Association study between β-defensin gene polymorphisms and mastitis resistance in Valle del Belice dairy sheep breed

Small Ruminant Research 136 (2016) 18–21

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Short communication

Association study between ␤-defensin gene polymorphisms and mastitis resistance in Valle del Belice dairy sheep breed Marco Tolone ∗ , Salvatore Mastrangelo, Rosalia Di Gerlando, Anna M. Sutera, Giuseppina Monteleone, Maria T. Sardina, Baldassare Portolano Dipartimento Scienze Agrarie e Forestali, Univeristà degli Studi di Palermo, Viale delle Scienze, 90128 Palermo, Italy

a r t i c l e

i n f o

Article history: Received 28 September 2015 Received in revised form 23 December 2015 Accepted 29 December 2015 Available online 4 January 2016 Keywords: ␤-Defensin genes Mastitis resistance Single nucleotide polymorphisms Valle del Belice dairy sheep breed

a b s t r a c t Mastitis is generally caused by bacteria, and it is the most common disease in livestock species. Defensins are peptides with a broad spectrum of antimicrobial activity and ␤-defensin genes have been studied in several livestock species due to their important role in the innate immune response. The aim of this study was to establish an association between polymorphisms in the ␤-defensin 1 and 2 genes and mastitis resistance in the Valle del Belice dairy sheep. Data consisted of 1855 and 2804 observations for case and control group, respectively. Six single nucleotide polymorphisms and seven haplotypes were selected for association studies with mastitis. In particular, polymorphism G1747A on ␤-defensin 1 gene was associated with susceptibility to mastitis, while polymorphism G1659A on ␤-defensin 2 gene was associated with resistance to mastitis. Haplotypes ACAGGG and GCAGGG were associated with resistance to mastitis, whereas haplotype ACGGGG was associated with susceptibility to mastitis. The present study has firstly suggested the possible associations of ␤-defensin gene polymorphisms with mastitis resistance traits and showed the presence of interesting haplotypes in Valle del Belice dairy sheep breed. Results from association analysis provided preliminary evidence that ␤-defensins could be used as candidate genes or molecular markers for the improvement of ovine mastitis resistance traits in Valle del Belice dairy breed. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Mastitis is the most common and costly infectious disease of mammary gland affecting dairy animals caused by bacteria. Mastitis alters the state of well-being and health of the animals, and it leads to economic loss mainly due to reduced milk yield and quality, veterinary treatments, milk disposal due to antibiotic treatments, and early culling. Moreover, milk and dairy products from animals affected by mastitis represent a significant risk for the consumers not only for the presence of the pathogens, in particular for dairy products made with raw milk, but also for the presence of bacterial toxins and the antibiotic residues (Mitchell et al., 1998). The contagious infection causes an increase in total somatic cell count (SCC) as a consequence of both leukocyte and epithelial cell numbers increasing, with or without clinical signs of mastitis. Mastitis remains a major challenge to the worldwide dairy industry despite the widespread implementation of mastitis control

∗ Corresponding author. E-mail address: [email protected] (M. Tolone). http://dx.doi.org/10.1016/j.smallrumres.2015.12.037 0921-4488/© 2015 Elsevier B.V. All rights reserved.

strategies (Bradley, 2002). Selection for genetic resistance to mastitis can be done directly or indirectly. Direct selection relates to the diagnosis of the infection, whereas indirect methods have been widely applied based on the evaluation of the degree of inflammation or of internal mammary lesions (Riggio and Portolano, 2015). Antimicrobial peptides are important and effective components of innate immunity and are being evaluated as possible alternatives to conventional antibiotics due to the fact that bacteria have not developed resistance against antimicrobial peptides because they target components that are within to bacterial structures (Lai and Gallo, 2009). These endogenous host-defense molecules are encoded by distinct genes and translated from mRNA templates (Ramanathan et al., 2002). Based on their common features, two major families of antimicrobial peptides have been characterized in mammals, defensins and cathelicidins, which are the part of the antimicrobial arsenal of the leucocytes (Ko´sciuczuk et al., 2014). Defensins are expressed in a variety of epithelial tissues, which serve as primary microbial interface sites (Kaiser and Diamond, 2000). They are cationic peptides, 18–45 amino acids residues in length, whose structure is stabilized by three intramolecular disulfide bonds formed by six strongly conserved cysteine residues

