Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China

Cryptosporidium and Giardia infections are common causes of diarrhea worldwide. To better understand the transmission of human cryptosporidiosis and giardiasis in Henan, China, 10 Cryptosporidium-positive specimens and 18 Giardia-positive specimens were characterized at the species/genotype and subtype levels. Cryptosporidium specimens were analyzed by DNA sequencing of the small subunit rRNA and 60 kDa glycoprotein genes. Among those genotyped, nine belonged to C. hominis and one C. felis, with the former belonging to three subtype families: Ia, Ib, and Id. The three Ib subtypes identified, IbA16G2, IbA19G2, and IbA20G2, were very different from the two common Ib subtypes (IbA9G3 and IbA10G2) found in other areas of the world. The distribution of Giardia duodenalis genotypes and subtypes was assessed by sequence analysis of the triosephosphate isomerase (tpi) gene. The assemblages A (eight belonging to A-I and four A-II) and B (belonging to six new subtypes) were found in 12 and six specimens, respectively. More systematic studies are needed to understand the transmission of Cryptosporidium and G. duodenalis in humans in China.     

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 μg ml⁻¹, this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6 ± 2.8% live trophozoites remaining after 24 h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 μg ml⁻¹ of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 °C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 μg ml⁻¹ gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.     

The CpA135 gene as a marker to identify Cryptosporidium species infecting humans

Of the 22 species currently recognized as valid in the Cryptosporidium genus, C. parvum and C. hominis account for most cases of human infections worldwide. However, C. meleagridis, C. canis, C. felis, C. suis, C. muris, as well as the cervine, rabbit and monkey Cryptosporidium genotypes, have also been recognized as the etiologic cause of both sporadic and epidemic cryptosporidiosis in humans. Molecular methods are necessary to distinguish species and genotypes of Cryptosporidium, due to the lack of reliable morphological variations. The aim of this work was to determine the genetic polymorphisms in a fragment of the A135 gene in isolates of C. parvum, C. hominis, C. meleagridis, C. canis, C. muris, C. andersoni and the Cryptosporidium cervine genotype. Primers were designed on conserved regions identified on a multiple alignment of the C. parvum, C. hominis and C. muris sequences, the three species for which information is available at the genome level. PCR amplification and direct sequencing of a 576 bp fragment revealed the presence of numerous single nucleotide polymorphisms (SNPs) among the species/genotype tested. The genetic variability was exploited to design a PCR-RFLP assay useful for a rapid identification of the most important human pathogens in the genus Cryptosporidium.     

Subtypes of Cryptosporidium spp. in mice and other small mammals

DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene has been used extensively in subtyping Cryptosporidium hominis in humans and Cryptosporidium parvum in humans and ruminants. In this study, nucleotide sequences of the gp60 gene were obtained from seven Cryptosporidium species and genotypes related to the two species. Altogether, seven subtype families were detected, including four new subtype families. These data should be useful in studies of the transmission and zoonotic potential of cryptosporidiosis in mice and small wild mammals.     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

Giardia cysts and Cryptosporidium oocysts detection in the intake and rapid filter system in a water purification plant

Water samples, taken from the intake and rapid filter system of a water purification plant, were analyzed using an immunofluorescence antibody method for detecting the presence of Giardia cysts and Cryptosporidium oocysts. Giardia cysts and Cryptosporidium oocysts were found in the intake water from zero to 38.7 cysts/100 l and 1.7–50.5 oocysts/100 l with averages of 9.6 cysts/100 l and 19.4 oocysts/100 l. Giardia cysts and Cryptosporidium oocysts were also detected in the samples taken from the rapid filtration unit with mean concentrations of zero to 2.3 cysts/100 l and 0–2.5 oocysts/100 l, respectively. The efficacy of the rapid filter in suspended material and (oo)cyst removal was significant. The removal late was 56–97% for suspended material and 69–100% for the (oo)cysts.     

Evaluation of a modified semi-automated immunomagnetic separation technique for the detection of Cryptosporidium oocysts in human faeces

Routine diagnosis of Cryptosporidium infection relies on the oocyst as the diagnostic target. To improve analytical sensitivity, we modified and evaluated an immunomagnetic separation procedure for Cryptosporidium oocysts applicable to formed, liquid or mucoid human faeces. The analytical sensitivity of the method is <5 oocysts per gram, regardless of stool consistency.     

