The Rancho Sierra Vista Equestrian Center, home to about 367 horses, is the affected facility in Orange County. It has had 16 confirmed cases of the disease.
One horse has been put down, but authorities said the necropsy indicated it was highly unlikely its recumbency, which forced its euthanasia, was due to the infection.
The department confirmed a case in Riverside County on Tuesday.
Those premises were identified as Indio's Empire Polo Club, where a gelding was tested after displaying poor hind limb co-ordination and urine dribbling.
He lay down and had to be euthanized.
Veterinarian Michael Manno, providing an update on the Empire Polo Club website, said a 21-day quarantine has been implemented on the entire polo complex and the private ranches within its boundaries.
He said an isolation barn had been establish at the old showgrounds along Avenue 52 should suspected cases arise.
"It provides the perfect set up far away from our horses and gives us plenty of separation among the many stalls available there should we need them."
Signs of the disease include nasal discharge, poor co-ordination, hindquarter weakness, recumbency, lethargy, urine dribbling and diminished tail tone.
In May last year, 10 western states were hit by a major outbreak linked to the National Cutting Horse Association Western National Championship, in Ogden, Utah.