Limited effect of adaptive immune response to control encephalitozoonosis.

This study revises our understanding of the effectiveness of cell-mediated adaptive immunity and treatment against microsporidia using molecular detection and quantification of microsporidia in immunocompetent C57Bl/6 and immunodeficient CD4-/- and CD8-/- mice for the first time. We demonstrate an intense dissemination of microsporidia into most organs within the first weeks post-infection in all strains of mice, followed by a chronic infection characterized by microsporidia persistence in CD4-/- and C57Bl/6 mice and a lethal outcome for CD8-/- mice. Albendazole application reduces microsporidia burden in C57Bl/6 and CD4-/- mice, whereas CD8-/- mice experience only a temporary effect of the treatment. Surprisingly, treated CD8-/- mice survived the entire experimental duration despite enormous microsporidia burden. On the basis of our results, we conclude that microsporidia survive despite the presence of immune mechanisms and treatments that are currently considered to be effective and therefore that CD8 T lymphocytes represent a major, but not sole effector mechanism controlling microsporidiosis. Furthermore, the survival of mice does not correspond to spore burden, which provides new insight into latent microsporidiosis from an epidemiological point of view.

The genome of an Encephalitozoon cuniculi type III strain reveals insights into the genetic diversity and mode of reproduction of a ubiquitous vertebrate pathogen.

Encephalitozoon cuniculi is a model microsporidian species with a mononucleate nucleus and a genome that has been extensively studied. To date, analyses of genome diversity have revealed the existence of four genotypes in E. cuniculi (EcI, II, III and IV). Genome sequences are available for EcI, II and III, and are all very divergent, possibly diploid and genetically homogeneous. The mechanisms that cause low genetic diversity in E. cuniculi (for example, selfing, inbreeding or a combination of both), as well as the degree of genetic variation in their natural populations, have been hard to assess because genome data have been so far gathered from laboratory-propagated strains. In this study, we aim to tackle this issue by analyzing the complete genome sequence of a natural strain of E. cuniculi isolated in 2013 from a steppe lemming. The strain belongs to the EcIII genotype and has been designated EcIII-L. The EcIII-L genome sequence harbors genomic features intermediate to known genomes of II and III lab strains, and we provide primers that differentiate the three E. cuniculi genotypes using a single PCR. Surprisingly, the EcIII-L genome is also highly homogeneous, harbors signatures of heterozygosity and also one strain-specific single-nucleotide polymorphism (SNP) that introduces a stop codon in a key meiosis gene, Spo11. Functional analyses using a heterologous system demonstrate that this SNP leads to a deficient meiosis in a model fungus. This indicates that EcIII-L meiotic machinery may be presently broken. Overall, our findings reveal previously unsuspected genome diversity in E. cuniculi, some of which appears to affect genes of primary importance for the biology of this pathogen.

Prevalence and molecular characteristics of urinary and intestinal microsporidia infections in renal transplant recipients.

Transplant recipients have been identified as a new risk group for microsporidia infection. We characterize for the first time the prevalence of microsporidia in intestinal and urinary tracts of renal transplant recipients. Molecular examination of 86 patients showed that 25.5% of them were infected; 86% were confirmed to have pathogens in their urine and 45.5% in stool. Among positive patients, 32% had microsporidia confirmed in both urine and stool. Genotyping revealed Encephalitozoon cuniculi (59%) and Enterocytozoon bieneusi (23%) monoinfections as well as coinfections with both species (18%). Moreover, we found diarrhoea and fever as symptoms significantly associated with microsporidia presence. Our results indicate that microsporidial infection should be considered in the assessment of renal transplant recipients, especially in the urinary tract, even if asymptomatic. Molecular identification of microsporidia species is relevant because of their different susceptibility for treatment.

Significantly higher occurrence of Cryptosporidium infection in Roma children compared with non-Roma children in Slovakia.

