Survey and genetic characterization of wastewater in Tunisia for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp

The microbial diversity of wastewater used for irrigation and fertilization was assessed using specific polymerase chain reaction (PCR) assays to detect and genotype several pathogenic protists including Cryptosporidium spp., Giardia duodenalis, Cyclospora spp., Eimeria spp. and Enterocytozoon bieneusi. A total of 220 wastewater samples (110 raw, 110 treated) and 12 sludge samples were collected from 2005 to 2008 from 18 treatment plants located throughout Tunisia. Except for Cyclospora, which was detected only once, E. bieneusi (61%), G. duodenalis (28%), Cryptosporidium spp. (27%) and Eimeria spp. (45%) were frequently observed in wastewater and sludge. Sequencing of PCR products showed that C. hominis, C. andersoni, G. duodenalis sub-assemblage A-II and E. bieneusi genotypes D and IV were the most prevalent. An analysis of the distribution of 209 internal transcribed spacer sequences of E. bieneusi originating from wastewater at the 18 treatment plants showed a similar genetic diversity, regardless of the geographical location. The identification of these parasite species and genotypes and of host-specific Eimeria species indicates that the microbial quality of wastewater was impacted by humans, livestock and rodents. Given the public health risks that some of these parasites represent, guidelines on wastewater usage are needed to minimize human exposure to these pathogens.     

Infection rate of Giardia duodenalis,Cryptosporidium spp. and Enterocytozoon bieneusi in cashmere,dairy and meat goats in China

Cryptosporidiosis, microsporidiosis, and giardiasis contribute significantly to the high burden of zoonotic diarrhea worldwide. Goats constitute an important species in animal agriculture by providing cashmere wool, meat, and dairy products for human consumption. However, zoonotic pathogens with the potential to cause morbidity and to degrade production have been reported frequently in goats recently. The present study examined 629 fecal specimens from goats, including 315 cashmere goats, 170 dairy goats and 144 meat goats, in multiple cities of Shaanxi and Henan provinces, northwestern and central China, to investigate the infection rate and species/assemblages/genotypes of Giardia duodenalis, Cryptosporidium spp. and Enterocytozoon bieneusi. Of these samples, 274 (43.6%) were positive for three zoonotic pathogens, including 80 (12.7%), 104 (16.5%) and 179 (28.5%) for G. duodenalis, Cryptosporidium spp. and E. bieneusi, respectively. Infections with G. duodenalis, Cryptosporidium spp. and E. bieneusi existed in meat, dairy and cashmere goats, with the highest infection rate of each pathogen being observed in meat goats. DNA sequencing of the SSU rRNA gene from 104 Cryptosporidium-positive specimens revealed existence of Cryptosporidium xiaoi, and the zoonotic parasites Cryptosporidium parvum and Cryptosporidium ubiquitum. Genotyping of G. duodenalis based on the triosephosphate isomerase (TPI) gene identified parasites from zoonotic assemblage A in four cashmere goats and the animal-adapted assemblage E in a group of 76 goats that included cashmere, dairy and meat animals. Polymorphisms in the ribosomal internal transcribed spacer characterized E. bieneusi genotype CHG1 and a novel genotype named as SX1 in both dairy and cashmere goats, genotypes CHS7 and COSI in meat goats, the genotype CHG2 in dairy goats, and the human-pathogenic genotype BEB6 in dairy and meat goats. This is the first detailed study to compare infection rate of the zoonotic protozoan pathogens in cashmere, dairy and meat goats in China. Our research discovered Cryptosporidium spp. and E. bieneusi infections, each with zoonotic potential in meat goats, and G. duodenalis and Cryptosporidium spp. in cashmere goats raising a significant public health concern.     

