Evaluation of targets for Strongyloides genus specific molecular diagnosis in experimental strongyloidiasis.

Strongyloides venezuelensis has been used in different experimental studies, such as those aimed at the evaluation of diagnostic techniques for human strongyloidiasis, mainly the molecular diagnosis. In this study, three regions (genus, 18S and 28S targets) of Strongyloides ribosomal DNA were evaluated for the molecular diagnosis of experimental strongyloidiasis. Rats were infected subcutaneously with 400 or 4000 S. venezuelensis infective larvae (400iL3 and 4000iL3), and kept for 35 days. Fecal samples were collected daily to count eggs per gram of feces (EPG) and to perform the polymerase chain reaction (PCR). Egg count started on the 5th day post-infection (pi) and ended on days 33 and 34 pi, in 400iL3 and 4000iL3 groups, respectively. Based in EPG, fecal samples were selected from days 2, 5, 8, 11, 15, 23 and 35 pi for DNA extraction; PCR (genus, 18S and 28S); and sequencing. The PCR-28S products showed higher values of identity (95-100%) in the database with the Strongyloides sequences. Therefore, it is possible to reinforce the application of PCR-28S in the diagnosis of experimental and human strongyloidiasis.

Structural and quantitative alterations of gut microbiota in experimental small bowel obstruction.

OBJECTIVE: To investigate structural and quantitative alterations of gut microbiota in an experimental model of small bowel obstruction. METHOD: A rat model of small bowel obstruction was established by using a polyvinyl chloride ring surgically placed surrounding the terminal ileum. The alterations of gut microbiota were studied after intestinal obstruction. Intraluminal fecal samples proximal to the obstruction were collected at different time points (24, 48 and 72 hours after obstruction) and analyzed by 16s rDNA high-throughput sequencing technology and quantitative PCR (qPCR) for target bacterial groups. Furthermore, intestinal claudin-1 mRNA expression was examined by real-time polymerase chain reaction analysis, and serum sIgA, IFABP and TFF3 levels were determined by enzyme-linked immunosorbent assay. RESULTS: Small bowel obstruction led to significant bacterial overgrowth and profound alterations in gut microbiota composition and diversity. At the phylum level, the 16S rDNA sequences showed a marked decrease in the relative abundance of Firmicutes and increased abundance of Proteobacteria, Verrucomicrobia and Bacteroidetes. The qPCR analysis showed the absolute quantity of total bacteria increased significantly within 24 hours but did not change distinctly from 24 to 72 hours. Further indicators of intestinal mucosa damage and were observed as claudin-1 gene expression, sIgA and TFF3 levels decreased and IFABP level increased with prolonged obstruction. CONCLUSION: Small bowel obstruction can cause significant structural and quantitative alterations of gut microbiota and induce disruption of gut mucosa barrier.

Sporadic isolation of Tetratrichomonas species from the cattle urogenital tract.

Tritrichomonas foetus is a venereal trichomonad parasite which causes reproductive issues in cattle. No other trichomonads are known to be urogenital pathogens in cattle, but there are several reports of Tetratrichomonas and Pentatrichomonas isolates of unclear origin from the cattle urogenital tract (UGT) in the Americas. This study reports the first case of a non-T. foetus cattle urogenital trichomonad isolate in Europe. Molecular analysis of the internal transcribed spacer (ITS) 1-5.8S ribosomal RNA-ITS 2 and 18S ribosomal RNA loci suggest that the isolate is a Tetratrichomonas species from a lineage containing other previously described bull preputial isolates. We identified close sequence similarity between published urogenital and gastrointestinal Tetratrichomonas spp., and this is reviewed alongside further evidence regarding the gastrointestinal origin of non-T. foetus isolates. Routine screening for T. foetus is based on culture and identification by microscopy, and so considering other trichomonad parasites of the bovine UGT is important to avoid misdiagnosis.

A simple method for the cultivation of the "unculturable" asterinaceous fungi (Asterinales/Dothideomycetes).