M. Tolone et al. / Small Ruminant Research 136 (2016) 18–21

(Selsted et al., 1993; Nicholas and Mor, 1995). They are classified as ␣-, ␤- and ␪-defensins based on structure, size, and disulfide bonds patterns (Kaiser and Diamond, 2000; Selsted and Ouellette, 2005). Defensins are peptides that exhibit a broad spectrum of antimicrobial activity, often including both gram-positive and gram-negative bacteria, fungi and enveloped viruses (Kaiser and Diamond, 2000). Moreover defensins have been suggested as effector molecules in host defense, interacting with many target cells and tissues (Yang et al., 2002). In recent years, ␤-defensin genes have been studied in several livestock species due to their important role in the innate immune response (Luenser et al., 2005). More than 20 ␤defensins were found in cattle tissues, and several of them are expressed in the mammary gland (Bagnicka et al., 2010; Meade et al., 2014). In pigs, ␤-defensin 1 has been first characterized by Shi et al. (1999) and 11 more ␤-defensins were identified by Sang et al. (2006) in the same species. In goat, the precursors of ␤defensin 1 and ␤-defensin 2 have been characterized by Zhao et al. (1999). In sheep, only two ␤-defensin genes have been described: ␤-defensin 1 (SBD1) and ␤-defensin 2 (SBD2). Both genes have been mapped on chromosome 26 and consist of two exons and one intron of approximately 1500 bp (Huttner et al., 1998). SBD1 and SBD2 expression was mainly described in the gastrointestinal tract and trachea (Huttner et al., 1998). Few studies have been conducted on ␤-defensin genes in sheep. However, Souza et al. (2015) performed a genotyping study on SBD2 gene in Amazon sheep. Monteleone et al. (2011), in a sequencing polymorphisms discovery study on Valle del Belice dairy sheep, reported a total of seven single nucleotide polymorphisms (SNPs), two in SBD1 and five in SBD2 genes. Only two of the five SNPs identified in SBD2 sheep gene (positions 1659 and 1667 referred to GenBank Acc. no. U75251), within coding region, were nonsynonymous mutations and leading to amino acid changes. Indeed, the SBD2 sequence carrying these two polymorphisms showed changes in mRNA secondary structure and suggested a possible role of these SNPs in modulation of the immune response (Monteleone et al., 2011). Considering that resistance to mastitis has a genetic background and genetic improvement is possible (Tolone et al., 2012; Sender et al., 2013), the identification of genetic markers that allow the inclusion of mastitis resistance in selection programs would help to reduce the economic loss linked with this intra-mammary infection disease. Genetic association studies, in which the allele or genotypic frequencies at markers are determined in infected individuals and compared with those of healthy ones (control case), may be an effective approach to identify the role of common variants with modest effects. Therefore, the aim of the present study was to investigate whether there is an association between SNPs or SNP haplotypes at SBD1 and SBD2 genes and mastitis resistance in Valle del Belice dairy sheep breed.

2. Materials and methods 2.1. Animal and sampling The data consisted of 4659 observations from 299 Valle del Belice dairy ewes. The genotyping data of SBD1 and SBD2 genes used in this study belonged to the previous SNP discovery study of Monteleone et al. (2011). Information about sampling and genotyping protocols were reported in Monteleone et al. (2011). Milk samples were collected aseptically for each animal from the two udder halves in sterile containers for bacteriological analyses following an A4 recording procedure (ICAR, 2014). Standard procedures were used for isolation and identification of bacteriological colonies: 5% sheep blood agar plates, incubated at 37 ◦ C, and examined after 10–24 h and 36–48 h incubation. The bacteriological colonies observed were mainly: Staphylococcus aureus, coagulase negative staphylococci, Staphylococcus intermedius and other staphylococci; Streptococcus canis, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus agalactiae and other streptococci; Corynebacterium spp., Pasteurella spp., and Pseudomonas spp. Milk samples were considered infected if more than five colony forming units (CFU) per 10 ␮l of milk of one species of bacteria were isolated and they were

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Table 1 Positions and regions of single nucleotide polymorphisms (SNPs) identified in SBD1 and SBD2 genes in Valle del Belice dairy sheep breed. Gene

