RU2147317C1 - Strain of bacterium pseudomonas aeruginosa 4992, used for preparing vaccine against pseudomonosis in fur wild animals - Google Patents

Abstract

FIELD: biotechnology, veterinary science, microbiology. SUBSTANCE: invention relates to production of vaccine preparations against pyocyanic infection in fur wild animals, mainly in polar fox and fox. Strain of bacterium Pseudomonas aeruginosa 499₂ is deposited in collection of strain-pathogens of sicknesses in fur wild animals of museum of bacterial and viral cultures of fur farming and rabbit farming at number 499₂. Strain is referred to serovariant 02/05. Strain is cultured in Marten's broth or synthetic medium at 32 C for 18-20 h at constant shaking. After control of grown culture for toxicogenicity and proteolytic activity bacterial mass is separated and inactivated. Then aluminium hydroxide as an adjuvant is added and packaged. Invention ensures to prevent concealed loss in multiplication of fur wild animals in farms with pyocyanic infection being independently of serological pattern of pathogen. EFFECT: strain indicated above, enhanced effectiveness of vaccine, improved method of preparing. 3 ex

Description

 The invention relates to the field of biotechnology and veterinary medicine and is intended for the production of vaccines against pseudomonas infection of fur animals, mainly arctic foxes and foxes.

 Pseudomonas infection is ubiquitous, causing significant economic damage to animal farming and poses a danger to human health. In fur farms, enzootypes among minks, arctic foxes and foxes are recorded annually. Cases of the disease have been reported in rabbits, nutria, trochees. In recent years, a Pseudomonas infection has been observed in dogs of domestic maintenance.

 The pathology of Pseudomonas infection in both humans and animals is very diverse - damage to the reproductive system, septic processes in the body, damage to the gastrointestinal tract in young animals, damage to the mammary glands, etc. In foxes and arctic foxes, it occurs with lesions of the genitals and is accompanied by a violation of the pregnancy process - abortion, resorption of embryos, and the passage of females.

 The causative agent of pseudomonosis has serotypic variability, therefore the effectiveness of specific prophylaxis is determined by the completeness of the antigenic composition of the components included in the vaccine.

 Known vaccine strain Pseudomonas aeruginosa PA-7 of the second serotype used for the manufacture of toxoid vaccines against Pseudomonas aeruginosa (SU N 1202109, publ. 15.06.86).

 However, the known strain does not contain antigens of the pathogen pseudomonosis of fur animals and does not provide the formation of immunity against Pseudomonas infection of fur animals.

 The closest analogue is the sixth serotype strain Pseudomonas aeruginosa N 202, which is part of the inactivated vaccine against pseudomonosis of fur animals, mainly minks ("Veterinary preparations". Handbook, edited by D. F. Ozidze. M .: Kolos, 1981).

 However, the known production strain has unstable virulent and immunogenic properties and, moreover, does not prevent the pseudomonosis of arctic foxes and foxes caused by pathogens of other serovariants.

 The objective of the invention is to obtain a strain of Pseudomonas aeruginosa for the manufacture of an effective vaccine against pseudomonosis of fur animals, regardless of the serological profile of the pathogen.

The technical result of the invention is to obtain a new strain of Pseudomonas aeruginosa 499 ₂ , capable of producing the main pathogenicity factors in optimal values and allowing to produce a vaccine against any serovariants of Pseudomonas aeruginosa not only for arctic foxes and foxes, but also other animals and animals susceptible to this infection.

The inventive bacterial strain Pseudomonas aeruginosa 499 ₂ isolated from a puppy fox Z / s "Guryevsky" and deposited in the collection of strains - pathogens of fur-bearing animals of the Museum of Bacterial and Viral Cultures of the Scientific Research Institute of Fur Breeding and Rabbit Breeding them (NIIPZK) V.A. Afanasyev and has registration number 499 ₂ .

Strain Ps. aeruginosa 499 _{2 is} intended as a producer of proteolytic enzymes (alkaline protease and elastase) in the manufacture of a specific immune preparation against Pseudomonas aeruginosa and foxes.

The bacterial strain Pseudomonas aeruginosa NIIPZK N 499 _{2 is} characterized by the following features and properties.

Genetic markers:
Serovariant 02/05 (Habs)
Pyocinotype 682668
Strain 499 ₂ proteolytic, with adhesive activity against rabbit erythrocytes.

 Morphological properties. Industrial culture is a thin straight gram-negative sticks, 2-3 x 0.5 microns in size. The rods are movable, monotrichi, do not form a spore, in strokes are located singly and in rows.

