Real-Time PCR and LAMP Assays for the Detection of Spores of Alternaria solani and Sporangia of Phytophthora infestans to Inform Disease Risk Forecasting.

Real-time loop-mediated isothermal amplification (LAMP) assays for the detection of sporangia of the causal pathogen of late blight, Phytophthora infestans, and spores of the main causal pathogen of early blight, Alternaria solani, were developed to facilitate the in-field detection of airborne inoculum to improve disease forecasting. These assays were compared with an existing real-time PCR assay for P. infestans and a newly developed real-time PCR assay for A. solani. Primers were designed for real-time LAMP of P. infestans and A. solani. The specificity of the P. infestans real-time LAMP assay was similar to that of an existing real-time PCR assay: DNA of P. infestans was consistently amplified as was DNA of the taxonomically closely related species Phytophthora mirabilis, Phytophthora phaseoli, and Phytophthora ipomoea; no amplification of DNA from the potato pathogens Phytophthora erythroseptica or Phytophthora nicotianae occurred. Real-time LAMP and PCR assays were developed for A. solani, and the specificity was compared with an existing conventional PCR assay. Importantly, the A. solani real-time LAMP and PCR assays did not amplify the species Alternaria alternata. However, cross-reactivity with Alternaria dauci was observed with the real-time PCR assay and Alternaria brassicae with the real-time LAMP assay. The sensitivity of all assays for the detection of DNA extracted from sporangia/spores of the target pathogens was evaluated. The P. infestans real-time LAMP assay reliably detected 5 pg of DNA, equivalent to ∼1 sporangia per reaction. By comparison, 20 fg of DNA was detectable with the existing real-time PCR assay. In the case of A. solani, real-time LAMP detected 4.4 pg of DNA, equivalent to ∼1 spore per reaction, and real-time PCR detected 200 fg of DNA. In-field air samplers were deployed in two trial plots planted with potato: one infected with P. infestans, and the other infected with A. solani. Four additional samplers were located in commercial potato fields. Air samples were taken through the season, and detection of airborne inoculum of P. infestans and A. solani with both real-time PCR and LAMP was assessed.

Morphological variation in Myxobolus drjagini (Akhmerov, 1954) from silver carp and description of Myxobolus paratypicus n. sp. (Cnidaria: Myxozoa).

There is uncertainty in the identification of Myxobolus drjagini, the causative agent of silver carp twist disease, in the literature. An investigation of fish parasites in Lake Taihu, China, revealed several Myxobolus drjagini-like myxosporeans infecting the subcutaneous tissue of the head skin, the olfactory and oculomotor nerves in the cranial cavity, and the intrafilamental epithelium of the gills of silver carp Hypophthalmichthys molitrix (Valenciennes, 1844). Myxospores from the head skin and the nerves were identified as conspecific to M. drjagini based on morphological and molecular data; although the spores from each of the two organs presented morphological variations. SSU rDNA sequence analysis revealed that the sequence of M. drjagini previously deposited in GenBank (AF085179) was invalid. Myxospores from the gills were identified as Myxobolus paratypicus n. sp. The spores were oval, asymmetric in frontal view, 13.8 (12.9-14.9) µm long, 9.9 (9.2-11.1) µm wide, and 7.0 µm thick. Two pyriform polar capsules were unequal in size (ratio above 4:1) with slightly converging anterior ends, and the posterior end of the large polar capsule extended beyond the middle of the spore. The large polar capsule was 7.5 (6.2-8.2) µm long and 5.0 (4.2-5.6) µm wide; the small polar capsule was 2.7 (2.1-3.6) µm long and 1.4 (1.1-1.9) µm wide. Polar filaments were coiled with 7-8 turns in the large polar capsule. The SSU rDNA sequence of M. paratypicus n. sp. was not identical to that of any myxozoan available in GenBank and showed highest similarity with M. drjagini (96%) and Myxobolus pavlovskii (95%) collected from bighead carp and silver carp, respectively.

Morphological, histological, and molecular description of Myxobolus ompok n. sp. (Myxosporea: Myxobolidae), a kidney myxozoan from Pabdah catfish Ompok pabda (Hamilton, 1822) (Siluriformes: Siluridae) in India.