Giardia duodenalis: Biochemical characterization of an ecto-5′-nucleotidase activity

In this work, we biochemically characterized the ecto-5′-nucleotidase activity present on the surface of the living trophozoites of Giardia duodenalis. Two sequences of the 5′-nucleotidase family protein were identified in the Giardia genome. Anti-mouse CD73 showed a high reaction with the cell surface of parasites. At pH 7.2, intact cells were able to hydrolyze 5′-AMP at a rate of 10.66 ± 0.92 nmol Pi/h/10⁷ cells. AMP is the best substrate for this enzyme, and the optimum pH lies in the acidic range. No divalent cations had an effect on the ecto-5′-nucleotidase activity, and the same was seen for NaF, an acid phosphatase inhibitor. Ammonium molybdate, a potent inhibitor of nucleotidases, inhibited the enzyme activity in a dose-dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. The results indicate the existence of an ecto-5′-nucleotidase that could play a role in the salvage of purines.     

Cryptosporidium spp. in pet birds: genetic diversity and potential public health significance

To characterize the prevalence and assess the zoonotic transmission burden of Cryptosporidium species/genotypes in pet birds in Henan, China, 434 fecal samples were acquired from 14 families of birds in pet shops. The overall prevalence of Cryptopsoridium was 8.1% (35/434) by the Sheather’s sugar flotation technique. The Cryptosporidium-positive samples were analyzed by DNA sequence analysis of the small subunit (SSU) rRNA gene. Three Cryptosporidium species and two genotypes were identified, including C. baileyi (18/35 or 51.4%) in five red-billed leiothrixes (Leiothrix lutea), four white Java sparrows (Padda oryzivora), four common mynas (Acridotheres tristis), two zebra finches (Taeniopygia guttata), a crested Lark (Galerida cristata), a Gouldian finch (Chloebia gouldiae), and a black-billed magpie (Pica pica); Cryptosporidium meleagridis (3/35 or 8.6%) in a Bohemian waxwing (Bombycilla garrulus), a Rufous turtle dove (Streptopelia orientalis), and a fan-tailed pigeon (Columba livia); Cryptosporidium galli (5/35 or 14.3%) in four Bohemian waxwings (Bombycilla garrulus) and a silver-eared Mesia (Leiothrix argentauris); Cryptosporidium avian genotype III (3/35 or 8.6%) in two cockatiels (Nymphicus hollandicus) and a red-billed blue magpie (Urocissa erythrorhyncha); and Cryptosporidium avian genotype V (6/35 or 17.1%) in six cockatiels (Nymphicus hollandicus). Among the pet birds, 12 species represented new hosts for Cryptosporidum infections. The presence of C. meleagridis raises questions on potential zoonotic transmission of cryptosporidiosis from pet birds to humans.     

Periodic appearance of a predominant variant antigen type during a chronic Giardia lamblia infection in a mouse model

Giardia lamblia (syn. Giardia duodenalis, Giardia intestinalis) infections are associated with continuous antigenic variation of the parasite which is mediated by the parasite's major surface antigen, named variant surface protein. Offspring mice and corresponding mothers were infected with G. lamblia clone GS/M-83-H7 (expressing variant surface protein H7) and various parameters of this infection were assessed in a long-term follow-up investigation. Our experimentation revealed that variant surface protein H7-type trophozoites were replaced by new variant-type trophozoites during the early stage of infection (around day 8 p.i.), but the original variant-type re-emerged at at least two time-points during the later stages of infection (at days 22 and 42 p.i.). Such periods of variant surface protein H7-type trophozoite re-expansion were accompanied by transient production of intestinal IgA against variant-specific epitopes on a 314-aa N-terminal region of variant surface protein H7. At late stages of infection (between days 42 and 200 p.i.), most mice produced intestinal IgA against both variant surface protein H7 and other antigens of the parasite. At these stages, infection seemed to be resolved in most mice, but occasional reappearance of relatively high (at day 64 p.i.) or at least detectable (at days 80 and 120 p.i.) amounts of intestinal parasites indicated that G. lamblia GS/M-83-H7 infections in mice may enter into a latent chronic phase which is interrupted by sporadic breakthroughs of parasite growth.     

Identification of zoonotic Cryptosporidium and Giardia genotypes infecting animals in Sydney's water catchments

To identify the animal sources for Cryptosporidium and Giardia contamination, we genotyped Cryptosporidium and Giardia spp. in wildlife from Sydney’s water catchments using sequence analysis at the 18S rRNA locus for Cryptosporidium and 18S rRNA and glutamate dehydrogenase (gdh) for Giardia. A total of 564 faecal samples from 16 different host species were analysed. Cryptosporidium was identified in 8.5% (48/564) samples from eight host species and Giardia was identified in 13.8% (78/564) from seven host species. Eight species/genotypes of Cryptosporidium were identified. Five G. duodenalis assemblages were detected including the zoonotic assemblages A and B.     

