RU2005779C1 - Strain of bacterium leptospira interrogans of serogroup icterohaemorrhagiae, serovara copenhagent used for preparing of vaccine against leptospirosis - Google Patents

Abstract

FIELD: microbiology, medicine. SUBSTANCE: new strain L interrogans No. 460 shows expressed antigenic and immunogenic properties providing use of it for vaccine preparing. Strain is isolated from blood of patient with leptospirosis. After heating at 56 C for 2 h strain shows high immunogenic activity and prevents death of experimental animals infected with high dose of virulent Leptospirae (above 1000 lethal doses). Serum obtained from animals immunized with vaccine specimens, which are prepared from isolated strain, shows high protective efficacy. EFFECT: preparing of vaccine with high efficacy.

Description

 The invention relates to medicine and can be used in vaccine-serum case for the preparation of a vaccine.

 It is known that at present, strains of the serogroup Icterohaemorragiae are used to prepare the leptospirosis vaccine used to prevent leptospirosis in humans.

 The immunogenicity of the strains currently used to prepare the vaccine is apparently not high enough.

 The purpose of the invention is the production of antigenic, immunogenic and epidemiologically relevant strain of leptospira, suitable for the preparation of a vaccine.

 The strain was isolated in 1982 from the blood of a patient with human hemorrhagic leptospirosis. The leptospira of the Institute of Epidemiology and Microbiology of the Academy of Medical Sciences of the USSR N.F. Gammalei N 460.

 Characterization of the strain.

Morphological and cultural properties. Gram-negative spirochetes with characteristic morphology and mobility. Cultivated in a liquid nutrient medium TERSKIKH supplemented with 5% rabbit serum at 28 ^° C Lag phase lasts for two days, the exponential phase - 6 days, the generation time 22 h of the stationary phase, the largest concentration of (3,7 ± 0,2). ˙10 ⁷ leptospira in 1 ml.

Pathogenic properties. In experimental models, the strain was classified as highly virulent: LD ₅₀ , which was determined by intraperitoneal infection of golden hamsters weighing 30.0 g, is 5.6 · 10 ^° / (8.2 · 10 ¹ -2.5 · 10 ^° ). The denominator indicates the value of the limits in which the effect of the LD ₅₀ value is concluded with a 95% probability. The clinical manifestations of infection in experimental animals are characterized by the appearance of fever in 100% of animals, hemorrhages in 100% and jaundice in 80%.

Antigenicity. The antigenic activity of the strain is high: in response to a single intravenous administration to rabbits weighing 3.0 kg of a live leptospira culture containing 10 ⁸ microbial cells, antibodies appeared, the maximum of which occurred in the second or third week and the geometric mean titer was 51200.

Immunogenic activity. The strain has a pronounced immunogenic activity. Killed by heating for 2 hours at 56 ^{° C} at 3 ⁷ ˙10 it protects from death and 100% infection of guinea pigs, infected by Leptospira serogroup Icterohaemorrhagiae at a dose of 1000 times the lethal.

 A vaccine was obtained from the leptospira strain Ryabuhin, which was tested in an animal experiment.

 An example of the preparation and testing of a vaccine.

The culture of Leptospira strain "Riabukhin" grown on medium containing 3 and Terskikh ˙10 ⁷ microbial cells in 1 ml, was heated at ⁵⁶ ° C for 2 hours. The thus obtained Greta vaccine was administered to guinea pigs weighing 20 g at a dose of 150.0 1 ml intraperitoneally. In response to the introduction of the vaccine, antibodies appeared in all experimental animals, the average geometric titer of which in the microagglutination reaction was 1: 84. After 30 days, lectospira of the serogroup Icterohaemorrhagiae was administered to all vaccinated and control animals at a dose 1000 times the lethal. All immunized guinea pigs remained alive for 30 days, there were no symptoms of the disease, no leptospira was isolated from organs of slaughtered animals when plated on a nutrient medium. All control animals (20 guinea pigs), which received a nutrient medium instead of a vaccine, died with a vivid picture of ictohemorrhagic leptospirosis.

.Immunogenicity of the vaccine, studied by the method of passive protection on golden hamsters. Two hours before the injection of the lethal dose, the animals were injected intraperitoneally with guinea pig immune serum containing antibodies in a titer of 1: 40. The preventive activity of guinea pig serum vaccinated with experimental vaccine samples for golden hamsters, calculated by determining the ED ₅₀ , was

.

The study of prototype virulence, correlating with immunogenicity in an experiment on golden hamsters, showed that the LD _{50 of} the Odessa 853 strain is 1.0 · 10 ³ / (6.9 · 10 ³ -1.4 · 10 ³ ). The LD _{50 of the} strain "Rattus norvegicus 382" is 2.0 · 10 ⁴ / (1.4 · 10 ⁵ -2.9 · 10 ³ ).

, для штамма "Rattus norvegicus 382" ЕД₅₀=

.A study of the immunogenicity of prototypes by determining the protective activity of animal sera immunized with strains of Odessa 853 and Rattus norvegicus 382 showed that for pcs. "Odessa 853" ED ₅₀ =

, for strain "Rattus norvegicus 382" ED ₅₀ =

.

 Thus, the proposed strain has a more pronounced immunogenic properties compared with the prototypes and can be recommended for the construction of a vaccine. (56) Leptospiroses and medicin. Human and Vet. 1989. Coll Found, Marsel, Merienx, P. 119-135.

Claims (1)

 The bacterial strain Leptospira interrogans "Ryabuhin" serogroup Icterohaemorrhagiae serovar copenhageni NIIEM N460 used to prepare the vaccine against leptospirosis.