the medication is started within 1 hour of infection, and is continued for 3 consecutive days. There is also an untreated infected group, and an uninfected group. The results obtained show that the prevention of clinical signs and mortality was equally satisfactory for the groups medicated with the two highest doses of valnemulin (0.0125 and 0.025 percent), the three doses of tiamulin, and the concentration of tylosin. Lesions were seen in fewer chickens, and were severe with tylosin, followed by tiamulin, and then the two highest doses of valnemulin (0.0125 and 0.025 percent). The two lowest doses of valnemulin were less effective in this regard. The highest weight gains were for the groups medicated with tylosin; at once, but significantly less (p <0.05) was the group of uninfected birds, those with tiamulin, and those that received valnemulin in all but the lowest dose. The lowest weight gains were obtained with the groups medicated with the lowest dose of valnemulin and the non-medicated infected group, and they were not significantly different. MG was not recovered during the life or autopsy of the uninfected group, chickens with the highest dose of tiamulin and tylosin, and was isolated only from a bird in the highest dose group of valnemulin. A higher proportion of isolates from the other infected groups was made. The serological results did not reflect this entirely for tylosin, since there was a relatively high proportion of positive reactors in this group. Therefore, the two highest doses of valnemulin were as effective as the reference compounds in preventing infection with Mycoplasma gallisepticum in young chickens. 2. 2 Treatment: Groups of young male chickens are infected with a virulent strain of Mycoplasma gallisepticum (MG), and each group is medicated respectively with valnemulin in a concentration of 0.0125 percent, 0.025 percent, 0.05 percent, tiamulin in the same concentration, tylosin at 0.05 percent, and a combination of lincospectin, spectinomycin (Linco-Spectin) at 0.083 percent. There is also an infected group that is left untreated and an uninfected group. Chickens become infected at two days of age by injecting 3.4 x 105 organisms into each lung. The medication in drinking water is started 24 hours later for three successive days, with the exception of Linco-Spectin, with which the treatment was continued for 5 days. The results obtained show that the control of mortality, with no more than 5 percent, was better with the three concentrations of valnemulin, and with tiamulin at 0.05 percent. No lesions were observed in the chickens medicated with the highest and lowest doses of valnemulin, and in only a few that received tiamulin and tylosin. The weight gains for valnemulin at all concentrations were higher than for the tiamulin treated groups, and the same for the tylosin group. Mycoplasmas were not recovered at the end of the experiment at 23 days, from the three groups of birds with valnemulin and those that received tylosin. In the serological results, at this time, there were some positive factors in all the infected groups. Considering mortality, weight gain, MG isolation, and the results of serum agglutination tests, the two highest doses (0.025 percent and 0.05 percent) of valnemulin given in drinking water, they give results at least as good or better than tiamulin at 0.05 percent or tylosin at 0.05 percent, and much better than Linco-Spectin. Accordingly, the agent of the invention is useful in the therapy of chronic respiratory disease and arthritis in poultry associated with Mycoplasma gallisepticum infection. For this use, the effective dosage, of course, will vary depending on the particular salt employed, the mode of administration, the size and age of the animal, and the desired effect; for example, for prophylactic treatment, relatively low doses would be administered for a long time. However, generally satisfactory results are obtained when the agent is administered in the drinking water in dosages from about 0.005 to about 0.05 weight percent by volume, particularly from 0.01 to 0.03 weight percent, and in the feed 20 to 400 grams / metric ton, particularly from 20 to 200 grams / metric ton.

