Mastitis is a multi-factorial and costly problem affecting all milk-producing ruminants. The breed improvement programs have proven useful for the selection of milk production traits but are expensive and timeconsuming. Therefore, marker-assisted selection that supports fast and low-cost genetic progress and improves the accuracy of selection. In this regard, it is useful to study the genetic variations of candidate genes and their associations with milk production and somatic cell count (SCC) which have a high genetic positive correlation with mastitis (with an estimated average coefficient of 0.7) (Motwani, 2011). Daughters of sires that transmit the lowest SCC had the lowest incidence of clinical mastitis and the fewest clinical episodes during first and second lactations. The incidence of clinical mastitis and the number of clinical episodes per lactation may be reduced by selection for lower somatic cell score, longer productive life, shallower udders, deeper udder cleft, or strongly attached fore udders (Nash et al. 2000). Project directorate on Cattle, Meerut, India has developed a crossbred cattle “Frieswal” (5/8 Holstein Friesian and 3/8 Sahiwal) in collaboration with Ministry of Defense. Frieswal cows are being maintained at various military farms of the country. Presently the total population of Frieswal females at 37 military farms located in various agro-climatic regions of the country is 16874 that include1054 elite females. In a mature lactation of 300 days. Frieswal cows are producing 3542 kg of milk with 3.9 to 4.1% milk fat. (Lakshmi et al. 2010). The LTF, a ferric ion (Fe3+) binding glycoprotein, is found most notably in milk (Guo-Hua et al. 2007). The bovine LTF gene, a member of transferrin gene family, spans about 34.5 kb of genomic DNA, consists of 17 exons. Higher concentration of LTF in milk has been associated with lower occurrence of bovine mastitis. Some experiments have shown affinity of LTF with biomembrane. The antibacterial activity of LTF makes it a candidate gene for increasing resistance against infections of mammary gland (Seyfert et al. 1994). Some researchers reported that LTF concentration in milk and serum would change during the infection of mastitis which indicates that there is some association between LTF and mastitis. Therefore, observation on polymorphism of LTF gene by PCR-RFLP, and association between LTF and mastitis could give some novel insight into theory and practice (Zhao et al. 2008). In the present paper, polymorphism of LTF gene promoter and association between LTF and mastitis was investigated, by PCR-RFLP.
MATERIALS AND METHODS
Animals and experimental group
The quarter milk samples of 150 Frieswal lactating cows upto3rdlactation of similar nutrition, management conditions were screened for clinical mastitis by clinical examination of the udder as well as by strip cup test, and for sub-clinical mastitis by California mastitis test (CMT), (Schalm and Noorlander, 1957) and SCC. (Roger Mellenberger, 2000). For screening the cows, SCC was determined in quarter milk samples using Fossomatic TM Minor cell counter (Foss Electric, Hillerod, Denmark) as recommended by the International Dairy Federation (IDF, 1984) and as described by Gonzalo et al. (2003). The SCC value more than 500 cells/µL of milk was considered positive for sub-clinical mastitis. The California mastitis test (CMT) was performed on quarter milk samples as per the method described by Schalm and Noorlander (1957). After screening the 150 cows by employing CMT and SCC, only 10 cows were found clinically positive and therefore a total of 30 Frieswal cows 10 in each group were classified as healthy, sub clinically and clinically affected.
Sample collection and DNA extraction
Blood samples were collected in vacutainer tubes containing ethylene diamine tetra-acetic acid (EDTA) (1 mg/mL). Genomic DNA was extracted using standard protocol (John et al. 1991) and stored at -20 ˚C until used for assay. The concentration of DNA samples was estimated using UV-visible range spectrophotometer and diluted to 30 ng/μL before PCR amplification. All the DNA samples were in the range of 1.8 to 2 at 260/280 optical density (OD), indicating high purity. The DNA was also examined by loading samples on 0.8% agarose gel and visualizing the band under UV light with a Gel Doc 1000 system (BioRad) after ethidium bromide staining.
Polymerase chain reaction
The polymerase chain reaction was based on the procedure reported by Zhao et al. (2009). Forward (5’-CAC ATT ACA AGC AGG ATC TTT TGC TG-3’) and Reverse (5’CTG GCC AAT GAG CCC TAT ATG TGT-3’) primers were used to amplify a 1143 base pair segment of the bovine LTF locus. The reaction mixture consisted of 90 ng of template DNA, 10 pmole of each primer (1 µL each), 12.5 µL of 2X PCR mastermixjand 7.5 µL of DNAse free water. This solution was initially denatured at 94 ˚C for 10 min. followed by 35 cycles of denaturation (94 ˚C for 1 min), annealing (63 ˚C for 1 min), and elongation (72 ˚C for 1 min) and a final extension at 72 ˚C for 10 min. The PCR products were electrophoresed on 1.5% agarose gels in order to check the quality and specificity of DNA fragment amplification.
Restriction enzyme digestion
For PCR-RFLP analysis, the 1143 bp PCR products were digested with HinfI (Fermentas Life Sci.). Restriction fragments were separated by electrophoresis in a 2% agarose gel and their sizes were estimated using the molecular markers. The results were taken into account when the sum of all the restriction fragments for HinfI enzyme was in the range of 1143 bp ±100. Ten µL of PCR products was digested for 12h at 37 ˚C with 1 µL units of restriction enzyme. Digested products were separated by electrophoresis on a 3% agarose gel and visualized with ethidium bromide under UV light with a Gel Doc 1000 system (BioRad) after ethidium bromide staining.
