RU2279274C2 - Method for preventing secondary alveolar echinococcosis (hydatidosis) - Google Patents

Abstract

FIELD: veterinary and medicinal helminthology.

SUBSTANCE: the present innovation deals with introducing cell culture out of Echinococcus multiocularis protoscoleces mixed with ribotan in efficient quantities, thrice subcutaneously at 10-d-long interval.

EFFECT: higher efficiency of prophylaxis.

2 ex, 3 tbl

Description

The invention relates to veterinary and medical helminthology and can be used for immunoprophylaxis of secondary alveolar echinococcosis (hydatidosis).

Alveolar echinococcosis (hydatidosis), the causative agent of which is Echinococcus multilocularis, is one of the most dangerous helminthozoonoses, which pose a serious threat to human health and are relevant for veterinary medicine. The prevalence of cystic and alveolar hydatidosis in recent years has been increasing due to increased urbanization, increased population migration, the collapse of established economic relations, as well as due to the deterioration of social life and reduced sanitary and epidemiological control (Markin A.V., 1999; Musaev G.K. ., 1999 Kovalenko F.P. et al., 2002).

The degree of negative impact of this invasion on the health of infected people is difficult to assess. The disease in advanced cases is incurable, because, as a rule, in this period, in addition to significant liver damage, metastases are already present in other organs, and even liver transplantation may be ineffective. That is why the problem of prevention and treatment of this socially dangerous helminthozoonosis has been and remains one of the priority areas of helminthological science and practice.

In the fight against alveolar hydatidosis, two pathways are most acceptable: chemotherapy and vaccine prophylaxis.

Chemotherapy requires not only large doses of chemicals, but also a long (years) time of their use. But even in this case, it is not effective enough, since the development of the parasite is stopped, and not its final death.

Methods for the prevention of secondary alveolar hydatidosis include the use of specific and nonspecific stimulants that increase the resistance of animals and humans to infection, inhibit the growth of metacestodes of E. multilocularis and cause the death of the entire parasite population.

It is known that nonspecific resistance of mice to E.multilocutaris infection is achieved by relatively low doses of BCG vaccine (10 ³ -10 ⁷ colony forming units), which is expressed by significantly smaller sizes of parasitic nodes in vaccinated animals compared to control (Reuben, Tanner, 1978). However, at a dose of 10 ⁵ -10 ⁷ BCG units, mice developed tuberculous granulomas (Reuben et al., 1978)

An identical and even stronger effect with complete suppression of the growth of metacestodes of E. multilocularis was observed in experimentally infected cotton rats, which received a dose of 26.4 × 10 ⁶ units of BCG. This dose was well tolerated and 100% killed parasite larvocysts in experimentally infected Mongolian gerbils (Rau and Tanner, 1975).

However, the development of tuberculosis granules in mice observed at elevated doses of BCG requires more careful development of the dose of this vaccine and the development of treatment for tuberculosis (Thompson, 1976).

Protection against alveolar hydatidosis was also achieved by stimulating cotton rats with cell peritoneapal exudate obtained 3 days after intraperitoneal administration of phytohemagglutinin. This procedure significantly protected animals from the development of Echinococcus multilocularis larvocysts in the abdominal cavity and internal organs (Reuben, Tanner, 1983)

Closest to the claimed technical solution is the creation of specific genetic engineering vaccines, the development of which is at an early stage and includes primarily the cloning of the protective gene.

A recombinant vaccine is known, which is a fragment of the II / 3-IO gene of the metasestode of E. multilocularis, which encodes a species-specific antigen and expressed in a weakened vaccine strain of Salmonella typhimurium, which, after subcutaneous and oral administration, elicited a humoral and cellular immune response in mice and dogs. In both methods of vaccine administration, synthesis of antibodies against S. typhimurium antigens and the recombinant E. multilocularis antigen was observed, but peripheral blood lymphocyte proliferation into antigens was not observed. In response to the recombinant E. multilocularis antigen in dogs, local proliferation was noted only with subcutaneous administration of the vaccine (Gottstein et al., 1990).

Regarding the use of recombinant vaccines for parasitic diseases, including secondary alveolar and cystic hydatidoses, there is an authoritative opinion of most scientists about the prospects of further research in this direction, including questions such as dose development, methods of administering vaccines, studying the ability of vaccines to eliminate or kill parasites and the development of immune memory (Bessonov A.S., 2000).