SNP position

Region

SBD1

1747 A → G 1757 T → C

3 -UTR 3 -UTR

SBD2

89 C → T 1659 G → A 1667 G → A 1750 G → A 1761 G → A

Coding Coding Coding 3 -UTR 3 -UTR

included in case group (1855 observations) otherwise they were considered healthy and included in control group (2804 observations). 2.2. Statistical analyses The online software platform SHEsis (www.analysis.bio-x.cn/myAnalysis.php) was used to calculate allelic and genotypic frequencies and to infer haplotypes for case and control groups separately, and to conduct genetic association study. Only haplotypes with a frequency greater or equal to 0.03 were considered for the analysis. Haplotypes were inferred using the Partition–Ligation Combination-Subdivision EM as implemented in the SHEsis software (Shi and He, 2005; Li et al., 2009). The use of haplotypes allows multiple potentially causal variants to be tested simultaneously for association; moreover, haplotypes can be tested for association because they may be a proxy for untyped causal markers. Association analysis was performed using the Case Control procedure in SAS 9.2, with the overall association with genotype based on the Armitage trend test and odd ratios (OR) based on allele counts, reflecting additive effects. Allelic association tests assume that the two alleles per marker within each individual are independent i.e., that they are in Hardy–Weinberg Equilibrium (HWE). Armitage’s trend test and other tests that assume additivity of allele effects are alternatives that do not impose this assumption (Sasieni, 1997) and are, therefore, preferred. Under HWE, the allelebased test and the trend test are asymptotically equivalent. Association test is a 2 test of independence computed on a cross-classification table of outcome versus alleles or genotypes.

3. Results In Table 1, SNP positions and regions of SBD1 and SBD2 sheep genes were reported by Monteleone et al., 2011. As reported by these authors, the functional analysis of the novel identified missense mutations in SBD2 gene was obtained using SIFT program (Ng and Henikoff, 2003) while the evolutionary analysis of coding SNPs was obtained with PANTHER tool (Thomas et al., 2003). According to software results, we concluded that these mutations may not have effects on protein function. SNPs in the 3 -UTRs (i.e., at position 1747 and 1757 in SBD1, and at position 1750 and 1761 in SBD2) were analyzed with RNAstructure software (Mathews et al., 2004) to check if they could influence mRNA structure. According to software predictions, SBD1 SNPs did not affect the mRNA shape whereas both SBD2 SNPs (at position 1750 and 1761) changed the mRNA secondary structure when they are present alone. When these mutations are both present, mRNA folds as when only the SNP at position 1750 is present demonstrating that it has a greater effect on RNA folding. Therefore, the structural difference in SBD2 messenger RNA may be related to a possible role in translation efficiency with a modulation in the protein produced amount. After editing, the data consisted of 1855 and 2804 observations for case and control groups, respectively. Considering the linkage disequilibrium between the two SNPs on SBD1 gene, only the ones in position 1747 was considered for the analysis. Allelic and genotypic frequencies for case and control groups, for all SNPs identified in the SBD1 and SBD2 genes are reported in Table 2. Minor allele frequencies for all SNPs in the two genes ranged from 0.014 to 0.315 in case and from 0.012 to 0.343 in control group (Table 2). Haplotypes and their frequencies for case and control group are shown in Table 3. In both case and control groups, the results showed that ACGGGG haplotype has the highest frequencies (0.484) whereas the lowest one was found for GCAGGG (0.020) (Table 3).

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M. Tolone et al. / Small Ruminant Research 136 (2016) 18–21

Table 2 Genotypic and allelic frequencies of SBD1 and SBD2 single nucleotide polymorphisms in case and control group. SNP