Prevalence and molecular characterization of Giardia duodenalis in dogs in Aydin,Turkey

The purpose of the present study was to investigate the prevalence and molecular characterization of Giardia duodenalis among dogs in Aydin, Turkey. A total of 473 faecal samples from dogs were collected. The overall prevalence of G. duodenalis was 18.8%. Higher infection rates were observed in dogs younger than three months old, from shelters, and with diarrhoea. Faecal samples of 89 dogs, diagnosed Giardia-positive by microscopy, were found positive by nested PCR. The β-giardin nested PCR assay revealed assemblage B in all samples (100%), whereas 38 of the samples were mixed with assemblage A (42%). Sequence analysis of isolates indicated sub-genotypes A3 and B4 which have been previously detected in human isolates from Turkey. The results of the present study indicated the relatively high prevalence of giardiasis and the presence of the zoonotic sub-genotypes suggesting the important role of dogs as potential reservoirs of human infections.     

Prevalence and multilocus genotypes of Giardia duodenalis infecting pigs in Ogun state,Nigeria

Giardia duodenalis is an intestinal flagellated protozoan parasite that is infectious to humans and a wide range of animals worldwide. While varying prevalence rates have been reported in pigs worldwide, there are currently no published reports on the genotypes of Giardia infecting pigs in any African country. The present study is on the prevalence and genotypes of G. duodenalis in 209 pigs raised on four farms in Ogun State Nigeria. Using an enzyme-linked immunosorbent assay (ELISA) kit, Giardia duodenalis coproantigens were detected on all farms and in 25.4% (53/209) of pigs sampled. However, there was no significant influence (p > 0.05) of age, sex and stool consistencies of the pigs on the distribution of the infection. Genotyping of Giardia duodenalis in all ELISA-positive samples, achieved by the amplification of the small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi) and beta giardin (bg) genes, identified 14 and 37 assemblage B and E isolates respectively while mixed infection by both assemblages was recorded in two isolates. Novel nucleotide substitutions were identified in four assemblage B isolates at the ssu rRNA locus. Genetic diversity was observed among the assemblage B isolates after multiple alignment analyses of the gdh, tpi and bg sequences whereby sub-assemblages BII (n = 2), BIII (n = 9) and BIV (n = 3) were identified. The assemblage B isolates from pigs in this study were phylogenetically related to isolates from humans, marmoset and cattle while the assemblage E isolates were related to isolates from sheep, goats and cattle. These findings suggest that pigs in southwest Nigeria predominantly harbour G. duodenalis isolates that could be infectious to other animal species and to a lesser extent, isolates that may be of zoonotic importance.     

Enterocytozoon bieneusi genotypes in farmed goats and sheep in Ningxia,China

Enterocytozoon bieneusi is reported to be a common microsporidian of humans and animals in various countries. However, scarce information on E. bieneusi has been recorded in farmed goats and sheep in China. As such, we undertook molecular epidemiological investigation of E. bieneusi in farmed goats (Capra aegagrus hircus) and sheep (Ovis aries) in Ningxia, China. A total of 660 genomic DNAs were extracted from individual faecal samples from famed goats (n = 300) and sheep (n = 360), and then tested using a nested PCR-based sequencing approach employing internal transcribed spacer (ITS) of nuclear ribosomal DNA as the genetic marker. Enterocytozoon bieneusi was detected in 237 of all 660 (36%) faecal samples from goats (n = 89) and sheep (n = 148). Correlation analyses revealed that E. bieneusi positive rates were significantly associated with age-groups, seasons and locations (P < 0.05). The analysis of ITS sequence data revealed the presentation of eight known genotypes (BEB6, CD6, CHG1, CHG3, CHG5, CHS8, CM7 and SX1). Phylogenetic analysis of ITS sequence data sets showed that they clustered within Group 2, showing zoonotic potential. These findings suggested that goats and sheep in Ningxia harbor zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission. Further insight into the epidemiology of E. bieneusi in farmed animals, water and the environment from other areas in China will be important to have an informed position on the public health significance of microsporidiosis caused by this microbe.     

Prevalence of Giardia duodenalis Infection in Household Cats of Ahvaz District,South-West of Iran

Background

The occurrence of Giardia duodenalis in cats is of potential significance from both clinical and public health perspectives. The object of this study was antigenic detection of G. duodenalis in household cats of Ahvaz district, South-West of Iran.