Although asterinaceous fungi have been studied for many years, all previous attempts to isolate, cultivate, and propagate these fungi in vitro have failed. This paper provides the first reports of in vitro isolation of representative strains of species belonging to five fungi from different genera belonging to Asterinales. To confirm if the sequences of DNA obtained from the mycelia are the same obtained in the direct extraction, a phylogenetic analysis of nuc LSU rDNA was performed. This paper reports for the first time the success of in vitro culturing of asterinaceous fungi using the ascospores ejection technique, opening perspectives of studies of genetics, physiology, among other aspects of the biology for this very understudied group of fungi.

REDESCRIPTION OF CATHARIOTREMA SELACHII (MACCALLUM, 1916) JOHNSTON AND TIEGS, 1922 (MONOGENOIDEA: MONOCOTYLIDAE), EMENDATION OF MONOTYPIC CATHARIOTREMA JOHNSTON AND TIEGS, 1922, AND PROPOSAL OF CATHARIOTREMATINAE N. SUBFAM. BASED ON MORPHOLOGICAL AND NUCLEOTIDE EVIDENCE.

We herein redescribe the enigmatic Cathariotrema selachii (MacCallum, 1916) Johnston and Tiegs, 1922 based on the holotype, paratypes, and newly collected specimens infecting the olfactory organ of 5 shark species from the Gulf of Mexico (all new host records): scalloped hammerhead shark, Sphyrna lewini (Griffith and Smith, 1834) (Carcharhiniformes: Sphyrnidae); great hammerhead shark, Sphyrna mokarran (Rüppell, 1837); blacktip shark, Carcharhinus limbatus (Müller and Henle, 1839) (Carcharhiniformes: Carcharhinidae); spinner shark, Carcharhinus brevipinna (Müller and Henle, 1839); and Atlantic sharpnose shark, Rhizoprionodon terraenovae (Richardson, 1836) (Carcharhinidae). These specimens were morphologically indistinguishable from each other and from MacCallum's holotype and paratypes. Those sequenced had identical first internal transcribed spacer (ITS1) and large subunit ribosomal DNA (28S) nucleotide sequences. As such, C. selachii infects sharks of 2 orders (Carcharhiniformes, Lamniformes) and 3 families (Carcharhinidae, Sphyrnidae, Lamnidae) in the Northwestern Atlantic Ocean (type locality) and Gulf of Mexico (new records herein). This report is the first of new specimens of C. selachii in the Atlantic Ocean Basin in 95 yr and corrects long-standing error cascades and ambiguities concerning the morphology and systematic placement of C. selachii. Considering morphology and nucleotide-based phylogenetic evidence (28S, Bayesian analysis), we herein emend monotypic CathariotremaJohnston and Tiegs, 1922 and propose Cathariotrematinae Bullard n. subfam. for it and 4 other genera (all formerly assigned to Merizocotylinae Johnston and Tiegs, 1922). These genera comprise species infecting only the nose of sharks (monotypic Cathariotrema, SqualotremaKearn and Green, 1983 and SeptitremaKheddam, Chisholm, and Tazerouti, 2020 plus 3 species of TriloculotremaKearn, 1993) and nose of a chimaera (monotypic HolocephalocotyleDerouiche, Neifar, Gey, Justine, and Tazerouti, 2019). Cathariotrematinae differs from Merizocotylinae by having a 3-part attachment organ and by lacking open loculi that symmetrically encircle a cluster of >2 loculi in the center of the haptor. Monophyletic Cathariotrematinae (with sequences representing species of Cathariotrema, Triloculotrema, and Holocephalocotyle only) was sister to monophyletic Merizocotylinae, which together were sister to monophyletic Calicotylinae Monticelli, 1903. These subfamilies comprise a monophyletic group of monocotylids that have a double vagina and infect extrabranchial, enclosed niches (urogenital system, body cavity, olfactory chamber/nose) on their shark, ray, and chimaera hosts (all other monocotylids have a single vagina and infect the gill or body surfaces of rays only). Monocotylinae Taschenberg, 1879 and Decacotylinae Chisholm, Wheeler, and Beverley-Burton, 1995 were recovered as monophyletic. Heterocotylinae Chisholm, Wheeler, and Beverley-Burton, 1995 remained paraphyletic. We accept ParacalicotyleSzidat, 1970.

