The mitochondrial heat shock protein 60 (HSP60) is up-regulated in Onchocerca volvulus after the depletion of Wolbachia.

Wolbachia, a genus of endosymbiotic bacteria of filarial worms, represent novel targets for anti-filarial therapy. The efficacy of compounds against Wolbachia has been evaluated using antiserum raised against the 60 kDa heat shock protein (HSP60) which binds specifically to this protein in both Wolbachia and mitochondria. It has been shown that Wolbachia stains (using such specific probes) stronger than the mitochondria in untreated Onchocerca volvulus, whereas after the depletion of Wolbachia (with drugs) staining of the mitochondria is increased. Herein, immunogold electron microscopy showed that specific anti-HSP60 serum specifically labelled Wolbachia and filarial mitochondria, and that both have distinct localization patterns, thus allowing them to be differentiated. Immunohistochemistry of O. volvulus showed that HSP60 staining is increased in the mitochondria after Wolbachia depletion in the hypodermis, epithelia, muscles, oocytes, embryos, and developing spermatozoa. This could have been the result of the antiserum preferentially binding to the Wolbachia when they are present or due to increased expression of the protein in the absence of the bacteria. To address this, mRNA levels of filarial hsp60 in O. volvulus were measured. After the depletion of Wolbachia, the transcription of hsp60 was significantly greater (7.7 fold) compared with untreated worms. We hypothesize that the increased expression of HSP60 in the absence of Wolbachia is due to a disruption of the homeostasis of the endosymbiosis.

Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility.

Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.

Expression and subcellular localization of cpn60 protein family members in Leishmania donovani.

We have identified two diverged members of the cpn60 gene family in Leishmania donovani, causative agent of Indian Kala Azar. One of the genes, cpn60.1, although actively transcribed, is not expressed to detectable levels of protein in cultured L. donovani. The other gene, cpn60.2, which, compared with cpn60.1, shows a higher sequence conservation with the hsp60 genes from Trypanosoma brucei and Trypanosoma cruzi is expressed constitutively in cultured promastigotes. The abundance of the gene product, Cpn60.2, increases by 2.5-fold under heat stress and in axenic amastigotes of L. donovani. Cpn60.2 is also found enriched in mitochondrial cell fractions and localizes to the mitochondrial matrix. We conclude that Cpn60.2 is the major mitochondrial chaperonin in Leishmania.

Presentation by scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon.

This paper presents, for the first time, documentation by detailed scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon. Phase 1 is represented by the extracellular phase with mature spores liberated by the rupture of host cells. To infect new cells the spores have to discharge their polar filament. Spores with everted tubes show that these are helically coiled. When the polar tubules have started to penetrate into a host cell they are incomplete in length. The infection of a host cell can also be initiated by a phagocytic process of the extruded polar filament into an invagination channel of the host cell membrane. After the penetration process, the tube length is completed by polar tube protein which passes through the tube in the shape of swellings. A completely discharged polar tube with its tip is also shown. The end of a polar tube is normally hidden in the cytoplasm of the host cell. After completion of the tube length the transfer of the sporoplasm occurs and phase 2 starts. Phase 2 is the proliferative phase, or merogony, with the intracellular development of the parasite that cannot be documented by scanning electron microscopy. The subsequent intracellular phase 3, or sporogony, starts when the meronts transform into sporonts, documented as chain-like structures which subdivide into sporoblasts. The sporoblasts finally transform directly into spores which can be seen in their host cell, forming bubble-like swellings in the cell surface.

Septata intestinalis and Encephalitozoon cuniculi: cross-reactivity between two microsporidian species.

An infection with Septata intestinalis was diagnosed in a 35-year-old AIDS patient without diarrhoea. The diagnosis was based on morphological examinations of a duodenal biopsy specimen. Serum antibodies were detected reacting with spores of Encephalitozoon cuniculi. Spores of S. intestinalis and E. cuniculi stained with Brown Hopps Gram stain showed a red colour (Gram negative) and not a blue/black colour which was described for microsporidian spores in tissue.

Leishmania donovani heat shock protein 100. Characterization and function in amastigote stage differentiation.

We report the cloning and molecular analysis of the Leishmania donovani clpB gene. The protein-coding region is highly conserved compared with its L. major homologue, while 5'- and 3'-flanking DNA sequences display considerable divergence. The encoded mRNA has an unusually long 5'-leader sequence typical for RNAs, which are translated preferentially under heat stress. The gene product, a 100-kDa heat shock protein, Hsp100, becomes abundant only during sustained heat stress, but not under common chemical stresses. Hsp100 associates into trimeric complexes and is found mostly in a cytoplasmic, possibly membrane-associated, localization as determined by immune electron microscopy. Hsp100 shows immediate early expression kinetics during axenic amastigote development. In its absence, expression of at least one amastigote stage-specific protein family is impaired.

Dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient.

This report describes the first dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient. In view of clinical and epidemiological findings, our E. cuniculi isolate was deduced to be of the dog strain. The patient's occupational involvement with dogs indicates that canines should be considered as a reservoir of human infections for both microsporidial species. Furthermore, our report provides detailed clinical and radiological information on a rare case of a symptomatic pulmonary infection by E. cuniculi and its improvement after treatment with albendazole.