Considering the serious danger of the alveolar form of echinococcosis (hydatidosis) to humans and the prospects for the development of vaccine prophylaxis for this invasion, work was carried out to develop a method for the prevention of secondary alveolar echinococcosis (hydatidosis) in the mouse model E. multilocularis; rat - E. multilocularis using a specific antigenic preparation and a non-specific immunomodulator.

A specific antigen is the main component of immunopreparations that promote the development of humoral-cellular mechanisms of directed action, block the vital functions of the parasite and ensure the development of immunity to subsequent infection.

The purpose of the invention is to find means of immunization, providing the development of a protective immune response that protects against subsequent infection.

The essence of the invention lies in the fact that the protective effect against secondary alveolar echinococcosis (hydatidosis) is achieved by injection of an immunopreparation including antigens of cell culture of E. multilocularis protoscolexes (a specific component - hereinafter referred to as "cell" antigen), which is quantified by the protein and ribotan content new complex immunomodulator. The protein in the cell antigen was determined spectrophotometrically according to Layne (1957).

“Cellular” antigen is an antigenic metabolite of protoscolex cells isolated from secondary larvocysts of E. multilocularis and cultured in artificial nutrient media with the addition of the necessary growth factors for active cell proliferation in culture.

A method of obtaining a "cellular" antigen is described in detail (Berezhko V.K. et al. "Method for the production of diagnostic cestode antigen", patent for invention No. 2219551). The “cellular” antigen elicits an immune response in rabbits and reacts with a homologous antiserum, forming at least 5-6 antigen-antibody complexes in the form of precipitation bands in the agar gel immunodiffusion reaction. Immunization of rabbits with a “cellular” antigen together with ribotan activated the immune response, which was manifested by an increase in the number of detectable antigen-antibody complexes by 1-2 and more distinct agar gel precipitation bands with the same quantitative ratios of antiserum and antigen used in the immunodiffusion reaction.

Ribotan is a compound of low molecular weight (0.5-1.0 kDa) polypeptides and low molecular weight RNA fragments. This drug belongs to the group of immunomodulators of natural origin and has an effect on the T- and B-system of immunity, stimulates immunoreactivity to a specific antigen, the functional activity of macrophages, a subpopulation of T- and B-lymphocytes, as well as the synthesis of interferon and lymphokines. The drug is used in medical practice.

Immediately before conducting experiments on animal models, the necessary amount of an immunizing dose of the “cell” antigen of E. multilocularis (protein) in volume terms was determined and mixed with the calculated according to the instructions dose of the ribotan immunomodulator. The resulting immunotherapy was used to prevent secondary alveolar hydatidosis in animal models.

A single immunizing dose of the immunopreparation for mice was 0.2 ml (80 μg of protein) of the “cellular” antigen and 5 μl of ribotan; for rats - 0.45 ml (200 μg of protein) of the "cellular" antigen and 50 μl of ribotan.

The protective properties of the immunopreparation were studied in animal models with 3-fold subcutaneous administration with an interval of 10 days and subsequent verification infection of the immunized and control animals with invasive material Echinococcus multitocularis 14 days after the last immunization.

Examples of specific performance.

Example 1. The protective properties of the immunopreparation ("cellular" antigen + ribotan) were evaluated in experimental secondary alveolar echinococcosis (hydatidosis) in mice.

Studies were carried out on 48 white mongrel mice weighing 16-18 g, divided into 4 equivalent groups (Table 1).

The first group of mice was immunized subcutaneously 3 times with an interval of 10 days by immunopreparation at the rate of 0.2 ml (80 μg protein) of the “cellular” antigen and 5 μl of ribotan per mouse.

The second group of mice received subcutaneously 3 times with an interval of 10 days, 0.2 ml (80 μg of protein) of the "cellular" antigen and 5 μl of sterile saline.

The third group of mice received subcutaneously 3 times with an interval of 10 days, 0.2 ml of sterile saline and 5 μl of ribotan

The fourth group of mice (control) received 3 times, subcutaneously with an interval of 10 days, 0.2 ml of sterile saline.

