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.

Detection of coproantigen in early trichinellosis.

Trichinellosis has become undoubtedly worldwide in distribution. Its diagnosis relies largely on the serodiagnostic procedures which are of great value but unfortunately miss the enteric phase. This could be a serious diagnostic problem in the absence of corresponding epidemiological data and typical symptoms and signs of the disease. In this study the possibility of coproantigen detection, as an early diagnostic aid in trichinellosis, was investigated in mice experimentally infected with Trichinella spiralis. A modified double sandwich ELISA was developed using polyclonal antibodies raised in rabbits and guinea pigs against larval somatic antigens. The first detection of coproantigen was as early as the first day post infection, gradually increasing to reach its peak on the seventh day and then decreasing to disappear completely on the third week post infection. Another test, the coagglutination test (Co-A) was used, and this test confirmed the previous results. The finding of this study suggest that the coproantigen detection could be exploited to confirm ongoing early Trichinella spiralis infection. This fast and easy to use diagnostic method should improve the early infection in human.

Experimental conversion of virulent RH Toxoplasma gondii tachyzoites in vitro.

We aimed to induce conversion of RH-stain tachyzoites to bradyzoites by changing the pH of the culture medium. Alkalization of the medium to pH 8 induced morphological changes in the cultured tachyzoites. The majority of the organism increased in size and changed from a regular crescent shape to a rounded or ovoid shape. Cyst-like structures were formed. Using a computerized image analyser, significant differences in the size of the whole organisms and in their nuclei were observed compared to the control group. The converted organisms also showed significant differences from the control group by quantitative DNA analysis, and did not infect mice.

Experimental conversion of virulent RH Toxoplasma gondii tachyzoites in vitro

We aimed to induce conversion of RH-stain tachyzoites to bradyzoites by changing the pH of the culture medium. Alkalization of the medium to pH 8 induced morphological changes in the cultured tachyzoites. The majority of the organism increased in size and changed from a regular crescent shape to a rounded or ovoid shape. Cyst-like structures were formed. Using a computerized image analyser, significant differences in the size of the whole organisms and in their nuclei were observed compared to the control group. The converted organisms also showed significant differences from the control group by quantitative DNA analysis, and did not infect mice

Identification of Cyclospora cayetanensis in stool using different stains.

Cyclospora cayetanensis, a newly emerging coccidian protozoa is world-wide in distribution. In the present study, different concentrations and staining techniques were used for identification of Cyclospora. Formol-ether sedimentation and Sheather's sugar flotation were used as concentration techniques and the different stains used were: the modified Ziehl-Neelsen, Giemsa, safranin-methylene blue, modifications of trichrome stain, calcoflour white and finally phenol-auramine. The safranin stain was the best, as it stained all the oocysts of Cyclospora uniformly, besides being rapid and easily applicable in the laboratories. Phenol-auramine stained the oocysts well, where both the wall and internal contents fluoresced brightly. With the calcoflour white stain, only the wall of oocysts took that fluorescent stain. The modified Ziehl-Neelsen stained some of the oocysts well, yet great variability in the staining pattern was noticed. Cyclospora oocysts were not efficiently stained with either trichrome modifications or Giemsa stains.

A trial to enhance the efficiency of Cyclosporin A in the treatment of Schistosoma mansoni infection by immunization of mice.

Cyclosporin A (CsA) acts in two distinct modes against Schistosoma mansoni (S. mansoni) infection. It behaves in part as an antihelminthic and also as an immunomodulator. In this study, two methods were performed to increase the efficacy of the subcurative dose of CsA (30 mg/kg). These methods were, passive immunization where the drug was given simultaneously with rabbit antisera post infection and active immunization where the worm surface antigen was given prior to infection. The results showed that both methods led to enhancement of the curative effect of the CsA. However, the active immunization gave more significant reduction in liver worm and egg loads. Histopathological study showed small size and few number of hepatic granulomas in the passively immunized group while in the active group, there was just few and small collection of inflammatory cells. Moreover, the indirect fluorescent antibody test visualized damage to the worm tegument in both types of immunization. Thus, it could be concluded that active immunization increased the immunomodulatory effect of CsA in treatment of S. mansoni with less toxic effects by using a subcurative dose.

Role of anti-Trichinella spiralis antibodies and interferon-gamma in the protection against the enteral phase of experimental trichinosis.

