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Title: Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum Oocysts

item Jenkins, Michael
item EAGLESHAM, B - Cornell University
item ANTHONY, L - Cornell University
item KACHLANY, S - Cornell University
item BOWMAN, D - Cornell University
item GHIORSE, W - Cornell University

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/15/2010
Publication Date: 3/5/2010
Citation: Jenkins, M., Eaglesham, B., Anthony, L., Kachlany, S., Bowman, D., Ghiorse, W. 2010. Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum Oocysts. Applied and Environmental Microbiology. 76:1926-1934.

Interpretive Summary: Cryptosporidium parvum (Crypto for short) is an intestinal parasite known to contaminate municipal water supplies. It can cause serious illness to healthy individuals and death to individuals with compromised immune systems. The source of this parasite in the environment is feces from infected animals that shed the parasite in the form of a microscopic egg called an oocyst. This oocyst contains four infectious agents (called sporozoites), and protects them from environmental stresses and standard forms of disinfection. The wall of the oocyst is known to maintain the survival of the sporozoites within it for many months in soil and water. The oocyst wall also plays a role in its movement with water through soil and across landscapes. To better understand how the Cryptosporidium oocyst can survive environmental stresses and standard methods of disinfection an ARS scientist at the J. Phil Campbell, Jr., Natural Resource Conservation Center, and scientists and students at Cornell University systematically analyzed the chemical composition and structure of the oocyst wall. Analysis was undertaken on both whole oocysts and purified oocyst walls. Combining the use of the electron microscope, and various instruments and methods of chemical analysis a model picture of the oocyst wall was formulated. The oocyst wall was observed to be constructed in four thin laminated layers. The outside layer appeared to be a complex of polysaccharides composed of various sugars such as glucose, mannose, and galactose. The next inner translucent layer appeared to be composed of long-chain fatty acids, alcolols and wax-like compounds. Next to this inner layer of fats and waxes is a layer of proteins that give rigidity and form to the oocyst wall. The inner-most layer is composed of polysaccharides. The outside layer of the wall affects the movement of the oocyst through soil and across landscapes. The translucent inner layer controls the permeability of the wall and is affected by temperature--with low temperatures making the wall impermeable, and high temperatures making the wall permeable. The inner protein layer maintains the rigidity the shape of the oocyst, and the inner most layer may provide additional protection to the sporozoites. This is important information for scientists studying the survival and transport of this parasite in the environment. The information may be useful for developing new and improved practices for environmental protection agencies and managers of municipal water works.

Technical Abstract: The structure and composition of the oocyst wall are primary factors determining the survival of Cryptosporidium parvum oocysts outside the host. An external polymer matrix (glycocalyx) may mediate interactions with environmental surfaces and, thus, affect the transport of oocysts in water, soil, and waste water treatment systems. Whole oocysts and purified oocyst walls free of residual bodies were analyzed by light and transmission electron microscopy (TEM), by thin-sectioning, freeze-fracturing, freeze-substitution and with a variety of biochemical analyses to determine macromolecular composition. TEM thin sections indicated that the multi-layered oocyst wall consisted of an irregular outer electron dense layer, a translucent middle layer and two inner dense layers. The irregular outer dense layer and translucent middle layer appeared to cover a characteristic suture structure embedded in the inner layers. Freeze-fracture of whole oocysts produced uniformly smooth, particle-free fracture faces on convex surfaces that showed the suture as a zipper-like structure within the smooth fracture face. Weaknesses in the translucent middle layer observed as separations in thin sections indicated that the fracture likely occurred along the thin electron translucent middle layer of the wall. Freeze substitution displayed a substantial layer of glycocalyx polymers external to the weak translucent layer. Alcian Blue staining confirmed the presence of an ephemeral acidic polymer matrix. Thin layer chromatography detected glycolipids. Gas chromatography-mass spectrometry (GC-MS) determined glucose, galactose, mannose, and talose to be the principal carbohydrates in the oocyst wall. Fluorescent lectin-binding (Concanavalin A) confirmed the presence of mannose in the inner wall polysaccharides and glycocalyx. GC-MS detected medium and long chain fatty acids, hexadecanoic, 6-hexadecenoic, octadecanoic, 9-octadecenoic, and 11,14-eicosadienoic acid, and fatty alcohols, 2-decanol, dodecanol, tridecanol, 1-octanol, and 1-teraoctanol. Significant amounts of long-chain aliphatic hydrocarbons up to C39 with melting points ranging from 10' to 81'C were identified in chloroform-methanol extracts. Purified oocyst walls were stained uniformly with Magnesium anilinonaphthalene-8-sulfonic acid (Mg ANS) indicating the presence of hydrophobic proteins. Untreated purified walls contained 7.5% Lowry protein with five major bands in SDS-PAGE gels ranging from 11.0 to 87.5 kDa. After lipid extraction, the purified walls showed twelve bands ranging between 10.6 and 173 kDa. These results support a model of the oocyst wall as tough, flexible structure, which depends on layers of glycolipoproteins and lipopolysaccharide for strength and flexibility, which can affect survival of the oocysts in natural environments. The presence of waxy, long-chain hydrocarbons in a layer containing complex lipids covering the suture may also affect survival by allowing oocyst walls to be more rigid and less permeable at lower temperatures and more flexible and permeable at elevated temperatures based on the melting points of the long chain hydrocarbons. An extensive, but ephemeral glycocalyx consisting of easily removed, acidic polysaccharides could dramatically affect oocyst transport in natural environments and might explain the variable surface properties noted in some oocyst transport studies.