Methods

The prevalence of G. duodenalis was determined in fecal samples by two techniques: centrifugation-flotation and a commercial Giardia Antigen Test Kit (immunochromatography assay) in 150 household cats of different ages among referred cases to Veterinary Hospital of Ahvaz University from January 2008 to February 2010.

Results

Five out of 150 fecal samples (3.33%) were positive for antigen of G. duodenalis by immunochromatography assay. The prevalence was significantly higher in young cats less than 6 months (15.79%) compared with adult cats 6 months – 3 years (1.37%) (P=0.027) and above 3 years (1.72%) (P=0.044). The infection had more prevalence in diarrheic cats (17.39%) compared with non-diarrheic cats (0.79%) and the difference was significant (P=0.02) as well. The prevalence was higher in male cats (3.41%) than females (3.23%) and in the season of autumn (6.06%), but the difference was not significant between the prevalence of infection relative to host gender and season (P>0.05). Microscopy examination on fecal samples showed that 2% of the studied cats were positive.

Conclusion

The parasite antigen was present as a zoonotic infection in Ahvaz district, South-west of Iran. More sensitive techniques, such as immunochromatography assay, might yield more reliable results, in the detection of low levels of Giardia in fecal samples of cats.     

Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis

To address the source of infection in humans and public health importance of Giardia duodenalis parasites from animals, nucleotide sequences of the triosephosphate isomerase (TPI) gene were generated for 37 human isolates, 15 dog isolates, 8 muskrat isolates, 7 isolates each from cattle and beavers, and 1 isolate each from a rat and a rabbit. Distinct genotypes were found in humans, cattle, beavers, dogs, muskrats, and rats. TPI and small subunit ribosomal RNA (SSU rRNA) gene sequences of G. microti from muskrats were also generated and analyzed. Phylogenetic analysis on the TPI sequences confirmed the formation of distinct groups. Nevertheless, a major group (assemblage B) contained most of the human and muskrat isolates, all beaver isolates, and the rabbit isolate. These data confirm that G. duodenalis from certain animals can potentially infect humans and should be useful in the detection, differentiation, and taxonomy of Giardia spp.     

Genotyping of Enterocytozoon bieneusi (Microsporidia) isolated from various birds in China

Enterocytozoon bieneusi is a common opportunistic pathogen causing diarrhea in humans and animals. However, epidemiological data on E. bieneusi infections in birds are relatively scare worldwide, especially in China. To understand the prevalence and genetic diversity of E. bieneusi in birds and to assess the zoonotic potential of bird-derived E. bieneusi isolates, 194 fecal specimens from Gruidae, Anatidae and Columbidae in Heilongjiang Province, China, were analyzed by PCR and sequencing of the single internal transcribed spacer region of the rRNA gene. The average prevalence of E. bieneusi was 22.2%, with 12.5% for Gruidae, 15.9% for Anatidae and 44.0% for Columbidae. Altogether seven genotypes of E. bieneusi were identified, including four known genotypes—Peru6 (n = 29), BEB6 (n = 5), D (n = 3) and EbpA (n = 1)—and three novel genotypes named CHN-B1 (n = 1), CHN-B2 (n = 3) and CHN-B3 (n = 1). All the known genotypes obtained here were previously detected in humans. All the novel genotypes were clustered into the zoonotic group 1 in phylogenetic analysis. The results indicate that these birds may play a potential role in the transmission of E. bieneusi to humans.     

First survey of Cryptosporidium,Giardia and Enterocytozoon in diarrhoeic children from Wuhan,China