Cryptosporidiosis is considered to be a widespread world zoonosis. The occurrence of Cryptosporidium species was investigated in Roma children in a district of Eastern Slovakia and, at the same time, also in children of non-Roma parents. In total, 103 children (54 boys and 49 girls) between 0 and 14 years of age were involved in this study. Fifty-three were Roma children and 50 children represented a non-Roma control group. Fecal samples were examined: immunologically [enzyme-linked immunosorbent assay (ELISA) test to prove antigen in the feces] and by molecular analysis [nested polymerase chain reaction (PCR)]. After the sequencing of the PCR, the products were identified as species of Cryptosporidium muris. Based on the results, the relative risk (RR) of the Cryptosporidium infection occurrence was calculated and we came to the conclusion that the risk of Cryptosporidium infection was almost 12 times higher in the Roma children compared to the non-Roma children.

Activated CD8+ T cells contribute to clearance of gastric Cryptosporidium muris infections.

The role of CD4+ and CD8+ T lymphocytes in the development of a protective immune response against Cryptosporidium muris infection was studied by the reconstitution of severe combined immunodeficient (SCID) mice with well-defined populations of either naive or immune CD8+ or CD4+ T lymphocytes. Adoptive transfer of both naive and immune CD4+ T lymphocyte subpopulations protects SCID mice against cryptosporidiosis. Moreover, a significant biological impact of activated CD8+ T cells against gastric cryptosporidiosis was observed. The significant difference in the course and intensity of the infection in reconstituted SCID mice was found to be dependent on the protective function of both the CD4+ and CD8+ T-cell populations transferred. While SCID mice reconstituted with either immune or naive CD4+ or immune CD8+ T-cell subpopulations resolved the infection within 29, 37 and 51 days post-infection, respectively, those reconstituted with naive CD8+ T cells suffered from chronic infection similar to control SCID mice. Reconstitution with CD4+ T cells resulted in suppression of oocyst excretion and shortening of patent period in comparison with SCID mice reconstituted with CD8+ T cells. Thus, although CD4+ T cells are considered important in protective immunity, our results are the first to demonstrate the involvement of activated CD8+ T lymphocytes in the protection of mice against gastric cryptosporidiosis.

Molecular characterization of Cryptosporidium spp. in pre-weaned dairy calves in the Czech Republic: absence of C. ryanae and management-associated distribution of C. andersoni, C. bovis and C. parvum subtypes.

A total of 750 faecal samples of dairy calves at up to 2 months of age kept in various housing systems were screened for Cryptosporidium spp. infection using the aniline-carbol-methyl violet staining method. DNA was extracted from Cryptosporidium positive samples and from 150 randomly selected microscopically negative samples. Nested PCR was performed to amplify the partial SSU rRNA gene of Cryptosporidium that was subsequently digested by SspI, VspI and MboII restriction enzymes to determine the present Cryptosporidium species and genotype. In addition, the samples characterized as Cryptosporidium parvum were subsequently analyzed at the GP60 gene to determine the distribution of zoonotic subtypes. Sequence analyses and RFLP identified C. parvum in 137, Cryptosporidium andersoni in 21 and Cryptosporidium bovis in 3 samples. Neither mixed infections nor Cryptosporidium ryanae was detected. Sequencing of the GP60 gene from C. parvum-positive samples revealed all five subtypes of family IIa (A15G2R1, A16G1R1, A22G1R1, A18G1R1, and A15G1R1). The obvious management-associated distribution of Cryptosporidium spp. was demonstrated. Direct contact with adult animals was found to be a risky factor for C. andersoni and C. bovis infection. IIaA15G2R1 and IIaA16G1R1 were detected as major subtypes, whereas only the IIaA16G1R1 subtype was found in animals kept in boxes. Three of the five detected subtypes were previously associated with human cryptosporidiosis, and moreover, the IIaA15G1R1 subtype, previously reported in humans only, was detected in calves for the first time.

Microsporidiosis and Cryptosporidiosis in HIV/AIDS Patients in St. Petersburg, Russia: Serological identification of microsporidia and Cryptosporidium parvum in sera samples from HIV/AIDS patients.

