ACCEPTED AUTHOR VERSION OF THE MANUSCRIPT: Mining for the association of bovine mastitis linked genes to pathological signatures and Pathways

Background: Bovine mastitis is a common infectious disease with a serious threat to the dairy industry and public health. Mastitis is a polygenetic trait under the control of many genes. In the current study, our research attempted to address the role of mastitis-associated genes in various signalings including parasitic, viral, cancer and fungal diseases by using online bioinformatics software. Methods: We selected mastitis-associated genes from already published data and using online bioinformatics tools including DAVID and String classify the pathological role of relevant genes. A Venn diagram was used to show the status of overlapping genes among different biological function processes. Result: This study revealed that the genes gathered in published resources of mastitis were significantly correlated with Influenza A, Chagas disease, Leishmaniasis, Toxoplasmosis, Tuberculosis, Cancer signaling, Hepatitis B, Type I &II diabetes mellitus and Prion diseases biological pathways. Based on our findings, we concluded that mastitis-linked genes could be used as markers for many other diseases. Moreover, the Bioinformatics tools applied in the current study might be helpful in screening the genes involved in one disease and their association with other diseases as well.

Key words: bovine mastitis, bioinformatics tools, biological signalling, genetic markers Bovine mastitis, the inflammation of udder tissue, is associated with physical and chemical changes in milk and has been classified as an important disease in the dairy cow industry (Chen et al. 2019). Due to high culling rates, milk losses, increased treatment cost, labor costs, and negative influences on human health, bovine mastitis is considered a serious threat to the dairy industry (Gomes and Henriques, 2016). Furthermore, mastitis is an immunity and inflammatory-related disease controlled by several genes (Thompson-Crispi et al. 2014;Griesbeck-Zilch et al. 2009). The susceptibility of dairy animals to udder infection is heritable (Tolone et al., 2016); however, limited knowledge about the complex physiological and cellular processes that occur in the mammary gland in response to pathogens is available. Thus, today, researchers are more interested in identifying the genetic markers for disease susceptibility and selection of mastitis-resistant breeds (Spaan et al. 2013).
Transcriptome analysis is considered a powerful tool to pinpoint several aspects of mastitis including the molecular mechanism and disease pathology (Wiggans et al. 2017).
On the other hand, transcriptomic screening is also performed to identify the genes involved in the disease development, interaction of these genes during disease pathogenesis and their regulated biological signaling linked with the progression of infection (Jensen et al. 2013).
Identifying the commonalities between mastitis and other diseases will provide clues for the therapeutic targets for mastitis and other diseases as well. Several RNA-Seq and microarray datasets are available online and utilizing these datasets for genetic analysis will help us to screen the genetic markers and related signalings. Moreover, full knowledge regarding protein function and their specific interaction during disease progression is warranted (Lee et al. 2007). In this regard, String online-based software is widely in practice to establish the interaction of proteins with each other during disease progression (Szklarczyk et al. 2015).
In the current research we utilized some online software such as String, DAVID and GO for the mastitis-associated genes and noticed that these molecular signatures are also involved in the regulation of other parasitic, bacterial and viral related diseases signalling. Thus the current findings provide a baseline for already reported genes in mastitis and their rules in the regulation of other disease signalings.

Data collection and Gene's selection
The genes significantly (P< 0.05> regulated (down and up-regulated) in bovine mastitis reported by previous studies were selected for the current study (Table 1). In addition, the inflammation and immunity-related genes documented in mastitis were selected from metaanalysis studies (Sharifi et al. 2018;Sharifi et al. 2018;Younis et al. 2016). Moreover, the above-selected genes were analyzed for their association with other diseases using already well-established online software. All published studies that have discussed the mastitisassociated genes were screened through authentic sources including PubMed, ScienceDirect,

2009
). The association of signaling pathway was counted significant for which P-value <0.05.
The String is a web-based tool that is used for the mining of protein-protein interaction (Szklarczyk et al. 2015). The list of the mastitis-associated genes selected from already published studies was uploaded on String and run to analyze protein-protein interaction with the highest confidence level (0.9). A maximum number of interactions to show were set up with none (1 st and second shell) (Szklarczyk et al. 2015).

