Nucleotide sequence variants, gene expression and serum profile of immune and antioxidant markers associated with bacterial diarrhea susceptibility in Barki lambs
Authors: Darwish Asmaa , Ebissy Eman , Hafez Amani , Ateya Ahmed , El-Sayed Ahmed 
Published: October 2024

Abstract
Background
Despite the fact that diarrhea is more accurately described as a clinical symptom than a disease. Diarrhea is one of the most important issues in ovine medicine, particularly in lambs, and because of high morbidity and mortality rate, sluggish growth performance, and veterinary costs, it is believed to be a major source of economic loss. Salmonella and enterotoxigenic Escherichia coli are the most common and commercially significant agents responsible for diarrhea.

Objective
The objective of this study was to monitor the nucleotide sequence variations, gene expression, serum inflammatory and oxidative stress biomarkers in diarrheic lambs. Another aim was to identify different pathotypes and virulence genes of Salmonella and E. coli causing diarrhea.

Methodology
Blood samples were taken from 50 Barki who were diarrheal and 50 who appeared to be healthy, and then divided in 3 portions, with EDTA added to the first part for CBC, DNA and RNA extraction. The second sample received 5000 I.U. of heparin calcium, and a clean plain tube was used for the third component. The second and third sections were centrifuged to extract serum and plasma until the biochemical and immunological analysis was completed. Fecal samples were collected for bacteriological examination, and the bacteria were identified by PCR analysis. PCR-DNA sequencing was conducted for immune (SELL, JAK2, SLC11A1, IL10, FEZF1, NCF4, LITAF, SBD2, NFKB, TNF-α, IL1B, IL6, LGALS, and CATH1), antioxidant (SOD1, CAT, GPX1, GST, Nrf2, Keap1, HMOX1, and NQO1), and GIT health (CALB1, GT, and MUC2) genes in healthy and diarrheic lambs.

Results
Virulent genetic markers of pathogenic characteristics of E. coli (astA, Vt2e (Stx2e), CFA/I, groES and luxS) and Salmonella (invA, SopB, bcfC and avrA) were detected in all diarrheic lambs. PCR-DNA sequencing of immune, antioxidant and intestinal health genes found eleven single nucleotide polymorphisms (SNPs) linked to either diarrhea resistance or susceptibility in Barki lambs. Transcript levels of immune, antioxidant, and GIT health (CALB1, GT, and MUC2) genes varied between healthy and diarrheic lambs. Nucleotide sequence variation of the genes under inquiry between reference sequences in GenBank and those of the animals under investigation verified all identified SNPs. Significant (P = 0.001) erythrocytosis, neutrophilic leukocytosis, with lymphocytopenia were observed in diarrheic lambs. Significant (P = 0.001) increases in serum IL-1α, IL-1β, IL-6, TNF-α (90.5 ± 1.7, 101.8 ± 1.7, 72.3 ± 6.6, 71.26 ± 4.89 Pg/ml, respectively), serum Fb, Cp, Hp, SAA (230.7 ± 12.4 mg/dl, 6.5 ± 0.07 mg/dl, 2.5 ± 0.09 g/dl, 7.4 ± 0.4 mg/L, respectively), free radicals (MDA, NO), cortisol (6.91 ± 0.18 μg/dl) and growth hormone, with significant (P = 0.001) decreases in serum IL-10 (81.71 ± 1.05 Pg/ml), antioxidants (CAT, GPx), insulin, triiodothyronine (T3) and thyroxine (T4) in diarrheic lambs.

Conclusions
The study's findings provided credence to the theory that marker-assisted selection (MAS) could be used to predict and prevent diarrhea in Barki sheep by selecting lambs based on SNPs in genes linked to inflammation, antioxidants, and intestinal health. In order to establish an efficient management protocol and determine the most susceptible risk period for disease occurrence, gene expression profiles of the genes under investigation, pro-inflammatory cytokines and acute phase proteins may also be utilized as proxy biomarkers for lamb enteritis.