Mutations in CD14 gene causes mastitis in different breeds of buffalo as confirmed by in silico studies and experimental validation

CD 14 is an important pattern recognition receptor having innate immune function and has antibacterial activity. It binds with LPS of gram-negative bacteria, arachidonic acid, and lipoteichoic acid. Being a receptor, it binds with the pathogen with the help of other cytokines. Mutations in CD14 affect the binding ability which in turn affects the biological potentiality. The present study was conducted on 228 nos. of buffaloes pertaining to four different breeds as Murrah, Mehsana, Surti and Bhadawari. CD14 gene was characterized and polymorphism was detected through Single nucleotide conformation polymorphism. Association study was conducted for different variants of CD14 with mastitis in buffalo, detected through somatic cell count, california mastitis test. Eight variants of CD14 were detected and mutational hotspots were detected in bubaline CD14 with 58 number of non-synonymous SNP, out of which 18 were observed to be deleterious and 34 as thermodynamically unstable. In the present study, we had detected the mutations in CD14 gene and its association with the somatic cell score and other indicators for mastitis. In-silico studies were conducted to understand the molecular mechanism how the mutations affect the biological potentiality by analyzing different domains and structural analysis along with various post-translational modification sites. causing and deleterious with immunological properties and diseases. Eight variants of bubaline CD14 were identified, out of which variant D and F were identified to have most of the deleterious and thermodynamically unstable mutations, leading to more susceptibility to mastitis. Proper characterization may lead to future study for therapeutic application of recombinant CD14 wild type variant A, somatic gene therapy, transgenic animal production with gene insert from Variant A and marker-assisted selection for variant A for production of future stock of livestock with reduced susceptibility of diseases, particular mastitis.


Abstract
Background CD 14 is an important pattern recognition receptor having innate immune function and has antibacterial activity. It binds with LPS of gram-negative bacteria, arachidonic acid, and lipoteichoic acid. Being a receptor, it binds with the pathogen with the help of other cytokines. Mutations in CD14 affect the binding ability which in turn affects the biological potentiality.

Method
The present study was conducted on 228 nos. of buffaloes pertaining to four different breeds as Murrah, Mehsana, Surti and Bhadawari. CD14 gene was characterized and polymorphism was detected through Single nucleotide conformation polymorphism.
Association study was conducted for different variants of CD14 with mastitis in buffalo, detected through somatic cell count, california mastitis test.

Result
Eight variants of CD14 were detected and mutational hotspots were detected in bubaline CD14 with 58 number of non-synonymous SNP, out of which 18 were observed to be deleterious and 34 as thermodynamically unstable. In the present study, we had detected the mutations in CD14 gene and its association with the somatic cell score and other indicators for mastitis. In-silico studies were conducted to understand the molecular mechanism how the mutations affect the biological potentiality by analyzing different domains and structural analysis along with various post-translational modification sites.

Conclusion
Deleterious mutations were observed in CD14 gene which have significant effect on mastitis of buffalo. It may be employed for marker assisted selection, therapeutic application of recombinant CD14, gene therapy, transgenic animal production with wild type CD14 resistant to mastitis as future strategy.