To determine seroprevalence of the opportunistic organisms Cryptosporidium parvum and microsporidia (Encephalitozoon cuniculi, E. intestinalis, E. hellem, and Enterocytozoon bieneusi) in Russian HIV/AIDS patients, we evaluated 46 sera from HIV/AIDS patients from the S.P. Botkin Clinical Infectious Diseases Hospital, St. Petersburg, Russia. Five (10.9%) sera were seropositive for E. cuniculi and 19 (41.3%) were positive for C. parvum by ELISA. By IFAT, 6 (13.0%) sera were seropositive for E. bieneusi, 4 (8.7%) for E. intestinalis, and 9 (19.6%) for E. hellem. This study is the first report to estimate the prevalence of infection with Cryptosporidium and microsporidia among Russian HIV/AIDS patients.

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.

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.

Prevalence and pathogenicity of Cryptosporidium suis in pre- and post-weaned pigs.

A total of 4338 faecal samples, 135 of sows, 3368 of pre-weaned and 835 of post-weaned piglets from eight farms in South Bohemia, Czech Republic were collected and examined for Cryptosporidium infection. No sow, but 5.7% pre-weaned and 24.1% post-weaned piglets were positive for Cryptosporidium infection. No relationship was found between diarrhoea and Cryptosporidium infection in any of the different age groups (pre- and post-weaned piglets). Four piglets, which were sporadically shedding cryptosporidia in faeces, were necropsied. Neither clinical signs of diarrhoea nor macroscopical changes were found. Histologically, a moderate infection of cryptosporidia was detected in the glandular epithelium along the large intestine, with predisposition to the ansa centralis of the colon. No inflammatory response in the lamina propria was observed. Cryptosporidia were also commonly found in the glandular epithelium of submucosal lymphoglandular complexes in the colon. Cryptosporidium isolates from all farms were identified as Cryptosporidium suis using molecular markers (SSU rRNA). All of the C. suis strains obtained were larger [6.2 (6.0-6.8) x 5.5 (5.3-5.7) microm] than any isolate described so far [4.6 (4.4-4.9) x 4.2 (4.0-4.3) microm] and did not appear to be infective for neonatal BALB/c mice.

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.

Prevalence and pathogenicity of Cryptosporidium andersoni in one herd of beef cattle.

Over a 35-week period from January to July 2002, a breed of Hereford beef cattle (H) and their hybrids were monitored. Five hundred and ninety-nine individual fecal samples from calves and 96 samples from their mothers were examined. First excretion of Cryptosporidium andersoni oocysts in calves was found in the 9th week after the start of calving (a calf 63-day old). The prevalence of C. andersoni in calves ranged from 11.1 to 92.9% depending on age. The mean prevalence in their mothers was found to be 43.8%. The size of oocysts was 8.48 +/- 0.78 x 6.41 +/- 0.59 microm. Infection intensity in calves ranged from 32 000 to 4 375 000 oocysts per gram (OPG) and in mothers from 78 000 to 2 552 000 OPG. Three cases of abomasal cryptosporidiosis slaughtered at the age of 81, 157 and 236 days were examined histologically and ultrastructurally [transmission electron microscopy (TEM) and scanning electron microscopy (SEM)]. Cryptosporidium infection of the abomasum was located in the upper half of the mucosal glands in the plicae spirales of the fundus, corpus and near the ostium omasoabomasicum. Cryptosporidia were not located in the glandular epithelium of the pars pylorica in the abomasum minimally 10 cm from pylorus. Histopathological changes in the site of cryptosporidial infection in the abomasum had a non-inflammatory character and included distinctive dilatation of infected parts of the glands with atrophy and metaplasia of the glandular epithelial cells, goblet cell activation and mucus hyperproduction. The TEM revealed a relatively small number of Cryptosporidium life cycle stages attached to glandular epithelial cells. In SEM the inner mucosal abomasal surface appeared swollen but was never infected by cryptosporidia.

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%.