Occurrence of Virulence Genes Associated with Diarrheagenic Escherichia coli Isolated from Raw Cow’s Milk from Two Commercial Dairy Farms in the Eastern Cape Province, South Africa
Abstract
:1. Introduction
2. Experimental Section
2.1. Study Sites
2.2. Isolation and Identification
2.3. DNA Extraction
2.4. Detection of Virulence Genes in E. coli Isolates
Target Strain | Target Gene | Primer Sequence (5′–3′) | Conditions | Amplicon Size (bp) | References |
---|---|---|---|---|---|
E. coli | UidA | AAAACGGCAAGAAAAAGCAG ACGCGTGGTTAACAGTCTTGCG | An initial 2 min of denaturation at 94 °C followed by 25 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C for 2 min. Amplified products were held at 4 °C after amplification. | 147 | [26] |
EHEC | flicH7 | TACCATCGCAAAAGCAAC TCC GTCGGCAACGTTAGTGATACC | Initial denaturation at 95 °C for 15 min followed by 35 cycles of heat denaturation at 94 °C for 45 sec, primer annealing at 55 °C for 45 sec and DNA extension at 68 °C for 2 min. After the last cycle the samples were kept at 72 °C for 5 min to complete synthesis of all strands. | 230 | [27] |
EIEC | Ial | CTGGATGGTATGGTGAGG GGAGGCCAATTATTTCC | 1 cycle for 2 min at 50 °C, 1 cycle for 5 min at 95 °C, 40 cycles for 45 sec at 95 °C, 45 sec at 55 °C and 45 sec at 72 °C and a final extension step for 10 min at 72 °C to complete synthesis of all strands. | 700 | [28] |
EAEC | EagR | AGACTCTGGCGAAAGACTGTATC ATGGCTGTCTGTAATAGATGAGAAC | Initial denaturation at 95 °C for 15 min followed by 35 cycles of heat denaturation at 94 °C for 45 sec, primer annealing at 55 °C for 45 sec and DNA extension at 68 °C for 2 min. A final elongation step at 72 °C for 5 min. | 194 | [29] |
EPEC | EaeA | TCAATGCAGTTCCGTTATCAGT GTAAAGTCCGTTACCCCAACC TG | An initial denaturation step at 94 °C for 5 min, followed by 36 cycles of 94 °C for 35 sec, annealing at 62 °C for 30 sec and elongation at 72 °C for 1 min. A final elongation step at 72 °C for 5 min. | 482 | [28] |
ETEC | Lt | GCACACGGA GCTCCTCAGTC TCCTTCATCCTTTCAATGGCTT | An initial denaturation step at 94 °C for 5 min, followed by 36 cycles of 94 °C for 35 sec, annealing at 62 °C for 30 sec and elongation at 72 °C for 1 min. A final elongation step at 72 °C for 5 min. | 218 | [28] |
UPEC | PapC | GACGGCTGTACTGCAGGGTGTGGCG ATATCCTTTCTGCAGGGATGCAATA | An initial denaturation step at 94 °C for 5 min, followed by 36 cycles of 94 °C for 35 sec, annealing at 62 °C for 30 sec and elongation at 72 °C for 1 min. A final elongation step at 72 °C for 5 min. | 328 | [30] |
2.5. Gel Electrophoresis
3. Results and Discussion
3.1. Isolation and Identification
3.1.1. Biochemical Confirmation
Farms | Number of Samples | G (-ve) Rods | Cat (pos) | Ox (neg) | API 20E | uidA Gene | |
---|---|---|---|---|---|---|---|
Clinical Mastitis | Sub-Clinical Mastitis | ||||||
A | 100 | 100 | 100 | 100 | 100 | 72 | 50 |
B | 100 | 100 | 88 | 88 | 88 | 68 | 37 |
3.1.2. Molecular Characterization of E. coli
Location | Amplified Genes | ||||||
---|---|---|---|---|---|---|---|
uidA | fliCH7 | eae | lt | papC | Ial | eagR | |
Dairy farm A | 50 (57.5%) | 27 (54%) | 9 (18%) | 2 (4%) | 3 (6%) | 4 (8%) | 5 (10%) |
Dairy farm B | 37 (42.5%) | 16 (43.2%) | 9 (24.3%) | 5 (13.5%) | 1 (2.7%) | 2 (5.4%) | 4 (10.8%) |
Total | 87 (100%) | 43 (49%) | 18 (21%) | 7 (8%) | 4 (4.6%) | 6 (7%) | 9 (10.3%) |
3.1.3. Virulence Genes from E. coli Isolates in Raw Cow’s Milk
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Caine, L.-A.; Nwodo, U.U.; Okoh, A.I.; Ndip, R.N.; Green, E. Occurrence of Virulence Genes Associated with Diarrheagenic Escherichia coli Isolated from Raw Cow’s Milk from Two Commercial Dairy Farms in the Eastern Cape Province, South Africa. Int. J. Environ. Res. Public Health 2014, 11, 11950-11963. https://doi.org/10.3390/ijerph111111950
Caine L-A, Nwodo UU, Okoh AI, Ndip RN, Green E. Occurrence of Virulence Genes Associated with Diarrheagenic Escherichia coli Isolated from Raw Cow’s Milk from Two Commercial Dairy Farms in the Eastern Cape Province, South Africa. International Journal of Environmental Research and Public Health. 2014; 11(11):11950-11963. https://doi.org/10.3390/ijerph111111950
Chicago/Turabian StyleCaine, Lesley-Anne, Uchechukwu U. Nwodo, Anthony I. Okoh, Roland N. Ndip, and Ezekiel Green. 2014. "Occurrence of Virulence Genes Associated with Diarrheagenic Escherichia coli Isolated from Raw Cow’s Milk from Two Commercial Dairy Farms in the Eastern Cape Province, South Africa" International Journal of Environmental Research and Public Health 11, no. 11: 11950-11963. https://doi.org/10.3390/ijerph111111950