Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 54 issue 3 (march 2020) : 265-274

Variation in lactoferrin gene affects milk lactoferrin content and somatic cell count in murrah buffaloes

Arun Pratap Singh1,*, K.P. Ramesha2, M.A. Mir3, Ashwani Arya4, S. Isloor5
1Livestock Production Management, Krishi Vigyan Kendra, Ajmer-305 206, Rajasthan, India.
2Dairy Production Section, Southern Regional Station of Indian Council of Agricultural Research National Dairy Research Institute, Bangalore-560 030, Karnataka, India.
3Animal Genetics and Breeding Division, Indian Council of Agricultural Research National Dairy Research Institute, Karnal-132 001, Haryana, India.
4Krishi Vigyan Kendra, Mahoba-210 423, Uttar Pradesh, India.
5Department of Veterinary Microbiology, Veterinary College, Hebbal, Bangalore-560 024, Karnataka, India.
Cite article:- Singh Pratap Arun, Ramesha K.P., Mir M.A., Arya Ashwani, Isloor S. (2019). Variation in lactoferrin gene affects milk lactoferrin content and somatic cell count in murrah buffaloes . Indian Journal of Animal Research. 54(3): 265-274. doi: 10.18805/ijar.B-3773.

ABSTRACT

Lactoferrin plays an important role in antimicrobial defence and is a potential candidate gene for mastitis resistance. In the present investigation, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) studies were carried out in Murrah (Bubalus bubalis) buffaloes to detect single nucleotide polymorphisms (SNPs) of lactoferrin gene and to analyse the association between the observed polymorphisms with milk lactoferrin content and Somatic cell count (SCC). PCR-SSCP analysis revealed a total of 07 different variants in the partial coding region of the lactoferrin gene. PCR-SSCP analysis of exon 10 of lactoferrin gene revealed three and that of exons 4 and 5 revealed two unique patterns each, while all other exons exhibited monomorphic pattern. Comparison of nucleotide sequences of lactoferrin gene of the Murrah buffaloes with taurine reference sequence revealed a total of 14 point mutations, 09 of which were found to be in coding region. Conceptualized translation of nucleotide sequence revealed 07 amino acid changes. SSCP variants of exon 10 (P<0.01) had significant effect on milk SCC. The SSCP variants of exon 4 and exon 5 had significant (P<0.05) effect on lactoferrin content. The SCC and lactoferrin content in Murrah buffaloes was highest in 4th and above parity group. Stage of lactation had highly significant (P<0.01) effect on both milk SCC and lactoferrin content. There was a high and significant (p<0.01) correlation (0.741) between SCC and lactoferrin content in milk. The observed association between SSCP variants in lactoferrin gene with milk SCC and milk lactoferrin content can be used as prognostic markers for selection of animals for high lactoferrin content and low somatic cell count, as well as a marker of susceptibility/resistance to mastitis in Murrah buffaloes.

INTRODUCTION

Selection and multiplication of genetically superior germplasm is one of the most important steps in any livestock development programme. However, there are large number of constraints hampering these programmes including prevalence of diseases like mastitis, which hamper the expression of true genetic merit of production of these animals. Mastitis is a polygenic trait with very low heritability, so selective breeding of dairy buffaloes for reduced susceptibility/increased resistance against mastitis is difficult. In India every year about 3.84% buffaloes are affected with clinical and 24.43% with sub- clinical mastitis (Singh, 1994). The clinical and subclinical mastitis in buffaloes account for an annual loss of Rs. 6,962,900,000 and 17,233,200,000, respectively (Dua, 2001). Indirect selection based on somatic cell count, lactoferrin content and candidate gene markers will help to increase the efficiency of breeding programmes.
       
