Dylan Hartley has been left out of England's Rugby World Cup training squad as the hooker continues to struggle with a knee problem.
England co-captain Hartley has not played since December and the Northampton Saints man's prolonged period of inactivity appears to have scuppered his hopes of featuring in Japan later this year.
Flanker and former England skipper Chris Robshaw, scrum-half Danny Care and back-row Nathan Hughes are other high-profile omissions.
Alex Dombrandt, Lewis Ludlam, Ruaridh McConnochie and Val Rapava Ruskin have been called up to the senior squad for the first time.
The 29-man party does not include any players involved in the Premiership semi-finals, with those from Gloucester and Northampton to join the camp next week.
Players from champions Saracens and runners-up Exeter Chiefs will then come into the reckoning when Eddie Jones names his final training party on July 4.
On Wednesday, Exeter's Jack Nowell was backed to recover from ankle surgery in time to feature at the World Cup by Chiefs director of rugby Rob Baxter.
George Kruis and Mako Vunipola will report for medical treatment when the squad gather for a week at Pennyhill Park and the Lensbury on June 30.
England training squad:
Dan Cole, Tom Curry, Alex Dombrandt, Tom Dunn, Charlie Ewels, Ellis Genge, Joe Launchbury, Lewis Ludlam, Val Rapava Ruskin, Nick Schonert, Brad Shields, Kyle Sinckler, Jack Singleton, Sam Underhill, Mark Wilson.
Chris Ashton, Mike Brown, Joe Cokanasiga, Elliot Daly, George Ford, Jonathan Joseph, Jonny May, Ruaridh McConnochie, Dan Robson, Marcus Smith, Ben Te'o, Manu Tuilagi, Anthony Watson, Ben Youngs.
In for medical treatment
George Kruis and Mako Vunipola.