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The most efficient diagnostics of ovine epididymitis sheep by cft modification

03 May 2017
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Authors: Y. Kassymov, B. Isbussinov, D. Zainettinova, G. Mukhitdinova, Z. Mukhiddinova, D. Khussainov
Session: Biotechnology

The most efficient diagnostics of ovine epididymitis sheep by cft modification

 

Y. Kassymov 1, B. Isbussinov 2, D. Zainettinova 3, G. Mukhitdinova 4,

Z. Mukhiddinova, 5 , D. Khussainov 6

 

1. Doctor of  Veterinary Sciences, Professor, Kazakh National Agrarian University(KazNAU), Almaty e-mail: yerken.kassymov@mail.ru; 

2. Master of Veterinary Sciences, Department of  Biological Safety (KazNAU);

3 Master of Veterinary Sciences, senior lecturer of  Shakarim State University of Semey, e-mail: tnt_rani@mail.ru;

4. PhD of Veterinary medicine and Veterinary sanitary, Department of Clinical, veterinary medicine (KazNAU);

5. Master of Veterinary Sciences, Department of Obstetrics, surgery and reproduction (KazNAU)

6. Candidat of  Veterinary Sciences (PhD), As. Professor, Kazakh National Agrarian University(KazNAU), Almaty e-mail: doctor-vet@mail.ru

 

Abstract

The indicator system (offered by us) consisting of equal volumes of 2%  standardized suspension of  red blood cells and working solution haemolysin (in triple  titer) is universal for setting CF, LCF and CCF tests. Using this indicator system increases the sensitivity of the compliment fixation test for the diagnosis of brucellosis in sheep.

 

Key words: complement fixation reaction, epididymis of rams, erythrocytes

 

Introduction

Complement fixation tests (СFT), its long-term option (LCFT) are the main methods of diagnosis of infectious diseases of animals and humans. Existing regulatory and normative-technical documents (NTD) for the production of dry complement and statement of CFT and LCFT have several disadvantages, both normative and methodical [1, 2, 3].

For the diagnosis of  infectious diseases (brucellosis, ovine epididymitis, tuberculosis, chlamydia and etc.) are widely used complement fixation test (CFT), long complement fixation (LCFT) [4, 5], nowadays in Kazakhstan conglutination complex fixation test (CCFT) is named after Saiduldin reaction (SR) [6]. In these reactions, the slurry is applied erythrocytes of different concentrations: 2,5%; 3%; 2% respectively.

The procedures of improving the activity of the complement of donors are not provided, titration methods do not allow to objectively and accurately determine the quality (activity), i.e. dose of dry drug. Variety of components of reactions, different ratio of reagents, and absence of common methods of standardization makes difficult the comparison of results obtained in laboratories in different countries.

Our proposed research raises the output of active substance in donors, provides a standard titer in its industrial manufacturing and in the application of CFT and LCFT. Standardization of limiting components simplifies setting methodology of reactions, significantly reduces the cost and time for mass studies of blood serum, eliminates problems of self delay and increases the sensitivity of CFT and LCFT by 20-30% compared with the classical analogues of animal ovine epididymitis, as well as being the standard improvement of diagnosis of other contagious animal diseases. The results will be used in veterinary, research laboratories, biological industry.

 

Materials and methods

 

The material studies were different optical density of erythrocyte suspension (1.5%, 2%, 2.5%, 3%, 3.5%), and serologic studies of five series of dry and guinea pig complement serum samples of 20 patients with brucellosis of animals. Statistical analysis of the results of  research carried out by the standard technique [6].

Titration complement formulation serological reactions were performed according also to the total received techniques.

 

Results and discussion

 

Results of the study are shown in the Figure 1 and the Table 1-2.
In the first experiment were prepared by mixing the indicator system of equal volumes of  2% -s suspension of sheep red blood cells and hemolytic serum triple titres. In this case, standardization suspension conducted by photometery at photoelectrocolorimeter  FEC-M  or CFC- 42. 15-20 minutes prior to the study include stabilizers and green filter set.

