In vivo killing of Giardia trophozoites harbouring bacterial endosymbionts by intestinal Paneth cells: an ultrastructural study.

To date Paneth cells have not previously been reported to kill Giardia trophozoites and other protozoa in vivo. Here we report the first evidence for in vivo killing of Giardia trophozoites by intestinal Paneth cells. Transmission electron microscopic (TEM) examination of duodenal specimens taken from naturally infected mice revealed that only Giardia trophozoites harbouring peripheral bacterial endosymbionts were destroyed and lysed in the vicinity of the activated Paneth cells. Additionally, intestinal epithelium was more affected by Giardia harbouring bacterial endosymbionts than Giardia with no endosymbionts. Our findings imply that the bacterial endosymbionts within Giardia trophozoites have a role in both host protective and pathological mechanisms, probably through altering the trophozoite antigencity. These observations might shed light on the diversity in infectivity and host specificity of Giardia species.

Immunolocalization of an enterotoxic glycoprotein exoantigen on the secretory organelles of Cryptosporidium parvum sporozoites.

In this study, the fine ultrastructures of the secretory organelles of C. parvum sporozoites were demonstrated using transmission electron microscopy (TEM). Meanwhile, a previously identified enterotoxic 18-20 kDa copro-antigen (18-20 kDa CCA), associated with cryptosporidiosis in both human and calves, was isolated and immunolocalized on C. parvum sporozoites. Using immunoelectron microscopy and anti-18-20 kDa monospecific antibody demonstrated marked existence of the 18-20 kDa CCA on the apical organelles and at the trilaminar pellicles. An anterior extrusion-of this protein was demonstrated around the excysted and released sporozoites. However, non excysted sporozoites did not show this protein. Affinity blotting, with biotinylated jacalin, demonstrated the O-linked oligosaccharide moiety of this protein. The potential role of this protein in the host cell invasion and/or gliding motility remains unelucidated. However, its enterotoxicity, location and secretory nature suggest that it may be a target for neutralization or invasion inhibition of Cryptosporidium.

Intravascular detection of Giardia trophozoites in naturally infected mice. An electron microscopic study.

During routine transmission electron microscopic (TEM) examination of mice naturally infected with Giardia muris, an intense infection with Giardia trophozoites was demonstrated within intestinal and renal tissues. Examination of randomly taken sections from these heavily infected tissues revealed marked deep affection with mixed pathology. Duodenal sections were found loaded with Giardia trophozoites in intimate contact with necrotic gut cells. Some of these trophozoites were detected within central lacteal of damaged villi and nearby blood vessels. Interestingly, and for the first time to be demonstrated, morphologically identical G. muris trophozoite was detected in a renal blood vessel. An intense cellular immune reaction was obviously demonstrated with remarkable interaction between giant macrophages and the trophozoites particulates. Involvement of deep tissues by Giardia trophozoites and their presence within vascular channels could open up questions about the possible invasive and disseminative behavior of G. muris, particularly in heavily and naturally infected hosts.

An enterotoxic activity of 18-20 kDa Cryptosporidium parvum secretory coproantigen (CCA 18-20 kDa).

An 18-20 kDa Cryptosporidum coproantigen (18-20 CCA), had been detected in the stool of infected humans and calves. A purified, electroeluted and concentrated 18-20 kDa antigen was tested in Ussing chamber, and electric parameters were tested before and after the addition of this antigen. A significant increase in the short circuit current (Isc) was detected. The enterotoxic effectof 18-20 kDa CCA, was time and dose dependent, heat labile, and Cl- dependent. The detected change in the short circuit current was not detected when the 18-20 kDa antigen wa incubated with its monospecific antibodies. These results indicate an enterotoxic activity of the 18-20 kDa antigen secreted from the released parasite and detected in stool of infected humans and calves. Additionally, they may help in developing an appropriate anti-secretory therapy for the intractable diarrhoea caused by cryptosporidiosis.

Immunofluorescent detection of both Giardia lamblia and Cryptosporidium parvum using anti-Cryptosporidium oocyst antibodies.

