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THE IMPACT STUDY OF ANTIGENS CLASS I BoLA-A AND ALLELES GENE BoLA-DRB3 MHC SYSTEM FOR STABILITY AND COWS SUSCEPTIBILITY TO MASTITIS
Tatiana Suprovych, Mykola Suprovych
State agrarian engineering university in Podilya, Kamianets-Podilskyi, Ukraine.
ABSTRACT
In a population of cows Ukrainian black-pied dairy cattle 1 class antigens found BoLA-A and allele BoLA-DRB3 gene is associated with resistance and susceptibility to mastitis. The resulting model is observed immunogenetic status metric to determine the individual integrated assessment cows due to mastitis.
Key words: cattle, mastitis, Ukrainian black-pied dairy breed, major histocompatibility complex, alleles, statusmetriya.
INTRODUCTION
The mastitis cows' diseases in Ukraine covers 10 to 70% of the herd and 8 16% of cows gets sick 2 times or more during lactation [5]. The cows number with subclinical mastitis in 3 5 times exceeds the number of animals with clinical mastitis forms [1, 7].
The domestic science and practice is made significant progress in addressing the problem of mastitis in animals. It is developed and implemented in the production methods of early diagnosis, prevention and treatment of this disease through various antimicrobial drugs and physical therapy, but their effectiveness and consequences are not always satisfactory. The prolonged, widespread, and sometimes unsystematic use of chemotherapeutic agents has led to a decrease in the treatment effectiveness of this disease because of the drug-resistant formation strains of microorganisms that cause the inflammation development [5, 6].
The studies, which were held in many countries, showed that among a large number of factors, contributing to the occurrence of intramammary infection cows was the most significant animal genetic predisposition to the disease, and measure mastytoresistance sort depends on hereditary qualities parents [9, 12].
This is due to the interest of genetic markers, which allows the use of marker-associated selection and to predict the animals' health and their economic and useful qualities. An important research aspect in this area is the potential use of domestic animals as human diseases models.
The main problem in cattle breeding for resistance to mastitis a duration when assessing the cows, which can be spent several years. One of the methods for early detection of mastitis resistant animals are studying the associative links major histocompatibility complex antigens with susceptibility and resistance to this disease. The major histocompatibility complex (MHC) performs a variety of functions, of which the main ones are: antibody formation stimulation, the reaction of "graft versus host", immune response genes, the immune response restriction. Antigens MHC play the role of cell surface markers that are recognized by cytotoxic T-killer cells and T helper cells in complex antigens. The products genes MHC class I transplantation antigens are located on the surface of all somatic cells, except cells attack and cells certain types of the salivary glands. A special role is given to finding genes within the MHC, which affect the immune system and resistance to disease [4, 14].
Over the last decade greatly is increased researchers interest to study the possible association between alleles of exon 2 of the gene BoLA-DRB3 and different breeds' resistance (susceptibility) of cows to mastitis. It is accumulated a significant amount of information on the presence and the alleles distribution of the gene for different populations, which provides the analysis of possible use as genetic DNA markers for various diseases [11, 13, 15].
This work explores the impact of the use of molecular genetic methods, which are based on major histocompatibility complex diagnosis of mastitis in cows and breeding and
breeding work to improve the resistance of cattle.
MATERIALS AND METHODS
Because of years of study populations' cows Ukrainian black-pied dairy cattle and continuous further diagnosis is formed base with 649 cows on which conducted the definition of "informative" class I antigens BoLA-A system and investigated gene polymorphism alleles BoLA-DRB3 due to resistance and susceptibility to mastitis.
The identification of class I antigens BoLA-A two-stage standard system was carried mikrocytotoxic test for Kissmeyer-Nielsen in the modification for cattle.
The spectrum of gene alleles BoLA-DRB3 was studied by PCR, which was performed using pre-sets "GenPakR PCR Core", LLC "Izohen Laboratory". For restriction fragment analysis of exon 2 of the gene BoLA-DRB3 use restriction endonuclease Rsal, Haelll, BstYI (Xholl) firms "Promega", "New England BioLabs" and NGOs "SibEnzym". The restriction fragments were separated by electrophoresis in 4% agarose gel
[9].
The analysis of the results was performed using standard biometric parameters [2]. Individual assessment of susceptibility to diseases was carried by statusmetriya [8].
RESULTS
The study of the spectrum of antigens expression in groups of susceptible and resistant to mastitis cows can identify a number of "important" frequency antigen detection. In our studies in healthy animals often are detected antigens MSU A19 (0,551), A17 (0.536), A21 (0,429) and A14 (0,406). Antigens W2 (0,046), W20 (0,084) and W14 (0,130) are determined by the least often. Patients are often venerable cows antigens MSU A19 (0,533), A24 (0,424) and A14 (0,424), less often - W10 (0,132) and W20 (0,151).