The lactoferrin (iron-binding glycoprotein) gene was mapped to bovine chromosome 22, contains 17 exons and spans about 34.5 kb of genomic DNA (Schwerin, 1994). Lactoferrin (Ltf) is present in milk and synthesized by specific granules of polymorphonuclear leukocytes (Lonnerdal et al., 1995) and by glandular epithelial cells (Baynes and Bezwoda, 1994). Lactoferrin plays a key role in the defense mechanisms of the mammary gland, contributing to the prevention of microbiological infectious diseases (Lee et al., 2004). The Ltf concentration in bovine milk varies widely from 1.15 μg/mL to 485.63 μg/mL in milk of healthy animals (Hagiwara et al., 2003). In colostrums, Ltf concentration is high, varying between 1 and 5 mg/mL, and dry secretions, varying between 20 and 30 mg/mL (Stelwagen et al., 2009). Lactoferrin plays major role as antibacterial, antiviral, antitumor and immunomo dulatory molecule during infections of mammary gland (Gonzalez-Chavez, 2009). Various in-vitro and in-vivo studies have proved the antibacterial activity of lactoferrin especially against E.coli, P. aeruginosa and S. aureus (Lacasse, 2008). The anti-bacterial activity of lactoferrin makes it a candidate gene for increasing resistance against infections of mammary gland (Seyfert et al., 1994). The lactoferrin as biopreservant enhanced the shelf life of khoa by lowering the microbial growth (Shashi, 2011). Its concentration in bovine milk was significantly associated with SCC and stage of lactation (Harmon et al., 1975; Liu et al., 2010).
       
Somatic cell count (SCC) is a useful measure for detection of subclinical mastitis. Increase in leucocyte number in milk and mammary gland, as a response to the assaulting pathogens or to their metabolites leads to an increase in SCC (Atasever, 2012). Total SCC in normal buffalo milk varies from 50,000 to 375,000/mL (Silva and Silva, 1994). In buffalo total SCC is a valid indicator of udder inflammation and a value of 200,000 cells/mL should be used as the threshold value for early identification of an animal affected by subclinical mastitis (Tripaldi et al.,  2010). Threshold limit of 200×10³cells/ mL of milk had been recommended for subclinical mastitis Federation (IDF, 2005). In EU countries, according to the directive 92/EEC, bulk milk samples with SCC greater than 400×10³ cells/ mL of milk is advised not to be used for human consumption (Atasever and Erdem, 2010). There was a moderate to high genetic association (rg = 0.3–0.7) between SCC and clinical mastitis and it is also comparatively more heritable (h2 = 0.10–0.14) than clinical cases making it one of the most important selection tool for mastitis resistant animals (Mrode, 1998). Polymorphism in lactoferrin gene has association with susceptibility/resistance to mastitis (Zhao, 2008). The above information clearly indicates the importance of lactoferrin gene as a candidate for selection of mastitis resistant cows. However, information regarding the polymorphisms within the Bubalus bubalis lactoferrin gene is very scanty. The aim of the present study was to describe the locus specific polymorphisms within the bovine lactoferrin gene through PCR-SSCP and DNA sequencing, and its association with lactoferrin content and SCC in milk of Murrah buffaloes.

MATERIALS AND METHODS

Experimental animals and their management
 
A total of 174 lactating Murrah buffaloes maintained under semi-intensive system of management at commercial buffalo farms at Hosur in Tamilnadu state and Dharwad in Karnataka state were utilized as the experimental animals for the present study. Murrah is one of the best milch breeds among buffaloes (Bubalus bubalis) and found in Rohtak, Hisar and Jind districts of Haryana state and Nabha and Patiala districts of Punjab state in India. Blood (8-10 ml) samples were collected aseptically by jugular vein puncture using vacutainers containing EDTA as anticoagulant from randomly selected Murrah buffaloes after obtaining permission from Institute Animal Ethics Committee. 50 ml of milk samples were also collected from these buffaloes. The information on parity and stage of lactation was collected from the animal records.
 