F) Secondary infection with Paateurella muí toe ida, Actinobacillua (Haemophilua). pleurooneumoniae,? / or Haemophilus paraauia: Secondary infection with Pasteurella multocida, Actinobacillus (Haemophilus), pleuropneumoniae, and / or Haemophilus parasuis, can be diagnosed in a conventional manner, for example, as described in veterinary manuals, such as Taylor, DJ, in Pi s Diseases, 6th edition, (1995), Publ. DJ. Taylor, Glasgow, United Kingdom, on pages 185-187; 188-194; and 194-197. The beneficial activity of the agent of the invention in this use is determined, for example, as follows: The minimum inhibitory concentration (MIC) of valnemulin hydrochloride is determined against fresh isolates of Pasteurella multocida, Haemophilus pleuropneumoniae, and Haemophilus parasuis, which is they originate from porcine respiratory material, and are compared with tiamulin. Cultures of bacterial isolates comprising 10 P. multocida, 10 H. pleuropneumoniae, and 3 H, parasuis, isolated within the last five years of diseased pigs evaluated in post-mortem examinations are used. Three of the Pasteurella isolates were toxigenic strains of Pasteurella multocida. For H. pleuropneumoniae and H. parasuis, on the day before the test, a single group of each of the pure cultures is inoculated in 10 milliliters of Todd &; Hewitt (Unipath CM190) containing 1 percent donor calf serum, and 2 milligrams per milliliter of nicotinamide adenine dinucleotide (NAD). For P. multocida, on the day of the test, a single group of each of the pure cultures is inoculated in 10 milliliters of nutrient broth number 2 (Unipath CM67) containing 1 percent calf donor serum. Each of the prepared broth cultures are incubated for 18 hours at 37 ° C in 10 percent C02. After incubation, and on the day of the test, the 23 cultures are adjusted in broth down to 105-106 organisms per milliliter in sterile physiological saline, in preparation for the inoculation of the culture medium in the minimum inhibitory concentration. For each test compound, standard control cultures of Staphylococcus aureus from Oxford NCTC 6571 are also established, to validate the results of the minimum inhibitory concentration tests. In each case, the broth cultures established from the control organisms are identical to those of the organisms under test. Valnemulin is a hydrochloride, thymulin is used as the acid fumarate. On the days of the test, supply solutions of test compounds containing 3.2 milligrams / milliliter of active ingredient in sterile distilled water are prepared. Then, nine double dilutions are prepared in sterile distilled water, giving a range for each antibiotic of 320 micrograms down to 1.25 micrograms per milliliter to be incorporated in the medium of minimum inhibitory concentration. Sensitized agar (Oxoid CM409) containing lysed horse blood (Oxoid SR50) is prepared, according to the manufacturer's recommendations, and adjusted to a pH of 7.4 if necessary. For all batches of the minimum inhibitory concentration medium prepared for the minimum inhibitory concentration assays against the two Haemophilus species, 2 milligrams per milliliter of nicotinamide adenine dinucleotide is added to provide the necessary growth factor. 18 milliliters of molten agar (50 ° C) is mixed with 2 milliliters (one in ten dilutions) of each of the prepared dilutions of the test compound in 90-millimeter Petri dishes. Four replica plates of each dilution are prepared. For each assay of minimum inhibitory concentration, four replica dishes containing 18 milliliters of agar and 2 milliliters of sterile distilled water are also prepared to act as growth indicators. The plates of minimum inhibitory dry concentration are inoculated with 0.2 microliters of prepared organisms, those with P. Multivitamins are incubated in air at 37 ° C for 18 hours, and those inoculated with H. pleuropneumcniae and H. parasuis are incubated at 37 ° C for 18 hours at 10 percent C02. After inoculation, the growth of the organisms is initially examined in a visual manner, followed by microscopic examination in a stereo-zoom microscope. The minimum inhibitory concentration value is taken as the lowest concentration of the test compound in which no growth can be detected at the inoculation point in the four dishes by microscopic examination. The results obtained are as shown in Table 10: Table 10: Minimum inhibitory concentrations (micrograms / milliliter) ch = hfu hydrochloride = acid fumarate, It can be seen that valnemulin, compared to tiamulin, had an average increase of 2.5 times in its activity against P. multocida, an increase of 15.5 times against H. pleuropneumoniae, and a threefold increase against H. parasuis. Accordingly, the agent of the invention is useful in the therapy of secondary pneumonia in pigs associated with infection by Pasteurella multocida, Actinobacillus (Haemophilus), pleuropneumoniae, and / or Haemophilus parasuis. For this use, the effective dosage, of course, will vary depending on the particular salt employed, the mode of administration, the size and age of the animal, and the desired effect; for example, for prophylactic treatment, relatively low doses would be administered for a long time. However, generally good results are obtained when the agent is administered in a daily dosage of about 5 milligrams / kilogram to about 15 milligrams / kilogram of body weight of the animal, suitably administered to taste in water or food, or in Doses divided 2 to 4 times a day, or in a sustained release form. For most animals, the total daily dosage is from about 100 milligrams to about 1000 milligrams, preferably from about 100 milligrams to about 500 milligrams, once or twice a day. It can conveniently be administered as the only therapy. Preferred doses in drinking water are 0.01 to 0.05 weight percent by volume, particularly 0.01 to 0.025 weight percent, and in food, 100 to 400 ppm (grams / metric ton), particularly 100 to 200 ppm .