To 1 ml of the prepared suspension  of  red blood cells was added 9 mL of distilled water, the resulting lysed blood was poured into 10 ml of a cell in the other two cells (the same amount) was poured solvent (saline 1 ml and 9 ml of stilled water).

In the left cell holder we put the cell with the solvent, and the right - to the lysed blood include galvanometer and rotating the photometric wedges installed galvanometer pointer to 0, then the galvanometer off. Then, instead of the cell with the lysed blood cell holder to the right we put the cell with the solvent. We  swithed on a galvanometer and rotating the left measuring drum (which is preset to 0.00) galvanometer needle set to 0, then the galvanometer off. The optical density  of the solution is measured along the left side of the drum.

Using the standardization curve (Figure 1), in terms of the optical density of the concentration of the prepared 2% suspension scale of the optical density showed 0.27. When the optical density is less than 0.27 we added to the cooked slurry corresponding Graph number of red blood cells from the centrifuged precipitate.

 

 

0.37

0.36

0.35

0.34

0.33

0.32

0.31

0.30

0.29

0.28

0.27

0.26

0.25

0.24

0.23

0.22

0.21

0.20

0.19

0.18

0.17

0.16

0.15

0.14

0.13

Suspension from ships red blood cells %
 

0.97

1.01

1.10

1.17

1.25

1.32

1.40

1.47

1.55

1.62

1.70

1.77

1.85

1.92

2.00

3.08

3.15

3.20

3.30

3.40

3.45

3.52

3.60

3.67

3.75

Ships red blood cells (in ml), which Last Post Add 100 ml suspension, to get 2% suspension
 

0.5

2

0.49

0.45

0.41

0.37

0.34

0.30

0.26

0.22

0.19

0.15

0.11

0.07

0.04

Saline solution (in ml), which Last Post Add 100 ml suspension, to get 2% suspension

 

 

 

 

 

 

 

 

 

                 

    

 

5.18

9.17

12.92

19.4

25.8

29.0

33.6

38.8

43.32

48.5

 

Figure 1 The calibration curve of red blood cells standardization.

 

If the optical density is above 0.27, we added the appropriate amount of saline schedule into the prepared suspension. Shown in figure 1 calibration curve standardization erythrocyte suspension composed us special experiment by measuring the optical density of different concentrations of erythrocytes lysates [2].

In another test series examined the 5 dry complement various indicator systems. The results  of  titration of various series of dry complement are shown in Table 1.

 

Table 1 Titers of complement by using different the concentration of red blood cells
 

№ series of dry complement

with 2,5 % of red blood cells

(classic method)

with 2% of red blood cells (proposed method)

1

2

3

4

5

М (middle titer)

                 + %

- %

0,24

0,20

0,22

016

0,12

0,19

11,0

10,0

0,20

0,16

0,18

0,12

0,10

0,15

3,5

3,3

 

Titers complement of the proposed method of  setting the reaction ( 2%  red blood cells) were high. The average titer was 0.19  in the control and in the experiment – 0.15.

In another experiment to study the effect of different concentrations of sheep red  blood cells sensitivity CF test determined the limits antibody titers in sera from 20 ovine epididymitis. Thus  Brucella antibody titers in the proposed method of setting  the reaction was high (P <0.01).  Furthermore, when using a  2% erythrocyte complement consumption was lower (Table 2).
 

