A method of producing antigen for serological diagnosis of paratuberculosis from ruminant

 

(57) Abstract:

The method is designed to obtain antigen of a strain of Mycobacterium paratuberculosis VGNKI"KA"-DEPT. The strain is cultivated in an environment of Watson-RAID for 28 to 35 days. Grown bacterial mass is washed with distilled water, suspended in saline to a concentration of 70 - 90 mg/cm3and disintegrate ultrasound at 22 kHz for 15 to 16 minutes to Separate the cellular debris by centrifugation at 16 to 20 thousand rpm for 50 minutes. Supernatant concentrate thiomersal rate of 1:10000. The method allows to obtain more active and specific antigen be used to diagnose paratuberculosis ruminants in REID. The use of REED received the antigen will reveal 1.4-9.8 times more sick animals than RAC. The diagnosis of the disease is simple, intuitive, fast and cheap. table 1.

The present invention relates to the field of veterinary immunology, in particular, to a method for producing antigen for serological diagnosis of paratuberculosis from ruminant.

Paratuberculosis is a chronic infectious disease of ruminants, widespread throughout the world, including in stravato 2 - 5% of cases. More common asymptomatic form of the disease that can last for years.

For a correct diagnosis it is necessary to conduct comprehensive studies using allergic, pathologic-anatomic, bacteriological and serological methods.

Known antigens for the detection of humoral antibodies in the serum of patients with paratuberculosis animals in the reaction of complement fixation (RAC), as well as in the reaction immunodiffusion in agar gel (REED).

A method of obtaining antigen for serological diagnosis of paratuberculosis from ruminants, including the production of bacterial mass from a strain of Mycobacterium paratuberculosis N 18 (Vavricka collection of strains of microorganisms), mechanical demolition, removal of cell debris by centrifugation and drying of the supernatant (1).

The disadvantage of this antigen is the low sensitivity due to the fact that for more drug uses coarse mechanical destruction of the bacterial mass (mechanical press), which leads to a considerable loss of serologically active components. In addition, for polovich, that led to changes in some of its biological properties.

There is also known a method of producing antigen for serological diagnosis of paratuberculosis from ruminants, including the production of bacterial mass from strains of Mycobacterium paratuberculosis II-V N106 and Teps N 105 (Vavricka collection of strains of microorganisms), by growing them on a RAID environment within 45 - 60 days, filtering, washing residue from the environment with distilled water, the concentration by centrifugation at 2000 - 3000 rpm for 15 min, treatment with acetone, dialysis, adding thimerosal 1:5000, subsequent disintegration with ultrasound for 30 min, the deposition of cellular debris by centrifugation at 5000 rpm for 30 min, the separation of the supernatant with the content of 3.5 - 7 mg/cm3protein and freeze drying of the target product (2).

The disadvantage of this method is the low sensitivity of the obtained antigen because of its use in the RAC, as well as its low specificity due to the fact that up to 50% of the reactions in animals are false positive (non-specific).

This method is also time-consuming to obtain the target product.

In order isoi, as well as reducing costs for receiving.

This goal is achieved by the fact that the receipt of the bacterial mass is carried out strain of Mycobacterium paratuberculosis VGNKI "KA" DEPT, which is grown on the environment Watson-RAID for 28 to 35 days, after washing with distilled water bacterial mass suspended in saline to a concentration of 70 - 90 mg/cm3that disintegrate at 22 kHz for 15 to 16 min, precipitated cell debris by centrifugation at 16 to 20 thousand rpm for 30 - 50 minutes, after separation of the supernatant can thiomersal rate of 1:10000.

A new strain of Mycobacterium paratuberculosis obtained from isolates, isolated from the body of the patient by paratuberculosis goats. Strain through a temporary crops and serial passages adapted to growth on synthetic medium Watson-Reid.

The strain of M. paratuberculosis deposited in the collection of microorganisms of the all-Russian state research Institute for control, standardisation and certification of veterinary preparations and has a registration number KA - DEPT.

The strain of M. paratuberculosis VGNKI "KA"-DEPT characterized by the following features and properties.

Its taksonomicheskaya signs. When dyeing method Zn-staining smears - small straight or slightly curved ruby red sticks with 1-2 pellets collected in piles or lying isolated, length of 1-2 μm and a width of 0.3 to 0.5 microns.

Cultural properties. On elective egg environments (Levenshtein-Jensen, Gerrold, Finn-2) mycobactin 14 - 16 day incubation at 37 - 38oC forms a hemispherical colony of gray-white color. On this same environment without enrichment mycobactin growth is missing.

Adding an additional Lowenstein-Jensen with micractinium sodium salicylate in amounts of 1 mg/cm3, tubazide rate of 5 mg/cm3, streptomycin and ethambutol in the amount of 1 µg/cm3and 50 µg/cm3respectively, did not inhibit the growth of strain VGNKI "KA". The nature of the growth and morphology of colonies on löwenstein-Jensen with these drugs is identical growth on löwenstein-Jensen with mycobactin.

Enzymatic properties. Strain VGNKI "KA" not reduces nitrate to nitrite. Under the action of special reagents, the colour of the solution does not change. Causes hydrolysis of Tween-80. Under the action of enzymes change the color of the substrate from amber to pink-red. Catiii sera to the strains of "KA" forms 4 lanes precipitation, and with antisera to the strains of Mycobacterium paratuberculosis isolated from cattle, three bands of precipitation.

Virulent properties. In hamsters and white mice causes massive bacterial dissemination parenchymatous organs and intestines.

