UA61391A - Method for detecting causative agent of anthrax - Google Patents

Abstract

The method for detecting the causative agent of anthrax in air, water and the hard substrates comprises the microscopy of Gram-stained smears followed by seeding the contaminated material to the artificial nutritive media and infecting the susceptible laboratory animals. The suspension of the material to be analyzed (the powder, the washes taken from the objects or the sterilizing filters, etc.) is mixed with the luminescent composition. The mixture is then heated, stained, and centrifuged. Upon centrifugation the supernatant is decanted and the precipitate is washed. Then the smears are prepared according to the crashed drop technique and investigated under the luminescent microscope. The spores of anthracic bacillus are stained bright orange. The precipitate is seeded to the special nutritive medium of the immunodiffusion assay system employing the commercial antitoxic anti-anthracic sera.

Description

Description of the invention

A large number of research methods are used to diagnose the causative agent of anthrax (anthrax). 2 In accordance with the most common scheme (A.S. Labynskaya. Mikrobiologiya s o tekhnikoi mikrobiologicheskih isledsovna. - M.: medicine. - 1978. - 394 p.), the diagnosis of anthrax ip mimo is carried out as follows: on the first day of research, the a sample of blood, sputum or the contents of an anthrax carbuncle of a sick animal and prepare microscopic smears. One smear is stained according to Gram, the second - for the purpose of detecting capsule forms of bacilli according to Romanovsky-Gimaa. The basis for making a preliminary diagnosis of anthrax is 70 detections in samples of gram-positive single rods surrounded by a capsule or their chains and supports.

The main drawback of the method is the frequent absence of capsule forms, in connection with which the causative agent of anthrax can be confused with saprophytic anthracoid bacilli: Vas. Segeyz, you. thesis submitted You. tedaiPegisht, Vas. tusoidez, you apigasoiides, Vas. rzeidoapiygasiz. In this regard, for an accurate diagnosis, it is necessary to isolate a pure culture of the causative agent and perform a bioassay on sensitive laboratory animals, and then, after their illness, clinical examination and death, again isolate a pure culture and diagnose the causative agent of the disease. Thus, the total time required to obtain a probable result is 8-10 days, which does not meet the main requirement: establishing a diagnosis in the shortest possible time for establishing timely preventive and therapeutic measures.

Identification of the causative agent of anthrax is carried out using an anthrax bacteriophage test (N.G. Ipatenko.

Laboratory methods of research in anthrax // Veterinaria. - 1983, Mo7; N.G. Ipatenko

Your differentiation. apipygasiz from spore-forming and soil bacilli / Veterinaria. - 1995. - Mo7).

The reaction is quite specific and allows to differentiate it from pseudoanthrax bacilli. Despite the high sensitivity of this method of diagnosis and its various modifications, the bacteriophage test requires the preliminary selection of a pure culture of anthrax from pathological material or the environment, for 29 to 48 hours. In addition, time is needed for the preparation of a bio sample, which takes several more days. "

Another disadvantage of the method is that it diagnoses not only pathogenic but also avirulent strains. It should also be taken into account the fact that in nature there are pathogenic strains of the causative agent of anthrax that are not sensitive to bacteriophage and saprophytic bacilli that are lysed under the influence of anthrax bacteriophage (N.G. Ipatenko, V.A. Gavrilov, V.S.

Zelepukin et al. Siberian ulcer. - M: Kolos, 1996. - 3356 p.). --

For the purpose of accelerated diagnosis of the anthrax pathogen, the "amber necklace" test is used. At the same time, a three-hour broth culture of the microorganism under study is applied to the agar medium with penicillin, which is previously poured into Petri dishes, with a bacteriological loop. Smears for microscopy are prepared from the colonies growing on the mentioned medium. Colonies of ordinary bacilli grow on the control medium (without the addition of penicillin), and on the experimental medium - colonies formed by spherical cells connected by 3o in chains - "amber necklace". In the latter case, the diagnosis is anthrax. However, as in the previous analogue, the "necklace" method does not allow distinguishing pathogenic strains of anthrax from avirulent ones and requires additional evidence. In addition to the above, in nature there are virulent anthrax bacilli that are not sensitive to penicillin, as well as cereus bacilli, which can acquire a spherical shape under the action of this antibiotic. "