Taxonomic implications of Liassic ferns Cladophlebis Brongniart and Todites Seward from Hungary

Ferns are diverse and abundant in the Lower Liassic flora of the Mecsek Mountains in south Hungary. The morphogenus Cladophlebis Brongniart, 1849, and the genus Todites Seward, 1900, show a high diversity. In the examined material, most of the characteristics used in taxonomy vary, showing transitional forms that cast doubt on the separation of species established on the grounds of either a low number of specimens, a low number of features or uncharacteristic differences.     

Campylobacter,salmonella and chlamydia in free-living birds of Croatia

Campylobacteriosis, salmonellosis and avian chlamydiosis are zoonotic diseases in which birds have been suggested to play an important role as reservoirs. We have investigated the prevalence of Campylobacter and Salmonella spp. and Chlamydophila sp. in 107 free-living birds belonging to 25 species from 13 families from Croatia in order to examine the natural infections caused by these agents. Campylobacter jejuni-like organisms were isolated from 2 of 107 free-living bird species examined (1.9%). Salmonella was isolated from 8 fresh fecal specimens from free-living bird species (7.4%). These isolates were identified as S. typhimurium in 4 (3.7%), and S. enteriditis in 4 (3.7%) free-living birds. These samples originated from feral pigeons (Columba livia domesticus; n=14; 28.6%), rook (Corvus frugilegus; n=13; 15.4%), buzzard (Buteo buteo; n=12; 16.7%), black-headed gull (Larus ridibundus; n=8; 12.5%) and tawny owl (Strix aluco; n=8; 12.5%). The presence of Chlamydophila sp. was not detected in the free-living birds examined during this study. Epidemiological aspects and possible significance of the examined birds as a source of infections for domestic animals and humans are discussed.     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.     

Isariotins G–J from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes

Four new alkaloids isariotins G–J (1–4), together with the known isariotins, were isolated from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Compounds 1–4 exhibited antimalarial and cytotoxic activities.     

An extract of Pelargonium sidoides (EPs 7630) inhibits in situ adhesion of Helicobacter pylori to human stomach

Root extract from Pelargonium sidoides DC is used therapeutically as antimicrobial agent against infections of the respiratory system. In order to elucidate possible modes of actions we investigated the influence of P. sidoides root extract on microbial adhesion with Helicobacter pylori as model microorganism, a germ with a strong adherence to human stomach tissue. In an in-situ anti-adhesion assay intact human stomach tissue from patient resectates was incubated with fluorescent-labelled bacteria. Epithelial adhesion occurred in untreated samples and was quantified by fluorescent microscopy. Pre-treatment of the bacteria with Pelargonium extract showed good anti-adhesive activity. The antiadhesive effect was clearly dose-dependent in a range from 0.001 to 10 mg/ml. Within agar diffusion-test the extract had no direct cytotoxicity against H. pylori. The results show that the root extract from Pelargonium sidoides is a potent anti-adhesive agent against H. pylori and could therefore be a useful choice to avoid the first step of a bacterial infection.     

Cell death induction in Giardia lamblia: Effect of beta-lapachone and starvation

Giardia lamblia is a protozoan that parasitizes the small intestine of vertebrates. It is a cause of intestinal infection and diarrhea and infects millions of people worldwide. This protozoan presents many characteristics common to eukaryotic cells but it lacks organelles found in most eukaryotes (e.g., peroxisomes, typical Golgi complex and mitochondria). Also it presents mitosomes, a relic organelle that appears to be a mitochondrial remnant. Cell death in Giardia was induced by the drug β-Lapachone and by starvation. Giardia behavior was followed by scanning, transmission and fluorescence microscopy, quantification of cell metabolism using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide), changes in lipid rafts, using DiIC₁₆ and cholera toxin. Cell shrinkage, chromatin condensation, membrane blebbing and vacuolization provided ultrastructural evidence of apoptosis, whereas the myelinic figures in large vacuoles and LC-3 staining suggested an autophagic process. Lipids rafts were altered by drug treatment and co-localized with regions containing membrane blebbing. The treatment with β-Lap induced encystation. A search for sequence similarities in databases and protein alignments was carried out. Although Giardia is an amitochondrial organism, it presented some autophagic-like cell death characteristics and several, but not all, apoptotic characteristics, induced by β-Lapachone and starvation.     

Effect of dung burial by the dung beetle Bubas bison on numbers and viability of Cryptosporidium oocysts in cattle dung

Cryptosporidium oocysts were inoculated into fresh dung (∼1.2 × 10⁴ oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.