The frequency analysis and comparison of a whole spectrum of histocompatibility antigens on biometric indicators are revealed an association with mastitis incidence for these 6 antigens class 1 BoLA-A system: W2, W6, W31, W19, W15 and A13. The favorable relative resistance to mastitis cows of the study are BoLA antigens A17 and A6 (Table. 1).
The population genetics methods, that were used to determine the "informative" antigens, are fundamental flaw. It is located straightforward or one variant model in which the need to find a "main" antigen among dozens of others, and associate it with a sign of disease. However, predisposition to the disease can be seen as a sign of population only a particular group of animals, united based on this quality.
Antigens W(MSUA) The frequency of antigen f Criterion Match hi2 Relative risk RR Attributive risk AR Etiological fraction EF
healthy diseased
W2*** 0,046 0,237 50,02 6,381 0,2 0,117
W6*** 0,171 0,431 52,7 3,671 0,314 0,195
W8 0,267 0,280 0,136 1,067 0,018 0,009
W21 0,226 0,247 0,381 1,121 0,027 0,014
W10 0,151 0,132 0,487 0,854 -0,023 -0,012
W20** 0,084 0,151 7,152 1,943 0,073 0,040
W31*** 0,209 0,378 22,66 2,307 0,214 0,127
W44 0,183 0,184 0,003 1,011 0,002 0,001
W14 0,130 0,178 2,785 1,440 0,054 0,03
W19*** 0,270 0,444 21,59 2,165 0,239 0,143
W15*** 0,246 0,401 17,86 2,05 0,206 0,121
A1 0,287 0,247 1,334 0,814 -0,056 -0,029
A2 0,391 0,362 0,597 0,882 -0,048 -0,025
A3 0,307 0,270 1,105 0,833 -0,054 -0,028
A6*** 0,333 0,178 20,34 -2,315 -0,234 -0,112
A7 0,168 0,188 0,416 1,142 0,023 0,013
A8 0,209 0,247 1,333 1,242 0,048 0,026
A9* 0,148 0,217 5,249 1,599 0,081 0,045
A10 0,212 0,217 0,029 1,033 0,007 0,004
A11 0,403 0,388 0,147 0,94 -0,025 -0,013
A12 0,252 0,299 1,806 1,267 0,063 0,035
A13*** 0,304 0,503 26,71 2,316 0,286 0,176
End of table 1
Table 1.
Biometric performance antigenic spectrum cows Ukrainian black-pied dairy cattle and their relationship with mastitis
A14 0,406 0,424 0,229 1,079 0,031 0,017
A15* 0,275 0,352 4,425 1,429 0,106 0,059
A16 0,365 0,313 2,000 0,79 -0,083 -0,042
A17*** 0,536 0,286 41,5 -2,884 -0,539 -0,229
A18 0,359 0,411 1,831 1,245 0,081 0,045
A19 0,551 0,533 0,207 0,931 -0,04 -0,021
A21 0,429 0,395 0,782 0,868 -0,06 -0,031
A22 0,313 0,293 0,314 0,908 -0,03 -0,015
A23 0,374 0,393 0,242 1,083 0,03 0,016
A24 0,394 0,424 0,608 1,133 0,05 0,027
* P > 0,95; ** P > 0,99; ***P > 0,999; (according to the criteria)
More importantly, the practical approach is the need to give a clear description of each individual animal in the herd, which is given the possible resistance or susceptibility to disease. This is determined by personal set of antigens each animal. The transition from population analyzes to determine the individual resistance is based on status metric [8]. As a result status metric l soil data set obtained with 17 informative antigens and a linear ; model to determine the immune status of the cow (Z), which ; is as follows:
Z = 0,599 - 1,556W2 - 1,133W6 + 0,747W10 - 0,563W31 i - 0,33W14 - 0,657W19 - 0,695W15 + 0,231A1 + 0,387A3 i + 0,799A6 - 0,685A9 - 0,285A12 - 0,619A13 + 0,332A16 + 1 1,121A17 + 0,447A22 - 0,244A24
For two alternative states "patients - healthy" condition by conventional units of the object a1 = -0,075 and a2 = 0,188. If Z <-0,075 animal susceptible to mastitis, while Z> 0,188 resistant. If -0,075 < Z < 0,188 decision uncertain.
According to the index value at the antigens in the linear model antigens W2, W6, W15, W19, A3 and W31 are unfavorable, that their presence in the phenotype of the animal indicates its susceptibility to mastitis, and the A6 and A17 antigens favorable, that their presence influences cows resistance to disease. Antigens ranked on the effect on the resistance to mastitis at a rate Bi (Table 2). Antigen W2 is the most unfavorable and favorable A17 relative resistance to mastitis.
