STUDIES ON ECHERICIA COLI ISOLATED FROM MASTITIC CATTLE AND COMPARATIVE REVEALANCE TO HUMAN.

A total of 50 clinical mastitic milk samples, 42 sub clinical mastitic milk samples ( positive with California Mastitis Test) and 40 hand swabs from contact human were collected from different dairy farms at Gharbia governorate and investigated bacteriologically to isolate Ecshericia coli . Among clinical mastitic samples a total of 5 isolates E. coli (10%) , from sub clinical mastitic samples 3 isolates (7.4%) were detected, while 3 isolates (7.5% ) were recovered from contact human hand swabs. E. coli isolates were serotyped under 6 different O serotypes (O 27 and O 55 ) isolated from contact human while (O 6 , O 86 , O 114 , O 27 and O 157 ) isolated from mastitic milk . Antibiotic sensitivity revealed that all isolates were fully susceptible to enerofloxacin, ciprofloxacin, while all isolates were fully resistant to penicillin. E. coli serogrouped isolates were subjected to PCR for detection of Stx 1 and Stx 2 genes. 3 out of 7 serogrouped isolates (42.85%) were carried Stx 2 gene (O55 and O27 from contact human and O86 from mastitic milk) while Stx 1 gene was not detected . phylogenetic analysis for the sequence data of the Sxt2 gene of E. coli serogroupes revealed that Sxt2 gene isolated from mastitic milk of cattle is closely identical (100% identity) to Sxt 2 gene isolated from contact human. In Conclusion , isolation of STEC from cattle might have potential pathogenicity for human. So that contact human should use sound hygienic measures during milking and management of these animals to avoid zoonotic infection.

A total of 50 clinical mastitic milk samples, 42 sub clinical mastitic milk samples ( positive with California Mastitis Test) and 40 hand swabs from contact human were collected from different dairy farms at Gharbia governorate and investigated bacteriologically to isolate Ecshericia coli. Among clinical mastitic samples a total of 5 isolates E. coli (10%), from sub clinical mastitic samples 3 isolates (7.4%) were detected, while 3 isolates (7.5% ) were recovered from contact human hand swabs. E. coli isolates were serotyped under 6 27 and O 157 ) isolated from mastitic milk . Antibiotic sensitivity revealed that all isolates were fully susceptible to enerofloxacin, ciprofloxacin, while all isolates were fully resistant to penicillin. E. coli serogrouped isolates were subjected to PCR for detection of Stx1 and Stx2 genes. 3 out of 7 serogrouped isolates (42.85%) were carried Stx2 gene (O55 and O27 from contact human and O86 from mastitic milk) while Stx1 gene was not detected . phylogenetic analysis for the sequence data of the Sxt2 gene of E. coli serogroupes revealed that Sxt2 gene isolated from mastitic milk of cattle is closely identical (100% identity) to Sxt2 gene isolated from contact human. In Conclusion , isolation of STEC from cattle might have potential pathogenicity for human. So that contact human should use sound hygienic measures during milking and management of these animals to avoid zoonotic infection.
1207 It is primarily caused by an invasion of mammary tissues by pathogenic microorganisms through the teat canal resulting in physical, chemical, pathological changes in glandular tissues and milk (Quinn et al., 2002;Radostitis, 2007).
Ecshericia coli is one of the most common causes of bovine clinical mastitis . The incidence of E. coli mastitis has increased in some countries in recent years (Green et al., 2005). It is a major problem in lactating dairy cows (Kobori et al., 2004).
Environmental contamination with faces is the main source of mastitis-causing E. coli bacteria (Nemeth et al.,1994). The controls of mastitis in dairy herds are accomplished in part with the aid of Antibiotics (NMC, 1999). Public hazards associated with the consumption of antibiotic contaminated milk results in allergic responses, changes in intestinal flora and development of antibiotic resistant pathogenic bacteria (Thirapatsakun, 1999).
Virulence factors of the bacterial strain can give it a chance for colonization, multiplication and survival in udder in the face of host defense mechanism (Kaipainen et al., 2002). The shiga toxin producing E. coli (STEC) strains can cause mastitis in bovine and reduce milk quality for human consumption (Momtaze et al., 2012). Many studies concluded that the STEC strains are the most prevalent resources for milk poisoning ( Solomakos et al ., 2009). About 82% of the STEC strains of animal origin belong to similar serotypes detected in humans, and 51% of these belong to serotypes related to human infection with HUS (Blanco et al., 2004a). Also, other infection routes may occur through direct contact with carrier animals and indirect contact with contaminated environments (Keen et al., 2006).
The objective of this study was to apply bacteriological and molecular studies on Ecshericia coli isolated from mastitic cattle and comparative revealance to human contact.