DESCRIPTION OF THE FIRST KLOSSIA SPECIES (APICOMPLEXA: EUCOCCIDIORIDA: ADELEORINA: ADELEIDAE) INFECTING A PULMONATE LAND SNAIL, TRIODOPSIS HOPETONENSIS (MOLLUSCA: POLYGYRIDAE), IN NORTH AMERICA.

Snails identified as Triodopsis hopetonensis were collected (n = 18) from the University of Arkansas-Fayetteville campus in December 2018. Additional snails were collected in April 2019 (n = 9) and in September 2019 (n = 9). Kidney tissues were examined using light microscopy, and polysporocystic oocysts were observed. Sporulated oocysts (n = 2) measured 78 ± 3.4 µm × 76 ± 2.9 µm with an irregular oocyst residuum and contained an estimated 44-55 tetrazoic sporocysts. The sporocysts (n = 10) measured 13 ± 0.5 µm × 11 ± 1.5 µm with an indistinct, irregularly placed, sporocyst residuum and usually contained 4 sporozoites, although an octozoic variant was observed. DNA was extracted from the snail kidney tissues and used as a template for polymerase chain reaction (PCR). PCR was used to determine the infection status of the snails; 13 of 36 (36%) specimens were found to be infected with a new Klossia species, and only 3 (8%) of these infections were detected using light microscopy. The complete nuclear 18S ribosomal DNA (1,800 bp) and mitochondrial genomes (6,775 bp) were generated, and they differentiated this parasite from the type species Klossia helicina and support the description of this new Klossia species, Klossia razorbacki n. sp. This is the first Klossia species to be described from any North American snail.

Identification of a new species, Unicapsula aequilobata n. sp., and Unicapsula seriolae (Myxozoa: Myxosporea: Multivalvulida) in carangid fish from the South China Sea.

An examination of 18 fishes caught in the South China Sea detected two Unicapsula spp. in the myofibers of the trunk muscles of carangid fishes: Unicapsula aequilobata n. sp. in the Japanese scad, Decapterus maruadsi, and Unicapsula seriolae in the yellowstripe scad, Selaroides leptolepis. They formed thin filamentous pseudocysts of 0.9-2.0 (mean 1.4) mm by 0.03-0.06 (0.04) mm (n = 5) and 0.9-3.4 (2.1) mm by 0.02-0.05 (0.04) mm (n = 12), respectively. Myxospores of U. aequilobata n. sp. are composed of three equal shell valves and measured 6.7-8.5 (7.3) µm in length and 7.1-8.8 (7.6) µm in width, and contained a prominent polar capsule (PC) 3.2-3.8 (3.6) µm in diameter (n = 18) and two rudimentary PCs. A nucleotide sequence (5127 bp) of the ribosomal RNA gene (rDNA) array was obtained for the genetic characterization of this new species. Based on morphological and phylogenetic criteria, we erect U. aequilobata n. sp. as the sixteenth species in the genus Unicapsula. Nucleotide sequences of the 18S and 28S rDNA obtained from U. seriolae from the yellowstripe scad were almost identical (99.6-100% or 99.0-99.6%, respectively) to those from fish found in the seawaters around Australia and Japan. Consequently, this is a new host and geographical distribution records for U. seriolae. In addition, we illustrated the predicted secondary structure of the available 5.8S rDNA sequences of multivalvulid species, including those obtained from U. aequilobata n. sp., to assess the significance of interspecific nucleotide variations in this short rDNA unit.

Identification of 5S and 45S rDNA sites in Chrysanthemum species by using oligonucleotide fluorescence in situ hybridization (Oligo-FISH).

Fluorescence in situ hybridization (FISH) is a conventional method used to visualize the distribution of DNA elements within a genome. To examine the relationships within the Chrysanthemum genus, ribosomal DNA (rDNA), a popular cytogenetic marker, was utilized as a probe for FISH within this genus. Based on the genome data of Chrysanthemum nankingense, C. seticuspe and its allied genera in the Compositae(Asteraceae), we explored rDNA sequences to design oligonucleotide probes and perform oligonucleotide fluorescence in situ hybridization (Oligo-FISH) in eight Chrysanthemum accessions. The results showed that the majority of 5S rDNA signals were located in subterminal chromosome regions and that the number of 5S rDNA sites might be tightly associated with ploidy. For 45S rDNA sites, the number and intensity of signals differed from those of previously investigated Chrysanthemum resources. These findings may provide an optimally reliable method of examining the chromosome composition and structural variation of Chrysanthemum and its related species and allow researchers to understand the evolutionary history and phylogenetic relationships of Chrysanthemum.