In a parasitological survey of freshwater fishes near Meerut, Uttar Pradesh, India, myxozoan infections in Pabdah catfish Ompok pabda Ham. (Siluriformes: Siluridae) were found. Round plasmodia and scattered spores of Myxobolus ompok n. sp. were found in the kidney of the host. Plasmodia measuring 150-200 µm were located in the renal interstitium. Spores of Myxobolus ompok n. sp. were elongated pyriform, 13.6-14.4 (14.8 ± 0.42) µm long, 5.6-6.4 (6.5 ± 0.33) µm wide, and 5.2-6.4 (5.9 ± 0.43) µm thick with two equal polar capsules measuring 8.0-8.5 (8.2 ± 0.2) µm in length and 1.5-2.4 (1.8 ± 0.33) in width having six filamental turns. Both the morphology and DNA analysis of the 18S rRNA gene revealed that Myxobolus ompok n. sp. is distinct from previously described species of Myxobolus and shares no significant similarity with any other Myxobolus deposited in the GenBank database. Phylogenetic analysis inferred that this species showed the closest similarity to Myxobolus miyarii (KT001495). This is the first record of any Myxobolus sp. from O. pabda in India.

A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.

Background: Malaria elimination strategies require a thorough understanding of parasite transmission from human to mosquito. A clinical model to induce gametocytes to understand their dynamics and evaluate transmission-blocking interventions (TBI) is currently unavailable. Here, we explore the use of the well-established Controlled Human Malaria Infection model (CHMI) to induce gametocyte carriage with different antimalarial drug regimens. Methods: In a single centre, open-label randomised trial, healthy malaria-naive participants (aged 18­35 years) were infected with Plasmodium falciparum by bites of infected Anopheles mosquitoes. Participants were randomly allocated to four different treatment arms (n = 4 per arm) comprising low-dose (LD) piperaquine (PIP) or sulfadoxine-pyrimethamine (SP), followed by a curative regimen upon recrudescence. Male and female gametocyte densities were determined by molecular assays. Results: Mature gametocytes were observed in all participants (16/16, 100%). Gametocytes appeared 8.5­12 days after the first detection of asexual parasites. Peak gametocyte densities and gametocyte burden was highest in the LD-PIP/SP arm, and associated with the preceding asexual parasite biomass (p=0.026). Male gametocytes had a mean estimated circulation time of 2.7 days (95% CI 1.5­3.9) compared to 5.1 days (95% CI 4.1­6.1) for female gametocytes. Exploratory mosquito feeding assays showed successful sporadic mosquito infections. There were no serious adverse events or significant differences in the occurrence and severity of adverse events between study arms (p=0.49 and p=0.28). Conclusions: The early appearance of gametocytes indicates gametocyte commitment during the first wave of asexual parasites emerging from the liver. Treatment by LD-PIP followed by a curative SP regimen, results in the highest gametocyte densities and the largest number of gametocyte-positive days. This model can be used to evaluate the effect of drugs and vaccines on gametocyte dynamics, and lays the foundation for fulfilling the critical unmet need to evaluate transmission-blocking interventions against falciparum malaria for downstream selection and clinical development. Funding: Funded by PATH Malaria Vaccine Initiative (MVI). Clinical trial number: NCT02836002.

The parasite that causes malaria, named Plasmodium falciparum, has a life cycle that involves both humans and mosquitoes. Starting in the saliva of female Anopheles mosquitoes, it enters a person's bloodstream when the insects feed. It then moves to the person's liver, where it infects liver cells and matures into a stage known as schizonts. The schizonts then divide to form thousands of so-called merozoites, which burst out of the liver cells and into the bloodstream. The merozoites infect red blood cells, producing more schizonts and yet more merozoites, which continue the infection. To complete its life cycle, the parasite must return to a mosquito. Some of the parasites in the person's blood transform into male and female cells called gametocytes that are taken up by a mosquito when it feeds on that person. Inside the mosquito, male and female parasites reproduce to create the next generation of parasites. The new parasites then move to the mosquito's salivary glands, ready to begin another infection. Stopping the parasite being transmitted from humans to mosquitoes will stop the spread of malaria in the population. Yet it has proven difficult to study this part of the life cycle from natural infections. Here, Reuling et al. report a new method for generating gametocytes in human volunteers that will enable closer study of the biology of malaria transmission. The method is developed using the Controlled Human Malaria Infection (CHMI) model. Healthy volunteers without a history of malaria are bitten by mosquitoes infected with malaria parasites. Shortly afterwards, the volunteers are given a drug treatment to control and reduce their symptoms. The gametocytes form during this phase of the infection. At the end of the experiment, all the volunteers receive a final treatment that completely cures the infection. Reuling et al. recruited 16 volunteers and assigned them to four groups at random. Each group received a different drug regime. Roughly a week after the mosquito bites, all participants showed malaria parasites in their blood, and between 8.5 and 12 days later, mature gametocytes started to appear. This early appearance suggests that the parasites start to transform into gametocytes when they first emerge from the liver. The experiment also revealed that female gametocytes stay in the blood for a longer period than their male counterparts. These results are proof of principle for a new way to investigate malaria infection. The new model provides a controlled method for studying P. falciparum gametocytes in people. In the future, it could help to test the impact of drugs and vaccines on gametocytes. Understanding more about these parasites' biology could lead to treatments that block malaria transmission.

