BoLA-DRB3 gene as a marker of susceptibility and resistance of the Ukrainian black-pied and red-pied dairy breeds to mastitis

State Agrarian and Engineering University in Podilya, Shevchenko st., 13, Kamianets-Podilskyi, 32300, Ukraine. Tel.: +38-098-57-63-018. E-mail: suprovycht@gmail.com Suprovych, T. M., Suprovych, M. P., Koval, T. V., Karchevska, T. M., Chepurna, V. A., Chornyi, I. O., & Berezhanskyi, A. P. (2018). BoLA-DRB3 gene as a marker of susceptibility and resistance of the Ukrainian black-pied and red-pied dairy breeds to mastitis. Regulatory Mechanisms in Biosystems, 9(3), 363–368. doi:10.15421/021853


Introduction
Udder diseases of cows lead to significant economic loss for the dairy business. That loss is greater than any damage from all non-contagious diseases in cattle farming. Mastitis in cattle imposes a significant financial burden on milk producers (the dairy industry worldwide loses $16-26 billion annually) (Abdel Hameed et al., 2006). Dairy production in Ukraine is the main branch of cattle farming. In 2016, 10,329 thousand tons of milk were received from 2,100 thousand cows (Chajkova & Foshhij, 2016). Mastitis of cows represents the biggest challenge for milk producers. The analysis of data from different publications shows that cases of mastitis amount to 18.8-34.7% of livestock, including 3.62-12.6% of clinically manifested cases. The loss due to the disease is about $75 per animal (Hanjejev & Janchuk, 2011).
The search for disease resistance markers includes the screening of candidate genes that are associated with the disease resistance caused by DNA polymorphisms. The major histocompatibility complex plays a crucial role in the immune response, and MHC genes are promising candidate genes to establish associations with various diseases (Abdel Hameed et al., 2006). MHC is a polymorphic genetic system that is a cluster of closely coupled genes. It is responsible for the formation of the immune response, macrophage, T-and B-lymphocyte interactions and for the immunological homeostasis support in general. The binding of peptides to MHC molecules triggers acquired immune responses. Therefore, MHC molecule polymorphism forms the diversity of the immune response. In particular, the major histocompatibility complex genes (BoLA) of cattle play an important role in the resistance of the host to diseases (Ibeagha-Awemu et al., 2008).
The BoLA system is located on the chromosome 23. It includes three classes: class I (locus A); class II, divided into IIa (includes DRA, DRB1, DRB3, DRB2, DQA DQB et al. loci) and IIb (includes DOB, DYA, DYB, DIB et al. loci); class III (including TNF, 21-OH, C4, BF, HSP70-1 and -2, EAM, PRL et al. loci). The first two groups of loci encode surface molecules that are related to the induction and regulation of immune responses.
The BoLA-DRB3 gene encoding antigens of Class II MHC is quite polymorphic (Van Eijk et al., 1992;Sharif et al., 1998). It is located in the IIa region of the sublocus of the DR system BoLA. The gene consists of six exons encoding the leader and the terminal sequence, the hydrophobic transmembrane region, the cytoplasmic and extracellular domain of the protein. The second exon of the BoLA-DRB3 gene encoding the β 1 -domain of class II antigens is highly polymorphic. This is necessary for binding of a wide range of foreign antigens (Behl et al., 2012). Analysis of the BoLA-DRB3.2 gene polymorphism is performed by PCR-RLFP (54 alleles) or PCR-SBT (107 alleles according to www.ebi.ac.uk/ipd/mhc/group/BoLA).
The BoLA-DRB3.2 gene polymorphism has been studied in more than 30 cattle populations (Takeshima et al., 2014) and the research is on-going. The allelic spectrum of the gene for most commercial breeds has been identified. The greatest attention of researchers is given to the purebred Holstein in the USA and Canada Sharif et al., 1998;Rupp et al., 2007) and to the breeds in other countries with a significant proportion of heredity from the Holstein (Duangjinda et al., 2009;Yoshida et al., 2012;Sun et al., 2013;Takeshima et al., 2015;Nikbakht Brujeni et al., 2016). There are numerous reports on the study of allelic diversity of indigenous breeds: Norwegian Red (Kulberg et al., 2007), Russian Ayrshire and Black-Pied (Udina et al., 2003), Japanese Shorthorn (Takeshima et al., 2002), USA Jersey (Gilliespie et al., 1999). Considerable attention is given to the study of the BoLA-DRB3.2 gene polymorphism in indigenous and native breeds: China Wanbei (Lei et al., 2012), Philippine native cattle (Takeshima et al., 2014), cattle of South American (Giovambattista et al., 2001;Takeshima et al., 2015), Mongolian, Kalmyk and Yakut breeds (Ruzina et al., 2010), Iranian native cattle (Firouzamandi et al., 2010), Indian native cattle (Behl et al., 2009), Egyptian cows (Ibrahim et al., 2012).
