RU2539105C1 - Preparation for treatment of necrotic stomatitis of cattle - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: preparation for treatment of necrotic stomatitis of cattle comprises multivalent toxoid against clostridioses in sheep, culture liquid of the strain Escherichia coli A-5 with the content of microbial cells of 7.0·10⁸-1·10⁹ CFU/ml and the culture liquid of the strain Escherichia coli B-5 with the content of microbial cells of 7.0·10⁸-1·10⁹ CFU/ml in the ratio of 1:1:1.

EFFECT: invention enables to improve the efficiency of treatment and to reduce the treatment period.

3 tbl, 3 ex

Description

The invention relates to the field of veterinary medicine, in particular to means for the treatment of necrobacillosis of cattle.

Necrobacteriosis (necrobacteriosis) is a chronic infectious disease characterized by purulent-necrotic lesions of the skin and underlying tissues, localized mainly in the distal parts of the hind limbs, and in some cases in the oral cavity, genitals, udder, liver, lungs, muscles and other organs and tissues. The disease is common in all parts of the world, including in Russia. Necrobacteriosis is especially dangerous for cattle.

The causative agent of the disease - Fusobacterum necrophorum - a strict anaerobic, is a gram-negative, motionless, non-spore and capsule-forming rod, highly polymorphic. It is stored up to 60 in soil and manure, and up to 15 days in water and urine (http: // necrobacterin http://blogspot.ru/2010/04/necrobacteriosis.html).

A known drug for the treatment of animal necrobacteriosis (see RF patent No. 2295332, class IPC A61K 31/045, published March 20, 2007) contains chloramphenicol, tylosin, metronidazole, a solution of hydrochloric acid, stabilizers - sodium formaldehyde sulfoxylate and sodium sulfite and solvents are benzyl alcohol and 1,2-monopropylene glycol.

This drug is a multicomponent chemical preparation. It has a number of side effects (causes allergies, disorders of the gastrointestinal tract, etc.).

Known hyperimmune serum for the treatment of animal necrobacillosis (see RF patent No. 2195318 according to IPC class A61K 39/40, published on December 27, 2002), containing antibodies to Fusobacterium necrophorum in a titer of at least 1: 128 in the agglutination reaction and obtained by hyperimmunization of oxen producers of inactivated formalin leukocidin-exotoxin, which is a highly purified protein with a molecular weight of 18-20 kDa and isolated from the production strain Fusobacterium necrophorum "0-1" VIEV.

Serum provides a single administration of the creation of intense passive immunity against necrobacteriosis in cattle.

However, its effect is limited in time (no more than 10-14 days), since serum antibodies are eliminated, and repeated administration of serum can cause sensitization of the animal organism. In addition, the cost of serum is high, and treatment requires a significant amount of its introduction.

Closest to the claimed is the drug "Nekovak" against necrobacteriosis of cattle (see RF patent No. 2098127 according to class IPC A61K 39/116, publ. 10.12.1997) containing formalin-inactivated cultures of microorganisms Fusobacterium necrophorum, Staphylococcus aureus, Coryneb, Coryneb pyogenes, Clostridium perfringens type A, selected by the breadth of the immunogenic spectrum and selected according to the maximum immunogenic and antigenic activity. The drug is administered to animals subcutaneously in a dose of 5 cm ³ twice with an interval of 28-30 days.

However, this drug is multicomponent (contains 7 inactivated cultures of various strains of microorganisms) and does not contain toxin-forming components of microorganisms, which are crucial in the treatment of necrobacteriosis in cattle.

The prototype uses a set of cultures of microorganisms, including against the causative agent of necrobacteriosis (Fusobacterium necrophorum) and complicating microflora (Staphylococcus aureus, Corynebacterium pyogenes, Clostridium perfringens type A). The use of this vaccine in successful farms will protect animals from necrobacteriosis and complicating microflora. In dysfunctional farms, the therapeutic effect of using this drug will not be effective, since the presence of Fusobacterium necrophorum in the vaccine will provoke an infectious process in latent animals and exacerbate the course of the disease.

The presence in the preparation of other types of bacteria (Staphylococcus aureus, Corynebacterium pyogenes, Clostridium perfringens type A), which should neutralize the effect of complicating factors in necrobacteriosis, will not neutralize toxins.

In addition, the drug can cause inflammatory reactions at the injection site.

The invention is aimed at solving the problem of creating a drug for the treatment of necrobacteriosis with high therapeutic efficacy, taking into account the variety of toxin-forming factors due to the synergism of its components of microorganisms, which does not provoke an infection process in latently infected animals and inflammatory reactions while shortening the treatment time.

