Genotype diversity in the honey bee parasite Nosema ceranae: multi-strain isolates, cryptic sex or both?

BACKGROUND: There is great controversy as to whether Microsporidia undergo a sexual cycle. In the paradigmatic case of Nosema ceranae, although there is no morphological evidence of sex, some meiosis-specific genes are present in its reduced genome and there is also high intraspecific variability, with incongruent phylogenies having been systematically obtained. The possibility of sexual recombination is important from an epidemiological standpoint, particularly as N. ceranae is considered to be a major factor in the current disquieting epidemic of widespread bee colony losses. This parasite apparently originated in oriental honey bees, spreading out of Asia and Australia to infect honey bees worldwide. This study had three main objectives: i) to obtain genetic markers that are not part of known multi-copy arrays for strain determination; ii) to shed light on the intraspecific variability and recombination of N. ceranae; and iii) to assess the variability in N. ceranae populations. The answers to these questions are critical to understand the capacity of adaptation of microsporidia. RESULTS: Biallelic polymorphisms were detected at a number of specific points in the five coding loci analyzed from European and Australian isolates of N. ceranae. Heterozygous genotypes were abundant and cloning experiments demonstrate that they reflect the existence of multiple alternative sequences in each isolate. The comparisons of different clones and genotypes clearly indicate that new haplotypes are generated by homologous recombination. CONCLUSIONS: The N. ceranae isolates from honey bees correspond to genotypically distinct populations, revealing that individual honey bees may not be infected by a particular clone but rather, a pool of different strains. Homologous recombination implies the existence of a cryptic sex cycle yet to be described in N. ceranae. There are no diagnostic alleles associated with Australian or European origins, nor are there differences between the two hosts, A. cerana and A. mellifera, supporting the absence of biological barriers for N. ceranae transmission. Diversity is high among microsporidia of both these origins, and the maintenance of a high heterozygosis in the recently invaded European populations, could hypothetically underlie the stronger virulence of N. ceranae observed in A. mellifera.

Microsporidia detection and genotyping study of human pathogenic E. bieneusi in animals from Spain.

Microsporidia are ubiquitous parasites infecting all animal phyla and we present evidence that supports their zoonotic potential. Fecal samples taken from domestic (cats and dogs), farm (pigs, rabbits and ostriches) and wild animals (foxes) from different provinces of Spain were evaluated for microsporidia infection by light microscopy and PCR. After Microsporidia species identification, E. bieneusi genotypes were additionally studied by sequence analysis of the ITS region. Eighty-five samples out of 159 exhibited structures that were compatible with microsporidia spores by Webers stain with 37 of them being confirmed by PCR. Microsporidia species identified included E. bieneusi, E. intestinalis and A. algerae. We report the first diagnosis of E. intestinalis and E. bieneusi in ostriches and A. algerae in pigs. We also provide new information on the molecular characterization of E. bieneusi isolates both in rabbits and ostriches. All of the E. bieneusi genotypes identified belonged to the zoonotic group of genotypes (Group I) including genotypes A (dogs), I (pigs), D (rabbits and foxes) and type IV (ostriches). Our results demonstrate that microsporidia are present in domestic, farm and wild animals in Spain, corroborating their potential role as a source of human infection and environmental contamination.

Ribosomal gene polymorphism in small genomes: analysis of different 16S rRNA sequences expressed in the honeybee parasite Nosema ceranae (Microsporidia).

To date, few organisms have been shown to possess variable ribosomal RNA, otherwise considered a classic example of uniformity by concerted evolution. The polymorphism for the 16S rRNA in Nosema ceranae analysed here is striking as Microsporidia are intracellular parasites which have suffered a strong reduction in their genomes and cellular organization. Moreover, N. ceranae infects the honeybee Apis mellifera, and has been associated with the colony-loss phenomenon during the last decade. The variants of 16S rRNA include single nucleotide substitutions, one base insertion-deletion, plus a tetranucleotide indel. We show that different gene variants are expressed. The polymorphic sites tend to be located in particular regions of the rRNA molecule, and the comparison to the Escherichia coli 16S rRNA secondary structure indicates that most variations probably do not preclude ribosomal activity. The fact that the polymorphisms in such a minimal organism as N. ceranae are maintained in samples collected worldwide suggest that the existence of differently expressed rRNA may play an adaptive role in the microsporidian.