DNA isolation
 
Genomic DNA was isolated by high salt method as described by (Miller, 1988). The quality and quantity of DNA was checked by agarose gel electrophoresis and UV spectrophotometer. The stock solutions were stored at -20°C and used for further analysis. The working solution was prepared by diluting the stock to 100 ng/µL for utilizing as DNA template in PCR.
 
Primers designing
 
Ten sets of primers were designed based on the 35384 bp sequence for Bos taurus lactoferrin gene (Ensembl RefSeq: ENSBTAG00000001292) by using primer 3 (http:/www.genome.wi.mit.edu/ cgibin/primer/primer-3www.cgi) software. These oligose were synthesized from Europhins MWG operon, Bangalore, India. The details of the primers, location, annealing temperature and the expected product sizes are summarized in Table 1.
 

Table 1: Primer sequences (5' to 3' sequences) used for amplification of lactoferrin gene.


 
PCR amplification
 
The Polymerase Chain Reaction (PCR) was carried out on about 50-100 ng of genomic DNA in 25 µl per reaction mixture. The PCR reaction mixture consisted of 200 µM of each dNTPs, 10X Taq DNA Pol assay buffer, 1U Taq DNA polymerase enzyme and 20 pM of each primer. The thermocycler conditions included with an initial denaturation at 94°C for 2 min, followed by 35 cycles with denaturation at 94°C for 30 sec with varying annealing temperatures based on primer set (Table 1), extension at 72°C for 1 min followed by a final extension at 72°C for 10 min. The PCR products were electrophoresed at 100 V in  1.5%  agarose gel in 1X  TBE buffer containing 0.5 μg/mL ethidium bromide along with a DNA molecular size marker. The gels were visualized and documented using Gel documentation system (Gel doc 1000, Bio-Rad, USA).
 
PCR-SSCP analysis
 
Amplified PCR products (10 µL) were further diluted in denaturing solution (95% formamide, 10 mM NaOH, 0.05% Xylene cyanol and 0.05% bromophenol blue, 20 mM EDTA) and heat denatured at 94°C for 8 minutes followed by rapid chilling on ice block for 20 minutes and loaded on 10% acrylamide: bisacrylamide (29:1) in 1X TBE buffer for 8 hours (200 V). The gels were silver-stained as described by Sambrook and Russell, 2001. Band patterns were characterized by the number of bands, mobility shifts and scored manually.
 
SNP identification
 
Representative PCR products giving unique SSCP patterns were custom sequenced using automated ABI DNA Sequencer (Amnion Biosciences Pvt. Ltd., Bangalore, India) to confirm the mobility shift in each pattern. Sequence data were analysed using Bio edit software Clustal W multiple alignments for detecting single nucleotide polymorphisms (SNPs) by comparing the observed sequence with the bovine lactoferrin gene reference sequence (Ensembl RefSeq:ENSBTAG00000001292).
 
Quantitative determination of Bovine lactoferrin (LTF) concentration in milk
 
The milk lactoferrin content was estimated using CUSABIO Bovine Lactoferrin ELISA Kit which is based on competitive inhibition enzyme immuno- assay technique. Lactoferrin concentration was calculated using the professional soft “curve expert 1.3”. Standard curve was created by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve fit and lactoferrin concentration was calculated.
 
Somatic cell count in milk
 
Milk somatic cell count in each milk sample was determined using NucleoCounter® SCC-100™ system (Chemometc, Denmark) following the protocol developed by Saleh and Faye, 2011.
 
Statistical analysis
 
In order to study the association of SSCP pattern with SCC and lactoferrin content in milk, the non genetic factors like parity and stage of lactation were also treated as source of variability. The association study of SSCP variants and effects of non-genetic factors like parity and stage of lactation on Somatic cell count and Lactoferrin content in milk was done by PROC GLM (SAS, 2011).
 