After 14 days, mice of all four groups were infected with protoscolexes and acephalocysts of E. multilocularis at a dose of 200 ± 20 ind. The experimental mice were slaughtered on day 70 after infection. The experimental results presented in Table 1 showed that 11 mice of the first group immunized with an immunopreparation remained uninfected, single larvocysts less than 1-1.5 mm in size in the mouse without invasive elements of E. multilocularis were found in one mouse.

Thus, the protection efficiency was 91.7%.

In the mice of the second group immunized with only the “cellular” antigen without ribotan, the protective effect was 75%; in three mice (25%) of 12, parasite cysts without germ elements were also found in the liver.

The mice of the third group immunized with ribotan alone, in most cases (9 mice) were infected and had numerous larvocysts in the liver and in the abdominal cavity up to 6-8 mm in size with the presence in some of them of germinal elements of alveolar echinococcus.

The mice of the fourth control group were all infected; numerous larvocysts were found in them in all internal organs and in the abdominal cavity up to 1.2 cm in diameter. In most parasite larvocysts, the formed protoscolexes were recorded.

Example 2. The protective properties of the immunopreparation ("cellular" antigen + ribotan) in experimental secondary alveolar echinococcosis (hydatidosis) in rats.

Studies were conducted on 36 white outbred rats weighing 180-200 g, divided into 4 equivalent groups.

The first group of rats was immunized subcutaneously 3 times with an interval of 10 days by immunopreparation at a dose of 0.45 ml (200 μg protein) of the “cellular” antigen and 50 μp of ribotan / rat.

The second group of rats was immunized 3 times subcutaneously with an interval of 10 days only "cellular" antigen at a dose of 0.45 ml (200 μg of protein) and 50 μl of sterile saline / rat.

The third group of rats was immunized subcutaneously 3 times with an interval of 10 days at a dose of 0.45 ml of sterile saline and 50 μl of ribotan / rat.

The fourth group of rats (control) received subcutaneously 3 times with an interval of 10 days, 0.5 ml of sterile saline.

14 days after the last injection, rats in all four groups were infected with invasive material (protoscolexes, acephalocysts) Echinococcus multilocularis at a dose of 600 ± 50 ind.

Slaughter of experimental animals was carried out on the 70th day after infection. The autopsy results presented in Table 2 showed that the rats of the first group immunized with the immunotherapy remained uninfected; the larvocyst E.multilocularis was not found in the internal organs and in the abdominal cavity, i.e. the immunotherapy showed a 100% protective effect. In 7 rats of the second group immunized with only the “cellular” antigen, parasite larvocysts were also absent, in two larvocysts up to 1.5–2 mm in diameter were found, in which there were no germ elements. The protective effect was 77.8%. Only in one rat out of 9 in the third group immunized with ribotan, parasite larvocysts were absent; in 8, E. multilocularis larvocysts were found both in the liver and in the abdominal cavity, ranging in size from 1.2 to 15 mm in diameter. In some large-sized cysts, the formed protoscolexes were recorded during microscopy. The rats of the fourth control group were all infected, in the internal organs and in the abdominal cavity they recorded multiple lesions ranging in size from 2.5 to 25 mm in diameter, most of which formed protoscolexes that retained their invasive properties were found, which was established by a subsequent bioassay on mice.

Information sources

1. Bessonov A.S. // Veterinary medicine. - 2000. - No. 11. - p. 3-6.

2. Markin A.V. // Theory and practice of controlling parasitic diseases. - 1999. - p. 152-154.

3. Musaev G.Kh. // Theory and practice of controlling parasitic diseases. - 1999. - p. 170-171.

4. Gottstein B., Muller N., Cryz S., Vogel M., Tanner J., Seebeck T. // Parasite Immunol. - 1990. - V.12, No. 2. - R.163-174.

5. Kovalenko F.P. et al. // Acta Parasitologica, VIII Europ. Microcolloquium of Parasitol. - 2002. - V.45, No. 3, - P.241-242.

