In the Sheep Breeding and Research Institute, an official organization affiliated with the Turkish Ministry of Agriculture and Forestry, which has an 80-year history in sheep farming and although it boasts considerable professional resources and personnel, the prevalence of neonatal diarrhea has been observed to reach concerning levels, such as 25.5%. As far as the authors are aware, no study has investigated the genetic factors contributing to resistance or susceptibility to neonatal diarrheas among farm animals at the molecular level. Nonetheless, there are noteworthy observations suggesting that despite harboring pathogens, many neonatal animals remain asymptomatic of diarrhea. For instance, in their case-control examination, Caffarena et al.31 found that while their pen mates developed diarrhea (case groups; n = 264), the control groups (n = 271) consisted of non-diarrheic calves testing positive for Cryptosporidium spp. (26.9%), bovine astrovirus (22.4%), Rotavirus (11.1%), Salmonella enterica (2.6%), or Escherichia coli (1.8%). In another investigation, Abdou et al.30 discovered that the prevalence of Group A rotaviruses in non-diarrheic cattle, sheep, and goats was 2.9%, 1.3%, and 2.8%, respectively. Zhong et al.32 provided a possibly more dramatic example when they showed that the relative prevalence of potentially pathogenic bacteria, like Bacteroides, in healthy lambs was more than double that in diarrhoeic pen mates using 16S rRNA sequencing of the gut microbiota .
These findings support the authors' conclusions, highlighting that variations in individuals' genetic backgrounds concerning innate immunity, coupled with other environmental factors like climatic conditions, management practices, and colostrum intake, significantly influence disease outcomes.
Our investigation delves into the intricate genetic landscape that underpins immunity, with a focus on key genes identified through dual GWAS. The SLCO2A1 gene encodes a crucial prostaglandin transporter essential for the cellular uptake of Prostaglandins (PGs)33. These PGs play pivotal roles in chronic inflammation by amplifying cytokine effects on inflammatory cells and regulating gene expression, thereby promoting autoimmune inflammation through the differentiation and proliferation of Th1 and Th17 cells34. Additionally, SLCO2A1 facilitates prostaglandin E2 (PGE2) secretion by macrophages, modulating neutrophil clearance from inflammatory sites35.
The N-acetylated alpha-linked acidic dipeptidase-like 2 (NAALADL2) gene is implicated in various molecular functions and diseases, including intestinal Behçet’s disease36, inflammation, apoptosis, and cardiovascular pathology in Kawasaki disease37. Moreover, NAALADL2 plays a role in Epstein–Barr virus infection, which is associated with disorders such as multiple sclerosis and lupus38.
The GOLM2 gene, also referred to as CASC4 and identified as a potential cancer susceptibility gene39, has been linked to reduced overall survival rates in ovarian cancer40. Recent studies conducted during the pandemic have highlighted the GOLM gene family, including GOLM1 and GOLM2, identifying them as potential causal plasma proteins associated with SARS-CoV-2 and suggesting their involvement in increased susceptibility to COVID-19 infection41,42.
The SDK2 gene is associated with circulating serum soluble gp130 (sgp130), an antagonist of the inflammatory response in atherosclerosis mediated by interleukin 643. The role of SDK2 in immunity is indicated by its altered stage-specific expression during HIV-1 infection 44. Additionally, it has been demonstrated that SDK2 is differentially expressed upon experimental infestation by the protozoan Amyloodinium ocellatum in European seabass tissues45.
Complement receptor (CR) type 2 (CR2/CD21) exhibits expression primarily during both the immature and mature stages of B cell maturation. In conjunction with CD19, CR2 plays a pivotal role in enhancing mature B cell responses to foreign antigens46. Research indicates that deficiency in CR2 could alter antibody production and lead to the deposition of immune complexes, thereby influencing the humoral immune response47. Moreover, CR2 has been linked to the recognition of foreign DNA during host-immune responses, highlighting its role in immune surveillance and pathogen response48.
The CEP350 gene has been implicated in the transcriptional response of macrophages triggered by Mycobacterium tuberculosis49 and displays distinct expression patterns in erythema nodosum leprosum50. Additionally, CEP350 proteins interact with viral proteins of SARS-CoV-2 and exhibit altered expression upon infection51.
CTNND2 undergoes downregulation in the later stages of Cytomegalovirus infection in humans52. Notably, both CEP350 and CTNND2 genes are similarly implicated in the macrophage transcriptional response to Mycobacterium tuberculosis49.The YDJC gene plays a pivotal role in autoimmune diseases by interacting with UBE2L3 promoters and co-bound transcription factors53. Furthermore, the involvement of the YDJC gene has been observed in other diseases such as Coeliac Disease54, idiopathic inflammatory myopathies55, and autophagy-dependent intracellular pathogen defense56.
The significant role of the SLC superfamily in drug transportation is widely recognized. As a member of this family, SLC22A8 encodes the protein OAT3, influx transporter protein, which primarily facilitates the uptake of substrates into cells57. The SLC22A8 gene is also implicated in various biological processes, including survival and immune infiltration in clear cell renal cell carcinoma (ccRCC), a prevalent form of renal malignancy worldwide58. Additionally, prior evidence suggests a link between acute neuroinflammation and alterations in the levels of prostaglandin E2, a substrate of SLC22A8. This association is accompanied by changes in SLC22A8 levels59.