Production of a novel monoclonal antibody applicable for an immunochromatographic assay for Kudoa septempunctata spores contaminating the raw olive flounder (Paralichthys olivaceus).

Kudoa septempunctata, a myxosporean parasite of the olive flounder (Paralichthys olivaceus), causes foodborne gastroenteritis after ingestion of contaminated raw flounder. Available methods to detect K. septempunctata require expensive equipment, well-trained personnel, and lengthy procedures. Here we generated a novel monoclonal antibody (MAb 15G11) against K. septempunctata and used it to produce a prototype immunochromatographic assay (prototype Kudoa-ICA). Within 15min, the prototype Kudoa-ICA detected ≥1.0×105spores/mL in a spore suspension and ≥2.0×104spores/g of P. olivaceus muscle. The prototype Kudoa-ICA weakly cross-reacted with spores of K. lateolabracis and K. iwatai. cDNA sequence, expression, and western blot analyses revealed that MAb 15G11 detected an approximately 24-kDa protein encoded by a 573bp mRNA. The cDNA nucleotide and predicted amino acid sequences were not significantly similar to any sequence in the GeneBank database. Immunoelectron microscopy revealed that MAb 15G11 reacted with the sporoplasmic cells and mainly with the capsulogenic cells of the K. septempunctata spore. Although the Kudoa-ICA was weakly cross-reactive with two other Kudoa species, it detected >1.0×106spores/g of K. septempunctata in P. olivaceus muscle, which is the criterion used to indicate a violation of the Food Hygiene Law of Japan. We conclude that MAb 15G11 may be suitable for use in an immunochromatographic assay for screening P. olivaceus muscle contaminated with K. septempunctata at food distribution sites such as food wholesalers, grocery stores, and restaurants.

Redescription of Chloromyxum ellipticum Li & Nie, 1973 (Myxosporea: Chloromyxidae) infecting the gall bladder of grass carp Ctenopharyngodon idellus Valenciennes, 1844, supplemented by morphological and molecular characteristics.

The traditional taxonomy of the genus Chloromyxum Mingazzini, 1890 has been intensively challenged to be paraphyletic by recent ribosomal DNA (rDNA)-based phylogenetic analysis. Undersampling to get rich sequence data to infer more scientific phylogenetic relationships makes scientists conservatively assign all non-marine elasmobranch-infecting species as Chloromyxum sensu lato. Although complex ridge pattern on the spore surface observed by scanning electron microscopy was thought to be critical for the identification of Chloromyxum species, insufficient data also prevent this ultrastructural data to be a valid taxonomic feature for this genus. It is especial for Chloromyxum species to be reported in China. Molecular and ultrastructural characteristics are yet available for all 22 Chloromyxum species recorded in China. During the investigation of the diversity of coelozoic fish myxosporeans, Chloromyxum ellipticum Li & Nie, 1973 was found to highly infect the gall bladder of Ctenopharyngodon idellus Valenciennes, 1844 in Poyang Lake watershed of Jiangxi province, Eastern China. Here, we redescribed it by the currently recommended holistic approach of combining morphological, ultrastructural, and molecular characteristics. Mature spores were found floating free in the gall bladder, but no plasmodium observed. Spores are typical freshwater teleost-infecting Chloromyxum species, spherical or subspherical in lateral view, measuring 7.7 ± 0.08 µm (6.9-9.1) in length, 6.3 ± 0.09 µm (5.6-7.6) in width, and 5.8 ± 0.20 µm (5.2-6.3) in thickness. Four pyriform polar capsules, located at the anterior end of the spores, were equal in size, 3.3 ± 0.06 µm (2.2-4.1) long and 2.1 ± 0.03 µm (1.7-2.5) wide. Polar filaments coiled with four to five turns. Two equal spore valves are symmetrical, with 10-16 surface extrasutural ridges per valve, aligned along the longitudinal axis. The obtained partial 18S rDNA of C. ellipticum did not match any sequences available in GenBank. Phylogenetic analysis showed that C. ellipticum clustered firstly with Chloromyxum legeri with robust nodal support and grouped then with urinary system of freshwater teleost-infecting Chloromyxum clade, rather than other gall bladder of freshwater teleost-infecting clade.

