Ветеринария/2. Ветеринарная медицина

M. Kozhabayev

Southern-Kazakhstan Research Veterinary Station, Kazakhstan

Comparative Studying the Aspects of Theileric Antigens By Sulphurological Methods

At present the existing methods of the diagnostics of theileriosis base on account of epizootological conditions, the areal and seasonal diseases, clinical signs and the results of the microscopically studying, not to allow to reveal all animals- parasite-carriers. So the big scientific and practical importance has sulphurological methods, allowing not only to put diagnosis on theileriosis quickly, but also to study epizootological situation and the immunological condition of organism in dynamics of theileriogical process, the importance of humeral immunity and other aspects, and it is impossible to develop scientifically motivated measures of pest control, therapy and specifical maintains without knowledge.

M.I.Titushin (1967), M.P.Konyukhov, G.V.Poluboyarova (1967), N.I.Stepanova (1970) and others have informed about possibilities of using the sulphurological methods from investigations in studying Phlebotomize- Parasitical Diseases of large horned live-stock.

From existing sulphurological tests the Reaction of the Connecting Complement (RCC) is considered to be the most acceptable, specificity and validity of which is confirmed by N.I.Stepanova (1971). Studying of other immunological tests has not been conducted at all. There is only single report of possible usage on diagnostics of theileriosis and reaction of hemoglutination, delays of hemoglutination (M.I.Tutushin, 1969) and the tests of immunofluorescention (Schindler, Wokatsch, 1965).

Consequently, in studying theileriosis, among immunological tests RCC is used mostly. However, validity of the evidences of RCC depends on specificity and active usage of antigens. As a rule, the blood of sick animals serves in preparation of source material. The important stage in process of the preparation of antigens is separating the blood-parasites from erythrocytes concentrating and peeling from the incitants. For this reaction the saponin solution (Schindler, Mehlitz, 1968), the sulphuric ether and distilled water (N.I.Stepanova, 1970), the ultrasound (N.I.Stepanova, Cadir with cooper…, 1970: Prior,Kreier, 1972) are used. Liberated parasites are active in RCC, with the help of ultrasound, and in inoculation they can infect receptive animals.

By our materials we have prepared theileric antigens by using different methods and studied their sulphurological aspects. The blood taken in high parasitism and organs of experimental and spontaneous, sick animal have served as a preparation material for antigens. Erythrocytes washed in 0,15 M NaCl solution are destroyed by distilled water, sulphuric ether and low frequency waves on device USDN-1at frequency of the ultrasonic waves, equal to 22 kHz. That is done by adding the distilled water to the washed erythrocytes in correlation 1:5 or the sulphuric ether 1:3, and this solution is placed into refrigerator under 4º C for 1-2 hours accordingly. While using the ultrasound, a 50%- thread in 0,15 M sodium chloride solution is prepared from the erythrocytes and it is sounded in 0,5 liter vial for 4-6 minutes. The length of sounding necessary for full destruction of erythrocytes is selected in preliminary experiences earlier. After each minute of sounding, smears are prepared from the liquid, which are then colored as to Romanovsky, and examined under microscope for checking the efficiency of the sounding. For comparing the efficiency of the erythrocyte destruction and their influence on the activeness of got antigens, the only one animal’s erythrocytes are used. The liberated parasites are concentrated and washed in 0,15 M sodium chloride solution or the distilled water by centrifuging on angular centrifuge under 4000q for 40 minutes. The washing is repeated for 4-7 times till full brightening of above-sedimentary liquid. Then the 0,15 M sodium chloride solution, equal to have volume of washed erythrocytes, is added into the final sediment, shoo tilled for 4-6 hours and filtered through double layer of the gauze in sterile vial, preserved with mertiolat 1:10000. The antigen, from healthy animal’s erythrocytes, is served as a control sample.

For preparing antigens from shisogonal stage of the incitants- grenade bodies, the lymphatic glands and spleen of theileriosis-sick animal are used. Also the extractive antigens were prepared from them. For this, the organs are dispensed from face and shell, reduced and homogenized for 10 minutes more than 9000 turns/min.

The homogenate is prepared from homogenatic lymphatic glands and spleens by accompanying to it the 0,15 M sodium chloride solution or the distilled water and kept under 4º C during 3 days. Then it is centrifuged and above-sedimentary liquid is used as antigen. Totally 17 series of antigens are received from blood and organs of 11 animals.

Sulphurological aspects got from antigens have been studied in RCC with wheys of experimental, infected animals by theileriosis, brucellosis and noitiosis free whays. The whays were inactivated for 30 minutes in water bath under 57-58ºC. The reaction of connecting the complement has been put in volume 1ml. The phase of connecting has been lengthened till 30-40minutes, and the phase of hemoliz till 25-30minutes. The complement has been titred with 2-30 positive and negative whays. In the main experience the dose of complement has been increased to 10-15%. The results of reactions have been taken into account after its production immediately, and in 24hours they has been evaluated in crosses. Sulphurological peculiarities of antigens have been shown in the table.

