RU2589819C1 - VACCINE AGAINST INFECTIOUS BOVINE KERATOCONJUNCTIVITIS BASED ON ANTIGENS OF Moraxella bovis and Moraxella bovoculi BACTERIA - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention is intended for prevention of infectious keratoconjunctivitis in cattle. As antigens vaccine contains inactivated suspension of cells of Moraxella bovis “G97-VNIVI” bacteria strains with concentration of 100-120 bln. m.c. at 1 cm³ and Moraxella bovoculi “SH-CH6 No.-DEP” with concentration of 100-120 bln. m.c. at 1 cm³ taken in equal proportions, on a physiological solution, as an adjuvant-6 % aluminium hydroxide gel, inactivator is formalin in the following components ratio per 1 l of the vaccine: suspension cell strain of bacteria Moraxella bovis “G97-VNIVI” with concentration of 100-120 bln. m.c. in 1 cm³, cm³ - 90.0-100.0; suspension cell strain of bacteria Moraxella bovoculi “SH-CH6 No.-DEP” with concentration of 100-120 bln. m.c. in 1 cm³, cm³ - 90.0-100.0; aluminium hydroxide gel 6 %, cm³ - 90.0-100.0; formalin, cm³ - 4.0-5.0; physiologic saline, l - 1.

EFFECT: invention is highly effective for preventing infectious keratoconjunctivitis in cattle.

1 cl, 3 tbl, 1 ex

Description

The invention relates to the field of veterinary microbiology and biotechnology, in particular to the production and use of a biological preparation against infectious keratoconjunctivitis in cattle. The vaccine is intended for specific prophylaxis of infectious cattle conjunctivitis caused by the bacteria Moraxella bovis and Moraxella bovoculi.

Infectious keratoconjunctivitis of cattle (ICC) is an acute contagious disease characterized by lacrimation, conjunctival vascular hyperemia, photophobia, serous-purulent outflow, clouding and ulceration of the cornea, deformity of the eyeball in the form of keratogon or keratoglobus palatus, or keratogloidus. The disease is widespread in all countries of the world and causes significant economic damage to livestock farms due to a decrease in milk yield by 50%, an increase in body weight by 31-37%, mortality, as well as the costs of veterinary, sanitary and therapeutic measures. We found that the incidence of calves of 2-6 months of age in individual farms reaches 85%, among fattening livestock - 22% and among dairy herds - in the range of 8-10%.

The disease is caused by hemolytic strains of bacteria Moraxella bovis (Gaffarov Kh.Z., Valebnaya L.V., Spiridonov G.N. Infectious keratoconjunctivitis of cattle in the Middle Volga and Ural regions / Mater. International scientific-practical conference: Topical problems of young diseases in modern conditions - Voronezh. - 2002. - P. 188-191; Karaichentsev VN, Dunaev GV, Rusinov AF, Babenko OV Isolation of Moraxella bovis from young cattle cattle with infectious keratoconjunctivitis / Veterinary medicine. - 1992. - No. 2. - P. 26-27; Rusinov AF. Infectious keratococcus bovine conjunctivitis / Information Bulletin, Ukrainian Academy of Agricultural Sciences, Institute of Experimental Clinical Veterinary Medicine, 1995; Spiridonov GN, Infectious keratoconjunctivitis in cattle // Problems of prevention and control of especially dangerous , exotic and poorly studied infectious diseases of animals. - Proceedings of the international scientific production conference dedicated to the 50th anniversary of VNIIVViM. - Volume 2. - Veil. - 2008. - P. 195-197. Arora A.K. Studies on Moraxella bovis isolated from cattle affected with or convalescing from infectious bovine keratoconjunctivitis / Vet. Arhiv. - 1989. - V. 50. - No. l. - P. 17-23).

In recent years, it has been established that infectious keratoconjunctivitis in cattle can be caused by other representatives of the genus Moraxella, in particular bacteria Moraxella bovoculi (Angelos JA, Spinks PQ, Ball LM, George LW Moraxella bovoculi sp.nov., Isolated from calves with infectious bovine keratoconjun // Int J Syst Evol Microbiol - 2007 Apr; 57 (Pt 4): 789-95; Galvão KN, Angelos JA, Ulcerative blepharitis and conjunctivitis in adult dairy cows and association with Moraxella bovoculi // Can Vet J. - 2010 Apr; 51 (4): 400-2; Angelos JA, Lane VM, Ball LM, Hess JF Recombinant Moraxella bovoculi cytotoxin-ISCOM matrix adjuvanted vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis. / Vet Res Commun. 2010 Mar; 34 (3); Angelos JA, Ball LM, Differentiation of Moraxella bovoculi sp. Nov.f rom other coccoid moraxellae by the use of polymerase chain reaction and restriction endonuclease analysis of amplified DNA // J Vet Diagn Invest - 2007 Sep; 19 (5): 532- (4).