6. Layne E. // Methods Enzymol. - 1957. - V.3 - P.447-454.

7. Rau M.E., Tanner C.E. // Nature, London. - 1975. - V.256, No. 5515. - P.318-319.

8. Reuben J.M., Tanner C.E // Short. Commun., Warszawa. - 1978. - Sec E. - P.51-52.

9. Reuben J.M., Tanner C.E., Rau M.E. // Infect and Immun. - 1978. - V.21, No. 1. - P.135-139.

10. Reuben J.M., Tanner C. E // Parasite Immunol. -1983. - V.5, No. 1. - P.61-66.

11. Thompson R.C. // Z. Parasitenk. - 1976. - V.21, No. 1. - S.31-36.

Table 1. The results of the evaluation of the protective properties of the immunopreparation ("cell" antigen of E. multilocularis + ribotan) in secondary alveolar hydatidosis in mice. Group Numbers The number of mice in the group Immunizing agent Immunizing dose, mcg protein The frequency of immunization The interval between immunizations, days Dose of infection, ind. The number of infected mice,% The effectiveness of protection,% Note I 12 Immunotherapy 80 3 10 200 ± 20 1 (8.3) 91.7 In one mouse, single larvocysts were found in the liver with a diameter of 1-1.5 mm without invasive elements. II 12 "cellular" antigen + physical. solution 80 3 10 200 ± 20 3 (25) 75 Larvocysts of alveolar echinococcus up to 1.5 mm in diameter were found in 3 mice in the liver, and there were no germ elements. III 12 Ribotan + physical. solution - 3 10 200 ± 20 9 (75) 25 Nine mice recorded numerous larvocysts up to 8 mm in diameter with germ elements of E. multilocularis IV 12 Saline - 3 10 200 ± 20 12 (100) - All mice are infected, numerous larvocysts 1.5-12 mm in diameter in the internal organs and in the abdominal cavity with the presence of protoscolexes were found.

Table 2. The results of the evaluation of the protective properties of the immunopreparation ("cell" antigen of E. multilocularis + ribotan) in rats with secondary alveolar hydatidosis. Group Numbers The number of rats in the group Immunizing agent Immunizing dose, mcg protein The frequency of immunization The interval between immunizations, days Ex infection dose The number of infected rats,% The effectiveness of protection,% Note I 9 "cellular" antigen + ribotan 200 3 10 600 ± 50 - one hundred Rats are not infected. In the internal organs and in the abdominal cavity, larvocysts were not found. II 9 "cellular" antigen + sterile physical. solution 200 3 10 600 ± 50 2 (22.2) 77.8 Two rats were infected, larvocysts up to 2 mm in diameter were found in them, germ elements were absent. III 9 Ribotan + sterile physical solution - 3 10 600 ± 50 8 (88.8) 11.2 In 8 rats, numerous larvocysts were found both in the liver and in the abdominal cavity, from 1.2 to 15 mm in diameter, parasite protoscolexes were recorded in some IV 9 Sterile saline - 3 10 600 ± 50 9 (100) - All rats are infected, all have numerous larvocysts in the internal organs and in the abdominal cavity from 2.5 to 25 mm in diameter, the majority of the formed protoscolexes are found.

Table 3 Evaluation of the protective properties of the "cellular" antigen (ClAG) of protoscolexes Echinococcus multilocularis in complex with ribotan (P) in secondary alveolar hydatidosis in rabbits No pp Number of rabbits in gr. Immunizing agent Immunizing dose, mcg the multiplicity of immunization., days. The interval between immunization., Days. Dose of infection, ind. Number of infected female workers,% The effectiveness of protection,% Note I 9 CLAG + P 300,0 3 10 600 ± 50 1 (11.1%) 88.9% In one rabbit, small, single larvocysts in the liver were found to be 1.0-1.5 mm in diameter without invasive. elements. II 9 KlAG + physical. rr 300,0 3 10 600 ± 50 4 (44.4%) 55.6% Larvonists 1.5–2.5 mm in diameter were found in 4 rabbits, and there were no germ elements. III 6 Ribotan + physical. p-p 15 μl + 0.3 ml 3 10 600 ± 50 4 (66.7%) 32.3% In 4 rabbits, numerous echinococcus larvocysts with germinal elements were recorded in the abdominal cavity and in the liver. IV 6 Fiz. rr 0.3 ml 3 10 600 ± 50 6 (100%) - All rabbits are infected, numerous larvocysts up to 16-25 mm in diameter, in the abdominal cavity and in the internal organs, in most of them protoscolexes of the parasite, have been found.

Claims (1)

A method for the prevention of secondary alveolar echinococcosis (hydatidosis) in animals, which consists in the fact that the antigen of a cell culture from protoscolex Echinococcus multiocularis is mixed with ribotane in effective amounts and administered to animals three times subcutaneously with an interval of 10 days.