Observations

Genotypes

SNP1747 Case Control

1855 2804

AA 0.815 0.765

AG 0.130 0.175

GG 0.056 0.059

A 0.880 0.853

G 0.120 0.147

SNP89 Case Control

1855 2804

CC 0.757 0.767

CT 0.243 0.233

TT – –

C 0.878 0.883

T 0.122 0.117

SNP1659 Case Control

1855 2804

GG 0.416 0.381

GA 0.539 0.553

AA 0.045 0.066

G 0.685 0.657

A 0.315 0.343

SNP1667 Case Control

1855 2804

GG 0.972 0.975

GA 0.028 0.025

AA – –

G 0.986 0.988

A 0.014 0.012

SNP1750 Case Control

1855 2804

GG 0.534 0.544

GA 0.466 0.456

AA – –

G 0.767 0.772

A 0.233 0.228

SNP1761 Case Control

1855 2804

GG 0.853 0.844

GA 0.147 0.156

AA – –

G 0.926 0.922

A 0.074 0.078

Alleles

Haplotypes for case and control groups account for about 93% and 85% of inferred haplotypes, respectively. Haplotypes ACAGGG and GCAGGG were associated with resistance to mastitis (P < 0.001, OR = 0.694, 95% CI: 0.580–0.831, and P < 0.001, OR = 0.612, 95% CI: 0.466–0.804, respectively), that is ewes with these haplotypes developed udder inflammation significantly less frequently (approximately 30% and 17% for ACAGGG and GCAGGG haplotypes, respectively). Haplotype ACGGGG, conversely, was found associated with susceptibility to mastitis (P = 0.01, OR = 1.120, 95% CI: 1.027–1.221) (Table 3). On the basis of minor allele frequency for control group two SNPs, one on SBD1 and one on SBD2, were found associated with susceptibility and resistance to mastitis (Table 4). In particular, SNP1747 on SBD1 gene was associated with susceptibility to mastitis (P = 0.002, OR = 1.257, 95% CI: 1.111–1.422) while SNP1659 on SBD2 gene was found associated to resistance to mastitis (P = 0.001, OR = 0.880, 95% CI: 0.806–0.962) (Table 4).

4. Discussion Resistance to mastitis can be determined by several antimicrobial peptides including defensins (Bagnicka et al., 2007), but the basis for this association has never been adequately explained. The main objective of this study was to investigate the association between defensin polymorphisms and mastitis resistance using results of bacteriological analyses in Valle del Belice dairy sheep breed as indicators of infection. Considering that results on the association of gene polymorphisms with disease can be influenced by many factors we aimed to ensure the correctness of our results. First, all the studied animals were similar with respect to their breeding system and environmental conditions, age and season of lambing. Second, an efficient association test was used to determine the significance of the association between polymorphism and mastitis. The study of association analysis of candidate gene is a useful step for the knowledge of the genetic basis of productive traits. SNP1747 on SBD1 gene was associated with susceptibility to mastitis, whereas SNP1659 on SBD2 gene was associated with resistance. Among all identified SNPs, only these two SNPs, even if with low frequencies within our samples, were found in mutated homozygote condition. Further studies are needed to investigate how they are involved in susceptibility or resistance to mastitis, in particular for SNP1659 which causes an amino acid change Arg42/Lys42 (Monteleone et al., 2011). Haplotype analysis revealed that ACAGGG and GCAGGG haplotypes were associated with resistance to infection, whereas haplotype ACGGGG was associated with susceptibility (Table 3). Considering the results presented in Tables 3 and 4, ACGGGG was the most frequent haplotype in our population (48.4% and 45.2% in case and control group, respectively) whereas the two haplotypes with a positive effect against mastitis, were present at very low frequencies (0.050 and 0.020, respectively) (Table 3). Until now there was limited information on ␤-defensins role on mastitis resistance in sheep. To the best of our knowledge, this is the first time that a similar association study was conducted on dairy sheep; therefore, it was not always possible to compare our results with what is reported in literatures. In bovine species, the expression of most of the ␤-defensins genes was shown to be much higher in tissue derived from udders infected with coagulasepositive or coagulase-negative bacteria than from bacteria-free

Table 3 Haplotypes, absolute and relative frequency estimates (in brackets) and significance levels (Fisher’s P) of case-control comparison. Haplotype

Case

Control

Chi2

Fisher’s P

ORa [95% CI]b

ACGGGG ACAGAG GCGGGG ATGGGG ACAGGG ATGGGA GCAGGG

1793.94 (0.484) 818.35 (0.221) 235.88 (0.064) 197.46 (0.053) 187.28 (0.050) 135.59 (0.037) 75.05 (0.020)

2534.33 (0.452) 1171.66 (0.209) 394.52 (0.070) 274.26 (0.049) 393.59 (0.070) 195.64 (0.035) 180.59 (0.032)

6.626 1.136 2.033 0.645 16.004 0.101 12.653

0.010077 0.286573 0.153979 0.421824 6.41E-05 0.750629 0.000378

1.120 [1.027–1.221] 1.057 [0.955–1.171] 0.885 [0.749–1.047] 1.080 [0.895–1.304] 0.694 [0.580–0.831] 1.037 [0.829–1.296] 0.612 [0.466–0.804]

a b

OR, odd ratio. 95% CI, 95% confidence interval.