Giardia lamblia and Cryptosporidium parvum are two protozoal parasites proved to have a major role in gastroenteritis in humans. Both are documented to coexist in many waterborne parasitic transmission, as well as in outbreaks. In the present work, a polyspecific anti-Cryptosporidium oocyst antibodies were used for simultaneous detection of both parasites in human stool. Known positive formalinized human stool specimens of Giardia sp. (n = 10), Cryptosporidium sp. (n = 7), mixed infection (n = 3), and negative specimens (n = 20), were tested using direct fluorescent technique against the developed antibodies. All positive stool samples for Cryptosporidium and 9 out of 10 Giardia samples, or each alone showed fluorescence with variable intensities, while no negative sample harboured other parasites had fluorescence. This newly used polyspecific antibodies offer the advantages of screening large number of patients, particularly in outbreaks. Additionally, it represents a cheaper alternative for the most sophisticated and costly immunoassay kits using the monoclonal antibodies, with more or less the same diagnostic potentials.

Detection of Fasciola-specific excretory/ secretory (E/S) protein fraction band (49.5 kDa) and its utilization in diagnosis of early fascioliasis using different diagnostic techniques.

The objective of the present work is to evaluate Fasciola E/S antigens for diagnosis of early fascioliasis utilizing different diagnostic techniques. Using enzyme-linked immunoelectro-transfer blot (EITB), Fasciola-specific E/ S protein fraction band (49.5 kDa) was determined and electroeluted. The mono-specific antibodies against this specific fraction band were prepared by immunizing pathogen-free rabbit. Assessment of the prepared mono-specific antibodies in diagnosis of human fascioliasis was performed through the detection of E/S copro-antigens by enzyme-linked immunosorbent assay (ELISA) in stool eluates obtained from patients with confirmed fascioliasis, other parasites as well as from other healthy individuals. Serum samples were collected and tested to detect serum antibodies against Fasciola E/S antigen using EITB and counter immunoelectrophoresis (CIEP). Analysis of Fasciola adult worm E/S products by SDS/PAGE revealed a number of bands, the molecular weight (MW) of which ranged from 14-200 kDa; with three major bands (27.5, 32.5 and 55 kDa). Fasciola EIS 49.5 kDa protein fraction proved to be specific to F. gigantica. Cross reaction with S. mansoni was observed at higher MW (110-120 kDa). The sensitivity, specificity and diagnostic accuracy of EITB were 45.2%, 100% and 70.7%, respectively, while those of CIEP were 38.7%, 100% and 67.2%, respectively. ELISA technique using mono-specific anti-49.5 kDa to detect copro-antigens proved to be practical and reliable. It showed higher sensitivity (91.4%) and higher diagnostic accuracy (91.8%), while the specificity was 92.3%. In addition, ELISA had higher negative predictive value (88.9%) and fair positive predictive value (94.1%).

Intestinal opportunistic parasites among different groups of immunocompromised hosts.

The present study was designed to find out the prevalence of different intestinal opportunistic parasites in different groups of immunocompromised hosts; patients suffering from malignancy with or without immuno-suppressive drugs, or with chronic renal failure, or diabetes mellitus, or subjects under cortisone therapy. Examination of stool samples collected from 427 immunocompromised hosts revealed the detection of intestinal opportunistic parasites in 98 samples, with a prevalence of 23%. Infection with opportunistic parasites was higher in males than in females (32.6% versus 12.9%). There was statistically significant association of infection with G. lamblia and Cryptosoporidium in the mixed infection detected. The highest group affected with the opportunistic parasites was the group of patients under corticosteroid therapy (31.7%), followed by patients suffering from renal failure (28.8%) and malignancy (25.7%), while the least group affected were the diabetic patients (8%). The highest prevalence rate was 10.3% for G. lamblia, being followed by E. histolytica (7%), C. parvum (6.3%), Microsporidia (2.3%) and the least one was for Strongyloides (0.7%). No cases of Isospora belli infection was detected. Relation between presence of opportunistic parasites and risk factors was discussed and evaluated.