In the table it is seen that the antigens prepared from defeated erythrocytes by theileria have been more active, moreover their activeness have varied depending on the level of defeat of theileria-sick erythrocytes and on method of getting antigens. High parasitism promoted to preparing antigens of high activeness and with small anticomplemental aspects (series 3,7,8,9). While comparing the antigen series 6,7,8 got from one animal, the ultrasonic antigen series 8 were the most actively possessed. Using low-frequency ultrasound allowed to get enough active antigens from the erythrocytes even under low parasitical reaction (series 12), while under liaising sulphuric ether and distilled water this has not been ever managed (series 2,15,16).

The antigens got under the further wire for ultrasound in special containers in volume of 20ml., during 3,5,10,20, and 45minutes became less active or completely disappeared. In Romanovsky colored smears from antigens, wired for sound for 3minutes, separate undisturbed parasite corpuscles were existed: more longer wiring for sound brought almost full destruction of theileria.

Comparative Studying the Peculiarities of Theileric Antigens

Table 1

Sulphurological Aspects of Theileric Antigens

Antigen Series	Ingredients	Parasitical Reaction in %	Destroyed by	Antigen Titration	Antigen Control
					Anticomplementalness in Diluting		Hemotoxicness in Diluting
					1:5	1:10	1:5
1	Erythrocytes	40	Distilled water	1:10	++	-	-
2	~	40	~		++	++	-
3	~	70	Sulphuric ether	1:80	++	-	-
4	Lymphatic gland	-	-	1:5	+-	-	-
5	Erythrocytes	-	-	1:10	+++	-	-
6	Erythrocytes	80	Sulphuric ether	1:20	+	-	-
7	~	80	Distilled water	1:10	+	-	-
8	~	80	Ultrasound	1:80	+	-	-
9	~	48	~	1:160	+	-	-
10	Lymphatic gland	Extract	~	-	++	-	-
11	~	~	~	-	+	-	-
12	Erythrocytes	8	Ultrasound	1:10	+	-	-
13	Lymphatic gland	Extract		-	+++	-	-
14	Erythrocytes	Undefeated	Sulphuric ether	-	-	-	-
15	~	15	~	-	++++	++	-
16	~	35		1:5	+	-	-
17	~	90		1:20	++	-	-

Hem- toxic aspects were not revealed in all got antigens, but were noted expressed anticomplemental activeness in different degrees. However it was revealed in minimal antigen diluting and in using in RCC highly active antigens did not distort the results of reactions. Antigens from grenade bodies showed the low activeness and possessed the expressed anticomplemental characteristics. Extractive antigens did not possess the complement linking activeness.

Parallel with RCC unsounded theileria antigens were tested in reactions of double diffusional precipitation in agar body and in reaction of mitosis hemoglutination. Negative results were received in all cases.

Findings:

1. From all tested ways of the destruction struck by theileria erythrocytes, providing the reception of active theileric antigens, the best is disintegrating them by the low frequency ultrasound.

2. The activeness of theileric antigens got by means of ultrasound depends on erythrocyte’s degree of defeat by theileria, as well as on the degree of the ultrasound destruction. If the parasitic blood reaction is more denominated, so the antigens are more active. Theileria antigens destroyed to corpuscle condition have possessed the most activeness: further wiring the corpuscles reduces the activeness of the antigens up to its loss sharply.

3. Theileric antigens got from erythrocytes by means of ultrasound possess the highest complement linking activeness in RCC. The antigens from grenade body of lymph glands possess low activeness and expressed- anticomplemental characteristics. Extractive antigens do not possess the complement-linking activeness.

4. In reactions of double diffusional precipitation in agar body and in reactions of mitosis agglutination unsounded antigens are turned out inactive.

Literature:

1. Konyukhov M.P.,Poluboyarova V. “The Role of RCC and RDCC in Execution of Immunity in Animals Recovered from Theileriosis”. "Veterinary Medicine",10, 1967.

2. Stepanova N.I.” RCC- Methods of the Diagnostics and Differentiation of Blood Parasites’’. "Veterinary Medicine", 1,1968.

3. Stepanova N.I. “The Methods of Preparation Antigens for Diagnostics of Philobotomized Parasitical Diseases and Studies of Immunological Conditions of Sick and Recovered Animals”. The Works VIEV, t. 38, 1970.

4.TutushinM.I. “The Reaction and Delays of Hemoglutination Under Theileriosis”. "Veterinary Medicine", 6, 1969.

5. Schindler R., Wokatsch W. “Versuche zur Differenzirung der Theilerien-spezies des Rindes durch serologische Untersuchungen”. Z. Tropenmed. und ParasitoL, 1965, 16, 1.

6. Schindler R., Mehlitz D. “Serologische Untersuchungen bei der Theileria parva — Infektion des Rindes”. Z., Tropenmed. Und Parasitol., 1965, 19, 3.

7. P r i о r R. В., К r e i e r I. P.” Isolation of Plasmodium berghei by use of a contiminosis — flow ultrasonic sustem: a morphological and immunological evalution”. «Proc. Helmintol. Soc. Wach.», 1972, 39, Spec. Issue.

8. Cadir F. A., Higalgo R. I., Kuttler K.” Production of complement-fixation test antigen for serodiagnosis of theileriosis in white - tailed deer”. «Amer. I. Vet. Res.», 1970, 31, № 5.