Infectious keratoconjunctivitis of cattle caused by bacteria Moraxella bovoculi, we observed in the farms of the Republic of Tatarstan (Chistopolsky state farm technical school; Srednye Devyatovo LLC, Khaerby LLC Laishevsky and Yenali agricultural production complex of Apastovsky district), Republic of Bashkortostan named after SPK-Kolk Kalinin of the Dyurtyulinsky district), the Leningrad region (Yarovoye LLC of the Priozersky region), the Chelyabinsk region (Kalininsky Agrofirm LLC of the Bredinsky district).

Currently, the main method of combating infectious keratoconjunctivitis in cattle is its specific prevention using various types of vaccines (Hughes D. et. Al. Experimenally induced Bovine Infectious Keratoconjunctivitis: Effectivcnes of Intramuscular Vaccination with Viable Moraxella bovis Culture. // Am, J. Vet. Res., 1971, 32 No. 6 - p. 799-886; Pugh GW et al. Infections bovine keratoconjunctivitis in cattle vaccinated and medicated against Moraxella bovis befare parturition. // Am. J. Vet. Res., 1982 43 No. 2 - p. 320-325; Gwin R. Medicament and method for prodicing immunity the infectious bovine keratoconjunctivitis.// US Patent No. 4539.201. Of September 3, 1985). In the Russian Federation, an “Associated vaccine for the specific prophylaxis of infectious keratoconjunctivitis of cattle based on the antigens of bacteria Moraxella bovis and herpes virus type 1” has been developed and is being produced (patent RU No. 2264227, A61K 39/295, C12N 1/20, 7/00 A61Ρ 31/00, published on November 20, 2005). The disadvantage of this vaccine is its incomplete antigenic composition, namely the absence of the bacterial antigen Moraxella bovoculi, one of the most common pathogens of infectious keratoconjunctivitis in cattle, which makes it ineffective in farms where this pathogen prevails.

The technical result to which the invention is directed is to obtain a vaccine having increased antigenic and immunogenic activity, expanding the spectrum of action and the protective effect of the vaccine.

This is achieved by the fact that the vaccine against infectious keratoconjunctivitis in cattle based on the bacteria Moraxella bovis and Moraxella bovoculi contains inactivated cell suspensions of bacteria strains Moraxella boyis "G97-VNIVI" with a concentration of 100-120 billion mk. per 1 cm ³ and Moraxella bovoculi “CX-Ch6 No.-DEPT” with a concentration of 100-120 billion m. per 1 cm ³ , taken in equal proportions on physiological saline, as an adjuvant - 6% gel of aluminum hydroxide, as an inactivator - formalin in the following ratio of components per 1 liter. vaccines: cell suspension of the bacterial strain Moraxella bovis "G97-VNIVI" with a concentration of 100-120 billion m. in 1 cm ³ , cm ³ - 90.0-100.0; cell suspension of the bacterial strain Moraxella bovoculi "CX-Ch6 No.-DEPT" with a concentration of 100-120 billion m. in 1 cm ³ , cm ³ - 90.0-100.0; gel of aluminum hydroxide, 6%, cm ³ - 90.0-100.0; formalin, cm ³ - 4.0-5.0 and physiological saline, l. - up to 1.

For the manufacture of a vaccine associated for specific prophylaxis of cattle infectious keratoconjunctivitis based on the antigens of the bacteria Moraxella bovis and Moraxella bovoculi, we used the strains of bacteria Moraxella bovis "G97-VNIVI" (RF Patent No. 2145353; publ. 10.02.2000) and Morax that we patented "CX-Ch6 No.-DEPT" (RF Patent No. 2521651; publ. 07/10/2014), isolated from patients with infectious keratoconjunctivitis of calves.

The strain of bacteria Moraxella bovis "G97-VNIVI" is characterized by the following properties: these are short thick sticks 1.5-2.0 in length and 0.5-1.0 μm in diameter with rounded ends, arranged in pairs or in the form of short chains, no spores form, motionless, stained gram-negative. On blood ΜΠΑ at pH 7.2-7.6, after 24-hour incubation at 37 ° C, colonies with a diameter of 1 to 3 mm with a β-hemolysis zone 0.5 to 2.0 mm wide are formed. The colonies are smooth, round, shiny, grayish-white. On the BCH, after a 24-48 hour incubation at 37 ° C, turbidity of the broth is observed with the formation of a small precipitate. The optional aerobic strain does not ferment sugars, does not restore nitrates to nitrites, does not form indole, liquefies gelatin, i.e. possesses proteolytic activity, peptones litmus milk, gives a positive reaction to oxidase.