Background
Mastitis is an important economic disease of livestock. It is also considered as one of the most costly production related disease in a dairy industry. (Oviedo-Boyso et.al.2007).
Economic losses due to mastitis may reach upto 35 million dollars per year world wide ( Giraudo et al,1997;Pereira et al.2011). Subclinical mastitis is of greater concern than clinical mastitis, since incidences were 15 to 40 times more than that of clinical mastitis and it forms the basis of herd outbreaks (Boichard et al 2003;Detilleux et al, 2012). As no gross abnormality in milk and udder is noticed, subclinical mastitis goes unnoticed to the farmers. So, subclinical mastitis is considered more important due to a negative impact on the economy throughout the world. The figure for subclinical mastitis cases are approx.18.40% (Joshi and Gokhle 2006).The prevalence of subclinical mastitis with respect to different etiological agents have been identified (Ayano et al., 2013).
Economic losses due to mastitis may be due to depressed milk production, deterioration of milk quality, cost due to health management as treatment and vaccination, milk disposal due to antibiotic residues, advanced culling due to permanent damage to udder, addition labour requirement. There are certain other limitations as evolvement of antibiotic resistant bacteria, vaccinations are not cent percent due to evolvement of multistain organism, due to frequent mutation , by genetic shift and genetic drift. Other constraint are milk disposal due to its residual effect of antibiotic on the end consumers, worldwide concern for organic food production and ethical considerations of sufferings of animals has limited the antibiotic therapy. Study has been attempted with bovine mastitis prevention by inoculation of lactic acid bacteria at the dry off period ( Pellegrino et al., 2017). Thus host immune response and its immunomodulation may be considered as an alternative for disease resistance.
In case of mastitis, upon the entrance of bacteria, there is neutrophil infiltration in the mammary gland . Cell surface receptors as CD14 and TLRs stimulate cytokines and other immune effector molecules. LPS signalling is a crucial step mediated by LR4, CD14 and MyD2 in presence of LPB, leading to release of Cytokine (Schepper etal.,2008). The concentration of sCD14 is highest within a few day of parturition and significantly increases parallel with increase in SCC, challenged with E.coli LPS. (Lee etal., 2003). It is reported that Buffaloes is more resistant than other livestock species and have least number of LRR i.e, leucine rich repeat, it has been observed that lesser no of LRR are less susceptible to disease (Pal et al.,2010).
Manipulation of CD14 gene may be conducted in disease management in various ways, which aid in decreasing disease incidences. The future use includes use as a therapeutic agent; somatic gene therapy and transgenic disease resistant animal production.
The present investigation was aimed for cloning and sequencing of the CD14 gene of buffalo and identification of the mutant variants. The current study aims to identify the molecular mechanism how the mutations in CD14 causes diseases as mastitis.
This study was carried out in accordance with the recommendations of IAEC (Institute Animal Ethics Committee, Indian Veterinary Research Institute). The protocol was approved by the IAEC, IVRI.
DNA preparations: 10 ml blood was collected from each animal in aseptic condition and genomic DNA was isolated from blood samples by phenol-chloroform extraction method as described by Sambrook and Russel (1989) with few modifications. After the whole process, the DNA was taken in an Eppendorf tube and has been dissolved in TE buffer.

DNA amplification:
All PCR amplifications were performed in 25 µl reaction volume. Each reaction contained 3.0 μL 10X PCR assay buffer, 0.5 μL of 10 mM dNTP, 60 ng of each primer, 0.5 units of Taq DNA polymerase and nuclease-free water to bring the total volume to 25 µl. Around 100 ng of cDNA was used as the template. The details of primers for different fragments of GH gene are Forward: CD14--F ATGGTCTGCGTGCCCTACCTG and Reverse CD14-R GGAGCCCGAGGCTTCGCGTAA. Thermal cycling was performed on an ABI PCR machine initial denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 61°C for 35 sec, and extension at 72°C for 3 min were carried out for 35 cycles followed by final extension at 72°C for 10 min.
Gene sequencing and sequence analysis: The novel sequences were submitted to the National Centre for Biotechnology Information Genbank.
Three dimensional structure prediction and Model quality assessment The PDB structures of different variants of CD14 was developed through homology modeling using PHYRE2 server (Kelley et al , 2015). The three dimensional structures of both wild and mutant variant were visualized by PyMOL ( http://www.pymol.org/). Different moieties in mutant variant and domain for CD14 were also visualized through Pymol.

Protein-protein interaction network depiction
Proteins cannot function alone, rather works through a biochemical pathway. Hence it is important to understand the proteins which are interacting for an functional outcome.
Protein interaction was estimated through STRING 9.1. Interactions with score < 0.3 are considered as low confidence, scores ranging from 0.3 to 0.7 are classified as medium confidence and scores > 0.7 yield high confidence . The functional partners were depicted.

Prediction of post-translational modification sites in mutant CD14
In-silico analysis were employed for the detection of post translational modification sites of bubaline CD14. Protein dynamics and functioning greatly depends on these PTM. The protein phosphorylation was analyzed using a tool, specifically, Ser, Thr and Tyr residues which required for catalyzing its role. The prediction of glycation site was carriedout with NetGlycate 1.0 server (http://www.cbs.dtu.dk/services/NetGlycate/). The serine (Ser), threonine (Thr) and tyrosine (Tyr) residues with a score of >0.5 were depicted as to be glycated. Netphos 2.0 server ( http://www.cbs.dtu.dk/services/NetPhos/) was used for detection of protein phosphorylation sites.

Analysis of samples for clinical incidences for mastitis
The symptoms for clinical mastitis were udder inflammation, with the usual signs of heat, pain, redness and swelling, color alteration, and abnormal milk with flakes. The presence or absence of clinical mastitis was established by estimation of somatic cell count (SCC) and the California mastitis test (CMT).
Milk samples were collected aseptically in strip cup in Right fore (RF), Left fore (LF), Right hind (RH) and Left hind (LH) chambers for the detection of sub-clinical mastitis.