Targeting of Wolbachia endobacteria in Litomosoides sigmodontis: comparison of tetracyclines with chloramphenicol, macrolides and ciprofloxacin.

Endobacteria of the genus Wolbachia in filarial nematodes are related to Rickettsiaceae and can be depleted by tetracycline antibiotics. This depletion blocks female worm development as well as early embryogenesis, in contrast to the currently used microfilaricidal ivermectin which blocks only the last stage of embryogenesis. Since targeting Wolbachia is becoming an area of research for the treatment of human filariases, it was investigated if antibiotics other than tetracyclines are able to deplete Wolbachia from filariae. BALB/c mice infected with the rodent filaria Litomosoides sigmodontis were treated with erythromycin, chloramphenicol or ciprofloxacin. All drugs were well resorbed and resulted in serum levels clearly above breakpoint levels for bacteria susceptible to the respective antibiotic. However, contrary to tetracycline, none of these antibiotics depleted Wolbachia or altered worm development and fertility, as evidenced by immunohistology, immunoelectron microscopy and semiquantitative PCR.

Acute and long-term humoral immunity following active immunization of rabbits with inactivated spores of various Encephalitozoon species.

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. helleri, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections.

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction.

In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for species-specific detection of Encephalitozoon cuniculi. Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, and Enterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected with Enterocytozoon bieneusi (n = 9), Encephalitozoon spp. (n = 2), and Encephalitozoon intestinalis (n = 1) as well as stool spiked with spores of Encephalitozoon cuniculi and Encephalitozoon hellem and tissue cultures of Encephalitozoon cuniculi and Encephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected with Encephalitozoon cuniculi and Encephalitozoon hellem. Moreover, identification of Encephalitozoon spp. could be specified as Encephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates species-specific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.

Follicular dendritic cells productively infected with immunodeficiency viruses transmit infection to T cells.

Lymphoid organs have been proposed to function as the major reservoir for the human immunodeficiency virus type 1 (HIV-1). Within lymphatic tissues germinal centers represent foci of rapidly proliferating B cells governed by the interaction between B and T cells and follicular dendritic cells (FDC). Accumulating evidence suggests an important role of FDC in the pathophysiology of the acquired immunodeficiency syndrome. Direct proof for the infectibility of FDC with HIV-1 has been lacking until recently when we were able to demonstrate a CD4-independent infection of FDC in vitro. Here we report that in vitro HIV-1-infected human FDC do not only contain proviral DNA, but also produce the virus, and transmit the infection to T cells. Furthermore, electron microscopical studies on ex vivo isolated FDC from simian immunodeficiency virus (SIV)-infected rhesus monkeys revealed typical virus budding. In addition, FDC from SIV-infected rhesus monkeys transmitted the infection to T cells in vitro. Due to this central role within the immune response FDC may serve as preferential targets for HIV both by trapping of virions on their surfaces and by productive infection. During disease FDC become productively infected and may, thus, be regarded as crucial elements in viral dissemination.

Prevalence and clinical significance of intestinal microsporidiosis in human immunodeficiency virus-infected patients with and without diarrhea in Germany: a prospective coprodiagnostic study.

The prevalence of intestinal microsporidiosis among human immunodefiency virus (HIV)-infected persons with chronic diarrhea varies from 7% to 50%; thus, microsporidia are a significant source of morbidity and, occasionally, mortality among these patients. Anecdotal reports suggest that intestinal microsporidiosis is also an important infection in patients with AIDS in Germany. To determine the prevalence of microsporidiosis among HIV-infected patients in Germany, we performed a prospective coprodiagnostic study of 97 consecutive HIV-infected patients. Microsporidia were the most common enteropathogen identified in 18 (36.0%) of 50 patients with diarrhea and 2 (4.3%) of 47 patients without diarrhea (P < .001; chi2 test). Microsporidia were present in 60% of patients with chronic diarrhea and 5.9% of patients with acute diarrhea. The etiologic agent was Enterocytozoon bieneusi in 18 patients and Encephalitozoon intestinalis in two patients. The prevalence of intestinal microsporidiosis in this cohort of German patients with AIDS and diarrhea is one of the highest to be reported anywhere in the world. Microsporidiosis seems to represent one of the most important causes of diarrhea in HIV-infected patients in Germany and thus must be considered in the differential diagnosis for all AIDS patients presenting with diarrhea.

Rotavirus em Belem do Para, Brasil(estudo piloto). / Rotavirus in Belem do Para, Brazil (pilot study)

Utilizando-se as tecnicas de microscopia eletronica e ELISA, rotavirus foram detectados em 4 (16%) de 25 especimes fecais de criancas portadoras de quadro diarreico agudo. Em 2 desses 4 materiais, apenas rotavirus foram detectados, sem quaisquer outros enteropatogenos contamitantes; nos outros dois foram observadas associacoes de rotavirus com Klebsiella sp em um caso e com cogumelos em brotamento em um outro