Cryptosporidium muris in a reticulated giraffe (Giraffa camelopardalis reticulata).

Cryptosporidium spp. infection in captive exotic mammals was investigated using staining and molecular biological methods. A total of 323 fecal samples from 100 mammalian species (62 Artiodactyla, 33 Rodentia, 3 Perissodactyla, and 2 Paenungultata) in 4 zoological gardens in the Czech Republic was examined. Only in a reticulated giraffe (Giraffa camelopardalis reticulata) sample was Cryptosporidium sp. infection detected. The partial small subunit rRNA sequence obtained from the isolate was identical to sequences of Cryptosporidium muris in rock hyrax (Procavia capensis) and Bactrian camel (Camelus bactrianus). Neonatal BALB/c mice inoculated with 1 x 10(3) fresh oocysts of the C. muris giraffe isolate did not produce a detectable infection.

Sources of potentially infectious human microsporidia: molecular characterisation of microsporidia isolates from exotic birds in the Czech Republic, prevalence study and importance of birds in epidemiology of the human microsporidial infections.

A total of 287 faecal specimens of captive exotic birds from the orders Psittaciformes, Passeriformes and Columbiformes were randomly collected from Bohemian pet stores, avian breeders and avian keepers and were screened for the presence of human pathogenic microsporidia by polymerase chain reaction (PCR). Microsporidial DNA was identified in 115 faecal samples (40.1%). Single-species infection was detected in 36 birds (12.5%) for Enterocytozoon bieneusi, 36 birds (12.5%) for Encephalitozoon cuniculi and 18 birds (6.3%) for Encephalitozoon hellem. No Encephalitozoon intestinalis positive samples were identified. Moreover, co-infections were detected in 25 birds: E. bieneusi together with E. cuniculi in 14 animals (4.9%) or E. hellem in 11 cases (3.8%). E. hellem was present in 1A (5.2%) and three (0.3%) genotypes, E. cuniculi in I (2.4%), II (8.0%) and III (0.7%) genotypes and E. bieneusi in A (8.4%) and EbpA (10.8%) genotypes. Several of these genotypes have never been recorded in birds before. The results of this report suggest the low host specificity of E. bieneusi, E. hellem and E. cuniculi and describe 44 new avian hosts.

[Diet in rheumatic disease]. / Ernährung bei rheumatischen Erkrankungen.

The goals of dietary therapy in rheumatic diseases are alleviation of under- and malnutrition, inhibition of inflammation, prophylaxis of osteoporosis, as well as recognition and treatment of nutrient sensitivities or intolerances.Inflammation inhibition in these patients is improved by manipulating the omega-3/omega-6 fatty acids ratio in the diet. Reduction of dietary arachidonic acid is recommended. This polyunsaturated fatty acid is the main precursor of pro-inflammatory mediators which interact with chemokines und cytokines. Simultaneously, intake of anti-inflammatory omega-3 fatty acids is increased. Studies have shown that this dietary regimen results in an amelioration of symptoms in patients with inflammatory rheumatic diseases. Dietary therapy in rheumatic diseases is often complicated by concomitant diseases and physical handicaps necessitating interdisciplinary patient care, consisting of rheumatologists, nutritionists, physiotherapists and ergotherapists. Dietary therapy of rheumatic diseases is an adjuvant therapy that should be initiated after a patient is properly diagnosed.

Detection of Encephalitozoon cuniculi in a new host--cockateel (Nymphicus hollandicus) using molecular methods.

A total of 123 avian faecal specimens randomly collected in Bohemian commercial aviaries, Zoo parks and countryside were screened for the presence of human pathogenic microsporidia by both calcofluor M2R staining and polymerase chain reaction. Of these, no positive sample was detected using microscopical examination, and one isolate was detected by polymerase chain reaction and identified as Encephalitozoon cuniculi. Cockateel (Nymphicus hollandicus) represents a new avian host of this microsporidian.

Antibodies enhance the protective effect of CD4+ T lymphocytes in SCID mice perorally infected with Encephalitozoon cuniculi.

The role of antibodies in the immune response to microsporidiosis was studied using a novel anti-exospore monoclonal antibody (MAb) P5/H1, which recognizes surface antigens of Encephalitozoon cuniculi. The effect of the MAb on microsporidial infection in vivo was to prolong the survival of previously CD4+ reconstituted, perorally infected and intraperitoneally MAb-treated SCID mice. The MAb decreased the numbers of E. cuniculi spores in peritoneal smears obtained post mortem. These results suggest a possible role for antibodies in protection against perorally acquired E. cuniculi infection.

