PCR-RFLP: a targeted method to reveal host specific malacosporean infection profiles (Cnidaria: Myxozoa: Malacosporea)

Malacosporeans are a group of endoparasitic cnidarians (Myxozoa) that use freshwater bryozoans and fish as final and intermediate hosts, respectively. The malacosporean Tetracapsuloides bryosalmonae causes proliferative kidney disease (PKD), an emerging disease in aquaculture and wild fish populations, including threatened salmonids in Europe and the USA. Mixed infections of malacosporeans are often encountered, and a monitoring tool for screening of multiple malacosporean species in either their fish or bryozoan hosts is therefore desirable. We describe an inexpensive method that combines PCR amplification of the partial 18S rRNA gene (~260 bp) and a single-step restriction fragment length polymorphism (RFLP) method for identification of 10 malacosporean lineages and species. We demonstrate and test this methodology on a set of DNA extracted from malacosporeans infecting fish kidney and tissues sampled from bryozoan colonies and compare the results with Sanger sequencing of the same parasite DNA isolates. The PCR-RFLP and Sanger sequencing methods agreed in 100% of cases. The PCR-RFLP method offers a number of opportunities, including screening large panels of host tissue samples to gain insights into infection patterns, characterizing mixed infections, and confirming highly pathogenic T. bryosalmonae infections. The method can also be further refined as new sequence data become available for malacosporeans.

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Molecular identification ofSarcocystisspp. helped to define the origin of green pythons (Morelia viridis) confiscated in Germany

Parasitology ◽

10.1017/s0031182013001960 ◽

2013 ◽

Vol 141 (5) ◽

pp. 646-651 ◽

Cited By ~ 9

Author(s):

GASTÓN MORÉ ◽

NIKOLA PANTCHEV ◽

DALAND C. HERRMANN ◽

MAJDA GLOBOKAR VRHOVEC ◽

SABINE ÖFNER ◽

...

Keyword(s):

18S Rrna ◽

Sequence Data ◽

18S Rrna Gene ◽

Rrna Genes ◽

Rrna Gene ◽

Intermediate Hosts ◽

Apicomplexan Parasites ◽

Large Numbers ◽

Wide Range ◽

18S Rrna Genes

SUMMARYSarcocystisspp. represent apicomplexan parasites. They usually have a heteroxenous life cycle. Around 200 species have been described, affecting a wide range of animals worldwide, including reptiles. In recent years, large numbers of reptiles have been imported into Europe as pets and, as a consequence, animal welfare and species protection issues emerged. A sample of pooled feces from four confiscated green pythons (Morelia viridis) containingSarcocystisspp. sporocysts was investigated. These snakes were imported for the pet trade and declared as being captive-bred. Full length 18S rRNA genes were amplified, cloned into plasmids and sequenced. Two differentSarcocystisspp. sequences were identified and registered asSarcocystissp. fromM. viridisin GenBank. Both showed a 95–97% sequence identity with the 18S rRNA gene ofSarcocystis singaporensis.Phylogenetic analysis positioned these sequences together with otherSarcocystisspp. from snakes and rodents as definitive and intermediate hosts (IH), respectively. Sequence data and also the results of clinical and parasitological examinations suggest that the snakes were definitive hosts forSarcocystisspp. that circulate in wild IH. Thus, it seems unlikely that the infected snakes had been legally bred. Our research shows that information on the infection of snakes withSarcocystisspp. may be used to assess compliance with regulations on the trade with wildlife species.

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An Oligonucleotide-Ligation Assay for the Differentiation between Cyclospora and Eimeria spp. Polymerase Chain Reaction Amplification Products

Journal of Food Protection ◽

10.4315/0362-028x-62.6.682 ◽

1999 ◽

Vol 62 (6) ◽

pp. 682-685 ◽

Cited By ~ 32

Author(s):

KAREN C. JINNEMAN ◽

JUNE H. WETHERINGTON ◽

WALTER E. HILL ◽

CURTIS J. OMIESCINSKI ◽

ANN M. ADAMS ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Polymerase Chain Reaction Amplification ◽