Estimation of California mastitis test
California mastitis test is also an important test for the detection of mastitis following the protocol as described by Dhakal (2006). Milk is collected in plastic paddle with four chambers. Equal amount of mastitis reagent was used in each cup and mixed gently . A score of 1 or more was considered as positive. The mastitis reagent was prepared initially by suspension of sodium lauryl sulphate (3g) in 100 ml of warm distilled water at pH 8.0.
The reagent was finally prepared by ixing with bromocresol purple (1: 10000)

Estimation of rennet coagulation time
The rennet coagulation time (RCT) of the milk was estimated with 0.2% Rennet solution ( Bittante , 2011).

Estimation of somatic cell count
Somatic cells in milk is a positive indicator for mastitis. 0.01 ml of milk was spreaded evenly over the slide. Staining of the slides were conducted with Newman Lampert stain (containing 1.2 g methylene blue, 54 ml 95% ethyl alcohol, 40 ml tetrachloroethane, 6 ml glacial acetic acid). Microscopic view of the slides reveal deep blue nuclei against light blue background. The total number of cells were counted to be total number of cells in 25 fields x working factor (Guha et al., 2012).

Statistical analysis
Identified SNPs were associated with the phenotypic traits in terms of mastitis specific marker as Somatic cell count. The effect of genotype on mastitis related traits were estimated through ANOVA. The model used for analysis was: Da) of buffalo sequence as compared to cattle (39679.96 Da). The buffalo CD14 peptide sequence was characterized by one extra strongly basic amino acid and had two polar amino acids less than that of cattle. The nucleotide sequence comparison of cattle with buffalo revealed 22 nucleotide substitutions, out of which eleven were synonymous codon without any amino acid change. The peptide sequence comparison of buffalo CD14 with that of cattle revealed eleven amino acid substitution at 14, 62, 131, 134, 139, 143, 154, 209, 235-236, 277, 337th position. In the derived peptide sequence of CD14 of buffalo, six leucine-rich repeats (LRR) were observed (Fig 1), whereas a cattle has ten LRR. Six LRR have been identified and depicted in different colours (Fig 1), amino acid site 114-138 as wheat, 168-176 as pale green, 192-218 as light blue, 244-256 (pale yellow), 266-283 (light pink), 296-309 (blue-white). Buffalo CD14 molecule was predicted to contain 17.2% leucine, which is similar to mouse (17.66%) and higher than that of human (15.5%).
Analysis of derived peptide sequence revealed 373 amino acids which is GPI anchored at C-terminus near 353 amino acid position, about 7 amino acid after hydrophobic tail. Four putative N-linked glycosylation sites were observed in bubaline CD14.
3.2. Comparison of CD14 with related genes.
Comparison of CD14 gene with other related genes containing leucine-rich repeats, viz. by String analysis (Fig 2). The result was confirmed through analysis of biochemical pathway that these genes are related in TLR signalling pathway through KEGG analysis (Fig 3). Moreover, it is evident that each molecule responsible for innate immunity, either receptor or immune mediator acts through a cascade of mechanisms with interaction with other. Mutations in CD14 affect the binding ability which in turn affects the biological potentiality. Mutational hotspots were detected in bubaline CD14 with 58 number of nonsynonymous SNP, out of which 18 were observed to be deleterious and 34 as thermodynamically unstable. Eight different variants of CD14 gene have been identified in all the four breeds of buffalo with 58 SNPs, revealing a high degree of polymorphism. AA ( genotypic frequency 0.468) as most frequent and AG and AH as the least frequent genotypes were identified as, with very low frequencies (0.0174) for both the genotypes.

Identification of CD14 variants with SNPs
High degree of variability was observed for alleles B (81.3%), C (97.0%), D (86.0%), E (97.4%) and F (85.8%) compared to A allele of amplified CD14 nucleotide of buffalo, which may be due to coding for leucine-rich repeats, that confers the recognition and binding ability for a wide range of pathogens. However, an absence of any change of amino acid at the sites of glycosylation confirms that the vital functions of the CD14 molecule remained unaltered.
Breed wise differences for allelic frequencies for CD14 gene were observed.
None of the breed of buffalo under current study was observed to be resistant to mastitis. However the differences in susceptibility was observed for Breeds of buffalo. Among the four breeds under consideration, Surti was observed to be most susceptible to mastitis, A number of SNPs were identified out of which were observed to be synonymous and nonsynonymous (Table 1)