The bacterial strain Moraxella bovoculi "CX-Ch6 No.-DEPT" is characterized by the following properties: bacteria are gram-negative diplococci with rarely encountered cocci with a diameter of 0.7-1.3 microns, do not form a spore. On blood ΜΠΑ forms colonies of white color with a diameter of ≤1 mm, round, convex, with smooth edges, a zone of β-hemolysis. Optional aerob, does not ferment sugars, does not form indole. Does not thin gelatin; gives a positive reaction to oxidase and a negative reaction to a test with litmus milk. The strain is characterized by a complete set of antigens typical of bacteria of the genus Moraxella.

The bacterial strains of Moraxella bovis and Moraxella bovoculi actively form endotoxins, which transform into toxoids under the influence of heat and formalin. The produced toxin has hemolytic and necrotic effects. LD ₅₀ for white mice is 5 × 10 ⁸ microbial cells. Formalin inactivated cultures of Moraxella bovis strains “G97-VNIVI” and Moraxella bovoculi “СХ-Ч6 No.-DEPT”, when administered subcutaneously, induce the formation of specific antibodies in blood serum in titers 1: 1280-1: 5120, as well as the formation of specific immunity.

The antigens included in the vaccine in effective amounts provide a synergistic effect, manifested in an increase in tension and duration of immunity.

Example 1. Obtaining matrovodnogo seed strains of bacteria strains Moraxella bovis and Moraxella bovoculi

For the manufacture of the vaccine, take ampoules with lyophilized production strains of Moraxella bovis "G97-VNIVI" and Moraxella bovoculi "CX-Ch6 No.-DEPT", which are dissolved in 0.5 cm ^{3 of} MPB and transferred to Petri dishes with 10% blood ΜΠΑ. Cultivation is carried out for 16-18 hours, then the cultures are checked for compliance with morphological, cultural, enzymatic and virulent properties. If they correspond to the passport data, then they are used to obtain the bacterial mass necessary for the manufacture of the associated vaccine.

The strains are stored in lyophilized form. For this, a suspension of daily cultures of bacteria Moraxella bovis "G97-VNIVI" and Moraxella bovoculi "СХ-Ч6 No.-DEPT" with a concentration of 50 billion m ³ in 1 cm ^{3 is} mixed with a protective medium (skim milk with sucrose 5%, gelatin 1.5%) in a ratio of 1: 1, packaged in ampoules of 1 cm ³ , dried by sublimation, sealed ampoules under vacuum and stored at a temperature of + 2-8 ° C.

Example 2. Cultivation of the bacterial mass of each strain of bacteria Moraxella bovis separately and its inactivation.

To obtain the biomass of Moraxella bovis and Moraxella bovoculi cultures, ΜΠΑ containing 10% defibrinated sheep blood is used. For this purpose, the indicated medium is poured into sterile conditions into 1.5 liter mother flasks of 250-300 cm ³ , after solidification of the medium, the strains of these strains are seeded by adding 5 cm ^{3 of a} bacterial suspension containing 1 billion m to each mattress in 1 cm ³ . Crops are kept in a thermostat at a temperature of 37 ° C. After 36-48 hours, the grown colonies of the cultures of Moraxella bovis and Moraxella bovoculi are washed off with 0.9% sodium chloride solution, a suspension is prepared with a concentration of 100-120 billion m. in 1 cm ³ according to the bacterial or optical standard turbidity GISK them. Tarasevich. The culture is checked for purity, morphological and serological typicality.

Inactivation of bacterial suspensions is carried out with formalin. For this, formalin with a content of 37-38% active formaldehyde is added to the microbial suspension at the rate of 0.5 cm ³ per 100 cm ^{3 of} suspension. Stirred and placed in a thermostat for 72 hours. Then take a sample for completeness of inactivation, which is carried out by plating inactivated microbial suspensions on the blood ΜΠΑ.

Example 3. Preparation of a vaccine against infectious keratoconjunctivitis in cattle based on the antigens of bacteria Moraxella bovis and herpes virus type 1.

The vaccine is prepared from formalin inactivated production strains of bacteria Moraxella bovis "G97-VNIVI" and Moraxella bovoculi "CX-Ch6 No.-DEPT".

