VIRULENCE GENES PROFILES OF ESCHERICHIA COLI ISOLATES AND THEIR EFFECT ON QUANTITATIVE INTERLEUKINS EXPRESSION IN SUBCLINICAL MASTITIC COWS | ||||
| Journal of the Egyptian Veterinary Medical Association | ||||
| Volume 78, Issue 4, December 2018, Page 751-778 PDF (1.79 MB) | ||||
| Document Type: Original Research Articles | ||||
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| Abstract | ||||
| A longitudinal observational study was carried out in 10 Egyptian dairy herds (n= 2500) in different lactation seasons to explore the molecular dynamics of E. coli serotypes in subclinical mastitis with diagnostic significance of typing virulence genes. In addition to, quantification of the triggered cytokines expression as analytical biomarker to determine the severity and/or onset of infection. Subclinical mastitic cows (n= 1125) were diagnosed in two infectious forms: single (n= 635) and mixed (n=490), where 88% (n = 990/1125) of examined udders developed 280 bacterial isolates of both contagious and environmental pathogens. Environmental bacteria had higher incidences recorded 55.4% (n=155/280); 91.61%(n=142/155)and8.339% (n=13/155) for E.coli and coagulase negative Staphylococcus (CNS), respectively, while 44.6% (n=125/280) was reported for contagious bacteria. Clinically, multiple quarters infection per cow; were mostly caused by same E. coli clone. They were morphologically identified by Gram stain and Transmission Electron Microscope (TEM), biochemically characterized, their enter pathogenicity in infant mice determined, hemolysin patterns reported, Shiga-like cytotoxicity in Vero cell line and the cytotoxic dose 50% (CD50) were also determined. E. coli isolates (n=142) were serologically grouped under 11 serotypes and 17 clones. Furthermore, serotypes were characterized by PCR with primers pairs complementary to species-specific and virulence genes nucleotide sequences. PCR fragments of molecular sizes 1403 bp,180 bp, 255 bp, 384 bp, and 534 bp distinctive for 16S rRNA, stx1, stx2, eaeA, and hlyA genes, respectively, were amplified, sequenced, and then aligned against similar GenBank records to confirm the identities. QRT-PCR revealed up regulation in expression of IL-6 and IL-8 cytokines mRNA in milk somatic cells in response to E. coli infections. The folds increased were higher for IL-8 than IL-6. The mean of the fold increase calculated to be 3.971 ± 5.178 and 15.732 ± 4.146 for IL-6 and IL-8, respectively. The onset of the up regulation is bacterial- dependent due to cellular subset count increase which mediated immune response. The presented study illustrated the capability of the investigated cytokines as biomarkers of subclinical stage of mastitis. Nonetheless, quantification of expression of additional cytokines genes upon udder subclinical infection will improve the outline of these analytical sensors of mastitis, hence, comprehensive information on onset of infection even regarding asymptomatic individuals will be developed with regulatory update on udder health condition. In conclusion, diagnosis of subclinical mastitis should not dependent on bacterial colonies count per sample, but additionally on bacterial secretory excretory toxins detection and host cytokines response estimation. | ||||
| Keywords | ||||
| Subclinical Mastitis; Cytokines; 16S rRNA; Stx1; Stx2; hylA; eaeA; qRT-PCR; Egypt | ||||
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