Expulsion of adult Trichinella spiralis is the result of a complex immunologically mediated response. Experiments in mice demonstrated that IFN-gamma, released by mesenteric lymph nodes (MLN) during infection play a major role. However, the role of mucosal and serum antibody responses is thought to be limited. Working on the intestinal phase, this study investigated, the role played by antibodies, transferred from vaccinated infected animals, in the immune response in comparison to that obtained by IFN-gamma administration. Transfer of antibodies gave 82.7% protection, while IFN-gamma in a dose of 1 x 10(4) U gave 98.4% protection. The use of half the previous dose induced a protection of 58.3%. These data indicate that vaccination followed by infection could generate antibodies capable of producing a protective immune response against the intestinal phase of T. spiralis. This reached a level near to that obtained by IFN-gamma administration.

Detection of Microsporidia by different staining techniques.

Previous detection of Microsporidia relied mainly on electron microscopy and histopathology. Recently, non invasive methods were able to recognize this microorganism. In the present study, different stains were used as a means of diagnosing spores of Microsporidia in stool samples of immunosuppressed patients. The original modified trichrome stain (MTS) was used as a standard screening technique for all stool samples. Positive samples for Microsporidia were then stained with the trichrome blue stain, Didier's trichrome blue stain, acid-fast trichrome stain (AFT), modified Ziehl-Neelsen stain, giemsa stain and calcofluor white M2R stain. Both calcofluor and the AFT stains were most efficient. They could simultaneously detect coccidial oocysts and microsporidial spores. This is beneficial and time-saving in the diagnosis of stool samples of immunosuppressed patients, which usually contain more than one opportunistic protozoon. Both stains are easy to perform and require the least amount of staining and examination.

Study and characterization of different developmental stages of Leishmania major.

The complete developmental cycle of Leishmania major in axenic culture was achieved by simply changing the temperature whether sudden from 22 degrees C to 37 degrees C or stepwise 22 degrees C, 29 degrees C and then 37 degrees C. The morphology by light microscopy, GPI isoenzyme pattern and PCR amplification of minicircle of kinetoplast DNA of the different stages were studied. The amastigotes obtained from the foot pads of mice were compared to those obtained from axenic culture. The GPI isoenzyme pattern and the PCR amplification products showed distinct differences between the promastigotes and the amastigotes. The amastigotes of the two sources also showed differences after temperature changes.

Preliminary identification of an intestinal coccidian parasite in man.

An unidentified coccidian parasite was observed in saline faecal smears of five immunocompromised patients. Very few of these organisms were observed in the oocystic stage. They were oval in shape with rounded edges and contained two sporulated sporocysts, 7.5-9.5 um in diameter with a nipple like projection. They are smaller than sporocysts of Sarcocystis hominis and suihominis. For further identification, the modified Ziehl-Neelsen stain was used. The sporocysts showed variability in staining. Most of them were unstained while those stained were more or less rounded with well defined four crescent shape sporozoites and a residual body but with an unstained cyst wall. Sporocysts were positively stained by phenol auramine which had the advantage of staining the cyst wall. Other coccidial parasites encountered in this study were Cryptosporidium parvum (18.2%), Cyclospora cayetanensis (1.8%) and Isospora belli (0.9%) they were differentiated from this organism. SEM showed oval sporocysts with thickened ridges on its surface dividing it into plates which is a characteristic feature of the family Sarcocystidae. From the above characters, the features of this parasite is suggested to be a Sarcocystis like Protozoa.

Leishmania major: leishmanicidal activity of activated macrophages by interferon gamma or tumour necrosis factor.

Infective, stationary growth phase metacyclic promastigotes of Leishmania major were introduced to murine peritoneal macrophages and incubated with recombinant murine tumour necrosis factor (TNF) or with interferon gamma (IFN-gamma) in the presence of LPS. The leishmanicidal activity of macrophages was estimated by the visual counting of intracellular parasites as well as by the (3H) thymidine incorporation of the residual parasites. The culture supernatants were also collected for the estimation of nitrite (NO-2) and hydrogen peroxide (H2O2). The results showed that both (TNF) and (IFN-gamma) had a significant leishmanicidal effect, yet it was more pronounced with IFN-gamma. The production of NO-2 and H2O2 were also correlated with the increased leishmanicidal activity in the activated macrophages.