Another person also admitted to hospital with a fever on Thursday had also initially tested negative for the virus, the government said. This person had traveled in the same ambulance as Teresa Romero, Spain's only known Ebola sufferer.
The cases are two of four people hospitalised with suspected Ebola symptoms on Thursday including the passenger on the Air France plane who developed tremors and prompted Spanish authorities to evacuate the plane.
Spain is on high alert for the disease after Ms Romero, a nurse who cared for two Ebola-infected priests before they died, became the first person to contract the virus outside West Africa. She is gravely ill but stable.
Although the two people tested negative for the disease initially, they must have another test within 72 hours in order to be given the all clear, the government said.
The United States, which is deploying up to 4,000 troops to West Africa to help contain the disease, has asked Madrid for permission to use U.S. military bases in Spain in its operation, a Spanish Defence Ministry source said on Thursday.
A decision is due to be announced on Friday when Spanish Defence Minister Pedro Morenes meets his U.S. counterpart Chuck Hagel in Washington. Madrid was set to agree to the request to use the bases at Rota near Cadiz and at Moron de la Frontera near Seville in southern Spain, the source said.
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