Molecular studies on E.coli isolate from milk of mastatic cattle with special reference to associated biochemical changes in kaliobea governorate

This investigation was performed in Teaching hospital and farm of Benha university in Moshtohor the number of cows in this farm 80 dairy cows that 40 of them had clinical signs of mastitis (inflammation in teats ,pain in milking and milk decrease in amount and quality) .When examine these cows to identify the disease which cause these signs . California Mastitis Test (CMT) was performed to determine positive milk samples in the Mastitic targeted cows. 20 samples of early lactation stage cows out of 40 samples recovered from CMTpositive milk samples. Biochemical and PCR tests were performed to isolates E. Coli from positive milk samples (CMT) and determined three virulance genes, eae gene ,SXT1 and SXT2 . The significance of Escherichia coli-induced mastitis and biochemical changes associated to it in cows, due to the presence of virulence genes and wide range resistance to 20 antimicrobials, is concluded. E.coli cause biochemical changes in mastatic cow as (liver enzymes AST,GPT,TP, ant. oxidative enzymes as CAT, SOD,GST,LDand kidney function as urea and creatinine .E.coli has effect on inflammatory response in immunity system of mastitic cow by increase IL6,TNF and CRP.


Introduction
Mastitis is an inflammation of the mammary glands associated with physical and chemical and microbiological changes . It is considered the most important disease in dairy herds ( Acik et.al 2004) [1] . The most important causitave environmental mastitis pathogen is E.coli (Mokovee and Ruegge 2003) [2].Escherichia coli is a major etiological agent of intra-mammary infections (IMI) in cows, leading to acute mastitis and causing great economic losses in dairy production worldwide ( Blum 2015 [3]). Particular strains cause persistent IMI, leading to recurrent mastitis. Virulence factors of mammary pathogenic E. coli (MPEC) involved pathogenesis of mastitis as well as those differentiating strains causing acute or persistent mastitis (Burvenich et.al,2003 [4]) . The infection occur after bacteria entrance mammary gland via teat canal ,overcoming anatomical battier so they must evade the cellular and humeral defence mechanism of mammary gland to establish disease (Radostits et al., 2007[5] and Mbuk 2016)6.. Limited number of E.coli strains has ability to adhere and invade bovine mammary epithelial cell[s and cause persistant infection ,have severa l fimberia and fimberial adhesion that mediat adhesion to host epithelial cell through cell surface (MILANOV Dubravka 2015 [ 7] andDopfer et.al. 2000)[8]. This study was performed to :Detection the causitive agent of clinical matitis in cow by isolation of E.coli from milk of mastatic cow with special refrence to biochemical changes associated to it in infected cow .Charactarization of E.coli pathogen isolated from mastatic cow chemical and serlogicaly .Investigation of some virulance factor associated to E.coli isolated . Detection of E.coli attaching and enffacing (Intimin) eaeA,STX1 and STX2 virulence factors of E. coli comprise adhesins, which help the bacteria to adhere to and colonize mucosal surfaces, and toxins, which are proteins with the ability to disturb or modify the normal function of the host cell and to help the bacteria to cross the epithelial barrier and to invade the tissue (Kaper et al., 2004)[9]. Clinical E. coli mastitis can range from mild with only local signs to severe disease with systemic clinical signs. In severe cases the outcome can be acute tissue damage and complete loss of milk production or even the death of the diseased cow The severity of E. coli mastitis depends on the age of the cow and on the lactation stage, i.e. older cows and cows in early lactation are more susceptible to infection (Mehrzad et al., 2002)[10]. [10].
The general aim of this study was 1-To investigate host response to Escherichia coli infection represented in biochemical changes and immunity system reponse 2-To identify possible specific virulence genes and phylogeny types of E. coli associated with severity of clinical mastitis and the intramammary infection.

Samples
A total 40 of milk samples were collected from clinically mastatic cows from Quliobea governorate all samples collected in sterail macartins and perform (CMT) the positive samples will send as soon as possible to lab to be examination .Bacteriological examination of milk samples (Qurnn et.al 2002)[11 ]the collected samples were incubate aerobically at 370 C for 18-24 hrs then centrifugate at 3000 rpm /20 min the cream and supernatant layer were discarded and streak the sediment on blood agar ,maconcky agar and EMBagar . The plates were incubated aerobically at 37C0 for 24-48 hrs and examined for bacteriological growth. Suspected colonies appeared on different media were picked up and purified by subculture on fresh set of protective and preserved into semisolid agar for Identification of isolated m.o. According to colonial morphological and appearance ,growth characterization ,hemolytic patterns ,microscopically by Gimesa stain and biochemically changes according to (Boerlin et.al. 2003) [12] 4-PCR molecular identification DNA extraction. DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer's recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56 O C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer's recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

Oligonucleotide Primer
. Primers used were supplied from Metabion (Germany) are listed in table(3) PCR amplification. Primers were utilized in a 25-µl reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler. For stx1,2 duplex PCR, primers were utilized in a 50-µl reaction containing 25 µl of EmeraldAmp Max PCR Master Mix, 1 µl of each primer of 20 pmol concentration, 13 µl of water, and 8 µl of DNA template.

Analysis of the PCR Products.
The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of each PCR product were loaded in each gel slot. A Generuler 100 bp ladder (Fermentas, Thermo Scientific, Germany) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.  Volume-2 | Issue-7 | July,2017 | Paper-5

7-Tsi
A/A/ gas+H -H2S 100%     (Kaper et al., 2004) [9 ]who concluded that virulence factors of E. coli comprise adhesins, which help the bacteria to adhere to and colonize mucosal surfaces, and toxins, which are proteins with the ability to disturb or modify the normal function of the host cell and to help the bacteria to cross the epithelial barrier and to invade the tissue . There was a clear significant correlation between the CMT scores and the E. Coli , The presence of eae Intimin gene in E. coli involved in mastitis of dairy cows is of paramount importance , E. coli with Intimin gene are able to form small microcolonies on the surface of infected epithelial cells, followed by localized degeneration of the microvilli cumulating in an attaching and effacing (A/E) Elie et.al. (2015) [18]. all the E. coli isolates with the virulence genes stx and eae showed resistance to a higher number of antimicrobials than those which were stx-negative (Solomakos et al 2009) [30]. It is recommended in disease-control programs of dairy to study the E. coli involvement in mastitis, and to include in the surveillance the detection of virulence genes that are decisive in economic losses in vetrinarian .