Cultural properties. Aerob, the optimum growth temperature at 32-37 ^o C, retains the ability to grow and reproduce at 42 ^o C. It grows on almost all nutrient media with a pH of 8.0. On the surface of the MPB forms a characteristic grayish-silver film. Forms pigment pyocyanin. The best culture medium is Iensen's, enriched with salts and aminopeptide. On MPA forms S- and R-forms, with the predominance of S-forms.

 Biochemical activity. High acid-forming activity against glucose and galactose; under aerobic conditions it breaks down sugars to form gluconic acid without gas, liquefies gelatin, coagulates milk, and does not cause the formation of indole and hydrogen sulfide. It forms cytochrome oxidase, causes hydrolysis of acetamide.

Virulent properties. When administered intraperitoneally to white mice, the LD / 50 is 1.28 x 10 ⁷ KOE / ml.

 Antigenic properties. The strain produces alkaline protease, elastase, has adhesive activity.

 Toxigenic properties. In a liquid nutrient medium, it produces an alkaline protease (an indicator of hydrolysis of casein 320), elastase (RP 1:32). Adhesion titer in RSA 1:32.

Strain storage. At a temperature of 6 ± 2 ^o C in ampoules in a lyophilized state for more than three years; in test tubes in a column of semi-liquid agar prepared on Marten broth with the addition of 0.5% sodium glutamate and 1% glycerol, under liquid paraffin for three months; in test tubes on mowed agar no more than 14 days.

 Monitoring the status of the strain. Morphological, biochemical, cultural and virulent properties are monitored at least once every three months. Antigenic properties (protease and elastase) are tested before production of the immune preparation. The strain is pre-cultured in Iensen's environment.

 The rate of hydrolysis of casein with an alkaline protease in the diffusion reaction to casein agar should be at least 320, and the elastase titer in the reaction of the supernatant with monovalent anti-elastase serum in 1% Purifait agar should be at least 1:32.

 The invention is illustrated by the following examples.

Example 1. The bacterial strain Pseudomonas aeruginosa 499 ₂ cultured in Marten broth or synthetic medium at 32 ^o C for 18-20 hours with constant shaking on a shuttle machine. At the end of the incubation period, the culture is checked for cleanliness of growth in smears stained by Gram.

 Next, a sample is taken from the grown culture, centrifuged at 6000 rpm for 20 minutes. The supernatant is drained and examined for the content of: exotoxin A in a precipitation reaction (RP) with antitoxic serum and / or an alkaline protease in casein hydrolysis and / or elastase in a RP with monovalent antelastase serum. The titers should be: exotoxin A - not lower than 1: 4, alkaline protease - not lower than 1:32, elastase - not lower than 1:16.

 After controlling the grown culture for toxigenicity and proteolytic activity, the separated bacmass is inactivated by any known method and an adjuvant, for example aluminum hydroxide, is added.

 Example 2. Obtained in example 1, the vaccine is checked for harmlessness on 9 Arctic foxes. The vaccine is administered intramuscularly in the thigh area of the right paw in doses: three foxes, 1.0 ml each, three - 2.0 ml each and three - 3.0 ml each.

 Conduct daily monitoring of Arctic foxes for 7 days with individual probing of the injection site. Thermometry of arctic foxes is carried out twice on the 2nd and 4th days after drug administration.

The results of the study showed that all Arctic foxes behaved normally, completely ate food, aggressiveness was not observed. Body temperature was within the normal range - 37-39 ^o C. When probing the injection site of the vaccine, no swelling of the limb or soreness was detected.

 Example 3. The resulting vaccine is used to immunize Arctic foxes on a farm that is not favorable for Pseudomonas aeruginosa to study the effect of immunization on reproduction results, since the main pathology in Pseudomonas aeruginosa is manifested precisely during the reproduction period.

 In this household, there are cases of abortion, omission of females, and the birth of dead puppies among arctic foxes. Moreover, in aborted females in the last stages of pregnancy (one week before the gestation period), bacteriological examination revealed a culture of Pseudomonas aeruginosa 03 and 09 serovars.

 Obtained according to example 1, the vaccine is immunized with 50 female foxes intramuscularly at a dose of 1.5 ml. Another 50 females, similar in age, color, and conditions of keeping and feeding, serve as control.

 It was found that the use of the vaccine increased the fecundity of the foxes of the experimental group by 0.7 puppies. The vaccine prevented the resorption of the fruit in some females of the experimental group, while in some females of the control group the offspring was registered in only 4-5 puppies. This is most often due to resorption of the fetus in the first half of pregnancy.

Thus, the vaccine based on strain 499 ₂ will prevent hidden losses during the reproduction of fur animals in farms that are not successful in Pseudomonas aeruginosa.

Claims (1)

The bacterial strain Pseudomonas aeruginosa 499 ₂ used for the manufacture of a vaccine against pseudomonosis of fur animals.