Simultaneous detection of protozoa in the tissues of snakes by double in situ hybridization

Different methods have been established for the simultaneous detection of different pathogens in tissue samples, each with certain advantages and disadvantages. Chromogenic in situ hybridization combines specific molecular pathogen detection with microscopic evaluation of pathogen quantity, morphology and distribution, as well as associated tissue damage. Furthermore, only a minimum of usually costly technical equipment is needed. The aim of our study was to detect two different protozoa simultaneously in tissue samples using exclusively digoxigenin (DIG)-labeled probes and alkaline phosphatase-coupled anti-DIG-antibodies and the chromogens Vector Red and NBT/BCIP with standard protocols. Gastrointestinal tissue samples from 15 snakes infected with either one or two protozoan species were investigated. All expected protozoa stained clearly dark purple or bright red, respectively, depending on the chromogen used. This technique can be used in pathogenicity studies of various pathogens in any kind of tissue.