Myxobolus chushi n. sp. (Myxozoa:Myxosporea) parasitizing Schizothorax niger (Heckel), a native cyprinid fish from Wullar Lake in Kashmir Himalayas.

In the study, a new species, Myxobolus chushi n. sp. infecting gills of wild specimens of Schizothorax niger (Heckel) inhabiting Wullar Lake in Kashmir Himalayas, (J&K) India has been described based on morphology of the myxospore and using partial 18S rDNA sequencing. Pathological changes in the gills have been studied with the help of histological sections stained with Luna's method. Twenty fish specimens were examined, out of which four had oval, white plasmodia in gills measuring 2.0×0.5mm. The myxospores were spherical to ovoidal in shape with slightly attenuated posterior end, measuring 11.17±0.23 (10.60-11.40)µm in length and 9.14±0.06 (8.80-9.20)µm in width, having a prominent pore at the anterior end. The polar capsules were pyriform in shape, measuring 4.25±0.15 (4.00-4.40)µm in length and 2.38±0.27 (2.00-2.65)µm in width having polar filaments forming coils up to 5 in number. Parietal folds 9 in number present on the posterior part of the shell. The intensity of infection was recorded to be moderate as indicated by gill plasmodial index (GPI=2). The plasmodium was located in the vascular network occupying whole of the gill lamella therefore typed as intralamellar vascular type, LV3. Analysis of 18S small subunit (SSU) rDNA sequence of the isolate demonstrated 90% homogeneity with M. sp. KLT-2014 infecting scales of Labeo rohita from Myanmar and 89% with M. dermiscalis infecting scales of Labeo rohita from India.

Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay.

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm(-3)) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.

Nivicolous Stemonitales from the Austral Andes: analysis of morphological variability, distribution and phenology as a first step toward testing the large-scale coherence of species and biogeographical properties.

Nivicolous myxomycetes occur at the edge of spring-melting snow in mountainous areas. They are mostly considered cosmopolitan species morphologically and ecologically uniform across their entire distribution ranges. Thus, long-distance dispersal has been suggested to be the main mechanism shaping their ranges and geographical variability patterns. To test this hypothesis we conducted the first detailed analysis of morphological variability, occurrence frequency and phenology of nivicolous myxomycetes collected in the hitherto unexplored Austral Andes of South America (southern hemisphere = SH) in the comparative context of data from the northern hemisphere (NH). We used Stemonitales, the most representative and numerous taxonomic order in nivicolous myxomycetes, as a model. A total of 131 South American collections represented 13 species or morphotypes. One of them, Lamproderma andinum, is new to science and described here. Several others, L. aeneum, L. album, L. pulveratum, "Meriderma aff. aggregatum ad. int.", M. carestiae and "M. spinulosporum ad. int.", were previously unknown from the SH. Lamproderma ovoideum is reported for the first time from South America and Collaria nigricapillitia is new for Argentina. The fine-scale morphological analysis of all species from the study area and reference NH material demonstrated a high intraspecific variability in most of them. This suggests isolation and independent evolutionary processes among remote populations. On the other hand, the uniform morphology of a few species indicates that long-distance dispersal is also an effective mechanism, although not as universal as usually assumed, in some nivicolous myxomycetes. Analysis of nivicolous species assemblages also showed significant differences among major geographic regions in that the Stemonitales were significantly less common in the SH than in the NH. Furthermore, the occurrence of nivicolous species in summer and autumn, out of the typical phenological season, is recognized as a possible distinctive phenomenon for the SH populations.

Didymium xerophilum, a new myxomycete from the tropical Andes.

A new species of Didymium (Myxomycetes), D. xerophilum, is described, and some details of its life cycle are provided. The new species was collected during studies of arid areas of Argentina and Peru. It can be distinguished by the persistent funnel-shaped invagination of the peridium, the top of which appears as a deep umbilicus in closed sporothecae, and the calcareous hypothallus shared among several sporocarps. This combination of characters, with a circumscissile dehiscence of the sporotheca and a cream stalk packed with rhombic lime crystals, is unknown in other described species. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. Phylogenetic analysis with 18S rDNA sequences of different species of Didymium supports the distinct identity of this new species. Some collections of this myxomycete were made at up to 4600 m, an altitude almost unknown for this group of microorganisms.

Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona.

We investigated the occurrence of Cryptosporidium, Giardia, and Cyclospora at two wastewater treatment plants (WWTPs) in Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent wastewater samples were collected monthly, and protozoan (oo)cysts were concentrated using an electronegative filter, followed by the detection of protozoa using fluorescent microscopy (Cryptosporidium oocysts and Giardia cysts) and PCR-based methods (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis). The concentration of Giardia cysts in the influent was always higher than that of Cryptosporidium oocysts (mean concentration of 4.8-6.4×10(3) versus 7.4×10(1)-1.0×10(2)(oo)cysts/l) with no clear seasonality, and log10 reduction of Giardia cysts was significantly higher than that of Cryptosporidium oocysts for both WWTPs (P<0.05). Log10 reduction of Giardia cysts at the WWTP utilizing activated sludge was significantly higher than the other WWTP using trickling filter (P=0.014), while no statistically significant difference between the two WWTPs was observed for the log10 reduction of Cryptosporidium oocysts (P=0.207). Phylogenetic analysis revealed that G. intestinalis strains identified in wastewater belonged to two assemblages, AII and B, which are potentially infectious to humans. C. cayetanensis was also detected from both influent and effluent using a newly developed quantitative PCR, with the highest influent concentration of 1.2×10(4)copies/l. Our results demonstrated that these protozoan pathogens are prevalent in the study area and that efficacy of the conventional wastewater treatment processes at physically removing (oo)cysts is limited.

Early development and tissue distribution of Pseudoloma neurophilia in the zebrafish, Danio rerio.

The early proliferative stages of the microsporidian parasite, Pseudoloma neurophilia were visualized in larval zebrafish, Danio rerio, using histological sections with a combination of an in situ hybridization probe specific to the P. neurophilia small-subunit ribosomal RNA gene, standard hematoxylin-eosin stain, and the Luna stain to visualize spores. Beginning at 5 d post fertilization, fish were exposed to P. neurophilia and examined at 12, 24, 36, 48, 72, 96, and 120 h post exposure (hpe). At 12 hpe, intact spores in the intestinal lumen and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx and within hepatocytes were observed. Proliferative stages were visualized in the pancreas and kidney at 36-48 hpe and in the spinal cord, eye, and skeletal muscle beginning at 72 hpe. The first spore stages of P. neurophilia were observed at 96 hpe in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle. The parasite was only observed in the brain of larval fish at 120 hpe. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hpe indicates that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tube.

Coccidia in passerines from the Nevado de Toluca National Park, Mexico.

In this study, we found unsporulated coccidia oocysts in passerines from the Nevado de Toluca National Park, Mexico. We captured birds and took samples of their droppings during three field visits. We examined a total of 72 fecal samples and found unsporulated coccidia oocysts in 10 samples from five passerine species: Atlapetes pileatus (3), Cardelina ruber (1), Mniotilta varia (1), Oreothlypis celata (2) and Regulus calendula (3). This appears to be the first recorded study of unsporulated coccidia oocysts in passerine species from Mexico.

Description, culture and phylogenetic position of a new xerotolerant species of Physarum.

A new widespread myxomycete species, Physarum pseudonotabile, inhabiting the arid regions of the Eurasia, South and North America is described and illustrated. Tentatively assigned to Ph. notabile T. Macbr., a phylogeny based on the small ribosomal subunit (SSU) and elongation factor 1 alpha (EF1a) genes placed the new species in a clade far from Ph. notabile. Ph. pseudonotabile was found to be frequent in surveys based on the moist chamber culture technique with samples of litter, bark and herbivore dung collected in dry steppe and deserts of the Caspian lowland (Russia), Kazakhstan, Mongolia, China, Spain, Argentina and USA. The main morphological difference between Ph. pseudonotabile and Ph. notabile lies in spore ornamentation. Spores of the former species display irregularly distributed verrucae, whereas the latter species possesses spores with dense and regularly arranged spinulae. In addition, the ecological preferences of the two species differ. Ph. pseudonotabile inhabits the bark of living plants and ground litter in arid regions, whereas Ph. notabile is found on coarse woody debris in boreal and temperate forests. Although the new species appears to be closest to Ph. notabile morphologically, the phylogenetic analysis reveals Ph. pusillum and Ph. nivale as the closest relatives. In addition, the molecular investigations revealed a considerable amount of hidden diversity within species of Physarum with gray lime flakes. Currently we have only sufficient material to assess the morphological variation of Ph. pseudonotabile but expect that more taxa within this clade may emerge within studies combining morphological and molecular analyses.