Intestinal protozoan pathogens cause significant diarrhoeal diseases in children. However, to date, there has been limited genetic study of the intestinal pathogens Cryptosporidium, Giardia and Enterocytozoon in humans in China, with the exception of research in a small number of cities/provinces. In the present study, PCR-based tools were used to detect and characterise these protistan parasites from 500 children with a history of diarrhoea in Wuhan and environs, Hubei province, China. Genomic DNAs from faecal samples were screened for the particular protists by PCR utilising regions in the small subunit (SSU) of the nuclear ribosomal RNA, the 60 kDa glycoprotein (gp60), the internal transcribed spacer of nuclear ribosomal DNA (ITS) and/or the triose phosphate isomerase (tpi) genes as markers. Cryptosporidium meleagridis subtype IIIb (10/500, 2.0%), Giardia duodenalis assemblage A (7/500, 1.4%) and Enterocytozoon bieneusi genotype D (1/500, 0.2%) were identified in small percentages of the 500 samples. No significant gender- or age-associated differences in the prevalence of Cryptosporidium and Giardia infections were found. Future studies might focus on the occurrence of these protists in children as well as animals, with an emphasis on Cryptosporidium meleagridis in pets and agriculturally important birds, in different parts of Hubei province.     

Occurrence and molecular characterization of Giardia duodenalis in child population from Colombia

Giardia duodenalis is one of the most prevalent human intestinal parasite, with children living in developing countries being particularly at risk of infection. The occurrence and molecular diversity of G. duodenalis was investigated in stools specimens from 307 individuals aged one to nineteen years in Colombia. Samples were collected in three educational establishments (n: 163) and two hospital laboratories (n: 144) from urban and rural areas. Feces were concentrated using a biphasic sedimentation method and wet mounts of the sediment were examined by light microscopy. G. duodenalis assemblages and sub-assemblages were determined on positive samples by PCR of the triose phosphate isomerase (tpi), β-giardin (bg) and small-subunit (ssu) rRNA genes. G. duodenalis infection was detected by microscopy in 23 individuals (7.5%). The protozoan was more prevalent among specimens collected in educational establishments (11.6%) than in those obtained from hospital laboratories (2.8%). Infection was most common in individuals from urban areas and children aged 1–5 years. No significant association between diarrhea and infection could be demonstrated. Twenty Giardia-positive samples were successfully allocated to assemblage B (n: 11), sub-assemblage AII (n: 7), and assemblage A (n: 2). Results indicate the potential for transmission of G. duodenalis infection in children attending educational establishments and individuals from urban areas, where transmission seems to be primarily anthroponotic.     

High genetic diversity of Giardia duodenalis assemblage E in Chinese dairy cattle

Giardia duodenalis is a common protozoan parasite that can infect humans and animals. Although previous studies demonstrated that the assemblage E of G. duodenalis is prevalent in cattle, studies on its genetic diversity were mostly based on single loci and very few involved multilocus analysis. To better understand the genetic variability and structure of G. duodenalis assemblage E in Chinese dairy cattle, 651 multilocus sequences derived from nine provinces (Gansu, Guangdong, Henan, Jiangsu, Ningxia, Shaanxi, Shanghai, Sichuan and Xinjiang) of China were analyzed in this study. Results showed that a total of 220 haplotypes were identified in the G. duodenalis assemblage E, with a high haplotype diversity (Hd = 0.97225) and low nucleotide diversity (π = 0.00259). The genetic differentiation index (FST) and gene flow (Nm) results indicated low degree of genetic differentiation, implying frequent genetic communication. Combined with the analysis of molecular variance (AMOVA), genetic variation within populations (81.7%) was higher than that among populations (18.3%), indicating low degree of genetic differentiation between populations. Such low rates of gene differentiation supported no significant correlations with geographical divisions. Moreover, both negative Tajima's D and Fu's FS values of neutrality tests and unimodal curve of mismatch distribution analyses indicated that G. duodenalis assemblage E population in Chinese dairy cattle had experienced demographic expansion. Overall, these findings contribute to an improved understanding of the population genetics and evolutionary biology of G. duodenalis assemblage E and assist in its control in cattle.     

