Isolation of Escherichia coli from Ducks and Duck Related Samples

MATERIALS AND METHODS

Sample collection: A total of 150 duck and duck related samples were collected from a wet market and two duck farms in Penang, Malaysia within a 4-month period. The samples were duck intestines (48), wash water (water use for washing duck carcasses) (20) from a wet market and duck faeces (58), soil (24) from duck farms. The samples were stored under 4°C and transported to the laboratory where analysis was carried out immediately upon arrival for the presence of Escherichia coli.

Bacteriological analysis: Analysis for Escherichia coli was done using a modified method according to the Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM). One gram each of intestinal, faecal and soil samples were enriched in 9 mL EC broths. For wash water sample, 10 mL were enriched in 90 mL EC broth. Enriched EC broths were incubated for 24±2 h at 45.5°C. After which 10 μL aliquots of EC broths were streaked onto Levine's eosin-methylene blue and Eosin-methylene blue agars. The plates were then and incubated for 24±2 h at 37°C. Presumptive Escherichia coli colonies appear as dark centered and flat, with or without metallic sheen. One to three colonies showing such characteristic nature were picked from each plate and purified on plate count agar slants. They were identified and confirmed using Gram staining and biochemical tests such as Indole production, Voges-Proskauer (VP), Methyl red and Citrate reactions (popularly known as IMViC reaction). All media were purchased from Merck, Germany.

Statistical analysis: The data obtained were analyzed using Chi-square test for goodness of fit to determine whether significant variations existed between the samples examined for Escherichia coli. Chi-square (χ²) was defined as: χ² = (o-e)²/e where o is the observed result, e is the expected result and the data obtained were interpreted using Chi-square distribution table at 5% significant level (Fisher and Yates, 1963).

RESULTS AND DISCUSSION

The result for the occurrence of Escherichia coli in the duck and duck related samples examined are presented in Table 1. Of the 150 samples tested, 117 (78.00%) were positive for Escherichia coli. The occurrence of Escherichia coli was highest in duck faeces 87.93% (51/58), followed by duck intestines 81.25 (39/48), soil 70.83% (17/24) and washes water 50.00% (10/20) samples. However, statistical analysis using chi-square indicated no significant difference (p>0.05) among the samples. It is possible that, Escherichia coli in soil samples might have resulted from the defaecation of birds or the pathogen may be harboring naturally in the soil. For Escherichia coli to be present in wash water samples it is feasible that contamination might have taken place during carcass processing. This is because portable and hot water used for carcass scalding is not known to be a major reservoir for Escherichia coli. Escherichia coli were also found more in farm B, 90.45% (19/21) than farm A, 80.33% (49/61). This suggests that farm B was more contaminated with Escherichia coli, compared to farm A. Moreover, the percentage Escherichia coli positive for both farms were higher than in the wet market (72.06%) but did not differ significantly from each other. In general the prevalence of Escherichia coli in the samples examined were relatively high. Thus healthy ducks like other animals may carry Escherichia coli in their intestines which they may share during defaecation. These pathogens can survive in the soil or faeces and cross contaminate other samples or equipments on the farm. Under poor or faulty processing conditions Escherichia coli can be transferred from duck intestines to wash water and other food samples.

Escherichia coli O157:H7 was determined using the ability and inability of the Escherichia coli isolates to ferment sorbitol. Biotype 1 gave ++-- and biotype 2 gave -+-- for the IMViC reaction. Thus biotype 2 Escherichia coli strains were negative for indole production. Twenty three (17. 56%) of the isolates belonged to the biotype 2 and 108 (82.44%) were of the biotype 1. Similarly, out of the 131 Escherichia coli types observed, 82.44 and 17.56% were serotype O517 and O517:H7, respectively. Sixteen Escherichia coli O517:H7 was isolated from intestinal samples, four from faeces, two from soil and one from wash water samples. The identification and differentiation of Escherichia coli types O517 and O517:H7 is important because the presence of Escherichia coli O517:H7 suggests that pathogenic or diarrheagenic Escherichia coli may be present in the ducks we sampled.

Table 1:	Occurrence of Escherichia coli in ducks and duck related samples
*Number of positive isolates

Pathogenic Escherichia coli O517:H7 are threats to public health and Feng and Weagant (2009) indicated that, the analysis for pathogenic Escherichia coli requires that the isolates should first be identified as Escherichia coli before testing for their virulence markers. The pathogenic groups includes enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and others that are not yet well characterized; and O157:H7 is the prototypic EHEC most often implicated in illness worldwide (Nataro and Kaper, 1998; WHO, 2005; Feng and Weagant, 2009).

CONCLUSION

In general, the prevalence of Escherichia coli in the samples analyzed was relatively very high. It ranged from 50.00 to 88.89%. Most of the Escherichia coli isolates were of the type O157 which are usually non-pathogenic and belong to the biotype 1. Duck faecal samples had the highest occurrence for Escherichia coli while wash water samples showed the least contaminated. There is the potential for contamination and cross contamination of Escherichia coli from ducks to farming and processing equipments and to other food samples.

ACKNOWLEDGMENTS

This study was financed by the Postgraduate Research Grant Scheme (1001/PTEK1ND/843007) of the Universiti Sains Malaysia (USM). We also acknowledge with gratitude the support given by the Institute of Postgraduate Studies, USM to run this research. The first author expresses his sincere gratitude for the support provided by Liew and Aronal during sampling.