"Christian Traesch cannot take part in the World Cup in South Africa," the DFB said on their website.
The loss of the 22-year-old further limited Germany's options in midfield after injured captain Michael Ballack was ruled out of June 11-July 11 tournament in South Africa.
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Traesch twisted his right ankle in the eighth minute of Germany's 4-0 win over FC South Tirol and was taken to hospital for a scan which revealed damage to his ankle.
"This is a real shame for him, for such a thing to happen at this stage," coach Joachim Low said.
The injuries have narrowed down coach Joachim Loew's preliminary squad to just 25 players. He must announce a final 23-man squad by June 1.
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