Variant A versus variant C
Three non-synonymous SNPs were identified out of which T235 K was observed to be deleterious by Provean. I-mutant has predicted Y225C and T235K with reduced thermodynamic stability (Table 1). The 3D structure for CD14 variant C (Fig 6) Table 1. This high degree of mutations has an effect on its structure also as revealed by TM-align software. Structural alignment of CD14A with CD14 D has revealed a greater degree of variation, RMSD was observed to be 1.87. The structural alignment with the differing domain of CD14 had been highlighted in Fig 7. Sites for disulphide bond has been depicted as blue coloured sphere. Site for mutation L215P as red. L253R, D254L, L255H, S256N, N258C, S259P as yellow.
The domain of CD14 present at site L215P is leucine-rich repeat. It has already been reported that LRR in extracellular domain is responsible for the recognition of pathogens, hence any mutation at this site may lead to defects in coded protein causing impairment of the function of pathogen recognition.
The mutation of CD14 at site 253-256 codes for leucine-rich repeat, may cause a defect in pathogen recognition and the ultimate function may be impaired. It is to be kept in mind that CD14 is a pattern recognition receptor containing leucine-rich repeats (LRR). It is to be taken into consideration that whenever there is any mutation causing alteration of leucine moiety, the function of LRR is effected. The mutations at P273A, R277M, N280D, S282A, F283L site of CD14 gene of buffalo cause alteration of LRR domain.
The 3D structure depicting 3 D structural alignment of Variant A and variant D (Fig 7).

Variant A versus Variant E
The mutations identified in CD14 E are listed in Table 1. Seven synonymous mutations were observed out of which K296 L was predicted to be deleterious by Provean. 3D structure of Variant E of a CD14 molecule (Fig 8). The sites for disulphide bond was predicted in blue spheres. K296L was predicted in red. T209S in green, R277S yellow, V289L orange.
Results in I-mutant revealed a large decrease of thermodynamical stability for T209S, R277S, V289L, R337H. Hence the mutation at these sites will also lead to structural instability and ultimately decreased function.
T209S, R277S moieties are present in LRR region, which in turn is present for pathogen recognition. V289L moiety is present in leucine zipper site. Leucine zipper is responsible for dimerization and binding with TLR4, MD2 and its functioning.R337H is the site for the alpha helix.
Mutation of CD14 buffalo at the site K296L indicates an alteration in the major function of some of the domain as LRR, domain linker, and leucine zipper. This zipper site is very important for dimerization and pathogen binding.

Variant A versus Variant F
This high degree of mutations has an effect on its structure also as revealed by TM-align software. Deleterious mutations as revealed were L255P, S256N, H257P, S259L, L260P, C270K, W272H (Table 1). Structural alignment of CD14A with CD14 F has revealed a greater degree of variation, RMSD was observed to be 1.81. The structural alignment has been observed in Fig 9. The site for mutation as L255P, S256N, H27P had been depicted as red sphere.S259L and L260P had been depicted in magenta. C270K as cyan. W272H as grey. 255 to 257 aa of CD14 Comprises for LRR. Both C270K, W272H sites comprise of LRR domain. Site C270K encodes for a di-sulphide bond. Alteration in amino acid moiety at site 270, impairs di-sulphide bonding, which affects protein folding. Impaired protein folding, in turn, effects its function. Much structural dissimilarity has been observed as RMSD 1.81.

Variant A versus G
Seven non-synonymous mutations were observed out of which K296 L was predicted to be deleterious by Provean (Table 1). Structural alignment revealed differences with RMSD was observed to be 0.22 (Fig 10).Variant A as green, G by Cyan. The sites for disulfide bond was predicted in blue spheres. K296L was predicted in red.
Results in I-mutant revealed a large decrease of thermodynamical stability for T209S, R277S, V289L, R337H (Table 1). Hence the mutation at these sites will also lead to structural instability and ultimately decreased function.
T209S, R277S moieties are present in LRR region, which in turn is present for pathogen recognition. V289L moiety is present in leucine zipper site. Leucine zipper is responsible for dimerization and binding with TLR4, MD2 and its functioning. R337H is the site for the alpha helix.
Mutation of CD14 buffalo at the site K296L indicates an alteration in the major function of some of the domain as LRR, domain linker, and leucine zipper. This zipper site is very important for dimerization and pathogen binding. Structural dissimilarity with RMSD 0.22 has also been observed.
However, the mutations were observed to be thermodynamically stable as revealed by Imutant and none of the mutations were observed to be deleterious.
The alignment of all the variants of CD14 had been depicted in Fig 11, which clearly reveals the mutations, both synonymous and non-synonymous for bubaline CD14.