Pharmaconcern Roche in cooperation with the company Sepievi (USA) and some other researchers for the rapid diagnosis of Z 50 causative agent of anthrax, including as part of a biological weapon, propose to use the well-known method of PCR with (polymerase chain reaction) (Egedegikw O.M., KeItap O.A

Iz" diadpovov oi Ipiesbyope dizveazev. // Sp. Ipiyesi Oiv.- 001999,.- m. 29. - pp. 457-458;

NEru/LMmlam. papaeiizBiai sott/primlumapueroiTp/hepibi/5tp/ryyapri/op/oAgi. Lpdeh. sweat. 09.12.01). The optimism of the authors regarding the effectiveness of this method is difficult to share, due to the following reasons: first, for conducting

For the PLeR test, it is first necessary to isolate a pure culture of anthrax from the suspected material, then isolate and

I need to clean the DNA, which will take a lot of time; secondly, there is a high probability of obtaining a pseudo-positive result due to the presence of anthracoid DNA, or vice versa, a pseudo-negative result (inhibition of the reaction due to biological contamination); thirdly, the multi-stage method of purification and concentration of bacterial DNA, as well as the analysis itself, are very time-consuming and expensive and require the creation of complex (Te) 20 stationary molecular biological laboratories.

The closest to the method of diagnosis of the anthrax pathogen proposed by us are the following. and Ascoli precipitation reaction. Its essence is that the material suspected of having the causative agent of anthrax is extracted in a hot or cold way. The resulting extract in the amount of 0.2-0.3 cm 3 is layered on the same volume of transparent precipitating anti-anthrax serum in an Ulengut test tube. In the 29th case of a positive reaction (presence of anthrax bacillus antigen) within 2 minutes after combining the mentioned v. components, a characteristic milk-white ring of precipitation appears. However, this method has weaknesses, namely: a) the reaction can be positive even in the absence of viable bacilli and anthrax spores, which leads to economic losses (destruction of the entire batch of animal raw materials): b) provided a negative result is obtained from the precipitation reaction with the extract , obtained using the hot method, requires a repeated 60 analysis with a cold extract and vice versa; c) when grinding pieces of pathological material, fodder or other dense substrates in a mortar with sand, there is a danger of contaminating the air or surrounding objects, as well as people with anthrax spores; d) a standard antigen is required for the Ascoli reaction, which also complicates the biotest.

A well-known luminescence-serological method of detecting anthrax bacilli using highly specific and immune capsule anti-anthrax sera conjugated with a luminescent dye. The presence of the causative agent of anthrax is ascertained under a fluorescent microscope in the form of rods and/or their chains, which give a green-yellow glow. Capsules glow brighter. The main disadvantage of this method is that the glow can give anthracoid bacilli. Therefore, there is a need to confirm the diagnosis with a bioassay on laboratory animals.

The task of the invention is to develop a reliable method of diagnosing anthrax in any material: livestock raw materials, soil, water, air, including in biological weapons, which would be distinguished by the shortest time for making a final diagnosis, high sensitivity and ease of use, taking into account field conditions. 70 The set task is achieved as follows.

A 0.5-1.0 g sample of the material (powder, fodder, leather raw material, soil, etc.) tested for possible contamination with an anthrax pathogen is poured with a dye solution in a ratio of 1:2 - 1:55 of the following composition: acridine orange 1.0-1 .5, rectified alcohol 967 and dimexide - 20.0 each: 195 aqueous solution of caustic potassium - 1.0; 10956th onion decoction (onion) - 30.0, water distilled to 100.0. The test tube with 7/5 paper is heated for 30 min. at 80-90" C, cool, centrifuge, remove the supernatant. The precipitate is washed 2-3 times in 5-10 volumes of distilled water. After each addition of water, the sample is centrifuged at 3000 rpm for 5-10 min. After washing of the sediment, the supernatant liquid is poured off, and "squeezed drop" smears are prepared from the sediment and examined under a fluorescent microscope. The spores are bright orange in color.

To make a final diagnosis, an experiment with a test system is performed. It is prepared as follows. At the bottom of a clean bacteriological test tube, add 0.5-1.0 ohm of commercial antitoxic anthrax serum or "Antrakol" serum. A transparent agar-agar gel heated to 42-43" 195, prepared on a physiological solution (0.8595 aqueous solution of sodium chloride) is layered on the serum. The test tube is placed vertically until the agar solidifies. After that, 1-2 cm of C liquid nutrient medium, for example, meat-peptone broth with the addition of bovine blood serum (bovine blood serum) and glucose, into which 0.2-0.3 ml of the suspension of the colored sediment is added. After that, the test tubes are placed in a thermostat at "37" C for 12-15 hours.