Humoral response of chicken infected with the microsporidium Encephalitozoon hellem.

Chicken (Gallus gallus) were used as the experimental model for study of immune response against the microsporidium Encephalitozoon hellem (Didier et al., J Inf Dis 163:617-621, 1991) infection in birds. Two-day-old chicken were infected perorally or intraperitoneally with a dose of 10(7) spores of E. hellem. The anti-E. hellem immunoglobulin (Ig)A, IgY, and IgM antibody responses in sera and dropping sample extracts were determined by enzyme-linked immunosorbent assay. Results have shown specific antibody production in sera and intestinal secretions of infected birds. Chicken inoculated perorally developed the lowest antibody response. Microsporidian spores were not identified in the smears from cloacal swab samples of individual chicken. Intestinal segment cultures of perorally infected chicken cultivated in vitro showed the highest production of specific IgY and IgA antibodies in jejunum segments. In the further course of infection, the colon produced the highest amount of IgA, and the ileum and colon produced the highest amount of IgY.

Effects of a novel anti-exospore monoclonal antibody on microsporidial development in vitro.

The possible role of humoral antibodies in the immune response to microsporidiosis was studied using a novel anti-exospore monoclonal antibody (MAb) P5/H1 which recognised Encephalitozoon cuniculi. The effect of the P5/H1 MAb on microsporidial growth in vitro resulted in a reduction of the numbers of E. cuniculi spores in a Vero E6 cell-line culture. This reduction in the number of infected cells and the decrease of intracellular spores in infected cells was found when MAb P5/H1 was present in cultures, compared to cultures with an irrelevant isotype control MAb. Moreover, the presence of P5/H1 MAb increased the number of phagocytosed spores in macrophage cultures, and increased the activation of macrophages measured by nitrite oxide production. These results suggest a possible partial role of specific humoral antibodies in the protection against E. cuniculi infection.

Comparison of selected diagnostic methods for identification of Cryptosporidium parvum and Cryptosporidium andersoni in routine examination of faeces.

This study involved the comparison of suitability of different methods for routine diagnostics of Cryptosporidium spp. Two staining methods, one concentration-sedimentation method, seven concentration-floatation methods and one combined floatation-sedimentation method were compared. The methods were tested with two concentrations (1 x 10(5) and 1 x 10(6)/g) of C. parvum and C. andersoni. The methods were evaluated using light microscope, magnification 400x for concentration methods and 1000x for stained samples respectively. Specificity of both staining methods was 95-100%. Ziehl-Neelsen with P < 0.01 is more suitable for identification of C. andersoni and modified Milácek-Vítovec with P < 0.01 for identification of C. parvum. Concerning specificity and sensitivity, the floatation-concentration method by Sheather was found to provide the best results of all selected methods. The merthiolate iodine formaldehyde concentration (MIFC) method was the least specific one. The least suitable method concerning sensitivity and costs was the floatation method with caesium chloride (CsCl) with a specificity of 29%.

Helminth parasites of cats from the Vientiane province, Laos, as indicators of the occurrence of causative agents of human parasitoses.

A total of 55 domestic cats (Felis catus f. domestica) and one wild (Bengal) cat (Prionaluirus bengalensis) from the Vientiane Province, central Laos, were examined for helminth parasites with emphasis given to potential human parasites. The following species were found (parasites infective to man marked with an asterisk): Opisthorchis viverrini, Haplorchis pumilio, H. taichui, H. yokogawai, Stellantchasmus falcatus (Digenea); Spirometra sp., Dipylidium caninum, Taenia taeniaeformis (Cestoda); Capillariidae gen. sp., Toxocara canis, T. cali, Ancylostoma ceylanicum, A. tubaeforme, Gnathostoma spinigerum, Physaloptera preputialis (Nematoda); and Oncicola sp. (Acanthocephala). This study demonstrated that examination of cats may provide useful data on the occurrence of helminths which are potential causative agents of human diseases.

Monitoring of Cryptosporidium and Giardia in Czech drinking water sources.

In Czech raw water sources for drinking water supply, Cryptosporidium was found in numbers from 0 to 7400 per 100 liters and Giardia from 0 to 485 per 100 liters. The summer floods of 1997 probably brought the highest numbers of Cryptosporidium oocysts into one of the reservoirs sampled; since then these numbers decreased steadily. A relatively high number of Cryptosporidium oocysts was found in one sample of treated water. Repeated sampling demonstrated that this was a sporadic event. The reason for the presence of Cryptosporidium in a sample of treated drinking-water is unclear and requires further study.

The fate of Cryptosporidium parvum oocysts ingested by dung beetles and their possible role in the dissemination of cryptosporidiosis.

