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Viability estimation of the highly resistant oocysts of Cryptosporidium remains a key issue for the monitoring and control of this pathogen. We present here a simple 'one tube' quantitative PCR (qPCR) protocol for viability estimation using a DNA extraction protocol which preferentially solubilizes excysted sporozoites rather than oocysts. Parasite DNA released from excysted sporozoites was quantified by real-time qPCR using a ribosomal DNA marker. The qPCR signal was directly proportional to the number of oocysts excysted, and a power-law relationship was noted between oocyst age and the proportion excysting. Unexcysted oocysts released negligible amounts of DNA making the method suitable for estimating viability of as few as 10 oocysts.

Besides Aelurostrongylus abstrusus, other parasites belonging to the superfamily Metastrongyloidea, namely Oslerus rostratus, Troglostrongylus brevior and to the family Trichuridae, i.e. Eucoleus aerophilus (syn. Capillaria aerophila), have also been reported as agents of respiratory infection in domestic cats. A case of simultaneous infection by four feline lungworm species in Sardinia is herein described. An adult female cat (Felis silvestris catus), road-killed in the southeast part of Sardinia (municipality of Villacidro, province of Cagliari), Italy, was referred to the Laboratory of Parasitology of the Veterinary Teaching Hospital in Sassari. At necropsy, the lungs were examined and dissected under a stereomicroscope for the presence of parasites, and first-stage larvae (L1) of broncho-pulmonary nematodes were searched for in a faecal sample using the Baermann method. Parasites collected in the lungs were morphologically identified as A. abstrusus, E. aerophilus, and O. rostratus. In addition to the above species, L1s of Troglostrongylus spp. were detected at coproscopy but no adult specimen was found in the lungs. The morphological identification was confirmed by the molecular amplification and sequencing of cox1 mitochondrial gene, 18S and ITS2 ribosomal DNA. This finding stands as the first simultaneous infection by four feline lungworm species in the same animal, and as the first report of O. rostratus and E. aerophilus in Sardinia.

Kudoa neothunni is the first described Kudoa species having six shell valves and polar capsules, previously assigned to the genus Hexacapsula Arai and Matsumoto, 1953. Since its genetic analyses remain to be conducted, the present study characterizes the ribosomal RNA gene (rDNA) using two isolates from a yellowfin tuna (Thunnus albacares) with post-harvest myoliquefaction and a northern bluefin tuna (Thunnus thynnus) without tissue degradation. Spores of the two isolates localized in the myofiber of trunk muscles, forming pseudocysts, and showed typical morphology of K. neothunni with six equal-sized shell valves radially arranged in apical view: spores (n = 15) measuring 9.5-11.4 μm in width, 7.3-8.6 μm in suture width, 8.9-10.9 μm in thickness, and 7.3-7.7 μm in length; and polar capsules measuring 3.6-4.1 μm by 1.8-2.3 μm. In lateral view, the spores were pyramidal in shape without apical protrusions. Their 18S and 5.8S rDNA sequences were essentially identical, but variations in the ITS1 (62.4 % similarity across 757-bp length), ITS2 (66.9 % similarity across 599-bp length), and 28S (99.0 % similarity across 2,245-bp length) rDNA regions existed between the two isolates. On phylogenetic trees based on the 18S or 28S rDNA sequence, K. neothunni formed a clade with Kudoa spp. with more than four shell valves and polar capsules, particularly K. grammatorcyni and K. scomberomori. Semiquadrate spores of a kudoid species with four shell valves and polar capsules were detected from minute cysts (0.30-0.75 mm by 0.20-0.40 mm) embedded in the trunk muscle of a chub mackerel (Scomber japonicus) fished in the Sea of Japan. Morphologically, it resembled K. caudata described from a chub mackerel fished in the southeastern Pacific Ocean off Peru; however, it lacked filamentous projections on the shell valves of spores. Additionally, it morphologically resembled K. thunni described from a yellowfin tuna also fished in the Pacific Ocean; spores (n = 30) measuring 8.2-10.5 μm in width, 7.0-8.8 μm in thickness, and 6.1-6.8 μm in length; and polar capsule measuring 2.5-3.4 μm by 1.3-2.0 μm. The similarities of the 18S and 28S rDNA sequences between these two species were 98.5 % and 96.3 %, respectively. Simultaneously, the dimensions of cysts in the trunk muscle formed by K. thunni are clearly larger than those of the present species from a chub mackerel: 1.3-2.0 mm by 1.1-1.4 mm (n = 14) vs. 0.30-0.75 mm by 0.20-0.40 mm (n = 7), respectively. Thus, Kudoa scomberi n. sp. is proposed for this multivalvulid species found in the chub mackerel.

Two members of Aulopiformes (grinners) were examined for myxosporean parasites in their gallbladders. Synodus saurus and Aulopus filamentosus from Cretan Sea, Greece were infected by Ceratomyxa parasites which are described in this paper. Both species of Ceratomyxa are new and referred for the first time as Ceratomyxa cretensis n.sp. and Ceratomyxa filamentosi n.sp. The new species are described morphologically by light and scanning electron microscopy analysis, and characterized genetically by sequencing the small subunit (SSU) rDNA. The SSU rDNA sequences obtained from the two novel Ceratomyxa species were used for the construction of a phylogenetic tree with all the available Ceratomyxa SSU rDNA sequences.

Helicobacter pylori able to express green fluorescent protein, as well as an ATCC strain, and a clinical isolate of this pathogen were evaluated for their ability to survive predation by Acanthamoeba polyphaga. Ingestion was evaluated by microscopic observation of the GFP-H. pylori and BacLight™-stained cells. Following phagocytosis, the fate of cells was assessed by fluorescent in situ hybridization with an oligonucleotide targeting H. pylori 16S rRNA and by quantitative-polymerase chain reaction (qPCR) tests with primers to 16S rDNA. Fluorescent in situ hybridization tests were inconclusive with only a small percentage of amoebae apparently containing active intracellular H. pylori. Furthermore, no increase in bacterial cells was detected by qPCR. Additional research is required to elucidate the mechanisms by which amoebae phagocytize this important bacterial pathogen.

The objective of this study was to identify species of rhizobia (from the IPA 403 and IPA 49 isolates), to assess the physico-chemical characteristics of the biopolymers produced by these rhizobia and to determine the soluble intracellular proteins that are present in these rhizobia. The polysaccharides containing acetyl and pyruvic acid groups that were produced by different strains that had been cultivated in yeast extract mannitol (YEM) medium for 132, 144, and 168 h were evaluated for yield, viscosity, and concentration. Based on the analysis of their partial 16S rDNA sequences, both isolates were identified as Rhizobium tropici. The polymers produced in liquid YEM medium were recovered, dried and weighed to determine culture yield. Soluble intracellular proteins were identified through the techniques of 2D-PAGE and mass spectrometry for cultures that were cultivated for 168 h. The largest biopolymer yield and the highest viscosity and concentration of acetyl and pyruvic acids were obtained from the IPA 403 isolate after 168 h of culture. The proteins that were identified for the CIAT 899 isolate included elongation factor TU, a chaperone; GroE/GroEs and a putative glycosyltransferase, all of which catalyze the production of polysaccharides. For the IPA 403 strain, dinitrogenase and nitrogenase iron proteins were found. In the IPA 49 strain, glyceraldehyde-3-phosphate dehydrogenase was found along with two other proteins, the beta subunit of an electron-transferring flavoprotein and a dehydrogenase.

Biogenic origin of the significant proportion of coal bed methane has indicated the role of microbial communities in methanogenesis. By using cultivation-independent approach, we have analysed the archaeal and bacterial community present in the formation water of an Indian coal bed at 600-700 m depth to understand their role in methanogenesis. Presence of methanogens in the formation water was inferred by epifluorescence microscopy and PCR amplification of mcrA gene. Archaeal 16S rRNA gene clone library from the formation water metagenome was dominated by methanogens showing similarity to Methanobacterium, Methanothermobacter and Methanolinea whereas the clones of bacterial 16S rRNA gene library were closely related to Azonexus, Azospira, Dechloromonas and Thauera. Thus, microbial community of the formation water consisted of predominantly hydrogenotrophic methanogens and the proteobacteria capable of nitrogen fixation, nitrate reduction and polyaromatic compound degradation. Methanogenic potential of the microbial community present in the formation water was elucidated by the production of methane in the enrichment culture, which contained 16S rRNA gene sequences showing close relatedness to the genus Methanobacterium. Microcosm using formation water as medium as well as a source of inoculum and coal as carbon source produced significant amount of methane which increased considerably by the addition of nitrite. The dominance of Diaphorobacter sp. in nitrite amended microcosm indicated their important role in supporting methanogenesis in the coal bed. This is the first study indicating existence of methanogenic and bacterial community in an Indian coal bed that is capable of in situ biotransformation of coal into methane.

Toxoplasmosis, a protozoan disease caused by the coccidian parasite Toxoplasma gondii, is one of the most prevalent parasitic diseases of humans. Although most infections are subclinical and asymptomatic, it has a great importance with respect to immunocompromized hosts and congenitally infected newborns. The diagnosis of T. gondii infection has gained in great interest over the past decades with a wide variety of techniques being investigated from animal inoculation to advanced molecular techniques. This paper reviews and highlights the development and evaluation of polymerase chain reaction in the diagnosis of congenital toxoplasmosis in human population to present the redundant research on it in a chronologically simple approach.

Mezcal is an alcoholic beverage obtained from the distillation of fermented juices of cooked Agave spp. plant stalks (agave must), and each region in Mexico with denomination of origin uses defined Agave species to prepare mezcal with unique organoleptic characteristics. During fermentation to produce mezcal in the state of Tamaulipas, not only alcohol-producing yeasts are involved, but also a lactic acid bacterial community that has not been characterized yet. In order to address this lack of knowledge on this traditional Mexican beverage, we performed a DGGE-16S rRNA analysis of the lactic acid bacterial diversity and metabolite accumulation during the fermentation of a typical agave must that is rustically produced in San Carlos County (Tamaulipas, Mexico). The analysis of metabolite production indicated a short but important malolactic fermentation stage not previously described for mezcal. The denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes showed a distinctive lactic acid bacterial community composed mainly of Pediococcus parvulus, Lactobacillus brevis, Lactobacillus composti, Lactobacillus parabuchneri, and Lactobacillus plantarum. Some atypical genera such as Weissella and Bacillus were also found in the residual must. Our results suggest that the lactic acid bacteria could strongly be implicated in the organoleptic attributes of this traditional Mexican distilled beverage.

