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Pro-Russian separatists shot down a military transport plane Saturday in the country's restive east, killing all 49 service personnel on board, Ukrainian officials said.
It was a bitter setback for Ukrainian forces, which have struggled to suppress an armed insurgency by foes of the new government.
Nine crew and 40 troops were aboard the Il-76 when it went down early Saturday as it approached the airport at the city of Luhansk, the Ukrainian prosecutor general's office said in a statement.
That exceeded the loss of 12 soldiers including a general on May 29 when rebels shot down a troop helicopter near the eastern city of Slovyansk.
The incident underlines questions about rebel access to military gear. Ukraine has accused Russia of permitting three tanks to cross the border where they were used by rebels. Russia denies supplying the separatists.
Denis Pushilin, a leader of the separatist Donetsk People's Republic, told Russian state television Friday that rebels had the tanks but it was "improper to ask" where they got them.
The defense ministry's statement said that the rebels "cynically and treacherously" downed the plane using anti-aircraft guns and heavy machine guns. It expressed sympathy to the families of those killed "for their tragic and irreparable loss."
Alexei Toporov, defense spokesman for the self-proclaimed Luhansk People's Republic, said the aircraft was shot down after what he termed Ukrainian "occupiers" refused an ultimatum to abandon the Luhansk airport.
Luhansk is in Ukraine's east near the border with Russia, an area that has seen separatists seize government buildings and declare independence after holding disputed referendums.
Ukraine had claimed a success on Friday when troops retook some rebel-occupied buildings in the port city of Mariupol. No deaths were reported.
Before Saturday's incident, the Ukrainian health ministry had said at least 270 people had died in clashes between government forces and armed separatists.