Impact of DNA extraction and sampling methods on bacterial communities monitored by 16S rDNA metabarcoding in cold-smoked salmon and processing plant surfaces.

Amplicon sequencing approaches have been widely used in food bacterial ecology. However, choices regarding the methodology can bias results. In this study, bacterial communities associated with cold-smoked salmon products and their processing plant surfaces were monitored via sequencing of the V3-V4 region of the 16S rRNA gene. The impact of DNA extraction protocols, sampling methods (swabbing or sponging) and surface materials on bacterial communities were investigated. α and ß diversity analyses revealed that DNA extraction methods mainly influence the observed cold-smoked salmon microbiota composition. Moreover, different DNA extraction methods revealed significant differences in observed community richness and evenness. ß-Proteobacteria: Photobacterium, Serratia and Firmicutes: Brochothrix, Carnobacterium and Staphylococcus were identified as the dominant genera. Surface microbiota richness, diversity and composition were mainly affected by cleaning and disinfection procedures but not by DNA extraction methods. Surface community richness and evenness appeared higher when sampled by sponging compared to swabbing. ß-diversity analyses highlighted that surface topology, cleaning and disinfection and sampling devices seemed to affect the bacterial community composition. The dominant surface bacteria identified were mainly Flavobacteriaceae, ß-Proteobacteria and γ-Proteobacteria described as fish spoilers such as Acinetobacter, Pseudomonas and Shewanella. DNA extraction and sampling methods can have an impact on sequencing results and the ecological analysis of bacterial community structures. This study confirmed the importance of methodology standardization and the need for analytical validation before 16S rDNA metabarcoding surveys.

Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types.

Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.

Development of conventional multiplex PCR method for discrimination between Dispharynx nasuta and Cheilospirura hamulosa (Nematoda: Acuariidae) parasitizing poultry.

The poultry infections caused by Dispharynx nasuta and Cheilospirura hamulosa nematodes are difficult to be diagnosed by fecal examination because of their egg similarity. In this study, we analyzed DNA sequences of nuclear ribosomal 18S-ITS1-5.8S-ITS2-28S region of D. nasuta and C. hamulosa and developed conventional multiplex PCR method using species-specific primers for discriminating between the two species. The method amplified 455-bp and 319-bp fragments specific to D. nasuta and C. hamulosa, respectively, and did not produce them against the other chicken nematode species, Ascaridia galli, Oxyspirura mansoni, Heterakis gallinarum, Heterakis beramporia, and Heterakis indica, suggesting that the multiplex PCR is sensitive and available for species diagnosis.

Acroeimeria lineri (McAllister, Upton, Freed, 1988) Paperna and Landsberg, 1989 in Mediterranean Geckos (Hemidactylus turcicus): Oocyst Morphometrics, Endogenous Developmental Stages, and Molecular Sequencing Support its Placement into Acroeimeria.

Between June 2016 and June 2019, we surveyed 62 Mediterranean geckos, Hemidactylus turcicus, from Abu Rawash, Giza, Egypt, for the presence of endoparasites. In June 2016, we found 3 individuals to be infected with Eimeria lineri. We studied the morphology and inner structures of its sporulated oocysts, and the locations of its intestinal endogenous stages. We also extracted genomic DNA from these sporulated oocysts and successfully sequenced a 632-bp fragment of the 18S rRNA gene. Phylogenetic analyses using this partial sequence allowed us to support previous studies that assigned E. lineri to the genus Acroeimeria. Our consensus sequence was used to query similar 18S rDNA sequences from GenBank, and 14 sequences were selected. The phylogenetic analysis inferred by maximum likelihood and Bayesian inference methods gave similar results, as both separated the sequences into 2 clades: (1) a monophyletic group of Goussia species (from fish); and (2) a strongly supported clade that separated 4 Choleoeimeria species from a polyphyletic group of species that clustered A. lineri with 3 other Acroeimeria species and 3 Eimeria species from lizards, including Eimeria tiliquae from Tiliqua rugosa (Gray, 1825), Eimeria tokayae from Gecko gecko (L., 1758), and Eimeria eutropidis from Eutropis macularia (Blyth, 1853). Our study supports the placement of E. lineri into the Acroeimeria and contributes additional life history information toward understanding the evolutionary origin of the Eimeria-like species that have sporocysts without Stieda bodies in their oocysts and that infect saurian reptiles. We also support the concept that several traits (morphological, endogenous, and gene sequences) are both necessary and important for authors to include when making generic reassignments within the eimeriid coccidia.