Kyoto encyclopedia of genes and genomes (KEGG) pathways of selected genes by using a database for annotation, visualization, and integrated discovery (DAVID) bioinformatics resources
Our findings revealed that bovine mastitis-linked genes significantly alter genes signature involved in several pathological conditions signaling as mentioned in Figure 1. The genes involved in different pathways have been provided in Table 2. In addition, our results showed that the Bovine mastitis-associated genes significantly regulated Influenza A, Chagas disease, Toxoplasmosis, Leishmaniasis and Pertussis linked signaling. The level of significance for each signaling regulated by mastitis-associated genes is summarized in Table   2. Consistently, our result showed that NFKBIA, JAK2, MAPK, STATs, RELA, MYD88, TNF, FOS, TLR2, TLR4, AKT1, NFKB1, IL1A, IL1B and JUN were involved in the majority of other disease signalings ( Table 2).

Association of bovine mastitis associated genes with cancer signalings
We have also mined to confirm that mastitis-linked genes were associated with cancer signalings ( Figure 2). Up to our expectations, bovine mastitis-linked genes regulate signaling pathways known to be important in cancers like prostate, pancreatic, colorectal cancer, melanoma, etc. The genes involved in different types of cancer pathways have been summarized in Table 3.

Protein-protein interaction networks of differentially expressed genes
We also visualized the protein-protein network using String. The String analysis showed that the mastitis linked-genes network is merged at TLR signaling via MYD88 or concentrating on chemokine signaling. Another branch of signaling pathways is originating from JAK2/Stat1 or MAP kinases ( Figure 3).
The list of genes involved in immune response, inflammatory signalling, LPS signalling and innate immunity has been provided in Figure 4. We further confirm the over-lapping of several signaling pathways that share commonality with mastitis, as demonstrated in the Venn diagram. Here we observed that all the signaling pathways share a degree of similarity; however, none of the genes was common in all pathways ( Figure 5).

Discussion
Bovine mastitis is a complex trait caused by many agents, including bacteria, fungi, viruses, and physical factors. On the other hand mastitis is polygenetic traits and has controlled by many genes. Due to the advancement in the area of molecular and genomic technology, a major increase in biological and quality of data has occurred. Recently many genes have been screened by various useful bioinformatics methods such as GWAS, RNAseq, and microarray screening, which were associated with the development or resistance of bovine mastitis. In current research by using an online bioinformatics web, we find out that the genes involved in mastitis also play a role in other diseases signalling such as Leishmaniasis, Chagas disease, Legionellosis, Salmonellosis, Toxoplasmosis regulating signalling etc.
The wealth of bioinformatics applications in the field of life sciences, including proteinprotein interaction and biological pathways, has changed the paradigm of research and utilization of knowledge [Sousa et al. 2016]. Interestingly, our research showed that various diseases associated signaling, including different types of cancer pathways were regulated by mastitis-associated genes (NFKBIA, RELA, ATK1, MYD88 and PIK3CA). Several genes, such as ATK1, MET, and PIK3CA, were found to be involved in cancer (Scheffler et al. 2015). According to Fresno vara and his team's findings, AKT1 interacts with the lipid kinase family and gets inter into the inner membrane where it has been activated by PDK1 and PDK2. The activated AKT1 alters the functions of many substrates involved in cell survival cell cycle progression and cellular growth. That is why any alteration in the ATK1 gene may reinforce the process of cancer (FresnoVara et al. 2004). Similarly, NF-kappa-B signaling has vital role in various human disease processes, including inflammatory infections and cancers, while RELA is the key member of the NF-kappa-B complex. The negative regulation of RELA is significantly associated with endometrial cancer (Lu et al. 2015). These published studies verified the mastitis associated genes role in cancer, which is documented to show a link with cancer as well. The reason might be their correlation with apoptotic processes and immune modulation, which are the main parameter for cancer development.
In the current analysis, we noticed that mastitis-associated genes also play a major role in various bacteriological (Tuberculosis, Salmonellosis, Legionellosis), parasitic (Toxoplasmosis, Chagas disease, Leishmaniasis) and viral diseases (Influenza A, Herpes simplex infection) pathways. The possible explanation for this might be the involvement of some major processes such as apoptosis, inflammatory changes, and immune depression during mastitis, as shown in Figure 4. These are common phenomena, which can be observed in many diseases. However, the bioinformatics tools we used in the current research will be helpful to screen/predict one gene in many signaling pathways. The bioinformatics analyses revealed that the reported genes linked to mastitis have multiple roles in various diseases, which open a new gateway for future research. Although our research provides the basic information for mastitis-associated genes engagement in many biological signaling, however, it still needs practical functional validation by using animal mouse models or cell cultures.