G) Pneumonia in sheep, sheep, and cattle, associated by infection with Pasteurella aemolytica; Infection by Pasteurella haemolytica can be diagnosed in a conventional manner, for example, as described in veterinary manuals, such as Veterinary Medicine, 8th edition (1994), Editors Radostits, P.M., Blood, D.C. and Gay, C.C., Publ. BaiHiere Tindall, London, United Kingdom, on pages 748-770. The beneficial activity of the agent of the invention in this use is determined in vivo, for example, as follows: Clinical efficacy is determined from two formulations of valnemulin hydrochloride in the treatment pneumonia in calves, due to a controlled infection with Pasteurella haemolytica Al .

On day -6, 24 calves that have been taken a nasal sample, and that are free of P. haemolytica Al, are weighed and assigned to four treatment groups of six, balanced according to live weight, with a uniform distribution of effects between the groups. The calves are housed in individual pens, in a conventional calf breeding unit, where they are acclimated for 33 days before the assignment. Blood samples are taken twice during the phase before the study for the analysis of leukotoxin antibody as a possible indication of previous exposure to P. haemolytica Al. Treatment begins on day -1, approximately 27 hours before the time of treatment. stimulus: Group 1 - control; Group 2 - 2.5 percent injectable valnemulin hydrochloride (2.5 mg / kg, twice daily). Group 3 - premix of 10 percent valnemulin hydrochloride orally using the calf milk replacer (5.0 milligrams / kilogram, twice a day). All the calves are stimulated on day 0 by the endobronchial deposit of 30 milliliters of culture in broth of Pasteurella Haemolytica Al M4 / 1/3, Moredun Research Institute - 1.25xl07 colony forming units / milliliter), by means of a fiber optic bronchoscope. Clinical examinations are performed once a day on days -3 and -2, and twice a day on days -1 to 3. Clinical ratings are assigned to each of four parameters: rectal temperature, respiratory rate, nature of the breathing, and behavior. Injection sites are also examined. Any calves that are recumbent, and / or show depression and / or signs of respiratory failure, or have a total score greater than 5 in a single examination between days 0 and 3, are euthanized immediately in a humane manner by intravenous administration of a lethal dose of Pentobarbitone-sodium BP (Vet) at 20 percent weight / volume, and the autopsy is performed within 24 hours. The calves that survive until day 4 after the stimulation with P. haemolytica are euthanized on day 4 following the same procedure as for those who were euthanized between days 0 and 3. The lungs of all calves, and visually evaluated to determine the consolidated lesions and pleuritic adhesions. Lung tissue samples are cut from eight standard sites, and tested for the presence of P. haemolytica, in order to determine a value for the isolation index. Blood samples are taken from the heart of calves that die acutely, and are tested for the presence of P. haemolytica. Individual and group calf totals, group averages, and group means are calculated for all parameters: total clinical score, consolidated injury score, isolation rate, pleurisy score, and total score. Individual and group calf totals, group averages, and group means are calculated for all categories, forming a total clinical score: rectal temperature, respiratory rate, nature of breathing, and behavior. The basic analysis is applied by the Kruskal-Wallis test (Minitab 9) to determine if there is a difference between any group ratings for each of the parameters: total clinical score, consolidated injury score, isolation rate, pleurisy score , and total rating. The meaning of the Kruskal-Wallis test indicates that the groups are not all the same, nor are they all different. To test which groups are different, pairs of groups are tested using the Mann-Whitney test (Minitab 9). Compared with the untreated calves (group 1), it was found that the total clinical scores (p = 0.013) were significantly lower in the calves that had been treated with a premix of 10 percent valnemulin, at 5.0 milligrams / kilogram / Dosage twice a day by calf milk replacer for 5 days, starting 27 hours before stimulation with P. haemolytica Al. Total clinical scores were also reduced, but were not statistically significant, in calves treated intramuscularly with valnemulin 2.5 percent injectable at 2.5 milligrams / kilogram / dose twice daily for 5 days, beginning 27 hours before stimulation with P. haemolytica Al. There were no significant differences between the groups that received treatment when total clinical scores were compared.

Accordingly, the agent of the invention is useful in the therapy of pneumonia in sheep, sheep, and cattle, associated with infection by Pasteurella haemolytica. For this use, the effective dosage, of course, will vary depending on the particular salt employed, the mode of administration, the size and age of the animal, and the desired effect; for example, for prophylactic treatment, relatively low doses would be administered for a long time. However, satisfactory results are generally obtained when the agent is administered in daily dosages of about 5 milligrams / kilogram to about 15 milligrams / kilogram of body weight of the animal, suitably given to the taste in the food, or in divided doses of 2. to 4 times a day, or in a sustained release form. For most animals, the total daily dosage is from about 250 milligrams to about 3000 milligrams, preferably from about 250 milligrams to about 1000 milligrams, given once or twice a day, co-administered to the taste in the imento.