First report of zoonotic Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi in golden takins (Budorcas taxicolor bedfordi)

Genetic study of Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi at species/assemblage/genotype/subtype level facilitates understanding their mechanical transmissions and underpins their control. A total of 191 fresh faecal samples were collected from golden takins in China and examined using multilocus sequence typing (MLST). Cryptosporidium spp. was detected in 15 faecal samples (7.9%), including Cryptosporidium parvum (2/15) and Cryptosporidium andersoni (13/15). MLST tool identified C. andersoni subtypes (A1, A4, A4, A1) and (A4, A4, A4, A1), and C. parvum gp60 gene subtype IId A19G1. The prevalence of G. intestinalis infection was 8.9% (17/191) and assemblage analysis identified 14 assemblage E and three assemblage B. Intra-variations were observed at triose phosphate isomerase (tpi), beta giardin (bg) and glutamate dehydrogenase (gdh) loci within the assemblage E, showing seven, three and three new subtypes in respective locus. Ten and one multilocus genotypes (MLGs) were present in assemblages E and B, respectively. E. bieneusi infection was positive in 14.7% (28/191) of the examined specimens, with three genotypes known (BEB6, D and I) and four novel internal transcribed spacer (ITS) genotypes (TEB1–TEB4). The present study revealed, for the first time, the presence of zoonotic C. parvum IId A19G1, G. intestinalis assemblage B and E. bieneusi genotype D and four novel genotypes in golden takins in China. These findings expand the host range of three zoonotic pathogens and have important implications for controlling cryptosporidiosis, giardiasis and microsporidiosis in humans and animals.     

Detection and characterization of Giardia duodenalis and Cryptosporidium spp. on swine farms in Ontario, Canada

As part of the C-EnterNet surveillance program of the Public Health Agency of Canada, 122 pooled swine manure samples from 10 farms in Ontario, Canada were collected and tested for Giardia and Cryptosporidium. Giardia duodenalis cysts and Cryptosporidium spp. oocysts were detected using immunofluorescence microscopy. Nested-polymerase chain reaction protocols were performed to amplify the small subunit rRNA gene and the β-giardin gene for G. duodenalis, and the small subunit rRNA gene and the heat shock protein-70 gene for Cryptosporidium spp. The DNA amplicons were sequenced to determine genotypes and species. A mixed multivariable method was used to compare the presence of Giardia and Cryptosporidium in different stages of production. Both Giardia cysts and Cryptosporidium oocysts were present on all tested farms, with 50.8% of the samples positive for G. duodenalis and 44.3% positive for Cryptosporidium spp. by microscopy, and 66.4% and 55.7%, respectively, positive by polymerase chain reaction (PCR). No significant agreement was observed between microscopy and PCR method to detect Giardia and Cryptosporidium (p<0.05). The prevalence of Giardia in manure pits and finisher pigs did not differ (p>0.05), however, it was less frequent (odds ratio, OR=0.21 [0.07, 0.63]) among sows. Cryptosporidium was more likely (OR=3.6 [1.3, 9.9]) to be detected in manure pits and weaners (OR=3.3 [1.1, 10.0]) compared to finisher pigs, and it was less frequent (OR=0.06 [0.007, 0.55]) in sows than in finishers (p<0.05). DNA sequencing demonstrated that 92.1% of the Giardia isolates were Assemblage B and 7.9% were Assemblage E. The most prevalent Cryptosporidium were Cryptosporidium parvum (55.4%), and Cryptosporidium sp. pig genotype II (37.5%). These findings indicate that the occurrence of zoonotic isolates of G. duodenalis and Cryptosporidium is very high on swine farms in southern Ontario, and that there is a potential for transmission between swine and humans by means of cyst and oocyst contaminated water or foods.     

Prevalence of Giardia duodenalis assemblages and sub-assemblages in symptomatic patients from Damascus city and its suburbs