Network analysis using the KEGG database identified four main pathways. Among these, the B cell receptor (BCR) signaling pathway, the complement and coagulation cascade pathways, and the hematopoietic cell lineage pathway emerged as directly associated with immunity, playing critical roles in immune cell activation, regulation, and differentiation.
The B cell receptor (BCR) signaling pathway serves as a pivotal mechanism in orchestrating the immune response upon antigen encounter. Upon binding of antigens to the BCR, a cascade of events is initiated, beginning with the clustering of BCRs and culminating in the activation of genes crucial for B-cell function and differentiation60. This pathway is characterized by the rapid phosphorylation of key molecules such as Igα/β and intracellular signaling proteins like lyn, syk, and BLNK61. Such signaling triggers essential processes including B-cell proliferation, differentiation, and antibody production. Co-stimulatory signals provided by molecules like CD40 further potentiate the response, particularly in the proliferation of antigen-stimulated B cells. Regulation of the BCR signaling pathway is finely tuned, with molecules such as CD22 exerting inhibitory effects on IgG1 B-cell receptor signaling, thereby impacting B-cell development62. Continuous signaling through CD22 is crucial for the normal development and survival of B cells within lymphoid tissues (Akatsu et al., 2022). Additionally, B cells play a pivotal role in antigen presentation to CD4 + T cells, facilitating B-cell help and subsequent differentiation into memory or plasma cells (Kim et al., 2006). Furthermore, this pathway is implicated in the production of IL-10 by B-1 cells, thereby contributing to the regulation of immune responses63.
Complement, an integral aspect of the innate and acquired immune system, operates through a series of proteolytic cascades initiated upon encountering microorganisms64. Activation of complement triggers potent proteolytic cascades, leading to opsonization, pathogen lysis, and the induction of inflammatory responses via proinflammatory molecule production65. However, dysregulation of the complement system can occur in various diseases, causing the tightly controlled proteolytic cascade to become harmful66. The complement system serves a pivotal role in innate immunity, bridging it with acquired immunity67. It defends against foreign pathogens by generating complement fragments that facilitate opsonization, chemotaxis, leukocyte activation, and cytolysis68. Blood coagulation, another intricate cascade, involves coagulation proteins as its core components, orchestrating a series of reactions that culminate in the conversion of soluble fibrinogen to insoluble fibrin clots. At the heart of this process lies thrombin, which binds to fibrin during clot formation, thereby enhancing clot strength and stability69,70. Protease-activated receptors, such as those activated by thrombin, belong to the family of G protein-coupled receptors, serving as mediators of innate immunity responses71. Moreover, the kallikrein-kinin system represents an endogenous metabolic cascade, with its activation leading to the release of vasoactive kinins, including bradykinin-related peptides. This intricate system involves precursors known as kininogens and primarily tissue and plasma kallikreins. The pharmacologically active kinins, often regarded as either proinflammatory or cardioprotective, are implicated in numerous physiological and pathological processes72.
The hematopoietic cell lineage pathway governs the intricate process of blood cell development, beginning with hematopoietic stem cells (HSCs) endowed with the capacity for self-renewal or differentiation into two primary progenitors: common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). CLPs give rise to lymphoid lineage cells, including natural killer (NK) cells, T lymphocytes, and B lymphocytes, while CMPs differentiate into myeloid lineage cells, comprising various leukocytes, erythrocytes (red blood cells), and megakaryocytes responsible for platelet production, crucial for hemostasis. Differentiating cells express distinct surface markers indicative of their stage and lineage, facilitating their identification and characterization73,74.
In addition, network analysis revealed the bile secretion pathway that could have an indirect association with immunity. The bile secretion pathway orchestrates a sophisticated interplay of molecular determinants and signaling cascades crucial for maintaining liver function and whole-body homeostasis. Regulation of bile formation and secretion involves a complex network of factors, both in animal models and humans75. Central to this process are the membrane transport systems within hepatocytes and cholangiocytes, along with the structural integrity of the biliary tree. Hepatocytes, comprising the majority of liver cells, are responsible for generating primary bile within their canaliculi76. Moreover, beyond their traditional role in aiding lipid digestion, bile acids (BAs) function as vital signaling molecules, influencing lipid and glucose metabolism and modulating the gut microbiota composition77. Hence, one could speculate that bile formation and/or the composition of gut microbiota influenced by bile secretion may impact the pathogenesis of intestinal pathogens responsible for neonatal diarrhea.
In conclusion, the findings highlight a significant concern regarding the prevalence of neonatal diarrhea and its associated economic losses. Addressing neonatal diarrhea in sheep farming is crucial not only for the welfare of the animals, the sustainability, and productivity of the industry but also for public health and food safety. Our dual GWAS results demonstrate that marker-assisted selection could be a possible tool for improving genetic resistance to neonatal diarrhea, serving as a complementary approach to combating this complex disease. Further research into neonatal diarrhea's underlying causes and risk factors is warranted to develop targeted and evidence-based interventions.