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Protocol for in situ hybridisation (ISH) technique for the detection of E. leei in fish tissues

Early stages of E. leei were seldom found, and their detection at light microscope level is compromised by the difficulty of identification when using only morphological characteristics. In addition, myxosporean stages, differing morphologically from typical E. leei stages, were detected in epi-epithelial location in several fish. To get an accurate identification of the true E. leei stages and confirm the nature of suspicious early stages, a protocol or ISH detection was developed. The strategy used consisted of the design and synthesis of two DIG-labelled oligonucleotides and the optimization of conditions allowing the hybridization of the probes with specific complementary targets located in E. leei nuclear rDNA and cytoplasmic rRNA.

Using in situ hybridisation (ISH), we could conclude that stages located on the mucosa epithelium surface of the intestine of some recipient fish do not belong to E. leei but to a different myxozoan species. Scarce early or suspicious E. leei stages were confirmed outside of the target organs using ISH, and usually in fish with confirmed infections in the intestine. Thus, putative extra-intestinal stages, responsible for the penetration in the fish and colonization of intestine would be very rare in these experimental infections, although they could be have more significance in spontaneous infections.

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