 

Table 2 Effect of different concentrations of red blood cells to the level of sensitivity for the diagnosis of ovine epididymitis

 

№ investigated sera

Brucella antibody titers limit

With 2,5 % suspension of red blood cells (classic method)

With 2% a suspension of red blood cells (proposed a method)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

  

М ( middle titer)

             + %

             - %

1:10++++

1:40++++

1:40++

1:20++++

1:5++

1:20++++

1:5+++

1:20++

1:10++

1:5++++

1:80++++

1:160++

1:320++++

1:5+++

1:20+++

1:80++++

1:40+++

1:10++

1:20+++

1:5++++

 

1:46

20,6

17,1

 

1:20++

1:80++

1:40++++

1:40++

1:10++

1:40++

1:10++

1:40++

1:20++

1:10++

1:160++

1:160++++

1:640++

1:10+++

1:40++

1:160++++

1:80+

1:10++++

1:40++

1:10+++

 

1:81

29,2

22,6

 

 

These blood serum of patients with ovine epididymitis  animals (of 20 samples) used in the previous experiment, the study also exposed to extreme antibody titers in CF test using  2% and  2,5% of red blood cells (Table 3). The concentration of red blood cells is used in various productions CF test also normalized to optical density. Brucella antibody titer also was higher in the inventive process CF test  formulation (2% red blood cells), than at the classical variant. Mean antibody titers ovine epididymitis in these reactions were 1: 46; 1:81 respectively.

 

Conclusions

 

Thus posing CF and LCF tests with 2% suspension of sheep red blood cells increases the efficiency of the reaction in the diagnosis of ovine epididymitis . This reduces the complement consumption and increases the sensitivity of these serologic tests. The indicator system consisting of equal volumes of 2% suspension of  red blood cells and working solution haemolysin (in triple  titer) is universal for setting CF, LCF and CCF tests.

Standardization of limiting components simplifies setting a methodology of reactions, significantly reduces the cost and time for mass studies of blood serum, eliminates problems of self delay and increases the sensitivity of CFT and LCFT by 20-30% compared with the classical analogues of ovine epididymitis , as well as being the standard improvement of diagnosis of other contagious animal diseases. The results can be used in veterinary, research laboratories, biological industry.

 

References

 

1. Kassymov E. I. Production of dry conglutinin serum and its application in CCFT for the diagnosis of  bovine brucellosis. Dis. Alma-Ata, 1990. 170 p.(in  Russion).

2. Kassymov E. I. Method of quality control of complement. Prev. Patent №10472 Bull. №7. 16.07.2001. National Patent Office of Kazakhstan. (in  Russion).

3. Kassymov I. A method for diagnosing brucellosis in cattle. Prev. Patent №10478 Bull.№7. 16.07.2001. National Patent Office of Kazakhstan (in  Russion).

4. Alton G.G., Jones L.M., Angus R.D., Verger J.M. Techniques for the brucellosis laboratory. INRA, Paris, 1988.73-81.

5. Kassymov E. I.Measures to combat cow brucellosis’ Monograph Almaty. Kazakhstan. 2002, 225 p. (in Kazakh).

6. Kassymov E. I., Zhumagalikyzy S. An improved method of conglutinating test at diagnostics of  brucellosis and other infectious disease. World Academy of Science, Engineering and Technology 80. – Paris, September, 2011.

Bibliography and references
1. Kassymov E. I. Production of dry conglutinin serum and its application in CCFT for the diagnosis of bovine brucellosis. Dis. Alma-Ata, 1990. 170 p.(in Russion). 2. Kassymov E. I. Method of quality control of complement. Prev. Patent №10472 Bull. №7. 16.07.2001. National Patent Office of Kazakhstan. (in Russion). 3. Kassymov I. A method for diagnosing brucellosis in cattle. Prev. Patent №10478 Bull.№7. 16.07.2001. National Patent Office of Kazakhstan (in Russion). 4. Alton G.G., Jones L.M., Angus R.D., Verger J.M. Techniques for the brucellosis laboratory. INRA, Paris, 1988.73-81. 5. Kassymov E. I.Measures to combat cow brucellosis’ Monograph Almaty. Kazakhstan. 2002, 225 p. (in Kazakh). 6. Kassymov E. I., Zhumagalikyzy S. An improved method of conglutinating test at diagnostics of brucellosis and other infectious disease. World Academy of Science, Engineering and Technology 80. – Paris, September, 2011.

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