Immunochemical studies of Toxocara canis proteases.

Proteases of nematodes play a crucial role in larval molting and, in addition to their active role in egg hatching, proteases are also considered a crucial factor in tissue invasion and connective tissue remodeling. In Toxocara canis, proteases play important roles throughout the complex life cycle. They can degrade components of a model of extracellular matrix, basement membranes and different physiological substrates. In the present study, measurements of the proteolytic activity of the perivitelline fluid (PF) surrounding Toxocara canis embryos at different stages of development, the hatching fluid (HF) surrounding the infective larvae, as well as the excretory secretory (ES) products of the larvae in the culture media were performed. Measurements were made using casein as substrate following the Sigma non-specific protease activity assay. The results showed that enzyme activity increased as the embryo matured. The infective larvae were found to continuously produce proteases in the surrounding HF and ES products after in vitro cultivation indicating that Toxocara canis proteases might be important for the worm in the egg and the host. Optimal enzymatic activity was found at pH 8. Incubation of the antiserum from infected mice with the HF and ES products decreased their proteolytic activities, suggesting that there may be a link between the proteases present in these fluids and the immune response.

Immunochemical studies of Toxocara canis proteases

@#Proteases of nematodes play a crucial role in larval molting and, in addition to their active role in egg hatching, proteases are also considered a crucial factor in tissue invasion and connective tissue remodeling. In Toxocara canis, proteases play important roles throughout the complex life cycle. They can degrade components of a model of extracellular matrix, basement membranes and different physiological substrates. In the present study, measurements of the proteolytic activity of the perivitelline fluid (PF) surrounding Toxocara canis embryos at different stages of development, the hatching fluid (HF) surrounding the infective larvae, as well as the excretory secretory (ES) products of the larvae in the culture media were performed. Measurements were made using casein as substrate following the Sigma non-specific protease activity assay. The results showed that enzyme activity increased as the embryo matured. The infective larvae were found to continuously produce proteases in the surrounding HF and ES products after in vitro cultivation indicating that Toxocara canis proteases might be important for the worm in the egg and the host. Optimal enzymatic activity was found at pH 8. Incubation of the antiserum from infected mice with the HF and ES products decreased their proteolytic activities, suggesting that there may be a link between the proteases present in these fluids and the immune response.

Encephalitozoon intestinalis: A new target for auranofin in a mice model.

Despite the fact that many approaches have been developed over years to find efficient and well-tolerated therapeutic regimens for microsporidiosis, the effectiveness of current drugs remains doubtful, and effective drugs against specific targets are still scarce. The present study is the first that was designed to evaluate the potency of auranofin, an anti-rheumatoid FDA approved drug, against intestinal Encephalitozoon intestinalis. Evaluation of the drug was achieved through counting of fecal and intestinal spores, studying the intestinal histopathological changes, measuring of intestinal hydrogen peroxide level, and post therapy follow-up of mice for 2 weeks for detection of relapse. Results showed that auranofin has promising anti-microsporidia potential. It showed a promising efficacy in mice experimentally infected with E. intestinalis. It has revealed an obvious reduction in fecal spore shedding and intestinal tissue spore load, amelioration of intestinal tissue pathological changes, and improvement of the local inflammatory infiltration without significant changes in hydrogen peroxide level. Interestingly, auranofin prevented the relapse of infection. Thus, considering the results of the present work, auranofin could be considered a therapeutic alternative for the gold standard drug 'albendazole' against the intestinal E. intestinalis infection especially in relapsing cases.

A new scope for orlistat: Effect of approved anti-obesity drug against experimental microsporidiosis.

As the current therapies for intestinal microsporidiosis are either inconsistent in their efficacies or hampered by several adverse effects, alternative antimicrosporidial agents are being sought. The present study is the first that was designed to evaluate the potency of orlistat, an approved anti-obesity drug, against intestinal microsporidiosis caused by both Enterocytozoon bieneusi and Encephalitozoon intestinalis. Results were assessed through studying fecal and intestinal spore load, intestinal histopathological changes, viability, and infectivity of spores from treated animals. Results showed that orlistat has promising antimicrosporidia potential, with better results in E. intestinalis than E. bieneusi. The animals that received orlistat showed statistically significant decrease in the fecal and intestinal spore load, when compared to the corresponding control infected nontreated mice. The results were insignificant compared to fumagillin and albendazole. Light microscopic examination of stained intestinal sections revealed amelioration of the pathological changes and decreased inflammatory cells detected in the control infected nontreated mice. Spores encountered from stool of orlistat-treated E. bieneusi and E. intestinalis mice showed low viability and significant reduction of infectivity versus their control. Thus, considering the results of the present work, orlistat proved its effectiveness against the intestinal microsporidial infection.

Effect of ozone on the viability of some protozoa in drinking water.