The test tubes are viewed in penetrating light on a black background, as when recording the Ascoli reaction. If the anthrax pathogen is present in the studied material, a precipitation ring is formed in the agar gel. In the process of growth on a nutrient medium, only anthrax bacilli produce and release a toxin to the outside (beyond the cell wall), which diffuses into the agar gel and is bound by the antibodies of the precipitating serum. Fo

Anthracoid bacilli do not form a precipitation disk.

Thus, as a result of the laboratory tests we have proposed, a preliminary diagnosis can be made in 30-60 minutes, and a final diagnosis in 12-15 hours. "-

Examples of implementation of the method

Example 1. The powder suspected of containing anthrax spores is placed in a test tube in the amount of 0.3 g. - 20.0, dimexide - 20.0, 1956 aqueous solution of caustic potassium 1.0, 1095 bulb decoction - 30.0, distilled water - up to 100 cm3. The test tube with the mixture is heated at 807C for 20 minutes, cool, centrifuge, pour off the supernatant liquid. The precipitate is washed with 5 cm" of distilled water. After each addition of water, centrifugation is carried out at 3000 rpm for 7 min. In parallel, a similar procedure is carried out with cow's milk powder ( control), manufactured in accordance with GOST 13264 - 70 at the Yagotinsky z» milk factory (Kyiv region).

Microscopic preparations of the "crushed drop" type are prepared from the sediments of the test and control tubes and examined under a fluorescent microscope. Bright orange spores were found in the test tube, but not in the control tube. b In order to confirm the diagnosis, a special biotest is performed with a commercial anti-toxic anthrax serum. In test tubes with experimental and control sediments, add sterile physiological o solution, mix and inoculate 0.2 cm? suspensions for test systems, made as indicated in the description of the invention. The composition of the nutrient medium of the test system is as follows: 1 cm3 of meat peptone broth, food grade Z, inactivated bovine blood serum and 195 g of glucose. The tubes with the test system are placed in a thermostat at 377C for 12-15 hours. When recording the results of the immunodiffuse reaction after 12 hours in the test tube, a characteristic ring of precipitation was found. There is no precipitation disk in the control.

Example 2. Water from a surface source suspected of being contaminated with anthrax spores.

0.5 dm are taken into sterile bottles? water to be tested for contamination with anthrax spores.

Water is filtered through bacteriological Moss membrane filters (pore diameter - 0.3 μm) using a Seitz filter. After that, the membrane filters are turned over and washed with 100 cm? of filtrate The resulting suspension is heated in a water bath for 20 minutes. at 75"C, cool. Then a series of multiple dilutions is made in sterile tap water and the suspension is inoculated on Petri dishes with solidified agar medium (composition of 60 medium as in example 1). The Petri dishes are placed in a thermostat at 37"C. After 3-6 hours of incubation, the Petri dishes are examined to identify colonies characteristic of anthrax bacilli. These are flat matte gray rough (K-shaped) colonies with fringed edges or curly processes. The total number is counted the number of colonies of spore-forming microorganisms and the number of colonies morphologically similar to colonies of the causative agent of anthrax. Using a bacteriological loop, material is selected from the colonies for inoculation into a biotest tube 65 containing antitoxic anthrax serum (as in example 1). Examination of the tubes after 12 and 15 hours of incubation did not reveal a precipitation ring.Therefore, the tested water was not contaminated with anthrax spores, but contained spores of saprophytic bacilli.

Example 3. Investigation of a soil area suspected of being contaminated with anthrax spores.

Soil samples weighing up to 100.0 g are taken by random sampling. in sterile jars with lids. In the laboratory, large particles of plants, roots, etc. are removed from the soil sample, the soil is mixed, Zog is weighed and it is poured with ZOsm? 0.595 sodium pyrosulfate solution. The mixture is stirred for 20 minutes. and leave for 5 minutes.