There are many breeds for which polymorphism of the BoLA-DRB3 gene and its connection with diseases has not been studied. The present study is aimed at analyzing the polymorphism of the BOLA-DRB3 gene exon 2 of the most common Ukrainian dairy breeds, black-pied dairy and red-pied and establishing associations between the alleles of this gene and mastitis.

Materials and methods
The research was carried on farms in Khmelnitsky and Chernivtsi regions from 2009 until 2015. Blood samples from the UBPD breed (5 farms, n = 276) and the URPD breed (2 farms, n = 117) were examined. Practical studies we conducted at the Animal Genetics Laboratory at the Institute of General Genetics Vavilov Russian Academy of Sciences (Moscow) and in the Genetics Laboratory of the Institute of Animal Breeding and Genetics of National Academy of Agrarian Science of Ukraine.
The maximum accuracy of the alleles of the gene BoLA-DRB3 definition was achieved by using three independent approaches (Sulimova, 2004): -restriction analysis of PCR-RFLP amplification products; -allele-specific PCR (AS-PCR) with primers ER-17 and VD-19; -allele-specific PCR with primers HLO-07 and HLO-24d. Isolation of DNA was carried out using "DIAtomTMDNA Prep200" kits (Isogen Laboratory Ltd.) in accordance with the manufacturer's instructions. DNA isolated from fresh biological material (yield was 5-10 mg from 200 ml of whole blood) high molecular weight (40-50 bp) and pure (OD 260/280 nm = 1.6-2.0) substance. The DNA concentration was determined visually. With this purpose, 25, 50, and 100 ng of λ phage DNA and aliquots of solution with an unknown concentration were applied to 1% agarose gel.
Electrophoresis was performed in 1 x Trisborate (TBE) buffer (89 mM Tris-OH, 89 mM H 3 BO 3 , 2 mM EDTA) with ethidium bromide (1 μg/ml) added to the gel to stain DNA at a constant voltage of 120 V. To avoid contamination, electrophoresis was performed in a separate room.
The DNA concentrations of the test samples were determined by comparing the fluorescence intensity of the aliquots from solutions of unknown concentration and control λ phage DNA. For further nested PCR analysis, the exon 2 region of the BoLA-DRB3 gene, 284 bp in size, was amplified (281 bp for alleles with deletion) (Van Eijk et al., 1992).
Amplification. The first stage was started from DNA denaturation at 95 °C for 5 min. followed by 10 cycles with denaturation (94 °C for 1 min), annealing (62.5 °C for 2 min) and elongation (72 °C for 1 min) and a final extension at 72 °C for 7 min. The second stage was started initial denaturation (95 °C for 5 min), was followed by 35 cycles of denaturation (68 °C for 30 s), and annealing-extension (72 °C for 30 s) and a final extension (72 °C for 7 min). Contamination and self-priming controls were included in each PCR round and 5 µL of the last PCR product were electrophoresed on 1.5% agarose gels in order to check the quality and specificity of DNA fragment amplification.
For the restriction analysis of the exon 2 fragment of the BoLA-DRB3 gene, endonucleases RsaI, HaeIII, XhoII (Promega, USA, New England BioLabs and SibEnzim, Russia) were used. The restriction fragments were separated by electrophoresis in a 4% agarose gel (Fig. 1). Amplification of exon 2 of the gene by means of a PCR followed by the analysis of restriction fragment length polymorphism and comparison of DNA patterns obtained using the three specified restriction endonucleases allows the identification of 54 alleles of the BoLA-DRB3 gene.
