Method for simulating delayed hyperresponsiveness to mycobacteria bovis in guinea pigs

FIELD: medicine.

SUBSTANCE: what is presented is a method for simulating a delayed hyperresponsiveness to mycobacteria bovis. Avirulent mycobacteria bovis of the strain BCG are administered intracutaneously to albino guinea pigs produced by consanguineous mating of brother x sister (F2); that is followed by forming a group (groups) of animals showing various extents of an inflammatory response in reaction to the intracutaneous administration of mycobacteria bovis of the strain BCG 30-35 days later.

EFFECT: method is effective in studying the mechanisms of the delayed hyperresponsiveness and assessing the efficacy of antituberculosis agents.

2 tbl, 5 ex

 

The invention relates to the Allergy and immunology, in particular to the simulation of delayed-type hypersensitivity (GST) bacilli in the body of Guinea pigs. The proposed method can be used to assess the impact of immunobiological preparations for skin reaction GST; the study of the mechanisms of reactions GST; evaluation of anti-TB drugs.

There is a method of modeling the delayed-type hypersensitivity in Guinea pigs [OIE Manual of Diagnostic Test and Vaccines for Terrestrial Animals of the 6-th ed. - 2008. - Vol.l. -P. 693], which is that animals injected intramuscularly pathogenic mycobacteria physiological solution in the minimum infective dose, which leads to the slow progression of the infectious process.

The disadvantage of this method is epidemiological danger to personnel, the probability of scattering of mycobacteria in the environment, the difficulty in selecting the minimum dose of bacilli not leading to death of the animals earlier than planned for the main phase of the study.

There is a method of modeling the delayed-type hypersensitivity in Guinea pigs [the who expert Committee on biological standardization. 36th report. Technical report series 745. Who. Geneva. 1988 S.43], which is that animals in utilisee injected inactivated mycobacteria in Freund.

The disadvantage of this method is the development of GST on modified mycobacterial antigens, which reduces the specificity of modeling GST.

There is a method of modeling the delayed-type hypersensitivity in Guinea pigs, which was selected for the prototype [HBASAC - BCG combined vaccine for intracutaneous injection and preparation method thereof and, no US 7,579,003 B2, Aug. 25, 2009]. The method consists in intradermal administration of an avirulent M. tuberculosis, M. bovis strain BCG physiological solution. In this case, the antigenic composition used attenuated mycobacteria is a close relative to pathogenic mycobacteria that causes the simulation GST adequate infectious process.

The disadvantage of this method is the wide variability of skin reactions GST in response to PPD for mammals. Thus, in the experimental groups randenborgh Guinea pigs variability of skin reactions GST varies from 12,70% to 26,98%, (range from medium Gv=11-25% to a significant Gv>25% of values)that is not considered as a significant factor that can lead to inaccurate results when implementing the stated objectives, and the need to permutation of the experiment. In turn, the rate coefficient of variation characterizes the reproducibility of the experimental results, in an inverse relationship: the lower the coefficient ecient variations, the higher the reproducibility of research results [Gracin. Biometrics. - M, High school, 1990. with 50-51].

The technical challenge is to improve the reproducibility of the obtained results in the simulation of delayed-type hypersensitivity in Guinea pigs against M. M. bovis strain BCG.

The technical problem is achieved by the method of modeling of delayed-type hypersensitivity in Guinea pigs on mycobacteria, including intradermal injection albino female Guinea pigs received closely related interbreeding brother x sister (F2) avirulent mycobacteria M. bovis strain BCG, followed by the formation of groups(s) animals on the size of the inflammatory reaction in response to intradermal injection of Mycobacterium M. bovis strain BCG, 30-35 days.

The basis of the proposed method are studies conducted on children who were vaccinated avirulent mycobacteria M. bovis strain BCG (BCG vaccine), which was shown correlation between the intensity of skin reactions GST, developing in response to the injection of tuberculin, and the size of post-vaccination scar on the injection of mycobacteria M. bovis strain BCG [Klimenko LI, Polyakova T.S., Buczkowska IU/ quality Assessment of BCG vaccination on the development of post-vaccination mark / Zaporozhye medical journal, No. 1 (70). 2012. S]. When this correlation was obtained on children, suggests a similar dependence in laboratory animals - albino female Guinea pigs received closely related interbreeding brother x sister (F2). So, studies have demonstrated that between the size of the inflammatory reaction in response to intradermal injection of Mycobacterium M. bovis strain BCG, and inflammatory reaction to intradermal injection of PPD for mammals there is a positive correlation, corresponding to 0.85 (p>0,01). In turn, the high rate of correlation coefficient allows to obtain a group of animals with similar values of the inflammatory reaction in response to intradermal injection of PPD for mammals, when using interval estimates.

The claimed invention differs from the prototype in that sensitization number of animals is determined by the table 1 with the frequency (interval) and after development status GST, before conducting the research is conducted by the selection of animals received closely related interbreeding brother x sister (F2) (- ) size of an inflammatory reaction at the injection site mycobacteria M. bovis strain BCG, according to table 1.

Implementation of the proposed method is available in two versions.

When the first option is assumed and the user number of animals for sensitization no less than 15 goals in a 3 rooms interval inflammatory reaction to the introduction of M. bovis strain BCG and highest frequency interval (0,4).

In the exercise of the second option assumes the use of all rooms intervals, so for sensitization of animals used for at least 100 goals that will ensure the distribution of all intervals of the inflammatory response to the introduction of M. bovis strain BCG, and the number of animals at least 10 goals.

The method is as follows. Determine the number of animals (albino, female Guinea pigs received closely related interbreeding brother x sister (F2), weighing 500-600 g)required for carrying out the research (in this case n≥6; when n=6, the number of animals required for sensitization will meet 15), by the formula (I):

N=nf,(I)

where:

N - number of animals needed for sensitization;

n - number of animals needed for the production experiment;

f is the frequency interval (- valued).