Giardia duodenalis is one of the most important human enteric parasites worldwide and is endemic throughout the world with a vast range of mammalian hosts. However, there is limited information on the prevalent genetic variability of G. duodenalis in Syria. This study aimed to evaluate the predominance of G. duodenalis assemblages/sub-assemblages causing humans infection in the city of Damascus and its suburbs. 40 symptomatic giardiasis patients were recruited in this study. Fecal samples were genotyped using PCR/RFLP assay targeting the β-giardin and glutamate dehydrogenase (gdh) genes. HaeIII, BspL1 and RsaI restriction enzymes were used to differentiate between G. duodenalis assemblages/sub-assemblages. Our data showed that 65% of isolates were of assemblage A; 45% belonged to sub-assemblage AII and 20% to sub-assemblage AI. Assemblage B was detected in 27.5% of isolates; 12.5% fit in sub-assemblage BIV, 5% fit in sub-assemblage BIII and 10.5% fit in Discordant genotype BIII/BIV. Mixed genotypes (AII+BIII and AI+BIV) were identified in 3 isolates (7.5%). Significant correlation was found between Giardia AII sub-assemblage and weight loss symptom (P-value = 0.05) as well as between contact with domestic animals (cats, P-value = 0.027). Moreover, a significant correlation was found between sub-assemblage AI and livestock breeding (P-value = 0.000). In conclusion genotyping of human Giardia duodenalis isolates suggests anthroponotic transmission for the route of infection in Damascus and its suburbs. Further studies are needed to screen a wide geographic areas in Syria and to estimate the prevalence of G. duodenalis infection in our population.     

Multilocus genotyping of Giardia duodenalis in Malaysia

Giardia duodenalis is considered the most common intestinal parasite in humans worldwide. In Malaysia, many studies have been conducted on the epidemiology of giardiasis. However, there is a scarcity of information on the genetic diversity and the dynamics of transmission of G. duodenalis. The present study was conducted to identify G. duodenalis assemblages and sub-assemblages based on multilocus analysis of the glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes. Faecal specimens were collected from 484 Orang Asli children with a mean age of 7 years and examined using light microscopy. Specimens positive for Giardia were subjected to PCR analysis of the three genes and subsequent sequencing in both directions. Sequences were edited and analysed by phylogenetic analysis. G. duodenalis was detected in 17% (84 of 484) of the examined specimens. Among them, 71 were successfully sequenced using at least one locus. Genotyping results showed that 30 (42%) of the isolates belonged to assemblage A, 32 (45%) belonged to assemblage B, while discordant genotype results were observed in 9 specimens. Mixed infections were detected in 43 specimens using a tpi-based assemblage specific protocol. At the sub-assemblages level, isolates belonged to assemblage A were AII. High nucleotide variation found in isolates of assemblage B made subtyping difficult to achieve. The finding of assemblage B and the anthroponotic genotype AII implicates human-to-human transmission as the most possible mode of transmission among Malaysian aborigines. The high polymorphism found in isolates of assemblage B warrants a more defining tool to discriminate assemblage B at the sub-assemblage level.     

Giardia duodenalis infection and wastewater irrigation in Pakistan

The risk of Giardia duodenalis (Giardia) infection in farmers using untreated wastewater in agriculture was investigated in the city of Faisalabad, Pakistan, through a cross-sectional study. The study found a significantly increased risk of (asymptomatic) Giardia infection in wastewater farming households when compared with farming households using regular (non-wastewater) irrigation water (OR 3.3, 95% CI 2.5-4.4). Textile labourers who were employed in the city of Faisalabad but who lived in the same village as the wastewater farmers showed a risk of Giardia infection in between that of wastewater and non-wastewater farming households (OR 2.4, 95% CI 1.9-3.1). This study suggests that exposure to wastewater with high Giardia concentrations carries an increased risk for (asymptomatic) Giardia infection.     

Enterocytozoon bieneusi (Microspora): prevalence and pathogenicity in AIDS patients

Microsporidia are unicellular organisms, which lack mitochondria. They have prokaryotic-like ribosomes and characteristic spores containing an extrusible polar tube which serves as a passage for inoculation of the infectious agent (sporoplasm) into host cells. Clinically apparent infections in man appear to be limited to immunoprivileged sites or immunocompromised patients. One species, Encephalitozoon cuniculi, has been reported several times in patients with neurological disorders and once causing a fatal hepatitis in an AIDS patient. The most recently discovered species, Enterocytozoon bieneusi, is known only from the small intestinal enterocytes of patients with the acquired immunodeficiency syndrome, and is easily differentiated from other microsporidia by the precocious development of spore organelles in the sporont and by the poor development of the endospore layer of the spore wall. Although only about 40 cases have been reported, circumstantial evidence suggests that E. bieneusi may be the cause of a severe watery diarrhoea, which responds only temporarily to treatment with metronidazole.     

