Messi, who suffered a hamstring injury during Barcelona's 1-1 draw at Atletico Madrid in the first leg of the Spanish Super Cup on Wednesday, is expected to be fit by the time group leaders Argentina visit Asuncion for the match on September 10.
Higuain, top scorer in the South American qualifying competition with nine goals in 11 matches, was sent off against Colombia while midfielder Mascherano was shown a red card versus Ecuador in Argentina's last two qualifiers in June.
However, striker Sergio Aguero is back after missing the 2-1 win over Italy in a friendly in Rome last week.
Coach Alejandro Sabella, who named an 18-man squad, is also without suspended first choice centre backs Ezequiel Garay and Federico Fernandez who have two bookings in the competition.
Second-placed Colombia can catch Argentina at the top of the South American group if they beat Ecuador on September 6 while Sabella's team can secure a berth at the next year finals in Brazil with a victory in Paraguay four days later.
Argentina, who lead the standings on 26 points and have three matches to go, do not play on September 6 when the eight other nations have qualifiers, most of whom have four matches left.
Colombia have 23 points, Ecuador and Chile are on 21, while Uruguay and Venezuela have 16 in the nine-nation group. The top four qualify for the finals while the fifth-placed team goes into a playoff against an Asian qualifier for another spot.
Paraguay, World Cup quarter-finalists in South Africa in 2010, have had a poor campaign and are bottom with eight points. However, they gave Germany a fright in a 3-3 draw in a friendly in Kaiserslautern last week on new coach Victor Genes's debut.
Squad:
Goalkeepers: Sergio Romero (Monaco), Mariano Andujar (Catania)
Defenders: Pablo Zabaleta (Manchester City), Hugo Campagnaro (Inter Milan), Fabriccio Coloccini (Newcastle United), Marcos Rojo (Sporting Lisbon), Jose Basanta (Monterrey), Cristian Ansaldi (Zenit St Petersburg)
Midfielders: Ever Banega (Valencia), Lucas Biglia (Lazio), Augusto Fernandez (Celta Vigo), Angel Di Maria (Real Madrid), Ricardo Alvarez (Inter Milan), Erik Lamela (AS Roma)
Forwards: Lionel Messi (Barcelona), Sergio Aguero (Manchester City), Ezequiel Lavezzi (Paris St Germain), Rodrigo Palacio (Inter Milan)
(Reporting by Luis Ampuero; Writing by Rex Gowar in London; Editing byKen Ferris)
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