Epidemiological studies on Escherichia coli O157:H7 in Egyptian sheep
Mohammed Kamel, Diea G. Abo ElHassan & Amr El-Sayed
Tropical Animal Health and Production ISSN 0049-4747 Volume 47 Number 6 Trop Anim Health Prod (2015) 47:1161-1167 DOI 10.1007/s11250-015-0843-2
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Author's personal copy Trop Anim Health Prod (2015) 47:1161–1167 DOI 10.1007/s11250-015-0843-2
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Epidemiological studies on Escherichia coli O157:H7 in Egyptian sheep Mohammed Kamel 1,2 & Diea G. Abo El-Hassan 2 & Amr El-Sayed 2,3
Received: 19 January 2015 / Accepted: 27 April 2015 / Published online: 7 May 2015 # Springer Science+Business Media Dordrecht 2015
Abstract In the present work, the epidemiological role of apparently healthy sheep in transmission of Escherichia coli O157:H7 in different seasons was investigated. Fecal samples (convenience sampling) of apparently healthy farmed sheep (three farms, n=70) and from 15 wandering flocks fed on city wastes (n=80) in the Giza governorate were examined. The samples were collected in spring under mild weather conditions and during hot summer to be compared. Out of the 150 animals, 13 (8.7 %) were E. coli O157 shedders. The 13 ovine sorbitol-negative E. coli O157 were characterized by different PCR sets. The eae gene was detected in 11 isolate (85 %), stx1 in 3 isolates (23 %), stx2 in 8 isolates (62 %), and finally the hlyA in 11 isolate (85 %). Among the 13 isolates, 2 strains (15 %) were positive for eae, stx1, stx2, and hlyA as gene combination, one isolate (8 %) for eae, stx1, and hlyA, 5 isolates (38 %) for eae, stx2, and hlyA, 1 isolate (8 %) for eae and stx2, 2 isolates (15 %) contained eae and hlyA, 1 isolate (8 %) contained hlyA only, and finally, 1 isolate (8 %) did not contain any of these genes. None of the isolates showed the gene combination eae stx1, stx1 hlyA, or stx2 hlyA. The results indicated significant association of unfavorable weather and management conditions on O157:H7 shedding while the age or sex did not play any role in this process.
* Amr El-Sayed
[email protected] 1
Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany
2
Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, El Giza Square, Giza, Egypt
3
Laboratory of Molecular Epidemiology (LME), Faculty of Veterinary Medicine, Cairo University, El Giza Square, Giza, Egypt
Keywords E. coli O157 . Egypt . Feeding . Housing . Latex agglutination test . PCR . Sheep
Introduction The continuous evolution of new pathogenic strains of Escherichia coli through the acquisition of virulence genes carried on mobile genetic represents a great challenge for the agro-ecosystems and public health (El-Sayed 2000; Etcheverria and Padola 2013). One of the major emerged E. coli strains in the last few decades is the E. coli O157:H7 which is a serious food-borne pathogen responsible for great economic loses in the food industry worldwide. The pathogenicity of E. coli O157 is associated with a number of virulence factors, including two bacteriophage encoded cytotoxins called Shiga toxin 1 and 2 (stx1 and stx2) and their variants in addition to the outer membrane protein (intimin) which is encoded by the eaeA gene (Ayaz et al. 2014). The gastrointestinal tract of ruminants constitutes the main natural reservoir of E. coli O157:H7; however, infection in animals is typically asymptomatic (Nguyen and Sperandio 2012). The utilization of Sorbitol MacConkey agar medium (SMAC) is a simple, economic, rapid, and a reliable tool which permits the recognition of E. coli O157:H7 in stool cultures with a sensitivity of 100 %, a specificity of 85 %, and an accuracy of 86 % (March and Ratnam 1986). On the other hand, both commercial latex sets (Oxoid, Pro-Lab and Remel O157 latex reagents) are used as good alternatives to standard serologic methods for detecting E. coli O157:H7 (Sowers et al. 1996). The used kit (Oxoid, UK) in this study had a sensitivity and specificity of 100 % compared with the CDC reference antiserum (Sowers et al. 1996). In sheep, the number of epidemiological studies carried out on STEC strains is limited (Blanco et al. 2003; Rey et al. 2003;
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Martins et al. 2015). In Egypt, STEC was isolated from cattle (El-Sayed 2000), buffaloes and chicken (El-Jakee et al. 2012), humans (Selim et al. 2013) but not from camels (El-Sayed et al. 2008). The sheep in Egypt are usually offered poor ration such as the organic wastes and grass. Usually, they are kept with goats outdoors at the borders of large cities as wandering flocks which makes them exposed to heat stress and dryness conditions. These two factors are known to negatively affect the colonization of STEC in the gastrointestinal tract (GIT) and decrease its survival chances (El-Sayed et al. 2008). Therefore, the effect of diet and housing system on STEC isolation was investigated in the present work. In addition, and to our knowledge, there is no epidemiological or genotypical study on sorbitol-negative E. coli O157 in feces of healthy sheep in Egypt. The aim of the present work was to (1) investigate the presence of E. coli O157 in the feces of apparently healthy sheep; (2) detect the influence of different factors such as management, environment, sex, and age on the shedding and survival of E. coli O157; and finally, (3) molecular characterization of E. coli O157 in Egyptian sheep.