Water samples from household tanks in several districts of Alexandria, were examined for protozoa contamination and study to the effect of ozone and chlorine on their viability. The parasitic protozoa encountered were Giardia (56%), Cryptosporidia (50%), Blastocystis (12%), Cyclospora (9%) and Microsporidia (3%). Distilled water was treated by ozone or chlorine. Ozonated-water samples were prepared at a concentration of 1 ppm after contact times with ozone of three, five, seven and nine minutes. Chlorine concentrations were 4 and 8 ppm. Parasites were incubated overnight at room temperature with the treated distilled water. Viability of the parasites was assessed by examining intestinal sections of infected animals. The best results were obtained by using ozonated water after a contact time of nine minutes. Giardia, Cryptosporidia and Microsporidia were completely inactivated, while infectivity of Cyclospora and Blastocystis was markedly reduced. It is suggested that ozone at an appropriate concentration inactivate pathogenic protozoa in water.

Morphology, histochemistry and infectivity of Blastocystis hominis cyst.

Different morphological forms of Blastocystis hominis had been identified in human stool samples. These included both cystic and trophic stages. The latter was induced to encyst by keeping them in potassium dichromate solution for two weeks. Suspected of being the infective stage, cysts were studied in more detail as regards their morphology using both light and electron microscopy. Histochemistry and infectivity studies were also carried out. Light microscopy revealed the cysts to be ovoid or round 5-7 microm with thick cyst wall and a single nucleus. The induced cysts were morphologically similar to the cysts present in the stool. By electron microscope, the cyst wall was evident surrounded by an additional fibrillar coat. The cytoplasm contained one nucleus, many mitochondria, glycogen deposits and a number of variable sized vacuoles. Histochemical studies detected carbohydrates in the cyst wall and fat globules in the cytoplasm. Oral inoculation of albino mice with these cysts led to inflammatory changes in the large and small intestine. The parasite was found at the mucosal epithelium but with no invasion. Different forms of the parasite were detected in the lumen of the intestine.

Metacyclogenesis of Leishmania major in an acidic medium.

An acidic medium was used to stimulate the production of a homogeneous stationary phase promastigotes, morphologically and functionally similar to the in vivo infective form. They possessed a short (< 8 Um) and narrow (< 1.5 Um.) cell body with a flagellum twice or more its length. They were PNA, highly infective to peritoneal macrophages in vitro and expressed high phosphatase activity. By elevating the incubating temperature, almost all of these promastigotes were transformed to amastigotes.

Studies on a newly emerging protozoal pathogen: Cyclospora cayetanensis.

Cyclospora is frequently misdiagnosed with Cryptosporidia. Stool samples were collected from 150 immunocompromised patients and concentrated by the parasep faecal parasite concentration and the discontinuous percoll gradients methods. Wet mount examination was done and the parasite was successfully stained with modified Ziehl Neelsen (ZN) and safranin methylene blue stain. Cyclospora was detected in 4% of cases examined. Cyclospora was easily differentiated from Cryptosporidia by using the modified detergent ZN stain whereby Cyclospora resist staining and Cryptosporidia pick up the pink colouration. Scanning and transmission electron microscopic examination were done to the unsporulated Cyclospora oocysts. They appeared as spherical objects with an outer fibrillar coat, an indentation and sutures. These spherical objects also contained light and dark granules. In studying the possible sources of transmission of this parasite, sporulated and unsporulated oocysts were detected in tap water and lettuce heads which support the theory that water and food could be the sources of transmission of this parasite.

A study on patients with eosinophilia of suspected parasitic origin.

A study of 53 patients with eosinophilia of suspected parasitic origin was conducted. Investigations done for each case included, stool and urine examinations, absolute eosinophilic count and countercurrent immunoelectrophoresis (CIEP) using crude antigens of Fasciola hepatica, Schistosoma mansoni worms, Trichinella spiralis and Toxocara canis larvae, and hydatid fluid. Stool examination revealed six cases with Fasciola eggs, two with Ascaris lumbricoides and one with S. mansoni eggs. The eosinophilic count ranged from 682 to 10560 cell/mm3. 26 cases showed mild eosinophilia, 24 moderate and three marked eosinophilia. Out of the 53 cases examined by the CIEPT, 30 were positive with Fasciola antigen. Their eosinophilic count ranged from 770 to 10560 cell/mm3. Six cases were positive with hydatid antigen and had an eosinophilic count ranging from 825 to 2970 cell/mm3. Only one case was positive with S. mansoni antigen and its eosinophilic count was 935 cell/mm3. 16 cases were negative with the different antigens used and their eosinophilic count ranged from 682 to 2200 cell/mm3.

Schistosoma mansoni: a comparative study on two cercarial transformation methods.

An agar: gelatin plate method, incorporating varying concentrations of linoleate was devised to measure cercarial penetration and transformation in vitro. Schistosoma mansoni cercariae were stimulated over a wide range of linoleate concentrations, while the transformation process occurred over a narrow range. Penetration rates rose gradually until at linoleate concentrations of 0.3 mM and greater, where penetration approached 100% while transformation just started to occur. The latter then reached maximum (90%) at 3 mM. This optimal concentration of linoleate leading to maximal transformation was then compared with a mechanical transformation method in relation to proteolytic activity and nuclear content. It was found that, proteolytic activity was higher by the chemical than by the mechanical transformation method. The nuclear content decreased by both techniques. This decrease was greater when the mechanical transformation method was applied.