To solve the problem, a preparation for the treatment of cattle necrobacteriosis containing inactivated cultures of microorganisms according to the invention further comprises a polyvalent toxoid against sheep clostridiosis, and as culture of microorganisms it contains a culture fluid containing microbial cells of 7.0 · 10 ⁸ -1 · September ¹⁰ cfu / ml, the resulting culture of the strain Escherichia coli a-5, and a culture liquid containing the microbial cells 7.0 × 10 ⁸ -1 x 10 ⁹ cfu / ml, the resulting cultured anija Escherichia coli strain B-5 (DEP) at a ratio of polyvalent toxoid, culture fluids from strains of Escherichia coli A-5 and Escherichia coli B-5 1: 1: 1.

The sources of patent and scientific literature known to the authors do not describe a drug for the treatment of cattle necrobacteriosis based on a mixture of polyvalent toxoid against sheep clostridiosis with culture fluids obtained from E. coli A-5 and E. coli B-5 strains taken in 1: 1: 1 ratio.

The main etiological factor in necrobacteriosis at the initial stage of the disease is anaerobes, which multiply without oxygen and secrete potent toxins.

These toxins provoke the multiplication of other opportunistic and pathogenic microflora, including Escherichia. They, in turn, also secrete toxins and complicate the course of the underlying disease.

It is known to use for the treatment of necrobacteriosis a preparation containing Clostridium perfringens type A (see RF patent No. 2098127, class IPC A61K 39/116, publ. 10.12.1997).

On the other hand, it is known to use the culture supernatant of Clostridium perfringens type C to stimulate cross-immunity against Clostridium perfringens type A infection and, as a result, to protect animals from Clostridium perfringens type A infection (see RF patent No. 2462263, class IPC A61K 39 / 08, publ. 09/27/2012). In addition, it is also known to use a combination vaccine containing Clostridium perfringens type C culture supernatant and components of the Escherichia coli vaccine to increase the effectiveness of animal protection against Clostridium perfringens type A.

The authors proposed for the first time to use, as the main component of the drug for the treatment of necrobacteriosis, Clostridium anatoxin against sheep (containing Clostridium perfringens type C and D toxoids, Clostridium oedematiens and Clostridium septicum vaccines) in combination with toxin-forming strains Escherichia coli A-5 and thermostab coli B-5, producing a thermolabile toxin, which together provide the neutralization of toxins of microorganisms, especially the intestinal group (Escherichia), complicating the course of necrobacteriosis.

Thus, the essence of the invention lies in the effect of the drug not on the causative agent of the disease (Fusobacterium necrophorum) and not on the toxins that it emits, in contrast to conventional drugs for the treatment of necrobacteriosis, and its action is aimed at enhancing immunity against toxins released by complicating microflora, but namely: facultative and obligate anaerobes (clostridia - Clostridium perfringens types C and D, Clostridium oedematiens and Clostridium septicum) and intestinal aerobes that secrete thermolabile and thermostable toxins.

Thus, the unknown properties of the drug for the treatment of necrobacteriosis to neutralize the effects of toxin-forming complicating microflora without provoking an infectious process in latently infected animals allows us to conclude that there is a criterion of "inventive step" in the claimed invention.

Polyvalent toxoid against clostridiosis of sheep, manufactured by FSUE "Stavropol Biofactory" according to STO 00482861-0010-2006, is intended for subcutaneous administration to adult sheep and lambs. It consists of hydrated alumina and concentrated toxoids Clostridium perfringens types C and D, Clostridium edematiens and Clostridium septicum vaccine. It is a yellow liquid. Designed for the prevention of clostridiosis in sheep.

The strains of E. coli A-5 and E. coli B-5 were isolated from the parenchymal organs of a fallen piglet of 10 days of age, and are stored at the State Scientific and Research Institute of the Saratov Scientific Research Veterinary Institute of the Russian Academy of Agricultural Sciences (State Scientific and Research Institute of Saratov NIVI of the Russian Agricultural Academy).

The strain E.coli B-5 was deposited at the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines and Feed (FSI VGNKI): certificate of deposit No. 18707/15 of 10.10.2002.

The properties of the strains are presented in table 1.