Materials and methods Animals and sampling A total of 150 apparently healthy sheep from different localities in the Giza governorate were sampled and examined bacteriologically for the presence of E. coli O157. The samples were collected from two groups of sheep (farmed sheep vs garbage eaters, nomadic sheep). Sampled sheep from the first group (farmed sheep, n=70) belonged to three farms in Giza (on the border of Cairo in the Nile delta). These animals are kept on high-quality concentrate ration. The second group represented the free-living wandering flocks in the slum areas of Giza and fed on city wastes (15 flocks, n= 80). The sampled animals included both sexes and were of different ages (Table 1). Ovine fecal samples were collected by rectal retrieval, and each sample has its own glove to avoid cross-contamination. Each fecal swab was then taken aseptically on EUROTUBO collection swab (Citoswab) containing 9 mL modified tryptone soya broth (Oxoid, UK) with 20 mg/L novobiocin (Oxoid, UK) (mTSB+n) and transported on ice within 1–2 h to the lab where they were incubated at 37 °C for 24 h upon arrival according to the method recommended by Turutoglu et al. (2007)and Akanbi et al. (2011). Isolation and identification of E. coli O157 After incubation, the enriched samples were streaked on Sorbitol MacConkey agar media (Oxoid) containing
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0.05 mg/L cefixime and 2.5 mg/L potassium tellurite (Oxoid) (SMAC-CT) and incubated at 37 °C for 24 h according to Turutoglu et al. (2007) and Akanbi et al. (2011). For biochemical identification, all grown non-sorbitol fermenting colonies from all samples were subcultured on MacConkey agar media (Oxoid) and incubated at 37 °C for 24 h. The MacConkey agar plates were examined for red/dark pink colonies. Sorbitol-negative and lactose-positive isolates were biochemically screened using the Indole, Methyl red, and Voges–Proskauer and Citrate utilization (IMViC) tests according to Farmer (1995). The obtained results were confirmed by subjecting all sorbitol-negative E. coli colonies to slide agglutination using the E. coli O157 latex test kit (Oxoid) according to the manufacturer’s instructions for serotyping.
Molecular characterization The presence of the genes encoding for Shiga toxin (stx1 and stx2), enterohemolysin (hlyA), and intimin (eae) were determined by PCR. Bacterial DNA used for amplification was extracted from the bacterial colonies by boiling method according to Zschöck et al. (1998) and Inat and Siriken (2010) with few modifications. As each positive colony was subcultured on sorbitol MacConkey agar medium and incubated for 24 h at 37 °C, two colonies were then selected and suspended separately in 100 μL of sterile distilled water in microcentrifuge tubes, after which they were incubated at 95 °C for 10 min. The suspensions were then centrifuged at 9503×g for 10 min, after which the supernatant containing the DNA template was transferred into DNase/RNase-free microcentrifuge tubes and stored at −20 °C until used.
Multiplex PCR for E. coli O157:H7 PCR assays were carried out according to Fagan et al. (1999) The assay was performed in a final volume of 50 μL reaction mixture consisting of 2 μl of nucleic acid template prepared from fecal cultures (approximately 60 ng of DNA), 10 mM Tris-HCl (pH 8.4), 10 mM KCl, 3 mMMgCl2; 2 mM concentrations of each primer (Table 2), 0.2 mM concentrations of each 2′deoxynucleoside-5′-triphosphate, and 4 U of AmpliTaq DNA polymerase. The amplification was carried under the following conditions: an initial 95 °C denaturation step for 3 min followed by the consequent 35 cycles of 95 °C for 20 s (denaturation), 58 °C for 40 s (annealing), and 72 °C for 90 s (extension). The final extension step at 72 °C for 5 min was then kept at 4 °C (hold temperature).
Author's personal copy Trop Anim Health Prod (2015) 47:1161–1167 Table 1
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The feces samples taken in the Giza governorate
Acc. to origin
Acc. to age
Acc. to sex
Acc. to season
Acc. to housing and nutrition
El-Monib
Bolaq and Imbaba
>1 year
Male
Spring
Farms (high-quality ration mainly concentrates)a
Grazing (Garber and low-quality roughage)b
No
80
70
111
39
47
103
80
70
70
80
%
53.3 %
46.7 %
74 %
26 %
31.3 %
68.7 %
53.3 %
46.7 %
46.7 %
53.3 %