Prepare a suspension with a content of mg quantities of mycobacteria in physiological solution, corresponding to 0.2 mg/ 0.1 cm3by calculating the amount of biomass of M. M. bovis strain BCG and the volume of saline required prepared for what I required amount of suspension.

The amount of biomass of M. M. bovis strain BCG, required for sensitization of Guinea pigs, calculated by the formula (II).

M=0,2N,(II).

where:

M - biomass mycobacteria (mg), M. bovis strain BCG

0,2 - dose Mycobacterium (mg)required for one animal sensitization;

N - number of animals needed for sensitization;

The volume of saline needed to prepare a suspension, is calculated by the formula (III).

V=0,2N2,(III)

where:

V - volume of saline (cm3)required for the preparation of suspensions;

0,2 - dose Mycobacterium (mg)required for one animal sensitization;

N - number of animals needed for sensitization;

2 - the conversion factor calculated mass of mycobacteria M. bovis strain BCG in the volume of the suspension.

Then the animals intradermally in delirously site on the abdominal wall sensibiliser mycobacteria M. bovis strain BCG. 30-35 days number of animals and estavudina mycobacteria M. bovis strain BCG, determine the linear dimensions of the inflammatory response, (the sum of the longitudinal and transverse diameters, divided by 2), the results are logged and according to table 1 (column intervals inflammatory reactions to the introduction of M. bovis strain BCG) is formed group(s). These animals are used for research.

An example of implementation of the proposed method.

Example 1. The study assumes conducting research on one group of animals in the amount of 10 goals. The calculations are supposed to be implemented using a number of interval 3.

In table 1 (column frequency interval f) calculate the total number of animals required for sensitization. Thus, to obtain 10 animals, substitute the value 10 in the formula I, and the resulting calculated value of 25 goals.

Example 2. Prepared suspension of mycobacteria M. bovis strain BCG physiological solution. To do this, determine the number of mg of Mycobacterium required for sensitization group of animals (N=25), formula (II), the result is 5 mg. then determine the volume of saline solution, which is prepared suspension, according to the formula (III), and get a value of 2.5 cm3.

Example 3. Perform intradermal sensitization 25 goals albino female Guinea pigs received closely related interbreeding the brother x sister (F 2), weighing 500-600 g at a dose of 0.1 cm3in depilitory area of skin on the abdominal wall.

Example 4. 30-35 days after sensitization form a group of animals according to table 1, with values of inflammatory reactions to the introduction of M. bovis strain BCG, included in the range of 13.0 to 13.5 mm

Example 5. The formed group is experiment.

Table 2 shows the results of variations of allergic reactions GST prototype and the proposed method: group 1 formed randomly, without contributions inflammatory reaction at the injection site mycobacteria M. bovis BCG; group 2 formed according to the size of the inflammatory reaction to intradermal injection of Mycobacterium M. bovis BCG.

The data obtained show that the value of the coefficient of variation in the dose of the DPP for mammals significantly lower in the group of animals, formed in accordance with the proposed method, in comparison with the group generated by the prototype method (p>0,05).

Depending on the planned experiment the proposed method involves the following use cases.

1. When conducting a single comparative studies it is advisable to use a small number of animals of the Central rooms of interval 3, which provides a frequency interval equal to 0.4.

2. When conducting large-scale, including fu is of fundamental research, comparative studies, it is appropriate sheet not less than 100 animals that will provide the group the number of animals from 10 or more and all non intervals.

The result of the proposed method is to improve the reproducibility of research results in the assessment of the impact of immunobiological preparations for skin reaction GST, the study of the mechanisms of reactions GST and evaluation of anti-TB drugs.

Table 1
Intervals inflammatory reaction to intradermal injection of microbes M. bovis strain BCG and PPD for mammals
Interval numberThe intervals of the amount of an allergic reaction to the introduction of the DPP for mammals (mm)Intervals inflammatory reaction to the introduction of M. bovis strain BCG (mm)The frequency interval TH
138,0-39,510,0-10,50,1
211,0-12,50,2
343,0 and 45.5at 13.0 to 13.50,4
446,0 is 48.514,0 is 15.50,2
549,0-a 50.5of 16.0 to 16.50,1
Table 2
The variation of the values of allergic skin reactions GST in groups of Guinea pigs, formed in different ways
No.Group 1Group 2
LM25 ME5 ME1 MEAmounts.25 ME5 ME1 ME Amounts.
here
s
(mm)(mm)(mm)R-I (mm)(mm)(mm)(mm)R-I (mm)
1of 17.011,010,5a 38.514,513,011,0a 38.5
2a 21.515,513,050,015,512,511,039,0
3of 17.513,511,042,016,012,511,039,5
4 15,516,014,045,515,512,510,038,0
516,015,010,541,5of 17.011,010,5a 38.5
6of 17.018,513,048,516,0to 12.011,539,5
720,016,515,552,016,512,510,539,5
8of 17.512,547,015,515,09,039,5
919,015,013,047,015,014,010,539,5
1016,512,510,539,518,0to 12.09,539,5
Gv10,5614,7313,779,606,35a total of 8.747,291,45

Note: Gv - coefficient of variation

The amounts of p-I - aggregate reaction

The method of modeling of delayed-type hypersensitivity in Guinea pig is to Mycobacterium M. bovis, including intradermal injection albino female Guinea pigs received closely related interbreeding brother x sister (F2), avirulent mycobacteria M. bovis strain BCG, characterized in that the sum of the sensitized animals form a group(s) animals on the size of the inflammatory reaction in response to intradermal injection of Mycobacterium M. bovis strain BCG 30-35 days.



 

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