The fate of oocysts of Cryptosporidium parvum ingested by dung beetles and the possible role these beetles serve in the dissemination of cryptosporidiosis were tested on the following species: Anoplotrupes stercorosus, Aphodius rufus, and Onthophagus fracticornis. Ten specimens of each species were offered cattle dung supplemented with 5.9 x 10(6) oocysts of C. purvum. After 24 hr of feeding, the beetles were examined for the presence of oocysts on their external surfaces, in their gastrointestinal tracts, and in feces passed during the experiment. Results indicate that although many oocysts pass safely through the mouthparts and gastrointestinal tracts of the beetles, the majority of them are destroyed. Coprophagous insects can, therefore, be considered an important aspect in the ecology of gastrointestinal diseases of man and livestock, as both agents of control and dissemination.

Cytokine response to infection with the microsporidian, Encephalitozoon cuniculi.

The production of three cytokines, interferon gamma (IFN-gamma), interleukin 10 (IL-10) and interleukin 12 (IL-12), was measured after intraperitoneal infection of immunocompetent Balb/c mice and immunodeficient SCID mice with the microsporidian, Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923. High levels of IFN-gamma were detected in ex vivo cultures of peritoneal exudate cells (PEC) of Balb/c mice, a lower, but earlier IFN-gamma response was observed in PEC from SCID mice. The early IL-10 response was detected in ex vivo cultures of splenocytes from Balb/c but not from SCID mice, explaining a delay in the IFN-gamma response in Balb/c mice. IL-12 was detected in PEC cultures from SCID mice, indicating an alternative pathway of IFN-gamma production by NK cells stimulated by IL-12 derived from macrophages.

New cryptosporidium genotypes in HIV-infected persons.

Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes.

Encephalitozoon intestinale infection in an AIDS patient--a case report.

The first case of one of the most frequent intestinal microsporidians, Encephalitozoon intestinale, is reported from an AIDS patient in the Czech Republic. The patient experienced diarrhoea and was found to have microsporidia spores in stool. Species determination by electron microscopy confirmed the diagnosis of the microsporidian, E. intestinale. The CD4-count at the time of the diagnosis was 73 cells/mm3, IRI = 0.21. Only after symptomatic therapy and rehydration the patient stopped the complaining, and although he refused an antimicrosporidial therapy, the CD4-count one month later increased to 200 cells/mm3 and patient didn't suffered from diarrhoea. Six months after the first finding of microsporidia, the patient was admitted to the hospital care for progressive encephalopathy and developing wasting syndrome again with the intermittent diarrhoea. The patient was treated with albendazole at that time. Nevertheless, after 14 days of albendazole therapy, he still remained positive for E. intestinale spores in the stool (urine specimens remained negative for all the time). The patient died after a two-month hospitalisation and the apparent cause of death was purulent bronchopneumonia, wasting syndrome with microsporidiosis, and HIV encephalopathy. Generalised mycobacteriosis (MAC) was also found from the autopsy material.

The serological surveillance of several groups of patients using antigens of Encephalitozoon hellem and E. cuniculi antibodies to microsporidia in patients.

This study was undertaken to attempt to identify correlations between microsporidial seroprevalence data in man, clinical diseases and groups of people at the risk of HIV/AIDS infection. Groups of patients were selected according to the predilection of members of the genus Encephalitozoon for nervous and kidney tissue. Female prostitutes and alcohol and intravenous drug abusers were selected as groups at risk of HIV/AIDS infections. A total of 401 samples of human sera were examined for the presence of antimicrosporidial IgG antibodies by ELISA test with a titre of 600 considered borderline positivity. The highest occurrence of antimicrosporidial antibodies was found in the groups of alcohol abusers (16% from 43 patients), intravenous drug abusers (11% from 9 patients) and prostitutes (10% from 80 women) for E. cuniculi antigen and in the groups of psychiatric patients (14% from 44 patients), malaria patients (11% from 38 patients) and alcohol abusers (7% from 43 patients) for E. hellem antigen. The occurrence of specific antibodies of the six examined diagnostic units (glomerulonephritis chronica, pyelonephritis chronica, schizophrenia, dementia, multiple sclerosis and cerebral stroke) was statistically significant only in patients with pyelonephritis chronica and dementia (p < 0.05). No cases of microsporidial infection were found among the female prostitutes by parasitological examination, although one case of giardiasis was identified. Sera of patients with high anti-E. cuniculi and anti-E. hellem antibodies (titres in ELISA of 600 and above) were confirmed by Western blot using E. cuniculi and E. hellem polypeptides, respectively. These results suggest that the examined patients could show residual antibodies from past or latent infections.

