Pear Tree Nursery in Haddington has been closed after 28 cases of the bug were detected.
An outbreak of E.coli had led to the closure two nurseries in East Lothian.
NHS Lothian said a source had not been found, but tests were being done to identify the strain of the infection. "We have written to parents and carers of the children and to staff at the nursery to provide the most up to date information and ongoing health advice.
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