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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 email [email protected];
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
[email protected];
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 [email protected]
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 normativetechnical 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].
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Titration complement formulation serological reactions were performed according also to
the total received techniques.
Results and discussion
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
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
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.
2
5.18
9.17
12.92
19.4
25.8
29.0
33.6
38.8
43.32
48.5
Saline solution (in ml), which Last
Post Add 100 ml suspension, to get 2%
suspension
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
1
2
3
4
5
М (middle titer)
+%
-%
with 2,5 % of red blood cells
(classic method)
0,24
0,20
0,22
016
0,12
0,19
11,0
10,0
with 2% of red blood cells
(proposed method)
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
With 2% a suspension of red
blood cells (classic method)
blood cells (proposed a
method)
3
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++++
1:20++
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+++
1:46
20,6
17,1
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).
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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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