                         Yijkl= µ + Pi + Sj + Ek + eijkl
Where, 
Yijkl   is observation on the lth cow of kth genotype in jth stage of lactation and ith parity
µ       is overall mean
Pi      is fixed effect of ith parity where, i=1, 2, 3 and 4 (4th and above)
Sj     is fixed effect of jth stage of lactation (j=1, 2, 3,) where,   1: < 90, 2: 91-180, 3: >180 days
Ek    is fixed effect of kth genotype
eijkl  is the random error which is NID (0, σe2)
       
For those variants and factors that were found to be significantly associated, comparisons were performed using Tukey’s honestly significant difference (HSD) test to account for the multiple comparisons being made. Somatic Cell Count and lactoferrin content were transformed to a logarithmic (log10) scale in order to balance the distribution.

RESULTS AND DISCUSSION

In the present study ten exonic regions of lactoferrin gene in Murrah (Bubalus bubalis) buffaloes were generated using 10 sets of primers. BLAST analysis of entire coding sequence revealed percent homology of 97%, 97%, 93%, 93% and 77% with Bos taurus, Bos indicus, Capra hircus, Ovis aries and Homo sapiens, respectively. SSCP patterns of lactoferrin gene fragments were sequenced and aligned to identify the SNPs using Clustal W program.
 
SSCP variants
 
PCR-SSCP analysis of amplicons of exons 4, 5 and 10 showed polymorphism while the remaining exons 1, 2, 3, 6, 7, 8 and 9 were monomorphic. The analysis of exon 10 of lactoferrin gene revealed three unique SSCP patterns (Fig 7). Two unique SSCP patterns were observed for the exons 4 (Fig 1) and 5 (Fig 4). The frequencies of SSCP variants for each exon in 174 Murrah buffaloes genotyped in the present study are summarised in Table 2.
 

Fig 1: PCR-SSCP patterns of exon 4 of lactoferrin gene in Murrah buffaloes.


 

Fig 4: PCR-SSCP patterns of exon 5 of lactoferrin gene in Murrah buffaloes.


 

Fig 7: PCR-SSCP patterns of exon 10 of lactoferrin gene in Murrah buffaloes.


 

Table 2: Frequencies of SSCP variants of lactoferrin gene in Murrah buffaloes.


 
SNP identification
 
Representative samples were custom sequenced to confirm the mobility shift in each pattern. The retrieved sequences representing each of the unique PCR-SSCP patterns were further analyzed by comparing these sequences with bovine lactoferrin gene reference sequence for Bos taurus cattle using Clustal-W multiple sequence alignment tool (Fig 2, 5, 8) and DNA Baser for detecting Single Nucleotide Polymorphisms (SNP’s) (Fig 3, 6, 9) and their respective deduced amino acid variations (Table 3). Seven out of 09 SNPs observed in exonic regions changed the amino acid in the transformed products where as remaining two were silent mutations. In intronic region a total of five SNPs were detected (Table 3).
 

Fig 2: Clustal W multiple alignment sequence of exon 4 of lactoferrin gene in Bos taurus and Murrah buffaloes.


 

Fig 3: Sanger Trace Figures of SSCP variant sites of exon 4 of lactoferrin gene in Murrah buffaloes.


 

Fig 5: Clustal W multiple alignment sequence of exon 5 and flanking partial intronic regions of lactoferrin gene in Bos taurus and Murrah buffaloes.


 

Fig 6: Sanger Trace Figures of SSCP variant sites of exon 5 and flanking partial intronic regions of lactoferrin gene in Murrah buffaloes.


 

Fig 8: Clustal W multiple alignment sequence of exon 10 and flanking partial intronic regions of lactoferrin gene in Bos taurus and Murrah buffaloes.


 

Fig 9: Sanger Trace Figures of SSCP variant sites of exon 10 and flanking partial intronic regions of lactoferrin gene in Murrah buffaloes.


 

Table 3: Summary of Single nucleotide polymorphisms observed in lactoferrin gene in Murrah buffaloes.