Table 2.
The distribution of "informative" BoLA - antigens in cows Ukrainian black-pied dairy breed (N = 649)
BoLA antigens W (MSUA) f Bi hi2 RR AR EF
«unfavorable» W2 0,136 -1,556 50,0 6,381 0,2 0,117
W6 0,293 -1,133 52,7 3,671 0,314 0,195
W15 0,319 -0,695 17,9 2,05 0,206 0,121
W19 0,351 -0,657 21,6 2,165 0,239 0,143
A13 0,398 -0,619 26,7 2,316 0,286 0,176
W31 0,288 -0,563 22,7 2,307 0,214 0,127
«favor-able» A6 0,26 0,799 20,3 -2,315 -0,234 -0,112
0,419 1,121 41,5 -2,884 -0,539 -0,229
The distribution of alleles of the gene BoLA-DRB3 and its genotypes were studied by PCR. In this study, to amplify exon 2 genes BoLA-DRB3 two-stage method were using PCR using primers HLO-30, HLO-31 and HLO-32. The comparison of DNA patterns obtained using restriction endonucleases three Rsal, HaeIII and BstYI, allows us to identify 54 genes allele BoLA-DRB3 (Figure 1).
The study by PCR-RFLP methods and allele-specific PCR was revealed that the black population grizzled were
determined 28 of the 54 known alleles for the gene BoLA-DRB3.2, encoding MHC class II antigens of cattle.
The significant by hi2 criterion are eight alleles BoLA-DRB3.2, which have sufficient reliability for the studied biological objects. The degree of confidence study P = 0,99 *26 allele exhibit (hi2 = 7,13) and *36 (hi2 = 6,61). Six alleles have a minimum threshold reliability P = 0,95: *13 (hi2 = 5,65), *22 (hi2 = 5,02), *18 and *48 (hi2 = 4,82), *24 (hi2 = 4,33) and *11 (hi2 = 3,8).
M 51 52 54 65 66 71 72 M 31 32 M 76 7 7 78 79 80 81 82
m ~ 2M — aoa ^^ 200 3ÛO. ^ — — — 1-^1 — - » —-
m 1SU fg
ä. 50 fr — Z iQfl 75 5£ 35 1 10 Huelll, Chôma-pestrij KR5:
25 20 25 20 Ukraine, 2010.
15 15
10 10
Rsëtl, Cherno-Pestrij KRS, Ukraine. 2010, Xholl CbP. u, 2010
Fig. 1. Restriction analysis of amplification products of exon 2 of the gene BoLA-DRB3, were obtained DNA cow black-pied breed with different endonucleases
In terms of relative risk for significant association with susceptibility or resistance to mastitis with 17 alleles. In connection with the disease (RR > 2) point 8 alleles, namely: *41 (RR = 8,31), * 11 (RR = 6,83), *18 and *48 (RR = 5,25) *26 (RR = 4,62), *15 and *21 (RR = 3,38) and *24 (RR = 2,17); in connection with resistance to mastitis (RR < -2) indicate the following 9 alleles *36 (RR = -14,5), *13 (RR = -5,29), *16 and *25 and *31 ( RR = -3,17), *32 (RR = - 2,61), *01 and *22 (RR = -2,52) and *08 (RR = -2,05). Associated with the disease allele is considered for which the condition RR > 2 and hi2> 3,8. Total, there are 5 of these alleles *11 (RR = 6,83; hi2 = 3,8), *18 (RR = 5,25; hi2 = 4,82), *24 (RR = 2,17; hi2 = 4,33) *26 (RR = 4,62; hi2 = 7,13) and *48 (RR = 5,25; hi2 = 4,8). However, three of them follow through validation criteria for small samples. Therefore cannot be considered associated with susceptibility to mastitis following allele *11, *18 and *48.
Is associated with resistance to disease allele is considered for which the condition RR < -2 and hi2>3,8. Total there are 3 of allele *13 (RR = -5,29; hi2 = 5,65), *22 (RR = -2,52; hi2 = 5,02) and *36 (RR = -14,5; hi2 = 6,61). For allele *36 (P(A) = 0,031) were performed on the test hi2, suggests that no significant relationship with his cows to mastitis resistance.
The study of gene expression of exon 2 BoLA-DRB3 in black-pied cows' dairy Ukrainian breed shows that this population appears two alleles that have a close relationship with susceptibility to mastitis cows. This allele BoLA-DRB3.2:
*24 (RR = 2,17; P (A) = 0,117; hi2 = 4,33);
*26 (RR = 4,62; P (a) = 0,043; hi2 = 7,13).
