RU2145353C1 - Strain of bacterium moraxella bovis "g97-vnivi" used for preparing diagnostica and vaccines against infectious keratoconjunctivitis in cattle - Google Patents

Abstract

FIELD: veterinary science, agriculture, microbiology. SUBSTANCE: invention relates to a preparation used for diagnosis and prophylaxis of infectious keratoconjunctivitis in cattle. New strain of Moraxella bovis "G97-VNIVI" is deposited in microorganism collection of VNIVI. Strain retains S-form stably, it does not dissociate in multiple resowings and has the complete set of antigens that are typical for Moraxella bovis. Invention ensures to prevent the propagation of infectious keratoconjunctivitis in cattle. EFFECT: enhanced effectiveness of preparations, decreased economical loss. 1 tbl, 3 ex

Description

 The invention relates to the field of veterinary microbiology and immunology and for the isolation, identification and selection of the Moraxella bovis strain "G97-VNIVI" - the main causative agent of infectious keratoconjunctivitis in cattle, first established in the Russian Federation and CIS countries, which can be used for the manufacture of diagnosticums and vaccines against this disease.

 From the sources of scientific and technical literature it is known that in our country the first reports of epizootic outbreaks of the ICC that took place on the farms of the Gomel region and the North-Western zone of the RSFSR appeared only in the 70-80s (V. Bobryn "Infectious keratoconjunctivitis cattle in the farms of the Gomel region. "// Diseases of agricultural animals and birds, their prevention and treatment. // Collection of LVI, issue 39. - L., 1974, p. 167 - 179; Andriasyan V. B. "Infectious keratoconjunctivitis in cattle in the Northwestern zone of the RSFSR." // Infections these diseases of agricultural animals. // Collection of scientific works of LVI. - L., 1988, pp. 6 - 11), which describe only the clinical and epizootological characteristics of the disease and there is no data on the isolation, identification and production of pathogen strains , its etiological role in the occurrence of the disease and the results of a study of its biological properties.

 The lack of decoding of the etiology of the disease in our country, the lack of diagnostic methods and means for specific prevention of cattle CKI led to the appearance of foci that are permanently unfavorable for infectious keratoconjunctivitis and continued the trend of the further spread of the disease, causing significant economic damage due to reduced milk yield and weight gain due to animals partial or complete loss of vision.

 The aim of the invention is the isolation, identification and preparation of strain G97-VNIVI of the bacteria Moraxella bovis, the main causative agent of this form of pathology, obtained for the first time and until now unknown in the Russian Federation and CIS countries, circulating in cattle and causing infectious keratoconjunctivitis.

 Strain "G97-VNIVI" was isolated from a flush of an eye of a sick calf (with signs of acute conjunctivitis) of the Uchkhoz of the Kazan State Academy of Veterinary Medicine of the Republic of Tatarstan by 50-fold passage on blood agar and constant selection of cultures after each reseeding according to the S-form of colonies and the ability cause hemolysis of red blood cells in a blood agar medium characterizing their virulence. This strain is deposited in the collection of microorganisms of the special laboratory (bank) of the All-Russian Scientific Research Veterinary Institute. Registration number assigned to the strain 06/08/1998, N 1.

 The strain Moraxella bovis "G97-VNIVI" is characterized by the following properties.

 Morphological signs. The strain is a short thick sticks with a length of 1.5 - 2.0 and a diameter of 0.5 - 1.0 μm with rounded ends, usually observed in pairs or in the form of short chains. Old cultures progressively become pleomorphic; the microorganism strain is immobile, does not form a spore; it is painted gram-negative.

Cultural properties. On blood agar at a pH of 7.2 to 7.6, the Moraxella bovis strain G97-VNIVI, after a 24-hour incubation at 37 ^° C, forms colonies with a diameter of 1.0 to 3.0 mm in diameter with a beta hemolysis zone wide 0.5 - 1.0 mm. The colonies are round, convex in the center, shiny, translucent, grayish-white, slightly grown into the environment.

After 48-hour incubation, the colonies become 3-4 mm in diameter and come with smooth edges. The hemolysis zone doubles when incubated for 24 hours at 37 ^o C and an additional 24 hours at 25 ^o C.

On the tryptose-soy broth after 48 - 72 hours of incubation at 37 ^° C, there is a meager growth, which, as it ages, is slightly cloudy, but a precipitate forms which, when agitated, breaks down into coarse particles.

 When incubated in a reactor, the broth becomes turbid after 24 hours with the formation of a small amount of sediment. Growth is further enhanced by the addition of rabbit or calf serum in a 5% volume. The strain persistently retains the S-form and does not dissociate during repeated reseeding on nutrient media.

 Enzymatic properties. The strain "G97-VNIVI" - an optional aerobic, does not ferment sugars, does not restore nitrates to nitrites, does not form indole; dilutes gelatin, i.e. possesses proteolytic activity; the oxidase test gives a positive result, which is a characteristic feature for microorganisms of the family Neisseriaceae, genus Moraxella.

 During a 24-hour incubation, the selected strain of M. bovis in Lamus milk forms a dark blue band in the upper part of the tube, and upon further incubation, the medium progressively becomes more alkaline. Within 6 days, distinct 3 zones appear: in the upper part there is a dark blue liquid, the middle zone is lilac in color, curdled by consistency, and the lower layer is a coagulated casein of pale lilac color.

 Continued incubation leads to complete peptonization of litmus milk of a uniform bright pink color.

 Antigenic properties. The strain is characterized by a complete set of antigens typical of Moraxella bovis.

