Rotifers ingest oocysts of Cryptosporidium parvum

Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Eauchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.     

Swimming speed and Reynolds numbers of eleven freshwater rotifer species

We obtained data on the swimming speed of 11 freshwater rotifer species. These data were analyzed in terms of Reynolds numbers given the fact that rotifers are in the evolutionary boundary between the use of cilia and swimming appendages. Swimming speed ranged from 0.174 to 0.542 mm ^–1. Philodina acuticornis odiosa was the fastest rotifer in terms of absolute speed. However, Gastropus hyptopus, the smallest of the rotifers analyzed, was the fastest (2.849) in terms of body-lengths s^–1. Reynolds numbers (Re) among the 11 species analyzed varied from 0.023 to 0.301. Lecane furcata had the smallest Re, while Epiphanes senta had the highest Re. The two bdelloid rotifers analyzed in this work swam five times faster than they crept. The importance of Reynolds numbers and drag coefficients are discussed in view of the present results and data found in the literature.     

五种淡水轮虫种群增长参数的比较研究

在32℃培养条件下,实验研究了5种淡水常见浮游轮虫的种群增长参数。通过编制生命表计算其内禀增长率rm(h-1)、净生殖力(R0)和世代时间T(h)分别为:萼花臂尾轮虫-0.091、14.1和34.1;壶状臂尾轮虫-0.055、6.9和38.4;角突臂尾轮虫-0.035、4.9和50.9;臂尾水轮虫-0.038、4.3和46.1以及裂足臂尾轮虫-0.017、2.1和43.7。其中,萼花臂尾轮虫内禀增长率大,繁殖率高,可作为批量培养的首选种类。       

Using Amplified Fragment Length Polymorphisms (AFLP) to Study Genetic Variability in Several Freshwater Rotifer Species

We have used amplified fragment length polymorphisms (AFLP) to investigate the potential of this technique as a tool to measure genetic variability in eight species of freshwater rotifers: Brachionus calyciflorus, Lecane bulla, L. luna, L. quadridentata, Plationus patulus, Philodina acuticornis odiosa, Rotaria neptunia, and R. rotatoria. We used nine combinations of oligonucleotides. We observed a total of 806 amplified bands, 798 polymorphic and 8 monomorphic. The data were analyzed using cluster analysis with UPGMA, first within each set of oligonucleotide combination and finally using all nine combinations. Our best dendrogram clearly separated monogononts from digononts, and grouped the species of monogononts in the two genera. However, it grouped R. neptunia with P. acuticornis odiosa rather than with R. rotatoria. These results are discussed in view of recent works in the literature measuring genetic variability and discussing the phylogeny of the Rotifera.     

Some new and rare warmwater rotifers

Two new rotifer species, Lecane (Monostyla) aliger n.sp. and Proales pugio n.sp. are described from the Bahama Islands, Florida and California, and their autecology outlined. Some other rare rotifers are discussed which also prefer subtropical conditions. They are: Epiphanes clavulata, Epiphanes brachionus spinosus, Lecane crepida and Proalides tentaculatus tentaculatus. The existence of subtropical rotifer associations is discussed and supported by ecological data.     

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.     

Developmental stages in diapausing eggs: an investigation across monogonont rotifer species

Monogononts reproduce by parthenogenesis punctuated by events of mixis that generate ‘resting eggs’, which actually are embryos whose development is arrested. We document the nuclei number and position of the resting eggs of nine rotifer species (Brachionus plicatilis, B. calyciflorus, B. manjavacas, Plationus patulus, Epiphanes senta, E. chihuahuaensis, Rhinoglena frontalis, Lecane bulla and Sinantherina socialis) in the attempt to assess the stage at which dormant embryos are arrested. The morphology of the entire embryos was reconstructed visualising nuclear DNA using confocal microscopy, and their developmental stage identified. Two groups of species were identified: dormant embryos of one group possessed on average less than 30 nuclei, with low variation within and between species, and the stage may correspond to early gastrula; embryos in the second group contain relatively more nuclei, averaging around 40–60, with higher variation within species. The two groups of species seem not to reflect any phylogenetic relationship. Consequences of the dormant embryo stages are discussed.     

Detection of Giardia cysts with a cDNA probe and applications to water samples

Giardiasis is the most common human parasite infection in the United States, causing a lengthy course of diarrhea. Transmission of Giardia species is by the fecal-oral route, and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia species in drinking water through the Surface Water Treatment Rule. Current methods for detection of Giardia species in water rely primarily on microscopic observation of water concentrates with immunofluorescence techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 bp long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of the M13 vector with an insert was isolated from lysed host Escherichia coli XL1-Blue and used for production of the cDNA probe by nick translation with 32P-labeled nucleotides. Six different protocols were tested for extracting nucleic acids from the cysts. With the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates with dot blot hybridization assays.     

Detection of Giardia cysts with a cDNA probe and applications to water samples.

Giardiasis is the most common human parasite infection in the United States, causing a lengthy course of diarrhea. Transmission of Giardia species is by the fecal-oral route, and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia species in drinking water through the Surface Water Treatment Rule. Current methods for detection of Giardia species in water rely primarily on microscopic observation of water concentrates with immunofluorescence techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 bp long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of the M13 vector with an insert was isolated from lysed host Escherichia coli XL1-Blue and used for production of the cDNA probe by nick translation with 32P-labeled nucleotides. Six different protocols were tested for extracting nucleic acids from the cysts. With the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates with dot blot hybridization assays.     

Detection of Giardia intestinalis assemblages A, B and D in domestic cats from Warsaw, Poland

Giardia intestinalis is a complex species divided into 7 assemblages (A - G). Two of them (A and B) are infective for both humans and animals. In cats four assemblages can occur: A, B, D, and F Assemblages A and B infect either cats, dogs and humans, assemblage D infects cats and dogs and assemblage F only cats. The purpose of this study was to determine the prevalence and genotypes of G. intestinalis in cats from Warsaw. From November 2006 to March 2007 a hundred sixty samples of stool were collected and examined by light microscopy. G. intestinalis cysts were detected in 3.75% of samples. DNA extracted from positive samples was used as template for PCR-RFLP using Giardia specific primers and the amplicons were sequenced. A comparison of the obtained DNA sequences with the Giardia sequences in the GeneBank database revealed assemblage A in 1.25% of the investigated cats, assemblage B in 1.25% and D in 1.25%.     

Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA Sequences

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.     

落叶生境蛭态轮虫物种多样性及四种中国新记录种

为研究落叶生境蛭态轮虫物种多样性,对中国8省28个落叶样品进行了调查,采样范围涵盖中国热带、亚热带及温带地区,海拔高度从3m到1500m不等,共记录蛭态轮虫3科8属29种,其中中国新记录属1属,中国新记录种4种,将中国蛭态轮虫记录由88种增加到92种.研究获取了新记录种跛足突盘轮虫(Bradyscela clauda ...     

Antigenic conservation and variation in Giardia cysts from various vertebrate hosts.

Monoclonal antibodies produced against Giardia muris cysts reacted in indirect immunofluorescence with homologous cysts and cysts from a Giardia-infected wild Norway rat but did not cross-react with Giardia lamblia cysts of human, dog, or beaver sources. Another monoclonal antibody raised against Giardia simoni cysts from the Norway rat reacted with homologous cysts (rat) and cross-reacted with cysts from a cow. The demonstration of antigenic differences at the cyst surfaces of Giardia organisms of animal and human origin suggests that it is possible to identify the animal source of Giardia cysts according to their pattern of reactivity with monoclonal antibodies. Such identification would have a useful application in affirming the possible zoonotic transmission of animal source Giardia species to humans.     

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.     

Phylogenomics of Unusual Histone H2A Variants in Bdelloid Rotifers

Rotifers of Class Bdelloidea are remarkable in having evolved for millions of years, apparently without males and meiosis. In addition, they are unusually resistant to desiccation and ionizing radiation and are able to repair hundreds of radiation-induced DNA double-strand breaks per genome with little effect on viability or reproduction. Because specific histone H2A variants are involved in DSB repair and certain meiotic processes in other eukaryotes, we investigated the histone H2A genes and proteins of two bdelloid species. Genomic libraries were built and probed to identify histone H2A genes in Adineta vaga and Philodina roseola, species representing two different bdelloid families. The expressed H2A proteins were visualized on SDS-PAGE gels and identified by tandem mass spectrometry. We find that neither the core histone H2A, present in nearly all other eukaryotes, nor the H2AX variant, a ubiquitous component of the eukaryotic DSB repair machinery, are present in bdelloid rotifers. Instead, they are replaced by unusual histone H2A variants of higher mass. In contrast, a species of rotifer belonging to the facultatively sexual, desiccation- and radiation-intolerant sister class of bdelloid rotifers, the monogononts, contains a canonical core histone H2A and appears to lack the bdelloid H2A variant genes. Applying phylogenetic tools, we demonstrate that the bdelloid-specific H2A variants arose as distinct lineages from canonical H2A separate from those leading to the H2AX and H2AZ variants. The replacement of core H2A and H2AX in bdelloid rotifers by previously uncharacterized H2A variants with extended carboxy-terminal tails is further evidence for evolutionary diversity within this class of histone H2A genes and may represent adaptation to unusual features specific to bdelloid rotifers.     

Distribution of Giardia duodenalis genotypes and subgenotypes in raw urban wastewater in Milwaukee, Wisconsin

Giardia cysts in 131 raw wastewater samples from Milwaukee, Wis., were genotyped by sequence analysis of the triosephosphate isomerase gene which showed the presence of two distinct genotypes (assemblages A and B) of Giardia duodenalis. Of the 131 samples, 111 belonged to assemblage A, and the remaining samples belonged to assemblage B. A high degree of genetic polymorphism was evident within the assemblage B cluster, with 10 distinct subgenotypes identified, eight of which have not been reported before.     

Prevalence of Giardia sp. in a beaver colony and the resulting environmental contamination

The prevalence of Giardia sp. in a beaver (Castor canadensis) colony in Colorado was determined by the collection and analysis of fecal samples over a period of 14 mo. Environmental contamination was monitored through the use and analysis of water filter samples. Beaver shed cysts of Giardia sp. in their feces throughout the year with temporal variations in the prevalence, and became infected as kits and remained infected as juveniles and adults. Beaver served as amplification hosts for Giardia sp. and contaminated surface waters downstream from their dams in late spring and early fall. In slow moving waters the cysts of Giardia sp. settled rapidly. Muskrats (Ondatra zibethicus) were the only other species of wildlife shedding cysts of Giardia sp. on the study area.     

Infectivity of cryopreserved Giardia cysts for Mongolian gerbils (Meriones unguiculatus)

Although Giardia species trophozoites have been cryopreserved successfully, no report on the successful cryopreservation of cysts could be found. Using infectivity to Mongolian gerbils (Meriones unguiculatus) as a measure of cyst viability, we tested the viability of 4 strains of Giardia cysts that had been cryopreserved for 1-67 wk. Cysts were frozen in either Keister's medium or physiological saline, both containing 5% or 7.5% dimethylsulfoxide as the cryoprotectant. Viability of cryopreserved cysts was dependent upon the number of cysts inoculated, the length of time cysts were held at 4 C before cryopreservation, and the cryopreserving medium. Infection was established in gerbils by inoculating them with cysts that had been cryopreserved for up to 67 wk, cysts that had been held for less than 30 days before cryopreservation, and cysts frozen in Keister's medium. Saline appears to be unsuitable as a freezing medium for the cryopreservation of Giardia cysts.     

On Soil Rotatoria from a Lithotelma near Halali Lodge in Etosha National Park in N-Namibia,South Africa

A soil sample out of a dried rockpool (lithotelma) on a dolomit hill in the Etosha National Park, Namibia, was investigated for possible occurence of rotifers. Besides a numerous and rich microfaunae, e.g. Zooflagellata, Rhizopoda. Acari, Nematoda, Tardigrada and Copepoda, 24 rotifers were found. Until today, four so far unknown Bdelloidea could be described: Dissotrocha decembullata n. sp., Dissotrocha hertzogi aculeata n. ssp., Otostephanos jersabeki n. sp. and Philodina foissneri n. sp.     

Localization and neuroanatomy of catecholaminergic neurons in some rotifer species

We studied Dicranophorus sp., Platyias quadricornis (Ehrb.) and Rotaria tardigrada (Ehrb.). These rotifers, systematically distant from each other, show the same pattern of the catecholaminergic (CA-ergic) part of the nervous system. It is formed of a small (23–24), but steady number of neurons characteristic for each species. Three types of CA-ergic neurons are described. The sizes of neurons vary from to two to ten µm. The distribution of the brain neurons is correlated with body shape. Such a type of nervous system is topographically comparable to the concentrated orthogon of the flatworms.