H) Polyarthritis in pigs associated with infection by Mycoplaama hvoavnovíme: Mycoplasma hyosynoviae infection can be diagnosed in a conventional manner, for example, as described in veterinary manuals, such as Taylor, DJ, in Pig Diseases, 6th edition (1995) ), Publ. DJ. Taylor, Glasgow, United Kingdom, pages 172-173. The beneficial activity of the agent of the invention in this use is determined, for example, as follows: 1. Activity in in vitro tissue isolates (minimum inhibitory concentration test: tissue samples are obtained from the lungs of pigs from different flocks affected with enzootic pneumonia of pigs (PPE) .The samples are shipped on dry ice for cultivation. from lung samples initially isolated to recover Mycoplasma hyopneumoniae, they were again isolated for Mycoplasma hyosynoviae after it was noticed, from the lung samples submitted (restored at -70 ° C), during culture for Mycoplasma hyosynoviae, which some showed an exuberant growth of colonies that had the typical morphology of Mycoplasma hyosynoviae Three more isolates were recovered from other lung samples provided All isolates were identified serologically by disc growth inhibition with specific rabbit antiserum raised against Mycoplasma hyosynoviae A solid medium is used commercially available (Mycoplasma Experience Ltd.) to isolate? f. Hyosynoviae of lung tissue. A liquid medium (Mycoplasma Experience Ltd.) containing phenol red and arginine (pH 7.0) is used for the minimum inhibitory concentration test. Preparation solutions of the test compound are prepared in a concentration of 1 milligram / milliliter in deionized water, sterilized by filtration through 0.2 micron pore size membrane filters (Sartorius Minisart, N), and stored at - 20 ° C. For use in the minimum inhibitory concentration tests, the supply solutions are diluted in a liquid medium to double the final concentrations required. Minimum inhibitory concentration tests are performed according to the method of Tanner and Wu, Avian Diseases 36 (1992) 714-717. Active stimulus cultures are prepared, either from aliquots of 1 milliliter of broth cultures stored at -70 ° C, or from cultures stored in agar at -70 ° C. The stimulus cultures are diluted to give an objective titration of 103 to 105 units of color change / milliliter. Aliquots of 0.1 milliliter of stimulus inoculum are mixed with 0.1 milliliter aliquots of antibiotic dilution in microtitre cavities. Each microtitre cavity contains the non-inoculated medium at a pH of 7.6 (endpoint control), and inoculated stimulus controls free of antibiotic. All plates are sealed with adhesive film, and are incubated aerobically at 36 ° C. Minimum inhibitory concentrations are recorded when the color change in the stimulus control cavities matches the pH of the endpoint control. The minimum inhibitory concentration is the lowest concentration that shows no color change. The results obtained are shown in Table 11 for valnemulin hydrochloride and two reference compounds, tiamulin acid fumarate and enrofloxacin: Table 11 Sensitivity in vi tro of seven field isolates of Mycoplaama hyosynoviae The sensitivities of seven field isolates of Mycoplasma hyosynoviae to valnemulin are much higher than for the other two compounds, indicating that nt field isolates are extremely sensitive to valnemulin. Accordingly, the agent of the invention is useful in the therapy of polyarthritis in pigs associated with infection by Mycoplasma hyosynoviae. For this use, the effective dosage, of course, will vary depending on the particular salt employed, the mode of administration, the size and age of the animal, and the desired effect; for example, for a prophylactic treatment, relatively low doses would be administered for a long time. However, generally satisfactory results are obtained when the agent is administered in a daily dosage of about 5 milligrams / kilogram to about 15 milligrams / kilogram of body weight of the animal, suitably administered to taste in water or food, or in divided doses 2 to 4 times a day, or in a sustained release form. For most animals, the total daily dosage is from about 100 milligrams to about 1000 milligrams, preferably from about 100 milligrams to about 500 milligrams, once or twice a day. It can be conveniently administered as the only therapy. Preferred doses in drinking water are 0.01 to 0.05 weight percent by volume, particularly 0.01 to 0.025 weight percent, and in food 100 to 400 ppm (grams / metric ton), particularly 100 to 200 ppm ( grams / metric ton). For all those uses, the compound can be used in free base form or in a veterinarily acceptable salt form, for example, the quaternary salt, or especially in an acid addition salt form. These salt forms exhibit the same order of activity as the free base form. Examples of suitable acid addition salts are the acid fumarate, the fumarate, the naphthalene-1-sulphonate, and especially the hydrochloride. The agent of the invention can be administered orally, locally, or parenterally, and mixed with conventional chemotherapeutically acceptable diluents and vehicles, and optionally other excipients, and administered in forms such as tablets, capsules, or injectable preparations. It also forms an excellent additive for food mixes (such as a premix), or for drinking water. Preferred veterinarily acceptable carriers include, for example, pharmaceutical excipients commonly used as sugar, corn starch, lactose, cellulose, as well as grain vehicle systems and grain by-products such as ground rice husks, wheat debris and soy flour. in addition, solid diluents such as ground limestone, sodium sulfate, calcium carbonate, and kaolin, or liquid substances such as veterinarily acceptable oils (vegetable oils or mineral oils), propylene glycol, and polyethylene glycol. Suitably, these formulations are administered to the animal orally, preferably mixed in the feed in the form of a medicated feed, or medicated granules. For applications in drinking water, water solutions are used with or without solvents, such as ethanol, propylene glycol, polyethylene glycol, approved liquid surfactants, and sorbitol. For example, for intramuscular injection, the formulation is usually prepared as a solution in water, which may contain solvents such as ethanol or propylene glycol, or in a veterinarily acceptable oil. Examples of the acceptable oils are sesame oil, medium chain triglycerides (for example Miglyol), isopropyl myristate, and ethyl oleate. The formulation may contain preservatives, pH regulators, and other common excipients. Veterinary formulations for use in the present invention can be prepared by mixing the ingredients in the required proportions. The formulation is then packaged in an appropriate container ready for administration. The following example illustrates the invention: The agent of the invention is well tolerated. The acute toxicity was determined in the rat, of the compound of formula I in the hydrochloride salt form, with single doses of 1000 milligrams / kilogram and 2000 milligrams / kilogram orally administered to five male rats and five female rats per dosage group . The deaths occurred within 1 to 8 days. The D50 value obtained is > 1000 milligrams / kilogram orally.