LTF gene polymorphic variants with somatic cell count
The SCCt for LTF gene was analyzed using simple analysis of variance model as given below:
Yij= µ + Gi + eij
Yij: mean log10 SCC of the jth cattle for ith genotype.
µ: overall mean.
Gi: effect of ith genotype (i=1, 2, 3).
eij: random error.
RESULTS AND DISCUSSION
The quality and quantity of extracted DNA from analyzed samples was tested by electrophoresis on agarose gel where the DNA showed a single band. The DNA quantity was estimated using the molecular markers and the quantity for each band DNA sample was amplified and the PCR product was shown on agarose gel. As expected, the size of PCR product of the LTF gene was 1143 bp in length (Figure 2) (Zhao et al. 2008).
Results of RFLP showed the existence of sequence encoding LFT in all sampled cows. Identification of different genotypes for LFT requires an enzymatic digestion. In this case, digestion was carried out by restriction enzyme HinfI, an endonuclease, which cuts at the restriction sites. Different fragments were separated by PCR products in LFT gene promoter using HinfI. Among 30 Frieswal dairy cows included in this study, 2 allele genes and 3 genotypes were revealed in each group. Allele gene (681 bp) was defined as A, whilst allele gene (462 bp) was defined as B. Other related genotypes were defined as G/G, G/C and C/C, respectively (Figure 3). After digestion, allelic and genotypic frequencies were listed (Table 1).
Among healthy dairy cows, CC genotype was detected with a frequency of 0.33, Frieswal dairy cows with subclinical mastitis showed GG (0.17) as well as GC (0.50) genotype and the cows with clinical mastitis showed GC genotype. The allelic frequency of G and C was 0.42 and 0.58 respectively. The non significant Chi-square value in Frieswal cattle showed that the population was in Hardy Weinberg equilibrium.
Figure 2 Amplified PCR product of LTF gene of Frieswal cattle electrophoresed on 2% agarose
M: 100 bp DNA ladderLanes 1-8: are amplified PCR product
Figure 3 PCR-RFLP pattern of LTF gene digested with HinfI enzyme in Frieswal cattle
M: 100 bp DNA ladder
P: PCR product
Lane 4, 6, 14: GG genotype (681 bp)
Lane 2, 3, 5, 9, 10, 13: GC genotype (681, 462 bp)Lane 1, 7, 8, 12: CC genotype (462 bp)
The association of SCC with LTF gene polymorphism was determined by analysis of variance on somatic cell count by using one way ANOVA. The relationship between genotypes and SCC was evaluated (Table 1).The LTF gene polymorphism showed significant association with SCC. The cows of genotypes GG and GC had higher SCC than those of the CC genotype. LFT, a ferric ion (Fe3+)-binding glycoprotein, is found most notably in milk and one member of transferrin family. Higher the concentration of LTF in milk, lower the occurrence of bovine mastitis. In the preliminary study of LTF, LFT was thought to act bacteriostatic agent due to its avid Fe3+-ion binding capacity depriving growing microorganisms of their demands for ferric ions. With further research, it showed that the bactericidal and bacteriostatic mechanisms of LTF protein are very complicated. It can affect the integration of biomembrane in bacteria and results in the delivery of lipopolysaccharide from the membrane and increase the sensitivity of bacteria toantibodies and lysozyme (Guo-Hua Li et al. 2007). Wojdak et al. (2006) reported that homozygous individuals for A allele presented the lowest rates of somatic cells. Similar observations were reported by Zhao et al. (2008) indicating that cows with A/A genotypes were resistant to mastitis infection. The obtained values of SCC were in lakhs which were further transformed to log10 SCC (Figure 1). The mean SCC value of healthy cows and those with subclinical and clinical mastitis were found to be 4.81 ± 0.08, 6.23 ± 0.07 and 6.38 ± 0.06, respectively. The analysis of variance on somatic cell count was calculated using the one way ANOVA. The relationship between genotypes and SCC was evaluated (Table 2).
The LTF gene polymorphism showed significant association with the SCC. Cows of genotypes GG and GC had higher SCC than those of the CC genotype. The RFLP fragments and patterns of genotypes obtained in the present study are in agreement with RFLPfragments and patterns of genotypes as reported by Zhao et al. (2008) and Jemmali et al. (2011). Earlier, Xingping et al. (2007) studied association of Toll-like receptor 4 (TLR4) gene with mastitis and reported moderate polymorphism based on the value of polymorphism information content (PIC). The authors observed that cattle with allele A in T4CRBR1 exhibited lower SCC than in cattle with allele B which suggested possible role of allele A in imparting resistance against mastitis.
In the present essay, Frieswal cows were sampled to search polymorphism in the LFT gene. The SCC of milk samples and the PCR-RFLP of LTF gene was carried out by standard protocols. The results of PCR-RFLP showed three different band pattern in all samples implying three genotypes for the LFT locus. The Frieswal cows exhibiting RFLP pattern CC may have LTF genotype that are good genetic markers to indicate increased mastitis resistance while animals exhibiting GC and GG may have LTF genotypes as genetic markers for susceptibility to clinical and subclinical mastitis. The association between genotypes of LTF gene and somatic cell count was found to be highly significant (P<0.01). This can be used to design future studies to determine the association of LFT alleles with resistance to mastitis in Frieswal cows that may be used in genomic selection for breeding animals.
ACKNOWLEDGEMENTThe authors are thankful to In-charge, Military Dairy Farm, Jabalpur for having allowed collection of milk and blood samples from the lactating cows and also for giving access to data, pertaining to Friekswal cows.