Development of multiplex polymerase chain reaction for detection of Ehrlichia canis, Babesia spp and Hepatozoon canis in canine blood.

A multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of canine blood parasites, Ehrlichia canis, Babesia spp and Hepatozoon canis, from blood samples in a single reaction. The multiplex PCR primers were specific to E. canis VirB9, Babesia spp 16S rRNA and H. canis 16S rRNA genes. Specificity of the amplicons was confirmed by DNA sequencing. The assay was evaluated using normal canine and infected blood samples, which were detected by microscopic examination. This multiplex PCR offers scope for simultaneous detection of three important canine blood parasites and should be valuable in monitoring parasite infections in dogs and ticks.

In vivo culture of Cryptosporidium oocysts for laboratory use.

In the present study, we describe in vivo cultivation to produce oocysts. Seven-day-old mice were orally infected with 100,000-120,000 Cryptosporidium oocysts. On day 8 post-infection, the mice were killed by ether, and the small and large intestines collected. A simple extraction procedure was used and purified using Ficoll gradient centrifugation. After purification, the oocysts were preserved in phosphate buffered saline with antibiotic at 4 degrees C before use.

Detection of Toxolasma gondii in captive wild felids.

Toxoplasma gondii can infect all species of warm-blooded animals, including humans, and causes serious diseases in immunocompromized hosts. Live tachyzoites derived from serial passage in HeLa culture were used in the Sabin-Feldman dye test for detection of Toxoplasma gondii antibody in serum samples of 21 captive wild felids including one fishing cat (Prion nailurus viverrina), one leopard (Panthera pardus), two flat-headed cats (Prion nailurus planiceps), 6 tigers (Panthera tigris), two leopard cats (Felis bengalensis), two clouded leopards (Felis nebulosa), 3 pumas (Puma concolor), and 4 jungle cats (Felis chaus). Antibodies to Toxoplasma gondii were founded in 9 of 21 felids (42.8%). This study revealed that cell culture-derived tachyzoites can be used successfully as a source of live organisms in a gold standard Sabin-Feldman dye test, which is simpler, cheaper and less ethically sensitive than in vivo inoculation.

Cultivation of Giardia duodenalis in Mongolian gerbils.

The Mongolian gerbil (Meriones unguiculatus) is susceptible to infection with Giardia duodenalis trophozoites. Each animal was orally infected with 0.5 ml Diamond's TYIS-33 culture medium containing 10(6) trophozoites. Cysts were then collected and concentrated by sucrose gradient centrifugation. G. duodenalis cysts were first observed in feces on day 5 post-infection. The characteristic of G. duodenalis infection in gerbils was intermittent cyst release. The range in the number of cysts released per gerbil for a 4-hour collection period was 0-1.5 x 10(3).