In cases where it was impossible to determine the genotype of the animal using restriction analysis, the AS-PCR method was used (Fig. 2). Allelic variants differ because the 3'-terminal nucleotide of one of the primers hybridizes directly to the variable nucleotide (SNP position), which causes the presence or absence of PCR. The specificity of the reaction can be increased by introducing an additional, not paired, nucleotide in the second or third position from the 3'-end of the same primer or using a competitive PCR in the same tube. Allele-specific PCR was performed using ready-made sets of "GenPakR PCR Core" (Isogen Laboratory). The PCR products of the first round of nested PCR were used as a template. A more detailed description of the method for typing the DRB3-alleles was reported by other sources (Udina et al., 2003;Firouzamandi et al., 2010;Ruzina et al., 2010).
Cows which repeatedly suffered during 2-3 lactations from various forms of mastitis were considered susceptible to mastitis. Animals which had no disease during this period were considered resistant.
In order to detect which cows were resistant and which were sensitive to mastitis, we conducted regular monthly studies. Special control plates with four deepenings were used. In these deepenings 1 ml of milk from each udder quarter and equal amounts of reagent were added. If the reaction was positive, a jelly-like clot formed, which indicated the presence of at least 500 thousand somatic cells per 1 ml of milk. Clinical manifestations of mastitis were diagnosed during milking based on several factors, such as: proportionality of udder quarters, pain sensitivity, local and general temperature increases, swelling, udder induration, secretion and presence of blood or pus in the secretrion.
Allele frequencies for the experimental sample were calculated taking into account the number of homozygotes and heterozygotes. In order to identify the associations between BoLA-alleles and the disease, it is necessary to establish the strength of association and the statistical significance between the frequency of gene carriers in the groups of susceptible and resistant animals. The force of the associative relationship is determined on the basis of relative risk (RR -relative risk, f b -frequency of gene carriers among susceptible animals, f k -frequency of gene carriers in resistant animals): The RR value shows how many times the risk of developing the disease is greater in the presence of a certain allele in the genotype than in its absence (Zaretskaya, 1983).
Test χ 2 indicates a statistically significant difference between the frequency of alleles among susceptible and resistant animals. The allele is considered as associated with the disease if the condition RR ≥ 2 and χ 2 > 3.84 (P < 0.05) is satisfied. If the value RR ≤ 0.5, then the presence of the allele in the animal genotype indicates a close association with the disease resistance. In this case, in order to highlight a positive association, relative risk values are set as 1/RR with a minus sign. Test χ 2 makes sense if the sample involves at least 20 animals and the respective conditions are met (a, c -susceptible to animal disease, having or not having an appropriate allele; b, d -resistant to animal disease, having or not having an appropriate allele; N -sample size): (a+b)×(a+c)/N > 5, (a+b)×(b+d)/N > 5, (c+d)×(a+c)/N > 5, (c+d)×(b+d)/N > 5.
(2) If an allele is not detected in groups of susceptible or resistant animals, one of the values a or b is zero. Then the value of RR is determined by the Haldane formula (Klеinbaum et al., 1982). Woolf-Haldane correction is a modification of data, which allows for the verification of statistical significance in cases where one of the values is zero. Numeric 0.5 to warn the division by zero and make the calculation possible increases the 0 value.
Ukrainian red-pied dairy breed. We studied the allelic spectrum of this breed in two herds on farms in Khmelnitsky and Chernivtsi regions. Twenty-two alleles were detected in 117 blood samples (Table 2).
Among the cows resistant to mastitis, 27 alleles were found. The total frequency of eight "informative" alleles was 65.5%. The most frequent was BoLA-DRB3.2*22 allele. It was carried by 24% of cows. The peculiarity of this allele is that it forms a homozygous genotype most often spread throughout a whole population (40% of the total number of homozygotes detected).
The association between the disease and the allele is detected by comparing the frequencies of alleles in sick and healthy cows based on relative risk (RR), which reflects multiplicity of the association, that is, how many times the risk of developing the disease is greater in the presence of a specific allele in the genotype than in its absence. The significance of the detected association is determined by the test χ 2 . According to the relative risk associated with mastitis, there are 17 alleles, of which eight are associated with susceptibility and nine with resistance (Table 3). Notes: * -The Table shows the alleles for which one of the following conditions holds: 1) χ2 ≥ 3.8; 2) RR ≥ 2 or RR ≤ -2; 3) both conditions 1 and 2.
Due to the resistance to mastitis, it is necessary to pay attention to the allele *08 (RR = -2.05; P (A) = 0.074), which does not withstand the test χ 2 (P > 0.05) but meets the requirements of reliability by other criteria. In case of competent biological studies, it is permissible to lower the threshold of reliability, which makes it possible to use this allele in further analysis.