Example 1

The strain of M. paratuberculosis VGNKI "KA" can be grown in liquid synthetic medium Watson RAID at 37 - 38oC for 28 to 35 days. The bacterial mass is washed filter paper residue from the environment 2-3 times with distilled water. After washing the bacterial mass is suspended in sterile saline to a concentration of 70 mg/cm3and subjected to disintegration by ultrasound at 22 kHz for 15 minutes After disintegration with ultrasound conduct the deposition of cellular debris by centrifugation at 16000 rpm for 30 min and separate the supernatant containing the antigen with a protein concentration of 1.5 mg/cm3. Before filling and capping in the finished drug make the preservative thiomersal rate of 1:10000.

Example 2

The antigen is prepared analogously to example 1. Washed with distilled water bacterial mass from a strain of M. paratuberculosis VGNKI "KA" suspended in sterile saline, Astorga detritus carried out by centrifugation at 18000 rpm for 40 min and separated supernatant, containing the antigen with a protein concentration of 1.6 mg/cm3.

Example 3

The antigen is prepared analogously to example 1. Washed with distilled water bacterial mass from a strain of M. paratuberculosis VGNKI "KA" suspended in sterile saline to a concentration of 90 mg/cm3and subjected to disintegration by ultrasound within 16 minutes Deposition of cell detritus carried out by centrifugation at 20000 rpm for 50 min and separate the supernatant containing the antigen with a protein concentration of 1.8 mg/cm3.

Ready antigen is a yellowish liquid with a slight opalescence.

Antigen retains its serological properties within 24 months. store in a dry dark place at a temperature of 2 - 8oC.

Example 4

Learning activity 3 variants derived antigen in the reaction, immunodiffusion in gel (REED) with blood sera of patients with paratuberculosis animals. The reaction placed in Petri dishes (d = 100 mm). Cups are arranged on a horizontal surface and each make 16 cm3molten agar. Leave at room temperature until fully cured. Stamp punch in agar wells with one Central and six, the second needle. One Cup place 6 pieces for study of 36 serum samples.

In the Central hole making the antigen according to example 1, 2 or 3, in the peripheral subjects serum. Explore 18 serum samples of cattle, 39 blood sera of sheep and 59 of the blood sera of goats of farms affected with paratuberculosis the enteritis. In a Cup put the control with positive paratuberculosis hyperimmune rabbit serum (reference serum). Petri dishes covered with a lid and leave at temperatures below 20oC.

The reaction accounting visually in daylight or with light OI-19. The formation of one or more bands of precipitation between the Central hole to the antigen and peripheral holes with subjects sera after 18 - 48 hours after administration of the reaction indicates a positive reaction. In the absence of control with reference serum line precipitation results REED with test sera are not subject to assessment.

Upon receipt of positive results in REED recognize animal infected with the causative agent of paratuberculosis.

Set in REID, the activity of all three received antigens with issleduemyi of antigens.

Register with positive reactions in REED: 10 of 18 serum samples of cattle, 21 - 39 sera of sheep and 33 of 59 blood sera of goats. Thus, recognize, 55.6% of the cows, 53.8 per cent of sheep and 55.9% of the goats from the studied population infected with the causative agent of paratuberculosis in farms affected with this disease.

Example 5

Study the specificity of the antigen in REED in example 1 in respect of closely related pathogens of the Mycobacterium tuberculosis bullish view.

Similarly, as in example 4 are examined in REED 50 serum samples of cattle from tuberculosis insulator and 20 serum samples from animals with tuberculous lesions in organs at autopsy.

The investigated antigen with all sera does not form lines precipitation, which indicates negative REED and specificity of antigen.

Example 6

Study the specificity of the obtained antigen REED in example 3 with hyperimmune sera against brucellosis (13 samples), pseudotuberculosis, Campylobacter, leptospirosis (4 samples each serum), Yersinia (2 samples). With all sera analyzed antigen in REED does not form lines precipitation, which confirms its Speu 2 and commercial antigen, Subnavi.

Similarly, as in example 4, are examined in REED 18 serum samples of cattle, 38 serum samples of sheep and 91 serum of goats derived from households, disadvantaged by paratuberculosis the enteritis. At the same time, all the sera examined in RAC commercial antigen, Subnavi. The results of comparative studies of blood serum on paratuberculosis presented in the table.

The table shows that the proposed antigen significantly outperforms its activity known drug and identifies 1.4 - 9.8 times more sick animals than production antigen, Subnavi.

Thus, the inventive antigen is an active and specific drug.

The use of REED with the proposed antigen is available to any veterinary laboratory, because it doesn't require special equipment and highly qualified personnel.

The method is simple, intuitive, fast and cheap.

REED with the antigen can be applied directly in the field (on-farm, sheep herds).

Use for serological diagnosis of paratuberculosis from ruminant improved method will allow a timely manner the definition of damage from this disease.

Sources of information:

1. D. M. Sherman, K. J. F. Markham, F. Bates "Agar del immunodifusion testfor diagnosis et clinical paratuberculosis in cattle" J. Amer Veter Med. Association (AVMA), 1984, 185, N 2 p. 179 - 182

2. Patent Romania N 81670, class A 61 K 39/02, 19833

A method of producing antigen for serological diagnosis of paratuberculosis the ruminant animal, comprising culturing the bacterial strain of Mycobacterium paratuberculosis in culture medium, washing the bacterial mass with distilled water and subsequent disintegration with ultrasound, deposition of cellular debris by centrifugation and separation of the supernatant, characterized in that use strain of Mycobacterium paratuberculosis VGNKI "KA"-the DEPOT, which has been cultivated in an environment of Watson-RAID for 28 to 35 days, after washing with distilled water obtained bacterial mass suspended in saline to a concentration of 70 - 90 mg/cm3, disintegration by ultrasound performed at 222 kHz for 15 to 16 min, precipitation of cell debris by centrifugation is carried out at 16000 - 20000 rpm for 30 - 50 minutes, after separation of the supernatant can thiomersal rate of 1 : 10000.

 

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