Document Type

Thesis - University Access Only

Award Date

2003

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Michael B. Hildreth

Abstract

Echinococcus multilocularis is a zoonotic cestode which parasitizes the intestines of canines/felines during its adult stage. Microtine rodents serve as the intermediate host for this tapeworm species, and the metacestode stage is found primarily in the livers of this host forming hundreds of protoscoleces (i.e. future scoleces) within a multi-chambered hydatid cyst. Canines become infected by ingesting these proto coleces whenever they eat infected rodents. The adult stage releases egges into the canine's feces which are infectious to rodents. This cestode is also a possible threat to humans if the egg stage is ingested, causing a lethal disease known as AJveolar Hydatid Disease (AHD). Diagnosis of E. multilocularis in dogs/cats is very difficult. Dogs/cats don't exhibit symptoms even if large numbers of tapeworms are present. Also, these adult tapeworms are extremely small (1.2-mm to 9.7-mm) and their eggs are morphologically similar to those of non-zoonotic tapeworms (e.g. Taenia pisiformis). Considering these factors, fecal floats of intestinal contents are not useful for diagnosis. A promising new technique is being developed for diagnosing E. granulosus and E. multilocularis infections in dogs/cats. This technique, known as a fecal ELISA ( enzyme Jinked irnmunosorbent assay), requires 1) that the infectious agent continuously releases antigens into the host's feces, 2) that antibodies can be produced from these antigens which are specific for the infectious agent, and 3) that these antigens are very stable and resistant to digestion while in the host's intestine. Thus far, only antibodies to adult stage antigens have been tested in an Echinococcus-based fecal ELISA. Adult Echinococcus tapeworms must either be obtained from experimentally infected dogs or from naturally infected wild canines; these worms are also very small and difficult to isolate from the intestine. The adult stage is generally filled with infectious eggs, which provides a potential source for human infections if expensive precautions are not taken. These factors make it both difficult and dangerous to use this stage as a source of antigens for routine antibody production. In contrast, the protoscolex stage of E. multilocularis can be easily recovered in large numbers from laboratory-infected rodents. Additional rodents can also be infected through the serial passage of hydatid cyst material from previously infected rodents without using eggs from the adult stage. Therefore, it would likely be easier and cheaper to use the protoscolex stage as an alternative source of antigens. In a previous study, Sai Leela Sriram (1995) produced polyclonal antibodies to E. multilocularis protoscolex EIS antigens and determined, immunocytochemically, that one or more of these antibodies bound to the entire surface of adult E. multilocularis tapeworms. This indicates that at least one of these proteins released from the_protoscolex is antigenically similar to at least one surface component on the adult. Further characterization of the antiprotoscolex antibodies and protoscolex/adult shared antigens was needed in order to identify potential protoscolex proteins of diagnostic value in a fecal ELISA for E. multilocularis adults. Therefore, the primary purpose of this study was to determine the number, molecular weights and relative quantity of adult EIS proteins that are antigenically similar to those from the protoscolex. The lectin binding characteristics of these proteins were also determined because complex glycoproteins are generally more stable than non- or lightly-glycosylated proteins, and fecal antigens need to be relatively stable molecules. In addition to FJS antigens, identification and characterization of protoscolex and adult tegumental proteins is needed to compare them to the antigenically similar protoscolex EIS antigens. Components of the tapeworm tegument are continually sloughed from the worms, and therefore, likely to represent a major component of the EIS antigens. From the protoscolex stage, 13 proteins were identified by the Coomassie stain, 16 proteins were antigenically active, and 21 proteins contained at least one carbohydrate component. From all the proteins identified, 8 were stained by the Coomassie, antibody, and lectin stains. They are molecular weight (kilodaltons [kD]) proteins 62.5, 40.8, 36.1, 32.2, 26.5, 22.0, 15.3, and 9.3. This is significant because these proteins have antigenic properties, have at least one type of carbohydrate component, and had enough protein material to be identified by the Coomassie stri.in. After identifying the tegumental proteins, the 8 proteins were narrowed down to 6. These proteins had molecular weights (kD) of: 62.5, 40.8, 36.1, 32.2, 22.0, and 15.3. In the adult sample, the Coomassie stain identified 24 proteins, 16 were antigenically active, and 21 had carbohydrate components. From the proteins identified, 11 potential protein candidates are significant because both the Coomassie and antibody stains reacted with them. From these 11 proteins, 9 have carbohydrate components. Six of these proteins are possibly from the tegument. These 6 proteins are 62.5, 36.1, 32.2, 26.5, 19.4, and 15.3 based on molecular weights measured in kD. By comparing the proteins (based on molecular weights) from both the adult and protoscolex samples, a total of 4 proteins (62.5 kD, 36.1 kD, 32.2 kD, and 15.3 kD) have similar molecular weights which were stained with both the Coomassie and antibody stains. Also these protein are possibly from the tegument and it was detennined the proteins have carbohydrate components. Therefore, these proteins might represent logical candidates for the generation of monoclonal antibodies for possible use in a fecal ELISA for detecting adult E. multilocularis adults in its definitive host.

Library of Congress Subject Headings

Parasite antigens.
Dogs -- Parasites.
Cats -- Parasites.
Enzyme-linked immunosorbent assay.

Publisher

South Dakota State University

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Rights Statement

In Copyright