Although oyster mushroom (Pleurotus spp.) is a valuable food, cultivated worldwide on an industrial scale, still very little is known about the microbial dynamics during oyster mushroom substrate preparation. Therefore, the characterization of the microbial dynamics by chemical and biological tools was the objective of this study. During substrate preparation, enzymatic digestibility of the substrate improved by 77%, whereas the cellulose and hemicellulose to lignin ratios decreased by 9% and 19%, respectively. Fluorescein diacetate hydrolysis reached its minimum value at the temperature maximum of the process during the composting phase and exceeded the initial level at the end of the process. Fungal species played part in the initial mesophilic phase of the substrate preparation process, but they disappeared after pasteurization in tunnels at constant elevated temperatures. Changes in the microbiota showed a marked bacterial community succession during substrate preparation investigated by 16S ribosomal deoxyribonucleic acid-based terminal restriction fragment length polymorphism (T-RFLP). Mature samples represented the least variance, which indicated the effect of the standardized preparation protocol. The relation between mushroom yield and the bacterial community T-RFLP fingerprints was investigated, but the uniformity of mushroom yields did not support any significant correlation.

Four hyper-variable regions (HVR-I to -IV) found in 18S ribosomal DNA sequences were compared among 34 isolates of 15 species of the genus Strongyloides to evaluate their diagnostic value. HVR-I to -III were short, and plural species exhibit the same nucleotide arrangement. Meanwhile, HVR-IV had 23 to 39 nucleotides, showing species-specific arrangements, except Strongyloides ransomi and Strongyloides venezuelensis, which had the same nucleotide sequence in HVR-IV but were readily distinguished by the difference in HVR-I and -III. Isolates of Strongyloides stercoralis from humans of USA, Japan, and Philippines, chimpanzees, and dogs had an identical sequence in this region. Meanwhile, intraspecific polymorphism in HVR-IV nucleotide arrangement was observed among isolates of Strongyloides fuelleborni and Strongyloides callosciureus, presumably reflecting process of geographical dispersal and adaptation to the hosts.

Theileria sp. MK in sheep and goats were detected first time by polymerase chain reaction (PCR) and detection limit of PCR and reverse line blotting (RLB) were compared. A part of 18S ssu rRNA gene was amplified from blood samples that were taken from sheep and goats naturally infected with Theileria sp. MK by PCR. Detection limit of both PCR and RLB methods was one infected cell in 10(7) sheep erythrocytes. Nine hundred twenty field samples that had been tested previously by RLB were evaluated by the PCR assay. As found by RLB previously, 12 of 920 (1.30%) samples were detected as positive by PCR. Two positive PCR products, one of which was from sheep and the other from goat, were sequenced. These sequences were identical to the reported nucleotide sequence of Theileria sp. MK. It is concluded that the PCR described in this study will be useful for epidemiological studies and for discrimination between Theileria sp. MK and other Theileria species. In addition, PCR has superiority over RLB because of its ease of use and time period required.

Analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. To avoid bias, real-time RT-PCR is referred to one or several internal control genes. Here, we sought to identify a gene to be used as normalizer by analyzing three functional distinct housekeeping genes (lipL41, flaB, and 16S rRNA) for their expression level and stability in temperature treated Leptospira cultures. Leptospira interrogans serovar Hardjo subtype Hardjoprajitno was cultured at 30 degrees C for 7 days until a density of 10(6) cells/ml was reached and then shifted to physiological temperatures (37 degrees C and 42 degrees C) and to environmental temperatures (25 degrees C and 30 degrees C) during a 24 h period. cDNA was amplified by quantitative PCR using SYBR Green I technology and gene-specific primers for lipL41, flaB, and 16S rRNA. Expression stability (M) was assessed by geNorm and Normfinder v.18. 16S rRNA registered an average expression stability of M = 1.1816, followed by flaB (M = 1.682) and lipL41 (M = 1.763). 16S rRNA was identified as the most stable gene and can be used as a normalizer, as it showed greater expression stability than lipL41 and flaB in the four temperature treatments. Hence, comparisons of gene expression will have a higher sensitivity and specificity.

The temporal stability and change of the dominant phylogenetic groups of the domain bacteria were studied in a model plant-based industrial wastewater treatment system showing high levels of organic carbon removal supported by high levels of N2 fixation. Community profiles were obtained through terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA amplicons followed by sequencing. Bacterial community profiles showed that ten common terminal restriction fragments made up approximately 50% of the measured bacterial community. As much as 42% of the measured bacterial community could be monitored by using quantitative PCR and primers that targeted three dominant operational taxonomic units. Despite changes in wastewater composition and dissolved oxygen levels, the bacterial community composition appeared stable and was dominated by alpha-Proteobacteria and beta-Proteobacteria, with a lesser amount of the highly diverse bacterial phylum Bacteroidetes. A short period of considerable change in the bacterial community composition did not appear to affect treatment performance indicating functional redundancy in this treatment system.

By light microscopy, cysts of Sarcocystis sp. (cyst type I) from the goldeneye (Bucephala clangula) seemed filamentous with a smooth and thin (<1 microm) cyst wall. Ultrastructurally, the cyst wall surface was irregular with minute undulations of the primary cyst wall. These sarcocysts had type-1 cyst wall. Cystozoites were banana-shaped and measured 7.0-8.5 microm in length. By light microscopy, cysts of Sarcocystis sp. (cyst type II) from the mallard (Anas platyrhynchos) were ribbon-shaped, very long, and thin. On the surface of the wall (up to 1.5 microm), they had palisade-like villar protrusions closely crowded together. Electron micrographs showed villar protrusions (up to 1.3 microm in length) different in size and shape. The latter had short microprojections especially obvious in the oblique sections. Cystozoites were slightly bent with blunt ends, broader at one end, and measured 13.0-16.1 x 1.8-2.5 microm. Phylogenetic analysis based on the comparison of partial 28S rRNR gene sequences of Sarcocystis sp. (cyst type II) derived from the mallard, Sarcocystis sp. (cyst type I) and Sarcocystis sp. (cyst type III) both derived from the white-fronted goose (Anser albifrons) suggested that these sequences belonged to separate Sarcocystis species.