Giardia duodenalis sub-Assemblage of animal and human origin in horses

In order to evaluate infection occurrence and the potential zoonotic role of horse isolates of Giardia duodenalis, 431 individual fecal samples were genetically characterized by PCR tests –coupled sequencing and phylogenetic analysis. Thirty-seven (8.6%) animals resulted infected by different Assemblage. The presence of sub-Assemblage was assessed by characterizing the β-giardin gene for 16 of the 37 positive horses. Ten isolates showed 99.6% to 100% homology with the sub-Assemblage described as B1–2 and B1–6, three Assemblage A showed 99.8% homology with sub-Assemblage A1, while one Assemblage E displayed 98.8% homology with sub-Assemblage E3. Furthermore, one isolate characterized as Assemblage A showed 99.6% homology with the sub-Assemblage B1–2 and one characterized as E was 100% identical with sub-Assemblage B1–6. These results demonstrate the presence of both animal and human sub-Assemblage of G. duodenalis in horses from Italy. Epidemiological and sanitary implications are discussed.     

Genotyping and molecular analysis of Enterocytozoon bieneusi isolated from immunocompromised patients in Iran

Microsporidia are known as opportunistic unicellular pathogens, particularly so in individuals with congenital or acquired immunodeficiency. Enterocytozoon bieneusi is one of the most common species infecting both immunocompromised and immunocompetent individuals. The aim of this study was to assess the distribution of E. bieneusi genotypes among immunocompromised patients in Iran. From 329 stool samples referred for parasitological analysis during 2011–2014, 14 samples from immunocompromised patients proving positive for E. bieneusi by SSU rDNA analysis were selected. Genotyping was carried out using specific primers targeting the Internal Transcribed Spacer (ITS) region. Subsequently, all samples were sequenced and results queried against the GenBank database. Moreover, sequences were subject to phylogenetic analysis. The expected amplification product was generated for all samples. Genotype D was identified in patients with HIV +/AIDS, transplant recipients, and cancer patients, while Genotype E was identified only in cancer and HIV +/AIDS patients. Phylogenetic analysis revealed that there was no relationship between genotypes and types of immunosuppression, whereas most genotype D isolates grouped with those described previously from cattle, horses, birds, and humans. E. bieneusi genotype D appears to be the most frequent genotype in immunocompromised patients, while Genotype E was observed only in HIV +/AIDS patients and cancer patients, not transplant recipients.     

Host-specific segregation of ribosomal nucleotide sequence diversity in the microsporidian Enterocytozoon bieneusi

Enterocytozoon bieneusi is a unicellular enteric fungal pathogen and the most common cause of human microsporidiosis. The frequent detection of this organism in animals, including companion animals, livestock and wildlife, has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen. A partial sequence of the ribosomal internal transcribed spacer (ITS) has been widely used as a genetic marker for studying the molecular epidemiology of E. bieneusi. With the aim of comparing E. bieneusi ITS genotypes originating from different host species, and assess the potential for zoonotic transmission, E. bieneusi ITS sequences retrieved from GenBank were analyzed using two metrics of diversity, rarefaction and phylogenetic distance. In spite of the human ITS sample being geographically more diverse, ITS sequence diversity in animals exceeded that of humans. In both host groups much of the ITS diversity remains to be sampled. Using quantitative phylogenetic tests we found evidence for a partial but significant segregation of E. bieneusi ITS sequences according to host species. Host-specific segregation was confirmed by hierarchical analysis of molecular variation. To improve our understanding of the epidemiology of human microsporidiosis and strengthen the study of E. bieneusi populations, efforts to genotype additional E. bieneusi isolates from wildlife and companion animals should be prioritized and the geographic and species diversify of animal samples should be increased. Due to the possibility of genetic recombination in this species, additional unlinked genetic markers need to be developed and included in future studies.