 
Association of genetic variants with SCC and lactoferrin content
 
The mean Somatic Cell Count of milk (million cells/mL) and milk lactoferrin content (mg/L) in Murrah buffaloes was estimated as 0.16 ± 0.02 and 98.35 ± 7.26 respectively. The study revealed significant effect of SSCP variants of exon 10 (P<0.01) on SCC in milk. The SSCP variants of exon 4 and exon 5 had significant (P<0.05) effect on lactoferrin content in milk. Parity and stage of lactation had highly significant (P<0.01) effect on SCC and lactoferrin content. The Least-squares means for SSCP patterns in different exons, different parity and different stage of lactation on SCC and lactoferrin content are summarized in Table 4 and 5.  Among the three SSCP variants of exon 10 in buffaloes, pattern A had highest SCC. Somatic Cell Count (SCC) exhibited an ascending trend with the progression of parity and stage of lactation. SSCP analysis revealed two distinct patterns in exon 4 and exon 5 and lactoferrin content was higher in pattern B of exon 4 and pattern A of exon 5. Lactoferrin content in Murrah buffaloes was highest in 4th and above parity group and lowest in 1st parity. An increasing trend of lactoferrin content was observed in successive parity and stage of lactation.
 

Table 4: Least-squares means ± SE of somatic cell count (million cells/mL) in Murrah buffaloes.


 

Table 5: Least-squares means ± SE of lactoferrin concentration (mg/L) in Murrah buffaloes.


 
SSCP variants
 
Eight different variants within exons 6, 7 and 13 and their flanking intronic regions in the bubaline lactoferrin gene with the help of PCR-SSCP analysis, locus LtfE6 and LtfE13 revealed two SSCP variants while four different variants, viz. LtfE7-A, LtfE7-B, LtfE7-C and LtfE7-D, were observed at the locus LtfE7 (Kathiravan, 2010).
 
SNP identification
 
 A total of 26 point mutations, 13 of which were found to be in coding DNA region, conceptualized translation of nucleotide sequence revealed two amino acid changes, viz. serine to threonine and arginine to alanine within exons 7 and 13, respectively in the bubaline lactoferrin gene (Kathiravan, 2010).
 
Association of genetic variants with SCC and lactoferrin content
 
Lactoferrin is a key element in the host innate defense system with its antimicrobial properties, which include iron sequestration, direct lytic activities, and the ability of the molecule to impair the binding of microbes to host cells (Kutila et al., 2003; Valenti and Antonini, 2005). The present investigation was aimed to characterise lactoferrin gene in Bubalus bubalis (Murrah) buffaloes and to elucidate the genetic variation in the partial coding region of bubaline lactoferrin gene. Single Nucleotide Polymo- rphisms give a better chance to explain a part of the genetic variance of different traits through understanding of the biochemical or physiological mechanisms in which a candidate SNP might be involved. In the present study nine SNPs were detected in the exonic region of bubaline lactoferrin gene. PCR-SSCP analysis showed that exons 4, 5 and 10 are polymorphic. Perusal of Table 3 indicates high degree of mutations in lactoferrin gene in Murrah breed of buffaloes.
 
       
There was a high variability in lactoferrin gene and genetic variants are associated with SCC and lactoferrin content in Murrah buffaloes. The results observed in the present study supports the observed association between genetic variants and SCC by Wojdak-Maksymiec et al., (2006). Association between SSCP markers with clinical mastitis residuals and SCC in milk in bovines was reported by Lei et al., (2006). The observed association between genetic variants and SCC count in milk is in agreement with the earlier report in crossbred cattle (Rahmani, 2012).
       
