A Gimenez stain
performed on sections from the 3 birds revealed minute magenta bodies in the cytoplasm of inflammatory cells, consistent with elementary bodies of Chlamydophila psinaci (Figs 1 and 2).
Gimenez stain yields a clear cut staining of rikketsiae, which appears as pink/red whereas the underlying tissue is blue/green.
Although, Gimenez stain has been used to stain the various rickettsial bacteria such as Bartonella, Coxiella, etc (20), it has not been reported to be used for the detection of Wolbachia from mosquito ovaries.
A cell sample from each well was tested for infection at days 9 and 15 by using Gimenez stain
and PCR with 17 kDa and gltA primers.
Chlamydophila was detected by PCR and Gimenez stain of the splenic samples, but the extent of hepatic necrosis and the general absence of Chlarnydophila in necrotic lesions in the spleen and liver suggests that other agents may have contributed to the lesions, possibly reovirus.
Gimenez stains for the demonstration of chlamydial bodies were positive in 3 of 6 birds, although the number of infected cells was considered very low in comparison with birds that typically die from chlamydiosis.
Spleen and liver samples from the birds in all treatment and control groups were polymerase chain reaction negative for Cpsittaci nucleic acid, and organisms were not detected by Gimenez stain
Antigens were prepared by placing the rickettsial stains in L929 cells and HL60 or and DH82 cells, respectively; collecting the culture when Gimenez stain
or Wright staining showed positive results; ultrasonically crushing the culture; and purifying the bacteria by density ultracentrifugation.