Claims (13)

1. The use of the compound of the formula I in the form of free base or of veterinarily acceptable salt, in the therapy of veterinary diseases whose expression is improved by increasing the density of supply.

2. The use of the compound of the formula I as defined in claim 1, in free or veterinarily acceptable base form, in the therapy of enzootic pneumonia in pigs caused by Mycoplasma hyopneumoniae infection.

3. The use of the compound of the formula I as defined in claim 1, in the form of free or veterinarily acceptable base, in the therapy of colitis of pigs associated with infection by Serpulina pilosicoli.

4. The use of the compound of the formula I as defined in claim 1, in the form of free or veterinarily acceptable base, in the therapy of ileitis in pigs associated with Lawsonia intracellularis infection.

5. The use of the compound of the formula I as defined in claim 1, in free base or veterinarily acceptable form, in the therapy of chronic respiratory insufficiency and arthritis in poultry, associated with Mycoplasma gallisepticum infection.

6. The use of the compound of the formula I as defined in claim 1, in the form of free or veterinarily acceptable base, in the therapy of secondary infection with Pasteurella multocida, Actinobacillus (Haemophillus), pleuropneumoniae, and / or Haemophilus parasuis .

7. The use of the compound of the formula I as defined in claim 1, in the form of free or veterinarily acceptable base, in the therapy of pneumonia in sheep, sheep, and cattle, associated with infection by Pasteurella haemolytica.

8. The use of the compound of the formula I as defined in claim 1, in the form of free or veterinarily acceptable base, in the therapy of polyarthritis in pigs associated with Mycoplasma hyosynoviae infection.

9. The use according to claims 1 to 8, of the compound of the formula I, in the form of the hydrochloride acid addition salt. The use of the compound of the formula I as defined in claims 1 or 9, for the manufacture of a medicament for use in the therapy of veterinary diseases whose expression is improved by the increase in the density of supply. 11. A method of treating veterinary diseases whose expression is improved by increasing the density of supply, which comprises administering a therapeutically effective amount of the compound of formula I as defined in claims 1 or 9, to pigs that need such treatment. 12. A veterinary agent for use in the therapy of veterinary diseases whose expression is enhanced by the increase in the feed density, which comprises the compound of the formula I as defined in claims 1 or 9, together with at least one veterinarily acceptable vehicle or diluent. 13. A process for the preparation of a medicament for use in the therapy of veterinary diseases whose expression is enhanced by the increase in the density of supply, which comprises mixing the compound of the formula I as defined in claims 1 or 9 , together with at least one veterinarily acceptable vehicle or diluent.