The ascending trend of SCC and significant effect (P<0.01) of parity and stage of lactation in Iraqi buffaloes was reported by Al-Saadi et al., (2015). Similar finding was observed in cattle by Munoz et al., (2002). On contrary stage of lactation did not affect the SCC in Murrah buffalo (Singh and Ludri, 2001).The ascending trend in SCC could be attributed to the lesions caused by daily milking or progress of bacterial infections in different lactation stages because of a dilution effect (Reneau, 1986). Results are in consonance with other studies (Sharif and Mohammad, 2008; Madouasse, 2009). Increased risk of infectious pathogens with animals age, entering the udder and causing mastitis and also environmental and physiological factors related to the activity of immune system and working to reduce the animal’s resistance to diseases. This causes increase of white blood cells in the bloodstream and in the mammary tissue. The white blood cells penetrate barrier tissue of the gland and mixes with milk thus the number of somatic cells increases in different parities (Obrien et al.,  2009). SCC increased after 90 days in milk and then remained constant until the end of the lactation period in Italian buffalo (Giacinti, 2013).
       
The increasing trend of lactoferrin content and significant effect (P<0.01) of stage of lactation was observed in Italian buffalo whereas, parity effect was found to be non-significant (Giacinti, 2013). Cheng et al., (2008) found similar results in dairy cows. The immunity due to lactoferrin results from the fact that possible infection factors have a limited availability of iron (as well as other growth agents, such as phosphorus and zinc), since its concentration in an organisms fluids is reduced (Persson, 2010). Therefore, a less vulnerability to mastitis in buffalos might be partially due to the high level of lactoferrin in milk. The ascending trend of lactoferrin concentration during lactation is due to mammary gland involution in cows (Shamay et al., 2003) and dilution effect in goats (Hiss et al., 2008).
       
Our study indicated a high and significant (p<0.01) correlation (0.741) between SCC and lactoferrin. Various workers were in conformity with the above results (Piccinini et al., 2006; Cheng et al., 2008; Al-Majali et al., 2007). Lactoferrin content was elevated in mastitis and there was a close relationship between SCC and Lactoferrin content (Chen et al., 2004). According to Lindmark-Mansson et al., (2000, 2006), a higher SCC leads to increase of lactoferrin content in milk, and a close relationship between this milk component and the status of udder health has been reported by very high correlation coefficients (r = 0.962 and r=0.918 at P<0.001) between milk lactoferrin concentration and SCC. The increase of lactoferrin content proportional to SCC qualify lactoferrin as an acute phase protein in milk and also observed positive correlation (r=0.21) of lactoferrin with SCC (Giacinti et al., 2013). The relationships between Lactoferrin concentrations and SCC in buffalo is difficult to analyse; this is due to the differential data for buffalo milk SCC that leads to uncertainty about the level of SCC in buffalo milk that can be used to define the presence of an inflammation (Pasquini et al., 2003). Early detection of mastitis in buffaloes could be important for most dairy farmers to reduce production losses and to enhance prospects of recovery.

CONCLUSION

In summary the present study revealed high degree of genetic variation as indicated by different SSCP patterns which resulted into presence of nine SNPs in lactoferrin gene in Murrah buffaloes. The observed SSCP patterns in exon 10 were found to have significant effect on SCC in milk and exon 4 and exon 5 were found to have effect on milk lactoferrin content in Murrah buffaloes. The present study indicated the possibilities of using SSCP patterns as marker for milk SCC and lactoferrin content. However, further studies using large number of animals need to be carried out to validate the marker data before using them in the Marker Assisted Selection (MAS). Identification of genetic markers associated with SCC and  lactoferrin content might be helpful in improving buffaloes’ health by implementing appropriate buffalo-breeding programmes. Present study could be a step towards identification of genetic markers for selecting buffaloes for udder health and immunity.

ACKNOWLEDGMENT

The authors are thankful to Director ICAR-National Dairy Research Institute, Karnal and Head, Southern Regional Station of ICAR-National Dairy Research Institute, Adugodi, Bangalore for providing necessary facilities.

COMPLIANCE WITH ETHICAL STANDARDS

No coflict of interest exists.

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