Sampling:
A total noumper of 50 mastitic milk samples, 42 positive California mastitis milk samples and 40 hand swabs from contact human were collected from different dairy farms from Gharbia governorates.

Milk samples:-
Mastitis milk samples were collected aseptically into screw capped bottles and kept at 4oC until microbiological examination. Twenty five ml from each sample were homogenized with 225 ml of buffered peptone water (BPW) for pre-enrichment and incubated at 37oC for 24 h (Addis et al., 2011a).

Contact human hand swabs:-
Moistened sterile swabs were rolled over the palm of hands, finger tips , nails and area between fingers. Each swab was inserted in tubes containing BPW for pre-enrichment.

Bacterial isolation by cultivation:-
A loopful from the pre-enriched culture homogenate in BPW was streaked onto the surface of MacConkey`s agar, Eosin Methylene blue (EMB) agar media. The inoculated plates were incubated at 37°C for 24 to 48 hours then examined for bacteriological growth. Greenish metallic shinny colonies on the plates were purified on nutrient agar slants and incubated at 37°C for 18-24 hours for further identification. (Ojo et al., 2010).

Serological identification of E. coli isolates:-
The isolated strains of E. coli were identified serologically by using polyvalent and monovalent antisera for diagnosis of pathogenic serotypes according to Varnam and Evans, (1991 ).
1208 Extraction of bacterial DNA: DNA was purified according to QIAamp DNA mini kit instructions.

duplex PCR for identification of Shiga toxin genes (Stx1& Stx2):
Purified DNA of E. coli isolates was subjected to a duplex PCR for the identification of Shiga toxin genes ( Stx1& Stx2 ) using specific oligonucleotide primers according to (Dipineto et al., 2006) as shown in the table (1) and agarose gel electrophoreses according to (Sambrook et al., 1989) with agarose gel (1.5 g).
The PCR condition for amplification was conducted according to (Dipineto et al., 2006). Briefly, initial denaturation was performed at 94oC for 5 min followed by Secondary denaturation at 94oC for 30 sec., annealing at 58˚C for 45 sec. and extension at 72oC for 45 sec. No. of cycles (35) and the final extension was carried out at 72oC for 10 min.

Detection of Shiga toxin genes in E. coli serogroupes
E. coli serogrouped isolates were examined for detection of Shiga toxin virulence genes by duplex PCR. stx2 gene was detected in STEC isolates ( O27, O55 ) from contact human and in O86 from mastitic milk while stx1gene was not detected at all as shown in figure (1) and (2).
Results of sequencing of Sxt2 gene of E. coli isolated from cattle mastitis and contact human hand swabs: Figure ( 3 ) demonstrated the identity and the diversion percent against the selected sequences , it revealed that Sxt2 gene (MG656983) isolated from mastitic milk of cattle (sample 1) was closely identical (100% identity) to Sxt2 gene (MG656984) isolated from contact human (sample 2). However they were showed identity percentage of (99.7%) with E. coli strain SWUN4041 Stx2 (KP120720.  Figure 4 ) and the amino acid sequences ( Figure 5) which represent hypotheses about the evolutionary relationships among a group of sequences . In which the length of the horizontal line connecting one sequence to another was proportional to the estimated genetic distance between the sequences . The phylogenetic analysis in this study revealed that Sxt2 gene (MG656983) isolated from mastitic milk of cattle (sample 1) and Sxt2 gene (MG656984) isolated from contact human (sample 2) were found in the same very short branch as they were closely related to each other and that indicate identical sequences . On the other hand they were highly related to Sxt2 gene of Yak origin (KP120720.1), (KP120725.1), (KP120721.1) and (KP120719.1) and Sxt2 gene of human origin (EF441604.1), (KF932368.1) .  , 2004). Cattle are considered the main reservoir of this serotype. O157 is one of the most important STEC that causes severe disease in human and was reported by many other authors as a cause of mastitis (Lipman et al., 1995 ;Aly, 2006 ). In the present study E. coli was isolated from contact human hand swabs with percentage (7.5%). It was isolated from dairy workers in Egypt with percentages (11.1%) by (Awadallah et al., 2016 ), 16% by (Zeinhom and Abdel-Latef , 2014) and 20% by (Gwida and El-Gohary , 2013 ).