Evaluation of fecal DNA extraction protocols for human gut microbiome studies.

BACKGROUND: DNA extraction is an important factor influencing the microbiome profile in fecal samples. Considering that the QIAamp DNA Stool Mini Kit, one of the most commonly used DNA extraction kits, is no longer manufactured, this study aimed to investigate whether a new commercially available kit, the QIAamp PowerFecal Pro DNA Kit, yields comparable microbiome profiles with those previously obtained using the QIAamp DNA Stool Mini Kit. RESULTS: We extracted DNA from fecal samples of 10 individuals using three protocols (protocol P of the QIAamp PowerFecal Pro DNA Kit, and protocols SB and S of the QIAamp DNA Stool Mini Kit with and without an additional bead-beating step, respectively) in triplicate. Ninety extracted DNA samples were subjected to 16S rRNA gene sequencing. DNA quality measured by 260/280 absorbance ratios was found to be optimal in protocol P. Additionally, the DNA quantity and microbiome diversity obtained using protocol P were significantly higher than those of protocol S, however, did not differ significantly from those of protocol SB. Based on the overall microbiome profiles, variations between protocol P and protocol SB or S were significantly less than between-individual variations. Furthermore, most genera were not differentially abundant in protocol P compared to the other protocols, and the number of differentially abundant genera, as well as the degree of fold-changes were smaller between protocols P and SB than between protocols P and S. CONCLUSIONS: The QIAamp PowerFecal Pro DNA Kit exhibited microbiome analysis results that were comparable with those of the QIAamp DNA Stool Mini Kit with a bead-beating step. These results will prove useful for researchers investigating the gut microbiome in selecting an alternative protocol to the widely used but discontinued kit.

Molecular detection of small ruminant piroplasmosis and first report of Theileria luwenshuni (Apicomplexa: Theileridae) in small ruminants of Pakistan.

Theileriosis is a widespread and economically important disease of small ruminants in Pakistan. Ruminants are the intermediate hosts in the lifecycle of Theileria spp., with ticks of the family Ixodidae being the definitive hosts. To better understand the distribution and prevalence of theileriosis in Pakistan, a molecular survey was performed in small ruminants from the Lower Dir district of the Khyber Pakhtunkhwa province. A total of 200 healthy sheep and goats were screened from Maidan, Samar Bagh and Munda districts of district Dir Lower, Pakistan during December (2017) to April (2018). DNA samples were screened through nested PCR using universal primers. The amplified 492-498 bp amplicon was subjected to RLB analysis which was based on the hypervariable of the 18S rRNA gene to test for the presence of genotypes of Theileria in blood samples. A phylogeny was constructed to determine the species of Theileria genotypes. Nested PCR results indicated 53.5% prevalence of one or more Theileria genotypes in the blood of the host animal. From RLB assay, 27 animals (13.5%) showed infection with only a single species of Theileria while 80 animals (40%) showed coinfection by multiple Theileria spp. Based on the 18S rRNA phylogeny, the unknown genotype is of the species Theileria luwenshuni and is closely related to Chinese isolates. The present finding is the first report on molecular diagnosis of Theileria luwenshuni in small ruminants in Pakistan.

Distribution, genetic characteristics and public health implications of Triatoma rubrofasciata, the vector of Chagas disease in Guangxi, China.

BACKGROUND: Triatomines are natural vectors of Chagas disease and are mainly prevalent in the Americas. In China, previous data from decades ago showed that there were two species of triatomine bugs, Triatoma rubrofasciata and T. sinica. However, the distribution, genetic characteristics and public health implications of triatomines in China are still relatively unknown. In order to gain knowledge on the distribution, genetic characteristics and public health implications of the triatomines in Guangxi, China, an entomological-epidemiological study and genetic research was conducted. METHODS: Different methods were used to elucidate the distribution of triatomines in Guangxi including consultations with county-level Center for Disease Prevention and Control staff and village doctors, the distribution of educational material on triatomines though the internet and social media apps such as Wechat and QQ, and conducting manual inspections and light trapping to collect triatomines. The morphological characteristics of the collected triatomines were identified under light microscopy. The mitochondrial 16S rRNA, cytochrome b (cytb) genes and nuclear 28S rRNA gene were amplified, sequenced and used in phylogenetic analyses. RESULTS: A total of 305 triatomines were captured from 54 different sites in 13 cities in Guangxi. All collected bugs were identified as T. rubrofasciata based on morphology. Most triatomine collection sites were around or inside houses. Four triatomines bite cases were observed during the investigation indicating that triatomine bites are common, the bites can cause serious anaphylaxis and skin papules and urticaria, suggesting a systemic skin response. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from Guangxi and other countries showed that T. rubrofasciata sequences from different regions exhibit a high similarity, with no geographical differences. The phylogenetic tree based on the 16S rRNA and cytb genes showed that T. rubrofasciata sequences from different regions and continents were in the same cluster, indicating no differentiation among different geographical populations. CONCLUSIONS: Our study showed that T. rubrofasciata is widely distributed in Guangxi and that people are commonly bitten by this insect in some regions. This highlights the need to enhance surveillance for and control of T. rubrofasciata and to strengthen the monitoring of imported Trypanosoma cruzi in China. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from different regions and continents suggested that T. rubrofasciata populations exhibit high similarity, and the clustering in the phylogenetic analyses indicates that T. rubrofasciata has a close ancestor originating in the Americas.

Molecular investigation and phylogeny of species of the Anaplasmataceae infecting animals and ticks in Senegal.

BACKGROUND: Our study aimed to assess the diversity of the species of Anaplasmataceae in Senegal that infect animals and ticks in three areas: near Keur Momar Sarr (northern region), Dielmo and Diop (Sine Saloum, central region of Senegal), and in Casamance (southern region of Senegal). METHODS: A total of 204 ticks and 433 blood samples were collected from ruminants, horses, donkeys and dogs. Ticks were identified morphologically and by molecular characterization targeting the 12S rRNA gene. Molecular characterization of species of Anaplasmataceae infecting Senegalese ticks and animals was conducted using the 23S rRNA, 16S rRNA, rpoB and groEL genes. RESULTS: Ticks were identified as Rhipicephalus evertsi evertsi (84.3%), Hyalomma rufipes (8.3%), Hyalomma impeltatum (4.9%), R. bursa (1.5%) and R. muhsamae (0.9%). The overall prevalence of Anaplasmataceae infection in ticks was 0.9%, whereas 41.1% of the sampled animals were found infected by one of the species belonging to this family. We identified the pathogen Anaplasma ovis in 55.9% of sheep, A. marginale and A. centrale in 19.4% and 8.1%, respectively, of cattle, as well as a putative new species of Anaplasmataceae. Two Anaplasma species commonly infecting ruminants were identified. Anaplasma cf. platys, closely related to A. platys was identified in 19.8% of sheep, 27.7% of goats and 22.6% of cattle, whereas a putative new species, named here provisionally "Candidatus Anaplasma africae", was identified in 3.7% of sheep, 10.3% of goats and 8.1% of cattle. Ehrlichia canis and Anaplasma platys were identified only from dogs sampled in the Keur Momar Sarr area. Ehrlichia canis was identified in 18.8% of dogs and two R. e. evertsi ticks removed from the same sheep. Anaplasma platys was identified in 15.6% of dogs. Neither of the dogs sampled from Casamance region nor the horses and donkeys sampled from Keur Momar Sarr area were found infected by an Anaplasmataceae species. CONCLUSIONS: This study presents a summary of Anaplasmataceae species that infect animals and ticks in three areas from the northern, central and southern regions of Senegal. To our knowledge, our findings demonstrate for the first time the presence of multiple Anaplasmataceae species that infect ticks and domestic animals in Senegal. We recorded two potentially new species commonly infecting ruminants named here provisionally as Anaplasma cf. platys and "Candidatus Anaplasma africae". However, E. canis was the only species identified and amplified from ticks. None of the other Anaplasmataceae species identified in animals were identified in the tick species collected from animals.