Saline is taken and inactivated suspensions of bacteria Moraxella bovis and Moraxella bovis are added to it at a rate of 9-10 cm ^{3 of} each strain per 100 cm ^{3 of} vaccine preparation, as well as 6% aluminum hydroxide gel at a rate of 9-10 cm ³ and formalin 4 -5 cm ³ per 100 cm ^{3 of} vaccine. The pH of the vaccine is adjusted to 7.2-7.4 and packaged with thorough periodic stirring into bottles, which are then closed with rubber stoppers, wrapped in metal caps and labeled. Table 1 shows the composition of the vaccine.

Example 4. The control of the vaccine for sterility, safety and antigenic activity.

To test for sterility, from 6 bottles of vaccine of each series, cultures of 0.2 cm ^{3 are made} on MPB, ΜΠΑ, blood ΜΠΑ, MPPB under paraffin oil and Saburo agar, 2 tubes per bottle. Crops with all media are kept in a thermostat at 37 ° C, and with Saburo agar - at 20-22 ° C for 15 days. Culture media should remain sterile.

To test for harmlessness, 10 white mice with a live weight of 17-18 g are selected and a vaccine is injected subcutaneously at a dose of 0.5 cm ³ . The vaccine is considered harmless if the mice remain alive and clinically healthy for 10 days after administration of the vaccine.

The antigenic activity of the vaccine is monitored on 3 rabbits with a live weight of 2.0-2.5 kg, to which the drug is administered subcutaneously at a dose of 2 cm ³ twice with an interval of 14 days. 14 days after the last vaccination, blood was taken from the ear vein in rabbits and the serum was examined to determine the level of specific antibodies to the antigens of the bacteria Moraxella bovis and Moraxella bovoculi. Antibody titers of the bacterial antigens Moraxella bovis and Moraxella bovoculi should be in the range of 10.0-11.0 log ₂ in the ELISA.

Example 5. The effectiveness of the vaccine is evaluated in a dysfunctional farm by the number of cases of infectious keratoconjunctivitis in cattle. Production tests of experimental vaccine series were carried out in the Yenali agricultural production complex of the Apastovsky District; Khaerbi LLC and Srednyaya Devyatovo LLC, Laishevsky District, state farm technical school of the Chistopol District of the Republic of Tatarstan, SPK collective farm named after Kalinin and SEC collective farm. Of the Kirov of the Dyurtyulinsky district of the Republic of Bashkortostan, which are disadvantaged for infectious keratoconjunctivitis in cattle, where the incidence of calves up to 6 months of age was 30-56%, young animals up to 12 months old were 27-36% and young animals over one year old and an adult population were 8-22% . The vaccine was used for prophylactic purposes. In this case, calves from 1 to 6 months of age were vaccinated at a dose of 2.5 cm ³ , young animals from 6 to 12 months. at a dose of 4 cm ³ , young animals over one year old and adult livestock at a dose of 5 cm ³ . The results of the vaccine tests in these farms are presented in table 3. From this table it follows that the vaccine against infectious keratoconjunctivitis in cattle has a high immunogenic activity. Its prophylactic use in 6 stationary dysfunctional farms made it possible to completely eradicate the disease during the year in 4 farms, and in the other two to reduce the incidence to 1-3%.

Thus, the proposed vaccine against infectious keratoconjunctivitis in cattle based on the antigens of bacteria Moraxella bovis and Moraxella bovoculi has a wider spectrum of action, increased antigenic and immunogenic activity, the immunity in animals persists for 12 months. after vaccination.

Example 5. General information, biological properties, the order of use of the vaccine against infectious keratoconjunctivitis in cattle based on the antigens of bacteria Moraxella bovis and Moraxella bovoculi

General information

The vaccine is made from concentrated antigens of bacteria Moraxella bovis (strain "G97-VNIVI") and Moraxella bovoculi (strain "СХ-Ч6 No.-DEPT"), inactivated with formalin (0.5% by volume) with the addition of aluminum hydroxide as adjuvant (10 % by volume).

In appearance, the vaccine is a yellowish liquid with a white precipitate that breaks easily when shaken.

The vaccine is packaged in 100 and 200 cm ³ in glass bottles of the appropriate capacity, corked with rubber stoppers, reinforced with aluminum caps.

Vaccine bottles are packed in cardboard boxes with dividing walls to ensure their integrity. Each box contains instructions for using the vaccine.

The shelf life of the vaccine is 12 months from the date of release, subject to storage and transportation conditions.