Association of SNPs with phenotypic traits related to mastitis
Mutations in CD14 effect the binding ability which in turn affects the biological potentiality. Mutational hotspots were detected in bubaline CD14 with 58 number of nonsynonymous SNP, out of which 18 were observed to be deleterious and 34 as thermodynamically unstable.
Eight variants of CD14 were identified, where variant A was observed to be the wild type.
Phenotypic association with different indicator traits for mastitis were identified and listed in

Discussion
CD14 is an important molecule conferring innate immunity. CD 14 is a pattern recognition receptor-rich in leucine-rich repeat (LRR) with N-terminal hydrophobic pocket or groove responsible for binding with pathogens. It can bind with a wide range of substances as LPS, lipotechoic acid, mannuronic acid, which are an integral part of pathogens and releases a series of cytokines, which ultimately destroy the pathogen. Extensive variability among CD14 has been observed between the ruminant species (Pal et al., 2018). Most of the variations have been observed in LRR region, LPS binding and LPS recognition site. Since LRR region is the site for pathogen recognition, the variability among the LRR leads to recognition of the wide range of pathogens.
CD14 acts through TLR 4 signalling pathway and interacts with a series of genes as revealed by String analysis and KEGG analysis. CD14 shRNA was observed to have effect on gene expression of TLR4 signal pathway in monocyte and macrophages of buffalo (Li et al., 2014).
Six LRR had been identified in bubaline CD14. CD 14 is basically a receptor molecule with the N-terminal pocket having the receptor binding groove. There is wide variability in CD14 variants with changes in LPS binding site and LRR site.
In our current study, we have detected wide variability as eight variants of CD14. Most of the variants were observed in six LRR regions. Bubaline CD14 with nucleotide sequence from 587th to 854th position of the second exon of buffalo (coding for amino acid sequence 197th to 285th) was observed to be highly variable among the variants of bubaline CD14 and designated as Mutational Hotspot. This fragment codes for the leucinerich repeats (LRR) starting from 195-218, 220-245, 247-270, 273-285th codon from the start codon. Leucine rich repeat (LRR) are important domain of CD14 responsible for pathogen binding and pathogen recognition. CD14 is basically a receptor molecule with the ability to bind with a wide range of ligands as lipopolysaccharides (from gram negative bacteria),mannuronic acid (from Pseudomonas spp.) lipoarabinomammans (from Mycobacterium spp.), peptidoglycans (from Staphylococcus aureus). Hence the CD14 receptor site must be flexible enough to accommodate these ligands. Within the leucinerich repeats, the particular leucine moiety was observed to be almost unchanged and the variations were observed for other amino acids.
A total of 59 non-synonymous SNPs were identified, which exceeds the synonymous substitutions indicating positive selection. For nine SNPs, the wild-type amino acid had been substituted by different sets of amino acids corresponding to different variants of CD14. 24 sites were observed to be only thermodynamically unstable as revealed by Imutant software, seven sites as only deleterious by Provean software. Eleven sites were identified as both of unstable and deleterious mutations.
In our earlier study, differences was observed among the CD14 molecule of different breeds of buffalo. In Bhadawari breed, only three patterns were identified; AA genotype having the highest frequency (0.818), whereas the lowest frequency was identified as AB.
In Mehsana, the most frequent genotype was observed to be AA genotype (0.465), whereas AB (0.056) and AG (0.056) were found to be the least frequent genotypes. Murrah breed, the most frequent genotype was observed to be AA (0.596), whereas AB (0.105) and AD (0.105) were the least frequent genotypes. In contrast to other buffalo breeds, in Surti breed, the highest frequency was observed for AD genotype, and AA (0.071) and AH (0.071) genotypes were the least frequent. Since the frequency of homozygote (AA) was less compared to that of heterozygote(AD), a higher degree of heterozygosity among the population of Surti breed of buffalo was predicted. As Bhadawari possess the highest frequency homozygote (AA), the population was predicted to be more homozygous.
The frequency of A allele ranged from 0.909 (Bhadawari) to 0.5 (Surti). The allelic frequencies for both G and H were found to be too low (0.008) to be regarded as polymorphic (Pal et al., 2014).
There was no change at the position of N-linked glycosylation sites of buffalo CD14 predicted peptide sequence. N-linked glycosylation is essential for the polarity and solubility of protein or protein folding. It determines if the CD14 molecule will be available either in membranous and soluble form.
We had reported for the first time the polymorphism with 52 SNPs identified at the coding region of CD14 gene of buffalo. In our earlier studies, we could detect polymorphism of CD14 gene in buffalo (Pal et al., 2014), cattle (Pal et al., 2011) and goat (Pal et al.,2009, Pal et al., 2013 with mutational hot spot detected. Variations were also observed for CD14 among different ruminant species.
In the current study, we could study the molecular mechanism of a depressed phenotype of a mutant variant of CD14, which is reported for the first time. Two basic mechanisms were responsible , the first is the deleterious nature of the mutation and second is the decreased thermodynamic stability of the mutant CD14. The mutational hot spot was detected and variant CD14 D and F were mostly affected as they contain the large number of identified non-synonymous mutation, which was deleterious in nature and was of reduced thermodynamic stability. As evident from the study that most of the mutations The second factor as thermodynamical stability is also equally important. Decreased thermodynamic stability causes reduced half life of the biologically active CD14 molecule.
Thus the variant of CD14 with more number of non-synonymous mutations with deleterious or unstable mutations, will definitely has reduced phenotypic value.
Considering the polymorphic nature of the gene, a critical association study encompassing all regions of the CD14 gene in a large population might reveal its functional importance in Buffalo. Thus in this study, we had conducted association study of variants of CD14 gene with the traits for udder health or mastitis as somatic cell count, California mastitis test and rennet coagulation time and significant differences were observed.
From the in silico study and also from experimental validations, Variant A was observed to be the wild type. Association study of CD14 gene with the indicator traits for mastitis revealed significant differences and confirmed the above findings. Variant H was also observed to be the resistant variant. Out of three non-synonymous SNPs identified, all were observed to be neutral. CD14 variants as D and F were observed to be highly deleterious and have high somatic cell count, rennet coagulation time and strongly positive for California mastitis test and Variant G as deleterious to some extent.
Genetic polymorphism of CD14 gene and its polymorphism with disease genetics (mastitis and others) had been studied by a number of workers. Report indicated that CD14 SNPs regulate the innate immune response (Liu et al., 2012).
However, most of the studies were in the promoter region of CD14 gene. CD14 gene variants at C-260T were observed to be associated with coronary heart disease risk in human (Dai et al., 2016 ). CD14 gene polymorphism has also been associated with a variety of diseases as cancer (Wang et al., 2014), sarcoidosis (Fridlender et al., 2010), pulmonary tuberculosis ( -Taraco et al, 2007), asthma (Wang et al., 2005), ischemic heart disease (Zhang et al., 2009), mastitis (Selvan et al., 2014, Baro, D. 2012, alcoholic liver disease and chronic hepatitis C infection (Meiler et al., 2005), coronary artery disease (Li et al., 2015). CD14 has been identified as genetic risk factor for restenosis after percutaneous coronary intervention (Zholdybayeva et al., 2016 ). C-159T SNP of the CD14 gene promoter was observed to have effect on lung dysfunction in smokers (Zhou et al., 2009).

Rosas
Characterization of bovine CD14 and association studies were conducted to explore the surface expression on monocytes and polymorphonuclear neutrophils, an indicator of immune status (Ibeagha-Awemu, 2008). The effect of CD14 in inflammatory responses during mastitis had been depicted (De Schepper et al., 2008;Buitenhuis, et al., 2011).
Reports indicate association of CD14 SNPs with SCC Ibeagha-Awemul et al. 2008). SNPs in the CD14 Promoter decreased the ability of Sp Protein Binding and increases Transcriptional Activity ( LeVan et al., 2001). Meta-analysis was employed to study the association of CD14 with cancer risk (Wang et al.,2014), coronary heart disease risk (Pu et al., 2013) inflammatory bowel disease  and coronary artery disease (Li et al., 2015 ).
It is understood from the current study that the mutation in CD14 gene causes unstable and deleterious mutations, which ultimately causes depressed function as immunity leading to the occurrence of diseases, mastitis in the current case. The variability identified in the CD14 gene sequence and SNPs and variants may be identified at the day old stage and employed for marker-assisted selected to retain the wild-type for the production of a future disease-free stock of the farm. Wild-type CD14 identified may be useful for recombinant CD14 production for therapeutic use.   Structural alignment for CD14 A (green)with CD14F (orange) of Bubalus bubalis Figure 10 Alignment report for CD14 A with CD14 G of Bubalus bubalis