Pathogenic E. coli Virulence Traits Regarding Quantitative Cytokine Expression in Subclinical Mastitic Cows

The present study aimed to determine the molecular dynamics of E. coli serotypes regarding the diagnostic significance of typing virulence genes, in addition to the description of the intensity of the triggered cytokines’ response by the invading pathogen to determine the severity and/or onset of the infection. The survey included 300 cows in 10 dairy farms in Egypt, characterized by low SCC bulk milk tank (≤ 4×10 5/ ml) with CMT recorded grades of (+) & (++); and the influx of neutrophils from the blood to the udder. Environmental microorganisms were isolated from 16.66% of milk samples, noting that E. coli isolates were 90% (91 isolates) of them, however, other types of bacteria (10%) were also recorded. Ten serogroups were typed; O1, O8, O20, O25, O78, O127, O146, O153, O157 & O159, with prevalence rated 9.9%, 12.1%, 11%, 13.2%, 12.1%, 9.9%, 16.5%, 8.8%, and 6.5%, respectively. Furthermore, the isolates were morphologically identified by Transmission Electron Microscope (TEM), andbiochemically characterized. The enteropathogenicity in infant mice, hemolysin patterns, Shiga-like cytotoxicity in Vero cell line, and CD50 were all studied and determined. PCR fragments of molecular sizes of 180 bp, 255 bp, 384 bp, and 534 bp distinctive for stx1, stx2, eaeA, and hlyA genes, respectively, were amplified, sequenced, and then aligned against similar GenBank records to confirm the identities. To study the influences of proteomics [9 dominant bands; 106 to109, 80 to 83, 54 to 56, 41, 37, 32, 26-25, 22, and 7.8 kDa] on bacterial pathogenicity and host immunity mRNAs expression of IL-4 and IL-10 cytokines in milk, the somatic cells were quantified, then normalized with housekeeping gene (βActin), finally expressed as folds of induction. qRT-PCR revealed up regulation of target cytokine genes in response to E. coli infections; the means of the folds’ increase were calculated as 2.712 ± 5.178 and 11.231 ± 4.146 for IL4 and IL10, respectively (Means St. Dev. 2 ̄(∆∆ст). The present study not only indicated diverse incidence rates of the four molecular characterizing genetic/phenotypic traits, but also, the tendency for clonality which was proved by the proteomics’ variance between the pairs of the same clone of some E. coli serotypes; O1, O8, O127, O146, O153, O157 and O159, where α & β hemolysin expression were noted. Consequently, it was reflected on the udders initiating inflammatory responses. The onset and intensity of the up regulated immune cascade; especially IL4 & IL10, is an invasive pathovar dependent on the triggered cellular subset which mediated the immune responses. The adverse effects exerted by the secretory toxins on dairy herds as well as the consumers of byproducts are significant but undervalued, hence,the subclinical mastitis diagnosis is still dependent on the bacterial colonies’ counts per sample not the bacterial secretory excretory toxins nor the hosts’ cytokines response’s quantification. In conclusion: The virulence of E. coli serotypes found dependent on the less characterized proteomics than Stx1 and Stx2, yet their role remains to be discovered.