Mutations in CD14 gene may have significant effect on mastitis resistance/susceptibility of buffalo.

Background: CD14 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 from mastitis may be because of depressed milk production, deterioration of milk quality, cost for health management as treatment and vaccination, milk disposal due to antibiotic residues, advanced culling owing to permanent damage to udder, addition labour requirement. There are certain other limitations as evolvement of antibiotic resistant bacteria, vaccinations are not cent percent because of evolvement of multistain organism and frequent mutation, by genetic shift and genetic drift. Other constraint are milk disposal owing 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 CD14 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.

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,

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 (3 g) in 100 ml of warm distilled water at pH 8.0. 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.

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. Breed wise differences for allelic frequencies for CD14 gene were observed.

Identification of CD14 variants with SNPs
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, 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) 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. 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.   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.

Discussion
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). 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.  Wild type CD14 A of Bubalus bubalis 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