The detection of Giardia cysts in a large-scale wastewater treatment plant in Hamburg, Germany.

Giardia is one of the most common human enteric parasites that continue to be a major cause of diarrheal disease globally. Wastewater is an important source of Giardia transmission, and control of the pathogen by appropriate treatment of wastewater would limit its transmission. In this study the occurrence of Giardia cysts at various stages of the wastewater treatment plants was monitored for a period of 18 mo. Using immunomagnetic separation and immunofluorescence with monoclonal antibodies, cysts were detected in all samples throughout the sampling period at a concentration ranging from 50 to 7548 cysts/L. The overall removal efficiency of the cysts in the treatment plants was 78%. Seasonal analyses of results revealed that the pathogens (cysts) were most prevalent in influents and effluents during autumn and winter.

Occurrence of organic chlorinated pesticides and their ecological effects on soil protozoa in the agricultural soils of North Western Beijing, China.

The occurrence of ∑HCHs, ∑DDTs, protozoa abundance and their community structure in surface soils of orchards, vegetable lands, and barren lands in northern west outskirts of Beijing were detected in order to investigate the protozoa responses to low dose organic chlorinated Pesticides (OCPs) after long-term field-based exposure. Significant differences in total concentrations of HCHs and DDTs were found among the three general groups ranking in decreasing order of concentration from orchard>vegetable lands >barren lands. Ciliate was the rare group in surface soils of all the sampling groups. The abundance of flagellate, ciliate, and amoebae in vegetable soils were significantly higher than those in orchard soils. The abundance of all the taxa of protozoa was strongly negative correlated with the residue level of ∑HCHs and ∑DDTs (P<0.05) in agricultural soils. However, no negative correlation between the residue levels of OCPs and protozoa abundance was shown in both the orchard and the barren soils. This field study demonstrated a considerable long-term impact of the OCPs residue on the abundance of protozoa in soils, and that the abundance of soil protozoa was much more influenced by land use type in association with different soil properties.

Collection and cultivation of dictyostelids from the wild.

Dictyostelium discoideum is a commonly used model organism for the study of biological processes such as chemotaxis, cell communication, and development. While these studies primarily focus on a single clone, recent work has revealed a host of questions that can only be answered from studies of multiple genetically distinct clones. Understanding intraspecific clone conflict, kin recognition, differential adhesion, and other kinds of interactions likely to occur in the natural soil habitat can only come from studies of multiple clones. Studies of populations of wild isolates are also important for understanding the factors contributing to associations such as species co-occurrences and to observed inter- and intraspecific interactions such as those found between bacteria and D. discoideum. Natural isolates of Dictyostelium are easily found in soil and leaf litter in nearly all habitats. Here we describe a simple and successful method for isolating new wild clones from soil, then isolating single clonal strains, and storing them for future use.

Physarum andinum, a new nivicolous species of myxomycete from the Patagonian Andes.

A new nivicolous species of Physarum was discovered during the study of myxomycetes in the Patagonian Andes of South America. It is described herein under the name Physarum andinum. The species is characterized by stalked sporophores or more rarely sessile sporocarps or short plasmodiocarps. The sporocarps are strikingly large, reaching 2.6 mm tall and 3 mm diam when open, and have a peridium with three layers, the internal layer being clearly visible and opening separately. Physarum andinum was found at five localities in Argentina as well as in herbarium material collected about 100 y ago in Chile. The new species is reminiscent of the non-nivicolous species Physarum brunneolum, but the latter forms smaller sporophores, has darker spores and the three layers of the peridium are adhered and open together. The characters of the new species were examined under stereomicroscope, light microscope and scanning electron microscope and micrographs of relevant details are included.

A new species of Trichia from Australia.

A new species of Trichia (myxomycetes) was collected during surveys for myxomycetes carried out in Nothofagus cunninghamii forests in western Tasmania in May 2008 and a similar survey carried out in a N. morrei forest in New South Wales in May 2009. This new species, T. brimsiorum, is described and illustrated. It resembles T. decipiens in overall shape and size of the sporocarps but has smaller spores and the ornamentation of the capillitium is different.

Proteomic analysis of the cyst stage of Entamoeba histolytica.

BACKGROUND: The category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools. AIMS: Three main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools. METHODS: Cysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins. RESULTS: A total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets. MAJOR CONCLUSIONS: The proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts.