|Matthews, Benjamin - Ben|
Submitted to: Experimental Parasitology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/20/2002
Publication Date: 8/1/2003
Citation: Morimoto, M., Zarlenga, D.S., Beard, H.S., Alkharouf, N., Matthews, B.F., Urban Jr, J.F. 2003. Ascaris suum: cdna microarray analysis of 4th stage larvae (l4) during self-cure from the intestine. Experimental Parasitology. 104:113-121 (2003) Interpretive Summary: Modern molecular techniques have increased our knowledge of how genes regulate the expression of various proteins that control growth and development. These techniques have also been applied to infectious agents to try and determine the genes that are key to the disease process and how these genes and their products can be neutralized to control infection. The current study was designed to evaluate the genes that regulate the infectious process of an important worm parasite that infects both humans and swine. An array of genes from a stage of the worm infection that is critical to survival in the infected host were isolated and printed on glass slides. Genes from other stages of the worm were also isolated and labeled to determine which genes were turned on or off during the critical stage of development in the host. This technique provides a method to evaluate almost 2,000 genes at one time and can be expanded to evaluate many more. The results showed that genes that are important to muscle cell and reproductive tissue development in the worm were turned on in those worms that successfully infected the host. These results can be used to target these genes and their products to better control this important infection. This study is an example of how cooperative interaction between several laboratories at the Beltsville Agricultural Research Center can test a proof of a principal that genes from important pathogens can be evaluated to provide better strategies for control.
Technical Abstract: There is spontaneous cure of a large portion of Ascaris suum 4th-stage larvae (L4) from the jejunum of infected pigs between 14 and 21 days after inoculation (DAI). Those L4 that remain in the jejunum continue to develop while those that have moved to the ileum are eventually expelled from the intestines. Although increases in intestinal mucosal mast cells and changes in local host immunity are coincidental with spontaneous cure, the population of L4 that continue to develop in the jejunum may counteract host protective mechanisms by the differential production of factors related to parasitism. To this end, a cDNA library was constructed from L4 isolated from pig jejunum at 21 DAI, and PCR products derived from 1920 clones were verified by gel electrophoresis and printed onto glass slides for microarray analysis. Fluorescent probes were prepared from total RNA isolated from; 1) 3rd stage-larvae from lung at 7 DAI, (L3); 2) L4 from jejunum at 14 DAI (L4-14-J); 3) L4 from jejunum at 21 DAI (L4-21-J); 4) L4 from ileum at 21 DAI (L4-21-I, and; 5) adults (L5). Cy3-labeled L3, L4-14-J, L4-21-I and L5 cDNA, and Cy5-labeled L4-21-J cDNA were simultaneously used to screen the printed arrays containing the L4-21-J-derived cDNA library. Several clones showed differential gene expression and were grouped into 3 distinct transcription patterns. The data showed that sequences from muscle actin and myosin were highly expressed in L4-21-J as were a collection of un-annotated genes derived from a worm body wall-hypodermis library, and a testes germinal zone tissue library. These results suggest that only actively developing A. suum L4 are destined to parasitize the host and successfully neutralize host protective responses.