Variability in minimal genomes: analysis of tandem repeats in the microsporidia Encephalitozoon intestinalis.

Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia.

Familial porphyria cutanea tarda in Spain: characterization of eight novel mutations in the UROD gene and haplotype analysis of the common p.G281E mutation.

Porphyria cutanea tarda (PCT) results from decreased activity of uroporphyrinogen decarboxylase (UROD) in the liver. Deficiency in this enzyme results in accumulation of highly carboxylated porphyrins responsible for the disease. PCT usually occurs in adulthood and is characterized by cutaneous photosensitivity, hyperpigmentation, skin fragility and hypertrichosis. Familial PCT (F-PCT) occurs in 20-30% of patients in whom UROD gene mutations in heterozygosity decrease the enzymatic activity to about 50% in all tissues. The rare homozygous form of F-PCT (hepatoerythropoietic porphyria) has more severe clinical features and onset in childhood. In Spain, F-PCT is molecularly heterogeneous and the most frequent UROD mutation is p.G281E. In the present study, we searched for the molecular defect causing F-PCT in a group of Spanish patients and investigated whether the p.G281E mutation in the Spanish population came from a single or various origins. Among seventeen F-PCT patients, sixteen UROD mutations were identified, including eight novel ones: six missense (p.A23V, p.L78P, p.W180G, p.T196I, p.E278G and p.V279M), one frameshift (c.233delT) and one splice site mutation (c.774G>C). Prokaryotic expression studies showed the detrimental effect for each missense mutation, whereas reverse transcription-PCR and sequencing demonstrated that the novel splice site mutation caused exon 7 skipping. Moreover, haplotype analysis performed in Spanish families with the p.G281E mutation indicated that this lesion is associated with at least five haplotype backgrounds. These results extend knowledge on the molecular heterogeneity of F-PCT and suggest multiple origins of the p.G281E mutation.

Molecular characterization of human-pathogenic microsporidia and Cyclospora cayetanensis isolated from various water sources in Spain: a year-long longitudinal study.

Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis, Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae. C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.

Identification and characterization of microsporidia from fecal samples of HIV-positive patients from Lagos, Nigeria.

BACKGROUND: Microsporidia are obligate intracellular parasites that infect a broad range of vertebrates and invertebrates. They have been increasingly recognized as human pathogens in AIDS patients, mainly associated with a life-threatening chronic diarrhea and systemic disease. However, to date the global epidemiology of human microsporidiosis is poorly understood, and recent data suggest that the incidence of these pathogens is much higher than previously reported and may represent a neglected etiological agent of more common diseases indeed in immunocompetent individuals. To contribute to the knowledge of microsporidia molecular epidemiology in HIV-positive patients in Nigeria, the authors tested stool samples proceeding from patients with and without diarrhea. METHODOLOGY/PRINCIPAL FINDINGS: Stool samples from 193 HIV-positive patients with and without diarrhea (67 and 126 respectively) from Lagos (Nigeria) were investigated for the presence of microsporidia and Cryptosporidium using Weber's Chromotrope-based stain, Kinyoun stain, IFAT and PCR. The Weber stain showed 45 fecal samples (23.3%) with characteristic microsporidia spores, and a significant association of microsporidia with diarrhea was observed (O.R. = 18.2; CI: 95%). A similar result was obtained using Kinyoun stain, showing 44 (31,8%) positive samples with structures morphologically compatible with Cryptosporidium sp, 14 (31.8%) of them with infection mixed with microsporidia. The characterization of microsporidia species by IFAT and PCR allowed identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis and E. cuniculi in 5, 2 and 1 samples respectively. The partial sequencing of the ITS region of the rRNA genes showed that the three isolates of E.bieneusi studied are included in Group I, one of which bears the genotype B. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first report of microsporidia characterization in fecal samples from HIV-positive patients from Lagos, Nigeria. These results focus attention on the need to include microsporidial diagnosis in the management of HIV/AIDS infection in Nigeria, at the very least when other more common pathogens have not been detected.

Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees.

Nosema ceranae is currently one of the major pathogens of honeybees, related to the worldwide colony losses phenomenon. The genotyping of strains based on ribosomal DNA (rDNA) can be misleading if the repeated units are not identical. The analysis of cloned rDNA fragments containing the intergenic spacer (IGS) and part of the rDNA small-subunit (SSU) gene, from N. ceranae isolates from different European and Central Asia populations, revealed a high diversity of sequences. The variability involved single-nucleotide polymorphisms and insertion/deletions, resulting in 79 different haplotypes. Two sequences from the same isolate could be as different as any pair of sequences from different samples; in contrast, identical haplotypes were also found in very different geographical origins. Consequently, haplotypes cannot be organized in a consistent phylogenetic tree, clearly indicating that rDNA is not a reliable marker for the differentiation of N. ceranae strains. The results indicate that recombination between different sequences may produce new variants, which is quite surprising in microsporidia, usually considered to have an asexual mode of reproduction. The diversity of sequences and their geographical distribution indicate that haplotypes of different lineages may occasionally be present in a same cell and undergo homologue recombination, therefore suggesting a sexual haplo-diploid cycle.

Phylogenetic approach to the variability of the microsporidian Enterocytozoon bieneusi and its implications for inter- and intrahost transmission.

Enterocytozoon bieneusi is a microsporidian parasite that infects many vertebrate animals, including humans. The rDNA internal transcribed spacer (ITS) shows a hypervariable sequence; however, so far no clear information has been inferred about strain evolution in this species. We reviewed all the sequences described and performed a phylogenetic study. Four groups of sequences strongly differentiated from each other were detected, although most of the isolates (94%) corresponded to group I. The highly diverse sequences of this group were analyzed using median-joining networks. The host species (humans, pets, swine, cattle, birds, and wild animals) and the continents of origin of the isolates were considered. Central haplotypes in the network were obtained from very diverse hosts and geographical origins. The results show that although E. bieneusi has a broad host specificity, transmission is not completely free: some strains were able to circulate within a given host species and were only occasionally transmitted to another host. Additionally, while not relevant for swine or cattle hosts, geography seems to be a relevant factor for human infection by E. bieneusi.

Smoking but not homozygosity for CYP1A2 g-163A allelic variant leads to earlier disease onset in patients with sporadic porphyria cutanea tarda.

Porphyria cutanea tarda (PCT) results from decreased activity of hepatic uroporphyrinogen decarboxylase (UROD). Both sporadic and familial forms are characterised by typical cutaneous lesions triggered by genetic/environmental factors. Studies in rodents showed that cytochrome P4501A2 (CYP1A2) plays a central role in the synthesis of a competitive inhibitor of hepatic UROD, but there is little evidence in humans. The impact of smoking and CYP1A2 g-163C > A allelic variant upon first appearance of clinical signs was investigated in 102 patients (80 sporadic-PCT) and 150 healthy donors from Spain. We found an increase in the frequency of CYP1A2 g-163A allele in patients with PCT when compared with controls, although the more inducible A/A genotype had no effect on the onset age. In sporadic-PCT, smoking leads to earlier onset of clinically overt disease in moderate-to-heavy smokers (>or=10 cigarettes/day). In conclusion, this study provides evidence that smoking hastens the onset of cutaneous symptoms in sporadic-PCT patients.

Immunohistochemical study of cell proliferation, Bcl-2, p53, and caspase-3 expression on preneoplastic changes induced by cadmium and zinc chloride in the ventral rat prostate.