 Virulent properties. The strain has stable high virulence for white mice weighing 12-16 g: it causes their rapid mortality (from 1 to 3 hours) after intraperitoneal administration of fresh live culture at a dose of 0.5 ml.

 The strain actively forms endotoxin, which passes into the toxoid under the action of heat and formalin. The toxin produced has hemolytic and necrotic effects and is fatal to mice and rabbits.

Calves 1 - 2 months of age are relatively easily infected by introducing into the conjunctival sac a 24-hour culture in a dose of 0.5 ml containing at least 3 • 10 ⁶ microbial cells in the presence of natural or artificial ultraviolet radiation.

 The immunogenic activity of the selected strain "G97-VNIVI" is 2.1 - 2.5 times higher compared to a mixture of field crops of Moraxella bovis isolated from naturally sick animals, due to the usefulness of the antigenic structure and its higher biological activity.

Strain storage. The resulting strain was grown at 37 ^o C on meat-peptone blood agar for 24 hours, washed with sterile saline. A suspension of M. bovis at a concentration of 50 billion microbial cells in 1 ml is mixed with a protective medium (5% sucrose, 1.5% gelatin) in a 1: 1 ratio, packaged in 1 ml ampoules, dried by freeze-drying, ampoules are sealed under vacuum and stored at 2 - 8 ^o C.

 Example 1. The strain M. bovis "G97-VNIVI" is used to identify cultures isolated from sick animals with signs of keratoconjunctivitis in the agar gel precipitation reaction (RDP).

Hyperimmune serum is obtained in rabbits with a live weight of 3.0 - 3.5 kg by 5-fold subcutaneous administration of an inactivated culture of M. bovis with an interval of 7 days. Inactivation of the strain is achieved by maintaining the suspension of culture in a water bath at a temperature of 56 ^o C for one hour.

The antigen for the RDP is made from the strain "G97-VNIVI" grown on blood meat and peptone agar at a temperature of 37 ^o C for 24 hours. The grown colonies are removed, transferred to cold distilled water and resuspended. The concentration of cells of the microorganism should be not less than 5.1 • 10 ⁶ to 7.5 • 10 ⁸ . The resulting suspension was frozen three times at 60 ^° C. and thawed at 25 ^° C., centrifuged to precipitate coarse particles, and the supernatant was used as antigen in the RPD.

 The reaction is set according to the generally accepted method. To identify the antigen, a specific serum is poured into the central well, while the peripheral ones are tested antigens and antigen prepared from the M. bovis selected strain “G97-VNIVI”.

 Example 2. The strain M. bovis "G97-VNIVI" is used in the manufacture of antigen for the diagnosis of infectious keratoconjunctivitis by enzyme-linked immunosorbent assay (ELISA). Soluble antigen is obtained from M. bovis by sonication on an ultrasonic disintegrator. After disintegration, the suspension of M. bovis is centrifuged at 6 thousand rpm. The supernatant is aspirated into a test tube and trichloroacetic acid (TCA) is added thereto to a final concentration of 5%.

 The preparation is centrifuged, the supernatant is discarded, the precipitate is washed twice in a 5% TCA solution, then the precipitate is resuspended in distilled water and dialyzed against tap water for 18 hours, then lyophilized.

Hyperimmune serum is obtained on calves of 2-3 months of age by 5-fold subcutaneous administration of an inactivated culture of M. bovis with an interval of 7 days. Inactivation of the strain is achieved by maintaining the suspension of culture in a water bath at a temperature of 56 ^o C for one hour.

 The ELISA is carried out according to the generally accepted methodology. Conjugates are diagnostic antibodies against bovine immunoglobulins labeled with peroxidase. The reaction is carried out spectrophotometrically at a wavelength of 492 nm. For a positive result, the sensitivity coefficient is taken (the ratio of the optical density of the control reaction with negative serum) equal to or greater than 2.0.

 Comparative data on studies of calf blood serum by ELISA are presented in the table.

 The table shows that the strain M. bovis "G97-VNIVI" is suitable for the manufacture of diagnostic drugs by ELISA. In patients with calf keratoconjunctivitis, antibody titers to M. bovis are detected in ELISA in titers of 1: 640 - 1: 1280.

Example 3. The strain M. bovis "G97-VNIVI" is used in the manufacture of inactivated vaccines against infectious keratoconjunctivitis. To this end, the strain is grown on blood meat and peptone agar in Petri dishes for 24 hours, washed with physiological saline, the concentration of 6 billion / cm ^{3 of} microbial cells is established and inactivated by adding formalin to a final concentration of 0.5%. After sterility is established, aluminum hydroxide is added to an inactivated culture to a 1% concentration. The vaccine is packaged in 100 cm ³ in sterile vials.

The vaccine made from the strain M. bovis "G97-VNIVI" protects when administered twice from the death of 90 - 100% of white mice and rabbits infected with live cells of field cultures of M. bovis (with the death of 80 - 100% of unvaccinated animals) with the introduction of 0 5 - 2.0 billion microbial bodies. The vaccine, given twice subcutaneously with an interval of 30 days at a dose of 5 cm ^3, induces the formation in calves of specific antibodies in the blood serum of M. bovis in titers of 1: 400 - 1: 1600 in ELISA.

 Thus, the test results showed that the diagnostic serum for the Moraxella bovis strain has a higher activity than the serum for field cultures of bacteria Moraxella bovis of the genus Moraxella, and the above data indicate the possibility of using the claimed Moraxella bovis strain "G97-VNIVI" as a vaccine against infectious keratoconjunctivitis in cattle.

Claims (1)

 The bacterial strain Moraxella bovis "G97-VNIVI" used for the manufacture of diagnosticums and vaccines against infectious keratoconjunctivitis in cattle.

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