A two-step method to purify Ostertagia ostertagi eggs from feces: Sucrose flotation followed by density gradient centrifugation using lymphocyte separation medium

Authors: Tuo, Wenbin; Zarlenga, Dante; Hebert, Deborah - Van Hook; Miramontes, Eliseo; Fetterer, Raymond

Submitted to: Comparative Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/20/2015
Publication Date: 7/20/2015
Citation: Tuo, W., Zarlenga, D.S., Hebert, D.A., Miramontes, E.N., Fetterer, R.H. 2015. A two-step method to purify Ostertagia ostertagi eggs from feces: Sucrose flotation followed by density gradient centrifugation using lymphocyte separation medium. Comparative Parasitology. 82(2):275-279.

Interpretive Summary: A method to purify livestock gastrointestinal (GI) nematode parasite eggs is needed. The present study developed a two-step technique that will allow purification of fecal Ostertagia ostertagi eggs that are free from fecal contamination. Current techniques use sucrose and/or salt for parasite egg isolation, which do not completely remove feces from the isolated eggs. The method developed in this study uses sucrose to enrich parasite eggs in the first step and uses the lymphocyte separation medium (LSM) to final purify the eggs. The second step completely removes the fecal contamination and has a 100% recovery of the eggs enriched in the first step. This two-step method, or the Sucrose-LSM method, will have a broad application in isolating eggs from feces of livestock infected with various GI nematode parasites. This information should be useful to livestock parasitology researchers and the livestock industry.

Technical Abstract: Many modern day studies require parasite samples essentially free of environmental contamination. Techniques used to produce gastrointestinal nematode eggs from livestock are sufficient for biological studies, but fall woefully short of generating pure preparations for downstream molecular, biochemical and immunological studies. Consequently, a method to produce highly purified nematode parasite eggs free from fecal contamination is needed. The present study compared different procedures for egg isolation and attempted to improve the purity of Ostertagia ostertagi eggs isolated from cattle feces. The Wisconsin method using saturated sucrose has been used widely to enrich eggs from feces and is adequate for identification and counting. A more recently developed method using the combination of salt and sucrose is robust and consistent, and can be used to isolate eggs with much higher purity; however, residual fecal materials are still present in the egg preparations which could account for large levels of non-specific DNA and RNA contamination in the final samples. Large numbers of eggs can be harvested from the medium of overnight, in vitro-cultured adult worms; however, this method is labor intensive, involves euthanasia of animals and required the purification of adult worms to high purity and subsequent culturing to acquire the eggs. In addition, a large proportion of the eggs have initiated development and hatched during isolation. In the current method, Ostertagia eggs free from fecal contamination were secondarily purified using lymphocyte separation medium (LSM) from eggs previously enriched by the Wisconsin method. The egg recovery following the LSM step was 100%. The results indicate that this two-step method involving sucrose and LSM was simple, rapid, non-selective, and greatly improved the purity of Ostertagia eggs. This method will have broad application for isolating eggs of the superfamily of Trichostrongyloidea which include the most important nematode parasites infecting livestock.