A morphological and molecular comparison of Eimeria bovis-like oocysts (Apicomplexa: Eimeriidae) from European bison, Bison bonasus L., and cattle, Bos taurus L., and the development of two multiplex PCR assays for their identification.

The European bison, Bison bonasus is the largest terrestrial mammal in Europe; it is also on the red list, being recognized as vulnerable to extinction by the International Union for Conservation of Nature. The species suffers from low genetic variability, rendering it vulnerable to various environmental and biological threats. This study presents the first molecular confirmation of Eimeria bovis infection in European bison, and details a 1708 bp nucleotide sequence of the 18S rRNA gene in European bison-derived E. bovis (GenBank: MK691697). It also describes two multiplex PCR assays based on 18S rRNA gene for identifying Eimeria bovis oocysts and developmental stages in European bison and cattle. These yielded DNA banding patterns common for those of Eimeria spp. (250 bp for the first assay and 305 bp for the second assay) and species-specific E. bovis DNA in positive samples (344 bp and 586 bp, respectively). Both multiplex PCRs yielded bands characteristic of Eimeria spp. and E. bovis in samples containing DNA of oocysts from both bison and cattle. Moreover, convergent results were obtained for the DNA of the wall of colon in both assays, indicating the presence of developmental stages of Eimeria spp. other than E. bovis. Despite displaying the same sporulation time (four days), and similar general morphological features, the E. bovis oocysts derived from European bison were significantly narrower than those obtained from cattle (t = -6.19, p < 0.001), with a significantly higher shape index (length/width ratio) (t = 3.94, p <  0.001). The result provides further evidence for infection of European bison with a highly-pathogenic bovine protozoan, E. bovis.

Molecular identification of mosquitoes of the Anopheles maculatus group of subgenus Cellia (Diptera: Culicidae) in the Indonesian Archipelago.

This study reports the molecular differentiation of females of Anopheles maculatus s.l. collected in eight localities on five islands in the Indonesian Archipelago: Hargowilis and Hargotirto villages of Central Java Province, North Kalimantan Province, Sabang off the northern tip of Sumatra Province, Sumba Island of East Nusa Tenggara Province and Sulawesi Province. Analyses based on rDNA (ITS2 and D3) and mtDNA (COII) sequences revealed the presence of An. greeni for the first time in North Kalimantan, and at least one novel (previously unrecognized) species of the Maculatus Group in Central Java (Hargowilis). Despite the similarity of rDNA markers of specimens of An. maculatus s.l. from Central Java and Sulawesi, their COII sequences are highly divergent (3.3%), which might indicate the presence of a further new species. Specimens of An. maculatus s.l. from the other localities had identical rDNA sequences to most An. maculatus s.s. from mainland Southeast Asia, but moderate divergence in their COII sequences (1.2-2.1%). The latter might indicate there are further novel species within the Maculatus Complex. However, as the divergence at COII may be the result of geographical structuring within species related to the historical biogeography of the region, further studies are needed to shed light on this possibility.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections.

OBJECTIVE: The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. METHODOLOGY: Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). RESULTS: Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). CONCLUSIONS: The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

A New Species of Ascarophis (Nematoda: Cystidicolidae) Parasitizing Clinocottus analis (Pisces: Cottidae) from Baja California, Mexico.

A new species of nematode, Ascarophis morronei n. sp. (Cystidicolidae), is described from the stomach wall of the woolly sculpin Clinocottus analis (Cottidae) collected in the rocky intertidal from northwestern Baja California, Mexico. Collected nematodes were studied using both light and scanning electron microscopy. Sequence fragments for 18S rDNA molecular markers were obtained from the new nematode species, in order to test its position within the family Cystidicolidae under a phylogenetic context. Main characters distinguishing this new species include the reduced labia and the morphology of the eggs, distances of nerve ring and excretory pore from the anterior end, and left spicule of males. The new species described here is the second for the genus Ascarophis reported as adult in the Southern California Bight, and the first one recorded for the fish genus Clinocottus.