Table 4 Armitage trend test of association between SBD1 and SBD2 single nucleotide polymorphisms (SNPs) and mastitis. SNPs

Case obs.a

Control obs.

ORb [95% CI]c

Chi2 trend

df trend

Prob trend

SNP1747 SNP89 SNP1659 SNP1667 SNP1750 SNP1761

1855 1855 1855 1855 1855 1855

2804 2804 2804 2804 2804 2804

1.257 [1.111–1.422] 0.953 [0.839–1.084] 0.880 [0.806–0.962] 1.141 [0.794–1.639] 1.028 [0.931–1.134] 0.939 [0.803–1.099]

9.953 0.603 10.32 0.518 0.433 0.651

1 1 1 1 1 1

0.002 0.437 0.001 0.472 0.510 0.420

a b c

obs, observations. OR, odd ratio. 95% CI, 95% confidence interval.

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udders (Günther et al., 2009; Ko´sciuczuk et al., 2014) suggesting a role of these genes in the defense of cow mammary gland against mastitis. 5. Conclusion The present study has firstly suggested the possible associations of ␤-defensin gene polymorphisms with mastitis resistance traits and showed the presence of interesting haplotypes in Valle del Belice dairy sheep breed (ACAGGG and GCAGGG) for future studies. Results from association analysis provided preliminary evidence that the ovine ␤-defensin genes could be used as a candidate genes or molecular markers for the improvement of ovine mastitis resistance traits in Valle del Belice dairy breed. Further studies will be needed to use these SNPs for marker assisted selection (MAS) in larger populations and carried out to clarify and to verify the effects of these markers on ovine mastitis resistance traits in order to increase the proportion of individuals carrying SNPs and haplotypes associated with resistance to mastitis. This would have a positive implication for different aspects as a reduction in the use of antibiotics, higher animal welfare and a greater economic return for farmers. Conflict of interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Acknowledgement We acknowledge Dr. M.L. Scatassa (Istituto Zooprofilattico Sperimentale della Sicilia) for bacteriological analyses. References ´ Bagnicka, E., Strzałkowska, N., Flisikowski, K., Szreder, T., Józwik, A., Prusak, B., ˙ J., Zwierzchowski, L., 2007. The polymorphism in the ␤4-defensin Krzyzewski, gene and its association with production and somatic cell count in Holstein–Friesian cows. J. Anim. Breed. Genet. 124, 150–156. ´ ˙ ´ Bagnicka, E., Strzałkowska, N., Józwik, A., Krzyzewski, J., Horbanczu, J., Zwierzchowski, L., 2010. Expression and polymorphism of defensins in farm animals. Acta Biochim. Pol. 57, 487–497. Bradley, A.J., 2002. Bovine mastitis: an evolving disease. Vet. J. 164, 116–128. Günther, J., Koczan, D., Yang, W., Nürnberg, G., Repsilber, D., Schuberth, H.J., Park, Z., Maqbool, N., Molenaar, A., Seyfert, H.M., 2009. Assessment of the immune capacity of mammary epithelial cells: comparison with mammary tissue after challenge with Escherichia coli. Vet. Res. 40, 31. Huttner, K.M., Lambeth, M.R., Burkin, H.R., Burkin, D.J., Broad, T.E., 1998. Localization and genomic organization of sheep antimicrobial peptide genes. Gene 206, 85–91. ICAR (International Committee for Animal Recording), 2014. International agreement of recording practices. Available online: http://www.icar.org/wpcontent/uploads/2015/11/Guidelines 2014. (accessed on 26.12.2014). Kaiser, V., Diamond, G., 2000. Expression of mammalian defensin genes. J. Leukoc. Biol. 68, 779–784. ˙ Ko´sciuczuk, E.M., Lisowski, P., Jarczak, J., Krzyzewski, J., Zwierzchowski, L., Bagnicka, E., 2014. Expression patterns of ␤-defensin and cathelicidin genes in

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