Effects of an anti-exospore monoclonal antibody on microsporidial development in vitro.

In this study we evaluated the effects of the anti-microsporidial exospore monoclonal antibody 3B6, recognizing 3 Encephalitozoon species, Encephalitozoon intestinalis (Syn. Septata intestinalis), Encephalitozoon cuniculi, and Encephalitozoon hellem on microsporidial growth in vitro. Pre-treatment of spores for 24 h with mAb 3B6 resulted in 21-29% fewer infected host cells 4 days after inoculation of the cultures compared to cultures pre-treated with medium or an irrelevant isotype control mAb (P < 0.001). Fewer intracellular spores (1.2 +/- 0.2) in infected cells were found when mAb 3B6 was present in cultures compared to cultures with medium alone (4.3 +/- 0.8) or an irrelevant isotype control mAb (4.2 +/- 0.9; P < 0.001). This decrease appeared not to be dependent on time of exposure, mAb concentration, or presence of complement. It is concluded that antibodies, particularly those directed to potential neutralizing-sensitive epitopes on spores, may have a role in the control of microsporidial growth in vitro.

Simple diagnosis of Encephalitozoon sp. microsporidial infections by using a panspecific antiexospore monoclonal antibody.

Microsporidia (phylum Microsproa) have recently become recognized as common opportunistic protozoans in the United States and worldwide, particularly affecting immunodeficient patients. Microsporidian organisms within the genus Encephalitozoon are the cause of nephrologic, ophthalmic, pneumologic, gastroenteric, and systemic infections. However, diagnosis of the small spores by light microscopy is difficult, even with newly developed and improved staining techniques. We have developed an anti-Encephalitozoon species monoclonal antibody-based immunoassay for easy diagnosis. A hybridoma was produced and selected following one main criterion: recognition by immunofluorescence of all known Encephalitozoon spores affecting humans. The selected monoclonal antibody-secreting hybridomas were characterized by enzyme-linked immunosorbent assay, immunofluorescence, Western blot, and immunoelectron microscopy using Encephalitozoon species from fresh and fixed samples from patients and from in vitro cultures. In the immunofluorescence assay, one monoclonal antibody, termed 3B6, strongly recognized Encephalitozoon cuniculi, E. hellem, and E. intestinalis. Monoclonal antibody 3B6 bound to other microsporidia (Nosema and Vairimorpha spp.) without cross-reacting with any other parasite, including Enterocytozoon bieneusi, fungus, or bacterium tested. In immunoelectron microscopy assays, monoclonal antibody 3B6 bound to the exospore of Encephalitozoon species, while in Western blot assays, it recognized three to seven antigens with molecular masses ranging from 34 to 117 kDa. We have developed a sensitive and specific monoclonal antibody-based immunoassay to diagnose common microsporidian infections, particularly with Encephalitozoon species. This is a new tool for identifying spores in bodily fluids and biopsy samples and is an efficient diagnostic test. Additionally, monoclonal antibody 3B6 can serve to assess the prevalence of microsporidial infections in immunodeficient and immunocompetent patients.

Parasitic opportunistic infections in Czech HIV-infected patients--a prospective study.

To determine the frequency of opportunistic parasites, especially causative agents of microsporidiosis and cryptosporidiosis in HIV-infected patients in the Czech Republic, more than 75% of all Czech HIV-infected patients were examined during this study. Target staining techniques were used for parasitological examination of stool, sputum and urine of HIV-infected patients. In addition, their sera were examined by indirect ELISA technique with specific antigens from E. cuniculi, E. hellem and C. parvum. Specific antibodies to T. gondii were detected by CF test. In 2.1% of HIV-infected patients microsporidia E. bieneusi and E. intestinalis were found by parasitological examination, 5.3% of HIV-infected patients were seropositive to E. cuniculi and 1.3% to E. intestinalis were found by parasitological examination, 5.3% of HIV-infected patients were seropositive to E. cuniculi and 1.3% to E. hellem antigens in the high titre of 600. All blood donors (control group) were seronegative in these titres. C. parvum oocysts were found in one AIDS patient, 10.5% patients were seropositive to C. parvum in higher titres (600, 1800). Occurrence of T. gondii antibodies was not significantly greater in HIV-seropositive patients (27.5%) than in the control group (21.4%). P. carinii was detected in 13.6% of the patients. This study shows the quite low occurrence of opportunistic parasitoses among Czech HIV-infected patients. Although the high prevalence of specific antibodies against causative agents of microsporidiosis and cryptosporidiosis was recorded, the number of clinical cases was low.