Ayala was forced off during the first leg at the Riverside Stadium on Saturday evening - which ended 1-0 to Villa - and the Spaniard has not recovered in time for the fixture in Birmingham.
James Bree has been brought into the team having not featured in the 18-man squad in the North-East.
Aston Villa: Johnstone; Bree, Chester, Terry, Hutton; Jedinak, Hourihane; Snodgrass, Adomah, Grealish; Grabban
Subs: Bunn, Samba, Whelan, Bjarnason, Onomah, Hogan, Kodjia
Middlesbrough: Randolph; Shotton, Fry, Gibson, Friend; Clayton, Besic, Howson; Traore, Downing, Assombalonga
Subs: Konstantopoulos, Fabio, Cranie, Leadbitter, Harrison, Bamford, Gestede
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