It is forbidden to use the vaccine after the expiration date.

The vaccine is stored and transported in a dry, dark place at a temperature of 2 to 8 ° C.

The vaccine should be stored out of the reach of children.

A vaccine in vials without labels with an expired shelf life, with a violation of the integrity and / or tightness of the cork, with a changed color and / or consistency of the contents, with the presence of impurities, as well as vaccine residues not used within 4 hours after opening the vials, are rejected, they are disinfected by boiling for 30 minutes or treated with a 2% alkali solution or 5% chloramine solution (1: 1) for 30 minutes and disposed of.

Disposal of a disinfected vaccine does not require special precautions.

Biological properties

The vaccine causes the formation of an immune response in cattle to the causative agents of infectious keratoconjunctivitis - the bacteria Moraxella bovis and Moraxella bovoculi 14 days after a double administration of 12 months.

In 1 cm ^{3 of the} vaccine contains 10 billion inactivated microbial cells of bacteria Moraxella bovis and Moraxella bovoculi.

The vaccine is harmless, does not possess medicinal properties.

Vaccine Administration

The vaccine is intended for the prevention of infectious keratoconjunctivitis in cattle in threatened and permanently dysfunctional farms.

It is forbidden to vaccinate clinically sick and / or weakened animals.

Cattle are subject to vaccination, starting from 30-35 days of age and older.

The vaccine is administered subcutaneously in the middle third of the neck, twice with an interval of 14-15 days in doses: calves from 1 to 6 months - 2.5 cm ³ , young animals aged 6 months to a year - 4.0 cm ³ , adult animals - 5 , 0 cm ³ .

Before use, the vaccine is heated in a water bath to a temperature of 37-38 ° C, during the application, the vaccine bottles are periodically shaken.

The vaccine is administered in compliance with the rules of asepsis and antiseptics, sterile materials and instruments are used for administration. A separate needle is used for each animal. The injection site is treated with 70 ° alcohol.

Symptoms of the manifestation of infectious keratoconjunctivitis or other pathological signs with an overdose of the vaccine have not been established.

Violations of the vaccination schedule should be avoided, as this can lead to a decrease in the effectiveness of immunoprophylaxis of infectious keratoconjunctivitis. If the next vaccine is missed, immunization should be carried out as soon as possible.

When using a vaccine with these instructions, side effects, as a rule, are not noted. In individual animals, a slight increase in temperature and the formation of swelling at the injection site, which spontaneously disappears after 3-5 days, is possible a day after vaccination.

It is forbidden to use the vaccine associated with infectious keratoconjunctivitis in cattle, together with other immunobiological preparations, as well as with anthelmintics and insectoacaricides within 14 days before and after the next immunization.

Milk and products from vaccinated animals are sold without restriction, regardless of the timing of vaccination.

Personal Prevention

When working with the vaccine, the general rules of personal hygiene and safety precautions provided for when working with veterinary medicines should be observed.

All persons involved in the vaccination should wear special clothing (rubber boots, a bathrobe, trousers, a hat, rubber gloves) and be provided with goggles. In places of work there should be a first aid kit.

If the vaccine gets on the skin and / or mucous membranes, it is recommended to immediately rinse with plenty of clean water.

If the vaccine is accidentally administered to a person, the injection site should be treated with a 70 ° solution of ethyl alcohol and contact a medical institution.

Claims (1)

Cattle infectious keratoconjunctivitis vaccine based on the antigens of the bacteria Moraxella bovis and Moraxella bovoculi, characterized in that it contains inactivated cell suspensions of the bacteria strains Moraxella bovis "G97-VNIVI" with a concentration of 100-120 billion mk per 1 cm ³ and Moraxella bovoculi “CX-Ch6 No.-DEPT” with a concentration of 100-120 billion m. per 1 cm ³ , taken in equal proportions, on physiological saline, as adjuvant - 6% gel of aluminum hydroxide, as inactivator - formalin in the following ratio of components per 1 liter of vaccine:
Cell suspension of a strain of bacteria Moraxella bovis "G97-VNIVI" with a concentration of 100-120 billion m. in 1 cm ³ cm ³ 90.0-100.0 Cell suspensions of the bacterial strain Moraxella bovoculi "CX-Ch6 No.-DEPT" with concentration 100-120 billion m. in 1 cm ³ cm ³ 90.0-100.0 Gel of aluminum hydroxide, 6%, cm ³ 90.0-100.0 Formalin, cm ³ 4.0 - 5.0 Saline solution, l up to 1