This work was directed to evaluate immunoexpression of markers for apoptosis, resistance to apoptosis, and cell proliferation, as well as estimates of nuclear size in ventral prostate of rats treated with cadmium chloride and cadmium+zinc chloride because a possible protective effect of zinc has been postulated. The following variables were studied: volume fraction (VF) of Bcl-2 immunostaining, percentage of cells immunoreactive to proliferating cell nuclear antigen (LIPCNA) and p53 (LIp53), numerical density of caspase-3 immunoreactive cells (NV caspase-3), and estimates of volume-weighted mean nuclear volume (upsilonV). The LIPCNA and VF of Bcl-2 were significantly increased in the treated animals. The dysplasias (independent of their origin) showed a significant increase of the LIp53, NV caspase-3, and upsilonV in comparison with normal acini from treated and control animals. It can be concluded that cell proliferation is enhanced in long-term cadmium-exposed rats, and exposure to zinc combined with cadmium had no effect on any of the variables studied when comparing with normal acini. The increase of nuclear upsilonV could indicate a more aggressive behavior for pretumoral lesions.

Variability in infection efficiency in vitro of different strains of the microsporidian Encephalitozoon hellem.

The infection efficiency of different strains of Encephalitozoon hellem of human origin was tested in Vero E6 cell cultures, scoring the number of infection foci (NIF) after 9, 14, 20, and 24 days of inoculation. The results revealed a strong interaction of the strain type with time: different strains showed different proliferative dynamics. Number of infection foci was lower on the first sampling day for CDC: V257, EHVS-96, and PV6-96, with a subsequent increase at a higher rate for the first strain and lower for the latter. In contrast, PV7-96 showed the highest NIF at the first sampling, followed by a slight decrease. Since these strains were selected by their genotype for the polar tube protein (PTP)-1A, 1B, 1C, and 2C, respectively, it is tempting to suggest a major role of this protein in the differences detected, although the influence of other genes that hypothetically may also differ among the strains employed cannot be discarded. The different in vitro infection efficiencies raise the possibility that some strains of E. hellem will also produce more aggressive features in infected patients.

Presence of neuroendocrine cells during postnatal development in rat prostate: Immunohistochemical, molecular, and quantitative study.

BACKGROUND: This work was undertaken to study the prostate neuroendocrine cells (PNEC) during the post-natal development of rats. METHODS: Forty male Wistar rats (pre-pubertals, pubertals, young, and aged adults) were used for immunohistochemistry of chromogranin A (cgA), serotonin (SER), and protein gene product 9.5 (PGP9.5). They were also evaluated for numerical cell density (NV SER) and PNEC number per prostate (N SER). Five additional young adult rats were used for a RT-PCR study (mRNA cgA detection). RESULTS: Weak immunoreactivity to cgA was observed in pubertal rats. No PNEC immunostained to PGP 9.5 was observed. Cells expressing SER were detected in all the groups exclusively located in periurethral ducts. The NV SER increased significantly in pubertal animals. In aged animals, it decreased to levels observed in pre-pubertal rats. The N SER increased significantly from pre-pubertal to young adults, decreasing in aged adults. There was weak production of cgA mRNA, with more expression in the dorsal prostate. CONCLUSIONS: PNEC differ in rats when compared to humans: they are weakly immunopositive to cgA, do not express PGP 9.5, only show immunoreactivity to SER, and do not appear in acini. The changes in the amount of rat PNEC during the post-natal development suggest an androgenic influx. PNEC might regulate the contractility of periurethral ducts.

Intraspecies genotype variability of the microsporidian parasite Encephalitozoon hellem.

Seven isolates of Encephalitozoon hellem from human immunodeficiency virus-positive patients were genotyped through a series of markers: the internal transcribed spacer (ITS) of ribosomal DNA, the polar tube protein (PTP) gene, and two intergenic spacers (IGS-TH and IGS-HZ) whose polymorphism is newly reported. The genome markers were all analyzed at three levels: PCR amplification followed by polyacrylamide gel electrophoresis, single-strand conformation analysis (SSCA), and DNA sequencing. The polymorphisms detected involve insertions/deletions and point mutations. SSCA can distinguish any pair of sequences, even those differing by a single base pair. The different isolates studied fit into the previously described ITS genotype 1A, except one which seems to be a 2A derivative variant (2D). When PTP and the new markers IGS-TH and IGS-HZ were analyzed, most of the isolates displayed different genotypes, demonstrating that E. hellem has a strong intraspecies variability. A set of markers such as those used here may be very useful in genotyping of clinical samples and in the assessment of epidemiological relationships among E. hellem strains.