The phylogenetic relationships among the Ergasilidae genera are poorly understood. In this study, 14 species from four genera in the Ergasilidae including Sinergasilus, Ergasilus, Pseudergasilus, and Paraergasilus were collected in China, and their phylogenetic relationships were examined using neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods based on partial sequences of 18S and 28S ribosomal deoxyribonucleic acid, respectively. All the analyses suggest that the Sinergasilus and Paraergasilus are both monophyletic, but the Ergasilus is polyphyletic rather than monophyletic. Considering the relationships among the four genera, the phylogenetic analyses and subsequent hypothesis tests all suggest that Pseudergasilus clustered with some Ergasilus species may have a closer relationship with Sinergasilus rather than with Paraergasilus. It is proposed that the Sinergasilus and the Pseudergasilus species might have evolved from Ergasilus species.

The monophyly of Thaparocleidus Jain 1952 and its phylogenetic relationship with Pseudancylodiscoides were assessed using the D1-D2 domains of the large-subunit ribosomal deoxyribonucleic acid sequences, and taxonomic implications were discussed concerning the relative taxonomic importance of the characters of the reproductive complex and those of the haptoral scleties for future genus erection and combination within the Ancylodiscoidinae. Thaparocleidus contained divergent genetic lineages and was not resolved as a monophyletic group. The first lineage was represented by two species found on Pangasium sutchi (Pangasidae), and the second one contained 12 species all from Silurus astus (Siluridae). Three clades were observed for species from S. astus, consistent with results of morphological analyses, indicating that 12 Thaparocleidus species could be divided into three groups by morphological examinations of the male copulatory organ (MCO). Pseudancylodiscoides spp. was more closely related to Thaparocleidus spp. from S. astus, and morphologically, it was found that shapes of MCOs of these species were the same type but different from that of Thaparocleidus spp. from P. sutchi. Therefore, based on results of molecular phylogenetic analyses and morphological examinations, we propose to abolish Pseudancylodiscoides and to erect a new genus to accommodate Thaparocleidus species from S. astus and Pseudancylodiscoides spp. It is also suggested to erect another new genus to accommodate two Thaparocleidus species from the P. sutchi, whose MCO shapes are different from other Thaparocleidus species.

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types.

Eight bacterial isolates closely related to Diaphorobacter sp. were isolated from activated biomass surviving on wastewater laden with dyes and nitro-substituted chemicals and were identified by 16S rDNA sequence analysis. The isolates showed sequence similarity of 99-100% to other Diaphorobacter strains such as ZY 2006b, F2, NA5, PCA039, D. nitroreducens KSP4, and KSP3 and 98-99% sequence homology to D. nitroreducens NA10B (type strain JCM 11421). Neighbor-joining tree revealed that all the eight strains formed tight cluster together and also showed close clustering with other Diaphorobacter strains. Isolates demonstrated the ability to perform simultaneous nitrification and denitrification under aerobic conditions. Strains HPC 805, 815, 821, and 856 gave highest chemical oxygen demand removal (85-93%) and ammonia removal (92-96%), which correlated well with higher growth rates of the cultures. Simultaneously, complete removal of nitrate supplied in the medium in presence of ammonium and acetate (electron donor) was observed in addition to aerobic nitrite release from ammonium. Thus, the above strains showed ability to perform partial nitrification followed by further aerobic removal of common intermediate nitrite, which indicated their potential application in treatment systems for treatment of high-nitrogen-containing wastewaters.

Microbial fuel cells (MFCs) harness the electrochemical activity of certain microbes for the production of electricity from reduced compounds. Characterizations of MFC anode biofilms have collectively shown very diverse microbial communities, raising ecological questions about competition and community succession within these anode-reducing communities. Three sets of triplicate, two-chamber MFCs inoculated with anaerobic sludge and differing in energy sources (acetate, lactate, and glucose) were operated to explore these questions. Based on 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE), all anode communities contained sequences closely affiliated with Geobacter sulfurreducens (>99% similarity) and an uncultured bacterium clone in the Bacteroidetes class (99% similarity). Various other Geobacter-like sequences were also enriched in most of the anode biofilms. While the anode communities in replicate reactors for each substrate generally converged to a reproducible community, there were some variations in the relative distribution of these putative anode-reducing Geobacter-like strains. Firmicutes were found only in glucose-fed MFCs, presumably serving the roles of converting complex carbon into simple molecules and scavenging oxygen. The maximum current density in these systems was negatively correlated with internal resistance variations among replicate reactors and, likely, was only minimally affected by anode community differences in these two-chamber MFCs with high internal resistance.

Fasciolopsis buski, the zoonotic intestinal flukes of pigs in South and Southeast Asia, is commonly prevalent in regions across Northeast India. Populations of the fluke collected from different parts of the region exhibit variations in morphology. The main objective of our study was to provide molecular characterization of the parasite so as to supplement morphological criteria, using ribosomal DNA cluster (rDNA), which is flanked by more conserved internal transcribed spacer (ITS) regions. We describe herein the ITS sequences of the parasite collected from swine hosts of Assam region. The ITS sequences of both egg and adult origins were found to be identical in length and composition. Phylogenetically, F. buski resembles closely the other members of family Fasciolidae, showing significant expectation value in the alignment. The results corroborate that the ITS sequences are not stage specific and are conserved through different stages of development of the fluke, and thus could be used as species markers.

The Anopheles (Anopheles) Hyrcanus group of mosquitoes is very important for human health because some are malarial vector mosquitoes. Despite their pathological importance, some unidentified internal transcribed spacer 2 (ITS2) sequences have been reported from members of this group, and their phylogenetic relationships have been rarely understood. In the present study, 84 ITS2 sequences for the Hyrcanus group members were retrieved from GenBank. The detailed sequence comparison unambiguously revealed that the unknown1 sequences of Li et al. (Zootaxa 939:1-8, 2005), YM-2004 (or sp.2) of Ma and Xu [J Med Entomol 42(4):610-619, 2005], Anopheles sp. of Ree et al. (2005), and Anopheles lesteri reported by Gao et al. [J Med Entomol 41(1):5-11, 2004] are identical to Anopheles belenrae [Rueda (Zootaxa 941:1-26, 2005)] and that YM-2003 (or sp.1) of Ma and Xu [J Med Entomol 42(4):610-619, 2005] is closely related to A. lesteri. In addition, some candidate species that may be synonymized are suggested in addition to the possibility that A. lesteri may be divided into at least three types: A, B, and C. The neighbor joining, maximum parsimony, and maximum likelihood trees show that the 23 examined Hyrcanus group members could be divided into four subgroups. The phylogenetic relationships among them are generally well resolved with high bootstrapping values (60-100%), and they are consistently supported by all three trees without any conflicts. These results may strongly suggest that the morphology-based groupings of the Hyrcanus group members should be seriously reconsidered. Further study must be conducted to address the following issues: the three or more multitypified A. lesteri; the unidentified YM-2003 closely related to A. lesteri; and the synonymies of Anopheles peditaeniatus/Anopheles nigerrimus, Anopheles kunmingensis/Anopheles liangshanensis, Anopheles pullus/Anopheles junlianensis, and Anopheles engarensis/Anopheles kleini.

The liver fluke Fasciola hepatica is a worldwide distributed trematode parasite of great veterinary importance. In Spain, the prevalence of the infection is up to 56% in sheep. Its control is mainly based on the use of anthelmitics, although some resistant populations have been described. Genetic studies of the genus Fasciola have been focused on the 28S ribosomal deoxyribonucleic acid (rDNA) gene, with no intraspecific variations and a few interspecific nucleotide differences. We have designed a specific polymerase chain reaction to amplify and sequence a 510-bp-long fragment of the gene to characterize several F. hepatica ovine isolates from the northwest of Spain. All the isolates rendered the expected results on the agarose gel, while sequencing revealed some differences in the 34 site. The anthelmintic-resistant specimens showed the same nucleotide differing of the sensitive ones. Comparing to other studies, our results conclude that there are some genetic variations in F. hepatica (at the level of the 28S rDNA), including heterozygous specimens. In conclusion, a genetic heterogeneity has been found in the F. hepatica populations of ovine origin in the northwest of Spain, using an assay that could be useful for further epidemiology and characterization studies.

Blood smears and purified trypanosome from freshwater fishes yellow catfish (Pseudobagras fulvidraco) and common carp (Cyprinus carpio) captured from Niushan Lake, Hubei Province were examined to determine whether all of their trypanosomes were Trypanosoma pseudobagri, a species of supposed host specificity and widespread existence across China. Trypanosomes occurred in 16/16 blood smears, and morphometric character analysis of trypanosomes from these smears showed that there were three morphospecies, Trypanosoma sp Carpio, T. sp Pseudobagri, and T. sp. 18S rDNA sequences of trypanosomes from 16 samples revealed three genetic groups among these fish trypanosomes. Group 1 was from C. carpio containing T. sp Carpio; groups 2 and 3 were from P. fulvidraco containing T. sp Pseudobagri and T. sp, respectively. The high similarity of morphometric characters and 18S rDNA sequences showed that T. sp Carpio and T. siniperca probably were the same species. T. sp Pseudobagri was the first occurrence in China. Sequence comparison showed that T. sp Pseudobagri sequence was most similar to that of clone Marv, whereas T. sp sequence differ from those of T. sp Carpio and T. sp Pseudobagri by 5.4 and 5.8%, respectively, and tentatively identified as T. pseudobagri. It was concluded that three species of trypanosomes, at least three genotypes occur in Niushan Lake fishes, and P. fulvidraco in this region appear to contain both types, although the identification of T. pseudobagri remains a problem.

The anaerobic treatment of saline effluents using halophilic and halotolerant microbial consortia is of major interest. Inhibition of anaerobic digestion is known to occur at high salt content. However, it seems that the suitable adaptation of an anaerobic sludge makes possible the treatment of saline wastewater. In this study, a non-saline anaerobic sludge was inoculated in two anaerobic batch reactors operating with a different substrate (distillery vinasse and ethanol) and subjected to increasing NaCl concentrations. The performance of the digesters appeared to be highly dependent on the nature of the substrate, and a similar level of inhibition (i.e. around 90% of the specific loading rate and specific methanogenic activity) was stated at 10 g l(-1) of NaCl with distillery vinasse and 60 g l(-1) of NaCl with ethanol. The characterization of the microflora and its adaptation to increasing NaCl conditions were also investigated using molecular tools based on the analysis of genomic 16S rDNA. The microbial communities revealed a high diversity that could be maintained in both reactors despite the increase in NaCl concentrations.

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day(-1). Butyrate, fed into the chemostat, was almost completely mineralized to CH(4) and CO(2) at dilution rates below 0.5 day(-1). The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day(-1) were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day(-1), whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.

Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9-11 log(10) copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides-Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r (2) = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50-800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments.

A myxosporean of the genus Chloromyxum sp., which sporulates in the kidney tubules, was detected in Atlantic salmon, Salmo salar L., and brown trout, Salmo trutta L., from three sites in Scotland. A comparison with other reports of Chloromyxum spores in the kidney of European salmonids suggests that they all refer to the same species, Chloromyxum schurovi [Shul'man BS, Ieshko EP (2003). Parazitologia 37:246-247]. Apart from Chloromyxum majori [Yasutake WT, Wood EM (1957). J Parasitol 43:633-637] which has a different geographical and host range, as well as tissue localisation, this is the only representative of the genus in the kidney of salmonids described so far. This study provides a re-description of C. schurovi including its myxosporean and actinosporean stage, i.e. Neoactinomyxum eiseniellae [Ormieres R, Frezil JL (1969). Protistologica 5:137-144], which was identified by 18S rDNA sequence identity. Results of phylogenetic analysis of the 18S rDNA sequence of C. schurovi and all other Chloromyxum species with published sequences showed that C. schurovi clusters with representatives of other myxozoan genera which inhabit the urinary system, whereas the other Chloromyxum species form a more basic clade together with another gall bladder parasite.

Cryptosporidium has been recognized as an emerging zoonotic agent of intestinal cryptosporidiosis leading to diarrhea, malabsorption syndrome, and weight loss in AIDS patients. In the present case, oocysts of zoonotic Cryptosporidium parvum were detected in the sputum and stool samples of an AIDS patient with a 3-month history of intestinal cryptosporidiosis. The oocysts were detected by modified Ziehl-Neelsen staining; confirmation was achieved by nested polymerase chain reaction (PCR), targeting the most polymorphic region of the 18S rRNA gene. Genotyping was done by restriction endonuclease digestion of the PCR product. The zoonotic C. parvum bovine genotype was identified in both intestinal and respiratory samples. Treatment with both azithromycin and paromomycin resulted in improvement of both intestinal and respiratory symptoms, as well as the elimination of the parasite. This is the first report of the identification of Cryptosporidium sp. oocysts in the respiratory samples obtained from an AIDS patient in Iran. Pulmonary cryptosporidiosis should be considered whenever an AIDS patient with intestinal cryptosporidiosis develops respiratory symptoms.

Three novel endophytic rhizobial strains (RRE3, RRE5, and RRE6) were isolated from naturally growing surface-sterilized rice roots. These isolates had the ability to nodulate common bean (Phaseolus vulgaris). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of 16S rDNA of these isolates revealed that RRE3 and RRE5 are phylogenetically very close to Burkholderia cepacia complex, whereas RRE6 has affinity with Rhizobium leguminosarum bv. phaseoli. Plant infection test using gusA reporter gene-tagged construct of these isolates indicated that bacterial cells can go inside and colonize the rice root interiors. A significant increase in biomass and grain yield was also recorded in greenhouse-grown rice plants inoculated with these isolates.

Bacterial strain Delftia sp. TBKNP-05, isolated by para-hydroxybenzoate enrichment technique, is capable of degrading di-n-butylphthalate (DBP) as a sole source of carbon and energy. Analysis of intermediates by thin-layer chromatography and high-performance liquid chromatography indicated the presence of monobutylphthalate (MBP), phthalate (PA), and protocatechuate (PCA). The washed cells grown on DBP and PA showed appreciable oxidation of DBP, MBP, PA, and PCA. The enzyme activities in cell-free extracts of Delftia sp. TBKNP-05 exhibited the presence of DBP esterase, MBP esterase, PA-dioxygenase, and PCA 4,5-dioxygenase. The PCA is metabolized by meta-cleavage pathway, leading to further mineralization of the compound in this bacterium.

Pseudomonas sp. OX1, an aromatic compound-degrading bacterium that was tentatively identified by conventional biochemical methods as P. stutzeri, has now been investigated at the molecular level to clarify its taxonomic position. Amplified ribosomal DNA restriction analysis and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis suggested that Pseudomonas sp. OX1 could not be classified as P. stutzeri. Phylogenetic analyses based on 16S rRNA and gyrB genes further confirmed that this strain belongs to the Pseudomonas (sensu stricto) genus, but not to the stutzeri species. The data obtained demonstrated that Pseudomonas sp. OX1 belongs to intrageneric cluster II and is related to the P. fluorescens-P. syringae complex.

A new Mycobacterium austroafricanum strain, IFP 2015, growing on methyl tert-butyl ether (MTBE) as a sole carbon source was isolated from an MTBE-degrading microcosm inoculated with drain water of an MTBE-supplemented gasoline storage tank. M. austroafricanum IFP 2015 was able to grow on tert-butyl formate, tert-butyl alcohol (TBA) and alpha-hydroxyisobutyrate. 2-Methyl-1,2-propanediol was identified as the TBA oxidation product in M. austroafricanum IFP 2015 and in the previously isolated M. austroafricanum IFP 2012. M. austroafricanum IFP 2015 also degraded ethyl tert-butyl ether more rapidly than M. austroafricanum IFP 2012. Specific primers designed to monitor the presence of M. austroafricanum strains could be used as molecular tools to detect similar strains in MTBE-contaminated environment.

Anaerobic co-digestion of food waste and biosolids was carried out in sequential batch and single-stage batch systems in four treatments. Methane yield, which was used as a functional process parameter, differed between treatments, with the single-stage batch system generating lower volumes than the sequential batch systems. Volatile fatty acid (VFA) concentrations and pH in the leachate also differed between treatments. VFA concentrations were highest and methane generation yields lowest in the single-stage batch system in comparison to the sequential batch systems. The anaerobic microbial community structure of the domains Archaea and Bacteria, determined by denaturing gradient gel electrophoresis, differed between treatments and was correlated to a number of environmental parameters such as pH, VFA concentration and methane generation rate. Methane generation rate was significantly correlated to the community structure of Bacteria but not Archaea. This indicated that the substrates that are produced by acetogens (Bacteria) are important for the growth and community structure of the methanogens (Archaea). Community structure of Archaea changed over time, but this had no observable effect on functional ability based on methane yields. Microbial diversity (H') was shown to be not important in developing a functionally successful anaerobic microbial community.

Molecular methods using genetic markers in the nuclear ribosomal DNA (rDNA) were established to identify and distinguish between two members within the Pseudorhabdosynochus lantauensis complex and two morphologically distinct congeners, Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus coioidesis, from different marine fish species and various geographical origins. Supported by selective DNA sequencing, it was demonstrated that the polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism analysis of the first internal transcribed spacer and restriction fragment length polymorphism analysis of a variable region (representing the D1-D3 domains) in the large subunit of rDNA achieved the identification and delineation of all four taxa examined. These PCR-based approaches provide useful complementary tools to traditional methods for the accurate identification of species within the genus Pseudorhabdosynochus (irrespective of developmental stage) and have major implications for studying the ecology, transmission, and population genetic structures of these and other related parasites and for the prevention and control of the diseases they cause.

Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.

To reveal the genetic diversity of Pasteurella pneumotropica, the 16S rDNA sequence and multiple alignments were performed for 35 strains (from 17 mice, 13 rats, 3 hamsters, 1 rabbit, and 1 guinea pig) identified as P. pneumotropica using a commercial biochemical test kit or PCR test and two reference strains (ATCC 35149 and CNP160). Each strain showed a close similarity with one of the following organisms: P. pneumotropica (M75083), Bisgaard taxon22 (AY172726), Pasteurella sp. MCCM00235 (AF224300), Pasteurellaceae gen. sp. Forsyth A3 (AF224301), and Actinobacillus muris (AF024526) on GenBank, and were divided into six clusters on a phylogenic tree. Two reference strains, P. pneumotropica biotype Jawetz and Heyl, were classified at both ends of the clusters. Our conclusion is that P. pneumotropica should be reclassified because of the very wide genetic diversity that exists.

A total of 23 blood samples from Red Hartebeest and 28 ticks of the subspecies Rhipicephalus evertsi were collected in Namibia during the summer 2003. For a Piroplasmida species, Theileria sp 95.7% of blood and 50.0% of tick samples (all R. e. mimeticus, none of R. e. evertsi were carrying the pathogen) were PCR positive. Sequencing showed infection from a re-emerging mild species of Theileria phylogenetically different from other African Theileria species such as Theileria parva, T. buffeli, T. annulata, T. taurotragi or T. mutans.

Intron origin and evolution are of high interest, yet the rates of insertion and loss are unclear. To investigate their spread, we studied ribosomal (r)DNA introns from the closely related lichens Physcia aipolia and P. stellaris. Both taxa are replete with rDNA spliceosomal introns and autocatalytic group I introns, many of which show presence/absence polymorphism when screened with the PCR approach. This initially suggested that Physcia could be a model for studying intron retention and loss. However, during the course of a population-level analysis, we discovered widespread intron presence/absence heterogeneity within lichen thalli. To address this result, we sequenced multiple clones encoding nuclear rDNA and the single-copy elongation factor-1alpha (EF-1alpha) from individual thalli. These data showed extensive rDNA heterogeneity within individuals, rather than the presence of multiple fungi within a thallus. Our results suggest that considerable care must be taken when interpreting intron presence/absence in lichen rDNA, an observation that has general implications for the study of rDNA intron evolution.

Activated sludge and acetate-fed granules were used as microbial inocula to start up two sequencing batch reactors (R1, R2) for phenol biodegradation. The reactors were operated in 4-h cycles at a phenol loading of 1.8 kg m(-3) day(-1). The biomass in R1 failed to remove phenol and completely washed out after 4 days. R2 experienced initial difficulty in removing phenol, but the biomass acclimated quickly and effluent phenol concentrations declined to 0.3 mg l(-1) from day 3. The acetate-fed granules were covered with bacterial rods, but filamentous bacteria with sheaths, presumably to shield against toxicity, quickly emerged as the dominant morphotype upon phenol exposure. Bacterial adaptation to phenol also took the form of modifications in enzyme activity and increased production of extracellular polymers. 16S rRNA gene fingerprints revealed a slight decrease in bacterial diversity from day 0 to day 3 in R1, prior to process failure. In R2, a clear shift in community structure was observed as the seed evolved into phenol-degrading granules without losing species-richness. The results highlight the effectiveness of granules over activated sludge as seed for reactors treating toxic wastewaters.

Twenty-four isolates of Tunga were collected from afflicted humans, dogs, cats, pigs and rats in Brazil. To investigate genetic diversity, a hypervariable section of mitochondrial 16S rDNA was amplified using PCR and subsequently sequenced. In order to compare results with another species of the genus Tunga, three isolates of the recently described Tunga trimamillata were also checked. Whereas eleven isolates (five from cats, three from dogs and three from humans) were of identical sequence, thirteen isolates collected from dogs, humans, pigs and rats showed differences in sequence up to 49%, so that the existence of one or more new species of Tunga may be presumed.

The DNA sequence information on avian malaria parasites of the genus Plasmodium is quite limited. At present, sequences of only 6 out of 34 valid species are available. However, sequence data of avian malaria parasites are particularly important with regard to the resolution of the phylogenetic relationships of the most virulent human malaria agent, Plasmodium falciparum. The question as to whether P. falciparum originates from avian or from mammalian parasites would contribute to our understanding of its biology and would probably facilitate the interpretation of experimental results. To add to the body of molecular data, we sequenced three genes (cytochrome b, 18 SSU rRNA, caseinolytic protease C) of different organellar origin of one of the most widespread avian malaria parasites, Plasmodium (Haemamoeba) cathemerium, which once used to be an important laboratory in vivo model in human malaria research. The analysis of the new P. cathemerium sequences in direct comparison with the rodent parasite P. berghei and the four human malaria parasites by pairwise distance calculation do not suggest a closer relationship of P. cathemerium to P. falciparum than to the other species involved.

A new aerobic Gram-positive bacterium designated TN58 producing antibacterial activities against Gram-positive and Gram-negative bacteria was isolated from Tunisian soil. The nucleotide sequence of the 16S rRNA gene (1516 bp) of the TN58 strain showed high similarity (96-98%) to the Streptomyces 16S rRNA genes, especially with that of Streptomyces lavendulae which produces the anti-tumor compound mitomycin C, and the cyclic peptide antibiotic, complestatin. Cultural characteristic studies, alignment data of the 16S rRNA gene, and analysis of the nucleotide sequence of a 2.2 kb genomic DNA fragment from TN58 strongly suggested that this strain could be an actinomycete and most probably belongs to the genus Streptomyces. Study of the influence of different nutritional compounds on antibiotic production showed that the highest antibacterial activities were obtained when glycerol at 1% (w/v) was used as sole carbon source in the presence of potassium. In analytical conditions, the application to supernatant culture of the TN58 strain of various extraction and purification steps led to the isolation of two pure active molecules having a retention time of 38.6 and 50.2 min, respectively. TN58 strain was untransformable with the Streptomyces cloning vector pIJ702 via classical polyethylene glycol (PEG) protoplast transformation and previously described Streptomyces electroporation procedures. Transformation was rendered possible by the electroporation technique only after utilization of a preculture medium without sucrose and a regeneration plate containing a low sucrose concentration.

Bacteria possessing high capacity to degrade gasoline, kerosene, diesel oil, and lubricating oil were screened from several areas of Hokkaido, Japan. Among isolates, two strains, WatG and HokM, which were identified as new strains of Pseudomonas aeruginosa and Serratia marcescens species, respectively, showed relatively high capacity and wide spectrum to degrade the hydrocarbons in gasoline, kerosene, diesel, and lubricating oil. About 90-95% of excess amount of total diesel oil and kerosene added to mineral salts media as a sole carbon source could be degraded by WatG within 2 and 3 weeks, respectively. The same amount of lubricating oil was 60% degraded within 2 weeks. Strain HokM was more capable than WatG in degrading aromatic compounds in gasoline. This strain could also degrade kerosene, diesel, and lubricating oil with a capacity of 50-60%. Thus, these two isolates have potential to be useful for bioremediation of sites highly contaminated with petroleum hydrocarbons.

Theileria lestoquardi and T. annulata can occur in similar vectors, and current available probes based on the 18S rRNA gene showed cross-reaction between the two species. However, we developed a species-specific RFLP test based on the MspI restriction enzyme, able to cut amplified products from T. lestoquardi only and to discriminate the two species in both tick and blood samples.

The relationship between 16S rRNA gene sequence-derived phylogeny and the bacterial production of diterpenoids from 18 isolates of marine bacteria belonging to the genus Saprospira was determined. Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified 16S rRNA genes of these isolates indicated four distinct phylotypes. The terpenoid metabolite profiles of each phylotype, determined by liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) analyses, indicated that diterpenoid production was restricted to phylotype A, which included the type specimen S. grandis Gross, and the sole member of the closely related phylotype B. The discovery of two new neoverrucosane diterpenoids produced by phylotype B has also been documented.

Sixty-four strains of Enterococcus spp. of different origin were identified using traditional phenotypic, biochemical and cultural tests, together with molecular tests. API 20 STREP tests identified the species at a preliminary level, only one strain remaining unidentified. Strains belonging to the genus Enterococcus were tested with genus-specific primers, while species-level identification was carried out with the 16S-23S rDNA intergenic region (ITS) and species-specific primers for E. faecium and E. faecalis. Those strains found to be negative with the species-specific primers were subjected to 16S rDNA partial sequencing.

A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.