Ukrainian red-pied dairy breed. Blood samples were taken from 117 animals. Fifty-nine cows were resistant and 58 susceptible to mastitis.
Twenty-one alleles were detected in the samples of diseased animals. Eight alleles with a frequency exceeding 5% were identified, which embrace in total 74.9%. The most common allele was BoLA-DRB3.2*07. It was detected in almost every third cow (32.8%), or in 21.6% of cases among all detected alleles.
Relative risk calculations show that 13 alleles of BoLA-DRB3.2 have a close association with mastitis, of which eight are associated with susceptibility and five with resistance to this disease (Table 4). The test χ 2 indicates five alleles that have a sufficient level of significance: *22 (P < 0.001); *07 (P < 0.01); *08, *24 і *45 (P < 0.05), of which only the latter cannot withstand a check for limited sample size. Note: * -the Table shows the alleles for which one of the following conditions is met: 1) χ 2 ≥ 3.8; 2) RR ≥ 2 or RR ≤ -2; 3) both conditions 1 and 2 are met.

Discussion
In our study we established that high allele diversity of BoLA-DRB3 gene is characteristic for UBPD livestock. This breed has a diverse genetic structure. Most researchers identify this breed as an open population. The UBPD cattle have similarity to Dutch, Estonian, Lithuanian, Moscow, black and red and other breeds. Nowadays when crossbreeding with Holstein bulls is spreading actively, the presence of 32 alleles BoLA-DRB3.2 is fully consistent with its genealogy. The same large number of RLFP-alleles has been found in Mongolian (35 alleles) and Kalmyk (34 alleles) breeds (Ruzina et al., 2010), as well as Iranian Sistani (30 alleles) cattle (Behl et al., 2009).
The Ukrainian red-pied dairy breed is the second commonest dairy breed in Ukraine after the black-pied one. It is derived from reproductive crossbreeding of the Simmentals (maternal breed) with red-pied Holstein. The breed develops according to the principle of open populations and also has a heterogeneous structure. In our study, 22 alleles were detected in URPB, which is significantly less than in UBPB. This result of our study is caused by limited range and the small sample size. Most commercial breeds have a wide range of alleles -from 13 in Canadian Jersey to 28 in Iranian Holstein (Behl et al., 2009). This number of alleles in these populations is likely a consequence of maternal breed features and selection actions.
Our study has identified alleles of cows susceptible and resistance UBPD and UBPB to mastitis. Significant research was carried out (Table 5) in detecting associations between alleles BoLA-DRB 3.2 and various aspects of mastitis, such as clinical and subclinical forms, specific pathogens (Staphylococcus aureus, Streptococcus agalactiae, E. coli) and somatic count cells Kelm et al., 1997;Sharif et al., 1998Sharif et al., , 2000Rupp et al., 2007;Duangjinda et al., 2009;Ruzina et al., 2010;Ibrahim et al., 2012). Sulimova, Ruzina et al. (2010) believes that generalization and analysis research of different authors indicate three groups of alleles BoLA-DRB3.2 in connection with mastitis: susceptibility -*16, *23 and *26; resistance -*07, *11, *13, *18 and *27; have opposite associations in different studies -*08, *22 and *24. Data indicate that alleles in differrent populations have opposite associations more: *11, *16, *22, *23 and *24. The conditions for the emergence of mastitis are very diverse. Many factors promote the development of mastitis, including the feeding environment, care conditions and genetic background. Infectious mastitis occurs because of the penetration of the udder by Streptococcus, Staphylococcus, coliform bacterium and other pathogens. Infection of the udder occurs through the galactogenous path (through the teat channel), the lymphogenous way (through the wounds of the teats and udder), less often by the hematogenous way (from other organs). Noninfectious mastitis is widespread. It occurs when milking is done incorrectly, when poor hygiene of the udder is maintained, when damaged milking equipment is used, etc. In milk of cows with mastitis, due to the development of the immune response to inflammation, SCC increases. Multifactorial effects influence different types of immune responses, which implies the peculiarity of its formation in different populations of cattle. Therefore, the possibility of finding deterministic BoLA-DRB3.2 alleles indicating resistance or susceptibility of cows to mastitis is unlikely for the whole world dairy herd.

Table 5
Summary of data on the detection of associations between alleles BoLA-DRB3.2 and various aspects of mastitis