The supernatant is drained and filtered through a sterilizing Seitz filter membrane. After that, the filter is washed 10 cm? of sterile tap water, centrifuged for 10 min. at 3000 rpm. The supernatant liquid is drained. The resulting precipitate is resuspended in a solution of a fluorescent dye and dyed as noted in Example 1. Similarly, a sample of control (known to be clean) soil is prepared. "Crushed drop" preparations are prepared from experimental and control sediments and examined under a fluorescent microscope. Orange-colored spores were found in the test and control soil samples. After that, they put a biotest with anti-toxic anthrax serum "Antrakol". Experimental and control test tubes with biosystems are inoculated, respectively, by 0.5 cm? experimental and control material and put them in a thermostat at 37"C. After 12 hours of incubation, a clear precipitation ring was found in the agar disk of the test sample. The control soil sample did not show a precipitation ring.

Example 4. A suspension of supports of the Vas strain is sprayed in a hermetic chamber of KPG-1. apigasiz After that, air is pumped from the chamber for 6 minutes with the help of a volunometric sampler from the atmosphere (Burkhard company, Great Britain), which is passed through a special bubble chamber with a 0.596 aqueous solution of the bacterial exopolysaccharide preparation "Baktosol" (certificate for the mark for goods and services Mo 7946 dated 12.07.2001).

The solution is heated at 80"7C for 20 minutes and centrifuged, the supernatant liquid is poured off, the sediment is washed three times, then it is mixed with a dye solution, dyed, as in example 1. From the sediment, "squeezed drop" preparations are prepared and microscopy is carried out under a fluorescent microscope. as a result, bright orange spores were found.

In order to confirm the preliminary diagnosis, with the help of a bacteriological loop, seed material is selected from the sediment, which is used to inoculate a test tube with a prepared test system and put it in a thermostat at 377C.

By visually recording the result of the reaction after 13 h. incubation revealed a clear disc of precipitation. Therefore, the sample of the studied air contains anthrax supports. --

Advantages of the proposed method Fo

Practically all known methods of diagnosing anthrax require mandatory selection of a pure culture of the causative agent, study of its morphological and cultural-biochemical properties, preparation of a biological sample with subsequent isolation of the causative agent from the organisms of dead laboratory animals, and its identification. From 7 to 10 days are spent on these "procedures", which does not allow timely organization of appropriate medical and preventive measures and leads to large losses. (Se)

Compared to known analogues, the proposed method of diagnosis has a number of advantages: 1. The time for making a preliminary diagnosis is 30 - 60 minutes, the final one - up to 12 - 15 hours, which allows you to quickly take all necessary treatment and preventive measures, especially in the case the use of "anthrax" supports as a biological weapon. 2. To make a final diagnosis (conducting a biotest with antitoxic anthrax serum), it is not necessary to isolate a pure culture of the causative agent of anthrax, it can be carried out with a stock culture. h C. The proposed method of express diagnosis of the causative agent of anthrax is simple, reliable and economical. For example, compared to PCR - a method that, moreover, often gives pseudo-positive or pseudo-negative results, it is hundreds of times cheaper and can be implemented in field conditions. 4. The proposed method of express diagnostics makes it possible to reduce as much as possible the loss of troops and (2) the population from anthrax infection with the use of appropriate bioweapons by means of mass immunization of people - the toxin-vaccine "Anthracol", which can be administered using an over-the-counter injector. (95) so 20

Claims (2)

The formula of the invention -. and 1. The method of diagnosis of the causative agent of anthrax in air, water and solid substrates, which includes microscopy of Gram-stained microscopic smears, sowing of contaminated material on artificial nutrient media and infection of sensitive laboratory animals with it, which differs in that the suspension of the investigated material (powder, washings from environmental objects, sterilizing filters, etc.) are mixed with a fluorescent mixture of a special composition: acridine orange 1.0-1.5; rectified ethyl alcohol 967 -20 and dimexide - 20.0; 1 bb aqueous solution of caustic potassium - 1.0; 10 95% onion husk decoction 20.0-30.0; water is distilled to 100 cm, heated to 80-90" and colored for 20-30 minutes, centrifuged for 3-6 min. at 3000 rpm, the supernatant liquid is drained, the sediment is washed 2-3 times in 5-10 volumes of 60 physiological solution, "squeezed drop" smears are prepared from the sediment and examined under a fluorescent microscope - anthrax spores are bright orange, the sediment is sown in the nutrient medium of a special immunodiffusion test system using known commercial antitoxic anthrax serums in order to detect the exotoxin of the anthrax pathogen. 2. The method according to claim 1, which differs in that the immunodiffusion reaction is carried out with the toxin-vaccine "Antrakol". b5 Official Bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2003, M 11, 11/15/2003. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. " "- Fo so "- (Se) shi s ;" (22) - (95) at 50 - 60 b5