Table 1 Properties of E. coli A-5 and E. coli B-5 strains Strain properties Strain E.coli A-5 Strain E.coli B-5 Cultural and morphological characters On MPB, cells grow diffusely; on MPA, colonies of S- and R-forms. On agar Endo raspberry colony On MPB, cells grow diffusely; on MPA, colonies of S- and R-forms. Endo agar colonies are dark red Enzymatic properties Microorganism: motile, ferments glucose, lactose, mannitol, maltose. Does not ferment sucrose. Microorganism: mobile, possesses catalase activity, gives an affirmative reaction with methylroth, produces indole, ferments glucose, lactose, arabinose, mannitol, maltose, xylose, sorbitol, dulcite. Toxigenic properties Produces thermostable exotoxin Produces thermolabile exotoxin. Antibiotic sensitivity Sensitive to: ampicillin, carbenicillin, chloramphenicol, furadonin, rifampicin. Sensitive to: polymyxin, streptomycin, chloramphenicol, rifampicin, carbenicillin;

Not sensitive to: tetracycline, oleandomycin, oxacillin, cefazolin, cefatoxin. not sensitive to: oxacillin, doxycycline, lincomycin, tetracycline, oleandomycin, Weakly sensitive to
polymyxin. ampicillin, penicillin. Virulent properties LD ₅₀ - 4.0 · 10 ⁹ m.k./cm ³ LD ₅₀ - 1.2 · 10 ¹⁰ shm.k. / cm ³ The death of white mice occurs within two days. The death of white mice occurs within 24 hours. Antigenic properties The culture is heterogeneous in antigenic properties. Some clones agglutinate serum 0-1 The culture is heterogeneous in antigenic properties. Some clones give agglutination with serum 0-4.

The drug is prepared as follows.

1. First, two culture fluids are prepared: on the basis of strains E.coli A-5 and E.coli B-5 with a content of microbial cells of 7 · 10 ⁸ -1 · 10 ⁹ CFU / ml each.

For this, daily diurnal broth cultures of strains are sown separately on a nutrient medium and their growth is monitored.

The criterion for choosing the preparedness of culture (toxin-containing) fluids with exactly such a number of microbial cells (7 · 10 ⁸ -1 · 10 ⁹ CFU / ml) was their maximum viability, due to different periods of production of the required amount of thermostable and thermolabile toxins.

It was experimentally established that the maximum cell viability (7 · 10 ⁸ -1 · 10 ⁹ CFU / ml in the culture fluid) during the cultivation of E. coli A-5 strain producing thermostable toxin was achieved at the initial stage of toxin formation, and in the culture fluid with culturing E. coli strain B-5, producing a heat-labile toxin, the maximum cell viability (7 · 10 ⁸ -1 · 10 ⁹ CFU / ml in the culture fluid) was achieved at a later stage.

2. Upon reaching the required number of microbial cells, formalin is added to each culture fluid, the mixture is stirred and placed in a thermostat for 14 days. Then check for sterility.

3. Mix in equal proportions the polyvalent toxoid against sheep clostridiosis and two inactivated sterile culture fluids with a given number of microbial cells. The components are thoroughly mixed and the suspension is poured into vials.

The finished product is a light straw-colored liquid with a white precipitate and a specific odor, and has a slightly acidic reaction. When shaking, an opalescent suspension is formed.

The drug is stored in a dry, dark place at a temperature of 2 to 15 ° C. Shelf life 18 months.

The drug is administered to animals 3-5 times intramuscularly at 7 ml per head in the area of the croup, regardless of the weight and age of the animals with an interval of 2-4 days.

Example 1

An example of the preparation of culture fluids from strains of E. coli A-5 and E. coli B-5.

We took two containers with a nutrient medium warmed up to 37 ° C, in one of which the daily broth culture of E. coli strain A-5 was introduced, and in the other, E. coli strain B-5. The cultures were introduced per 1 liter of medium, 1 ml of broth containing 3-5-10 microbial cells.

The culture medium on which the strain culture was seeded consisted of 4 components: a base, a sprat hydrolyzate digest, a glucose solution and a Hanks solution.

In this case, the basis of the nutrient medium was prepared as follows. 9 g of NaCl, 1 g of ammonium chloride (NH ₄ Cl) and 10 g of peptone were taken per 1 liter of distilled water, the solution was brought to a boil, filtered, sterilized at 126 ° C (1.5 atmospheres) for 30 minutes.

A sprat hydrolyzate digest was also prepared at the rate of 1 liter of distilled water. To do this, took 40 g of fish paste, stirred in water, filtered, poured into vials and sterilized at 121 ° C (1 atmosphere) for 20 minutes.

A glucose solution (40%) was autoclaved at 112 ° C (0.5 atmosphere) for 20 minutes.