Discussion:-
Isolation of E. coli from the hand of contact human and dairy workers may be attributed to poor hygienic measures during milking and poor personal hygiene practices in dairy workers. These results indicate the possibility of transferring E. coli to milk and consequently increasing the risk of infection for milk consumers (El-Gedawy et al., 2014). Regarding to E. coli isolated from contact human serotypes O27 and O55 were isolated. Strain O55 is considered as enteropathogenic E. coli (EPEC) strain and was isolated from contact workers in Egypt by (Merwad et al., 2014). Also O55 and O27 were isolated from dairy cows, and hand swabs of dairy workers by (Awadallah et al., 2016). Severe gastrointestinal diseases in humans and complications such as the haemolytic uremic syndrome can caused by Shiga toxin-producing E. coli (STEC) and it is considered as an important group of food-borne pathogens. Chern et al. (2004) and Kobori et al., (2004). Also several authors recorded that Shiga toxins genes (Stx1, Stx2) and eae (intimin) gene are the most important virulence genes in E. coli strains isolated from bovine mastitic milk Kobori et al. (2004) , Wieler et al. (1996) and Paton et al. (1998). So that in the current study multiplex PCR protocol used for detection of stx1 and stx2 genes on isolated E. coli serogroups to confirm their pathogenicity. stx2 gene was detected in STEC isolates from contact human and mastitic milk. While stx1gene was not detected. Similar results was mentioned by (Singha et al., 2013) who reported that only one isolate was positive for shiga toxin gene (stx2), and none were harbouring stx1 gene from dairy cattle suffering from clinical/subclinical mastitis. While 1210 Merwad et al., 2014) detected the presence of stx1, stx2, both stx1 and stx2 in E. coli isolated from contact workers and milk. On the other hand , ( Murinda et al., 2004 ;Carneiro et al., 2006 ;Wenz et al., 2006) mentioned that E. coli strains isolated from cows with mastitis are negative for stx2 gene by PCR. (Osman et al., 2012) found that STEC isolates were not found in bovine mastitic milk in Egypt. While (Farhad et al., 2012 ;Bean et al., 2004) showed that the most common virulence gene detected in mastitic milk samples was stx1. In the present study the phylogenetic analysis revealed that Sxt2 gene (MG656983) isolated from mastitic milk of cows (sample 1) and Sxt2 gene (MG656984) isolated from contact human (sample 2) were found in the same very short branch as they were closely related to each other and that indicate identical sequences . On the other hand they were highly related to Sxt2 gene of Yak origin (KP120720.1), (KP120725.1), (KP120721.1) and (KP120719.1) and Sxt2 gene of human origin (EF441604.1), (KF932368.1) . This result agreed with (Asakura et al., 2000) who recorded that Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. (Murinda et al., 2004) investigated that E.coli isolates from cattle and human disease shared similar toxigenic profiles. These findings suggesting that the toxin of STEC from cows might have potential pathogenicity for human. So that contact human should use sound hygienic measures during milking and management of these animals to avoid zoonoticinfection

Conclusion:-
Detection and treatment of E. coli mastitis appear to be important for animal health and has a public health for contact human. No association between strain serotype and the presence of shiga toxin genes and clinical disease severity. Enerofloxacin and ciprofloxacin were the most effective antibiotics on treatment of E. coli mastitis. phylogenetic analysis revealed that Sxt2 gene isolated from mastitic cattle is closely identical (100% identity) to Sxt2 gene isolated from contact human. These findings suggesting that the toxin of STEC from cattle might have potential pathogenicity for human. So that contact human should use sound hygienic measures during milking and management of these animals to avoid zoonotic infection.