Also, the presence of two alleles is found, which indicates an association with resistance to mastitis cows. This allele BoLA-DRB3.2:
*13 (RR = -5,29; P (A) = 0,053; hi2 = 5,65);
*22 (RR = -2,52; P (a) = 0,12; hi2 = 5,02).
The susceptibility further analysis of to mastitis requires paying attention to allele *18 and *48 that have high relative
risk sufficient reliability for hi2, but cannot stand the check in accordance with the criteria of low frequency in the general population and the minimum detection in healthy animals. This limit, usually maintains a check for small samples by Fisher criterion, with a slight decrease in the overall reliability of the study.
CONCLUSIONS
Thus, in this paper for the first time the following results are found:
1. It is studied the spectrum of antigens BoLA-A, which revealed antigens W2, W6, W31, W19, W15, MSU A13 (P > 0,999), which was significantly more common in patients animals compared with healthy;
2. The linear model status metric immunogenetic status of the population, which is determined on the basis of individual integrated Z score for each animal: a value of Z> 0,188 animal resistant, and if Z <-0,075 susceptible to disease. In this model, was found most "favorable" (A6 and A17) antigens BoLA-A, whose presence in the phenotype of the animal increases resistance to disease, and "unfavorable" (W2, W6, W15, W19, A3 and W31), whose influence determines the sensitivity to cow mastitis, which allows use them as lymphocytic markers in connection with diseases of the breast;
3. It was found that cows Ukrainian black-pied dairy cattle determined 28 of the 54 possible alleles that typed PCR-RFLP and AS-PCR;
4. Analysis of gene allele frequencies BoLA-DRB3 shows that with resistance to mastitis is associated allele *13 and *22, and a close relationship with susceptibility to inflammation of the udder of cows showing allele BoLA-DRB3.2 *24 and *26.
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ХАРАКТЕРИСТИКА КОБЫЛ МОЛОЧНОГО ТИПА ЛИТОВСКОЙ ТЯЖЕЛОВОЗНОЙ
ПОРОДЫ
Чиргин Евгений Дмитриевич
кандидат биологических наук, доцент кафедры ТППЖ Марийский государственный университет
FEATURE OF MARES LITHUANIAN DAIRY TYPE HEAVY DRAFT BREED
Chirgin E. D., Candidate of Biological Sciences, associate professor Mari State University
АННОТАЦИЯ
В республике Марий Эл ученые Марийского государственного университета более тридцати лет занимаются селекцией кобыл литовской тяжеловозной породы по молочной продуктивности. За этот период времени молочная продуктивность кобыл достигла 4516-5532 кг молока за 210 дней лактации. Массовая доля жира в молоке кобыл колебалась от 1,60 % до 2,12 %, а коэффициент молочности составлял 429,0-675,7 кг. Были исследованы морфологические особенности вымени кобыл и выявлена связь формы и промеров вымени кобыл с их молочной продуктивностью. Телосложение молочных кобыл за это время стало менее массивным и чуть более угловатым, то есть понемногу проявлялись черты молочного типа телосложения, по образцу молочного скота. Выявлена положительная корреляция между молочной продуктивностью и обхватом пясти кобыл - от +0,07 до +0,99. Также положительной была зависимость между удоем и живой массой кобыл + 0,52. Не обнаружено корреляции между промерами тела и промерами вымени кобыл. Предполагается, что на базе данной популяции лошадей литовской тяжеловозной породы формируется внутрипородный тип молочных животных.
ABSTRACT
In the Republic of Mari El, the scientists of the Mari state University more than thirty years engaged in the breeding of mares Lithuanian Heavy Draft breeds for milk production. During this time period the milk yield of mares reached 4516-5532 kg milk in 210 days of lactation. Mass fraction of fat in milk of mares ranged from 1.60 % to 2.12 %, and the coefficient milking was 429.0-675.7 kg. Were studied morphological characteristics of the udder of mares and correlation of the shape and dimensions of the udder of mares with their milk productivity. Physique dairy mares during this time became less massive and slightly more angular, have little apparent traits dairy type of a Constitution, on the model of dairy cattle. There was a positive correlation between milk productivity and metacarpus mares - from +0.07 to +0.99. There was also a positive correlation between milk yield and live weight of mares + 0.52. Not found correlations between body measurements and the measurements of the udder of mares. It is assumed that on the basis of this population of Lithuanian Heavy Draft horse breed is formed interbreed type of dairy animals.
Ключевые слова: Молочное коневодство; корреляция; индекс молочности; молочный тип.
Key words: Dairy breeding; correlation; index milking; the milky type.