To obtain the required amount of nutrient medium, the components were mixed in the following percentages: base 71%; digest of sprat hydrolyzate 12%; glucose solution 3% and Hanks solution 14%.

Cultures of strains of E. coli A-5 and E. coli B-5 were incubated on this nutrient medium in an incubator at 37 ° C and monitored the growth of cultures.

Observation data are presented in table 2.

The name of the culture fluid The number of microbial cells, CFU / ml after 2 days after 4 days on the 7th day culture fluid from E. coli strain A-5 2.7 · 10 ⁵ -4.3 · 10 ⁵ 5.8 · 10 ⁸ -7.6 · 10 ⁸ 7.0 · 10 ⁸ -1.0 · 10 ⁹ culture fluid from E. coli strain B-5 3.5 · 10 ⁵ -4.2 · 10 ⁵ 5.5 · 10 ⁷ -8.1 · 10 ⁷ 7.0 · 10 ⁸ -1.0 · 10 ⁹

From table 2 it follows that when cultured on this nutrient medium, the required amount of toxins in both culture fluids from strains of E. coli A-5 and E. coli B-5 is reached on day 7.

When conducting studies on a different nutrient medium, the cultivation process may differ, but the indicated concentrations of microbial cells in culture fluids must be achieved, which determine the required amount of toxins for cultures of E. coli A-5 and E. coli B-5.

Upon reaching the required number of microbial cells, formalin was added to each container with culture fluid at the rate of 0.4% of the volume of the nutrient medium (in this case, 4 ml per 1 liter of medium), mixed and placed in a thermostat for 14 days at a temperature of 37 ° C.

Next, sterility samples were taken from each container.

Example 2

Proof of the ratio of the components of the drug.

At different departments of one of the dysfunctional farms, the sick animals were treated with a drug whose components were taken in different ratios.

In all cases (with a different ratio of components), the drug was administered three times in a dose of 7 ml per head with an interval of 3 days.

Branch number in the household Number of animals Number of recovered animals The ratio of the components of the drug clostridial toxoid: culture fluid from E.coli A-5: culture fluid from E.coli B-5: total in the department including sick goals % one. 798 23 eighteen 78.3% 1: 0.5: 0.5 2. 878 eighteen 12 66.6% 0.5: 1: 1 3. 421 10 9 90.0% 1: 1: 1

Thus, the greatest treatment effectiveness (90%) was achieved with the introduction of the drug with components taken in equal proportions 1: 1: 1.

In addition, selectively, in each department of this farm, additional treatment of clinically healthy animals was carried out with a preparation containing components in equal proportions: 50 goals were processed in 1 department, 60 goals in the second, and 3-30 goals. Processing was also performed three times with a dose of 7 ml with an interval between injections of 3 days.

Of the 140 treated healthy animals, no clinical signs of necrobacteriosis were recorded, which indicates the absence of an infection provocation in latently infected animals, in contrast to the use of known vaccines containing the causative agent of necrobacteriosis and causing the activation of the disease in dysfunctional farms .

At the injection site of the drug, inflammatory reactions were not registered in any case.

Example 3

Tests of the drug were carried out in 3 farms unsuccessful for necrobacteriosis, where 312 sick animals were recorded.

The drug (with the content of components in equal proportions) was administered three times in a dose of 7 ml per head with an interval of 2-4 days.

Out of 312 sick animals, 268 were cured at different stages of the disease, which amounted to 86%, while in one household therapeutic efficacy was 99%, in the second - 96%, in the third - 71%.

Thus, the claimed drug for the treatment of necrobacteriosis of cattle has high therapeutic efficacy, takes into account the variety of toxin-forming factors due to the synergism of its components of microorganisms, does not provoke an infection process in latently infected animals, does not cause inflammatory reactions at the injection site and reduces the duration of treatment is 2-2.5 times.

Claims (1)

A preparation for the treatment of cattle necrobacteriosis containing inactivated cultures of microorganisms, characterized in that it additionally contains a polyvalent toxoid against sheep clostridiosis, and as microorganism cultures it contains a culture fluid containing microbial cells of 7.0 · 10 ⁸ -1 · 10 ⁹ CFU / ml obtained from the cultivation of the strain Escherichia coli A-5, and a culture fluid containing microbial cells of 7.0 · 10 ⁸ -1 · 10 ⁹ CFU / ml obtained from the cultivation of the strain Escherichia coli B-5, when wearing polyvalent toxoid, culture fluids from strains of Escherichia coli A-5 and Escherichia coli B-5 1: 1: 1.