Pcr Amplification ◽

18S Rrna Gene ◽

Rrna Gene ◽

Chain Reaction ◽

Eimeria Spp ◽

Pcr Rflp ◽

Oligonucleotide Ligation Assay ◽

Polymerase Chain

An oligonucleotide-ligation assay (OLA) was developed and compared to a restriction fragment length polymorphism (RFLP) test for distinguishing between 294-bp polymerase chain reaction (PCR) amplification products of the 18S rRNA gene from Cyclospora and Eimeria spp. The PCR/OLA correctly distinguished between three Cyclospora, three E. tenella, and one E. mitis strains and the ratio of positive to negative spectrophotometric absorbance (A490) values for each strain ranged from 4.086 to 15.280 (median 9.5). PCR/OLA provides a rapid, reliable, spectrophotometric alternative to PCR/RFLP.

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Metabarcoding reveals low prevalence of microsporidian infections in castor bean tick (Ixodes ricinus)

Parasites & Vectors ◽

10.1186/s13071-022-05150-9 ◽

2022 ◽

Vol 15 (1) ◽

Author(s):

Artur Trzebny ◽

Justyna Liberska ◽

Anna Slodkowicz-Kowalska ◽

Miroslawa Dabert

Keyword(s):

Ixodes Ricinus ◽

Dna Analysis ◽

Sequence Data ◽

Pcr Amplification ◽

18S Rrna Gene ◽

Rrna Gene ◽

Microsporidian Species ◽

Encephalitozoon Intestinalis ◽

Host Seeking ◽

Low Prevalence

Abstract Background Microsporidia is a large group of eukaryotic obligate intracellular spore-forming parasites, of which 17 species can cause microsporidiosis in humans. Most human-infecting microsporidians belong to the genera Enterocytozoon and Encephalitozoon. To date, only five microsporidian species, including Encephalitozoon-like, have been found in hard ticks (Ixodidae) using microscopic methods, but no sequence data are available for them. Furthermore, no widespread screening for microsporidian-infected ticks based on DNA analysis has been carried out to date. Thus, in this study, we applied a recently developed DNA metabarcoding method for efficient microsporidian DNA identification to assess the role of ticks as potential vectors of microsporidian species causing diseases in humans. Methods In total, 1070 (493 juvenile and 577 adult) unfed host-seeking Ixodes ricinus ticks collected at urban parks in the city of Poznan, Poland, and 94 engorged tick females fed on dogs and cats were screened for microsporidian DNA. Microsporidians were detected by PCR amplification and sequencing of the hypervariable V5 region of 18S rRNA gene (18S profiling) using the microsporidian-specific primer set. Tick species were identified morphologically and confirmed by amplification and sequencing of the shortened fragment of cytochrome c oxidase subunit I gene (mini-COI). Results All collected ticks were unambiguously assigned to I. ricinus. Potentially zoonotic Encephalitozoon intestinalis was identified in three fed ticks (3.2%) collected from three different dogs. In eight unfed host-seeking ticks (0.8%), including three males (1.1%), two females (0.7%) and three nymphs (0.7%), the new microsporidian sequence representing a species belonging to the genus Endoreticulatus was identified. Conclusions The lack of zoonotic microsporidians in host-seeking ticks suggests that I. ricinus is not involved in transmission of human-infecting microsporidians. Moreover, a very low occurrence of the other microsporidian species in both fed and host-seeking ticks implies that mechanisms exist to defend ticks against infection with these parasites. Graphical abstract

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Molecular Characterization ofCryptosporidiumspp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

Journal of Tropical Medicine ◽

10.1155/2016/6834206 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6 ◽

Cited By ~ 12

Author(s):

Jasem Saki ◽

Masoud Foroutan-Rad ◽

Reza Asadpouri

Keyword(s):

Molecular Characterization ◽

Nested Pcr ◽

18S Rrna ◽

Restriction Enzymes ◽

18S Rrna Gene ◽

Rrna Gene ◽

Wild Rodents ◽

Nested Polymerase Chain Reaction ◽

Pcr Rflp ◽

Southwest Of Iran

Background. Rodents could act as reservoir forCryptosporidiumspp. speciallyC. parvum, a zoonotic agent responsible for human infections. Since there is no information aboutCryptosporidiuminfection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization ofCryptosporidiumspp. in this region.Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method usingSspIandVspIrestriction enzymes was carried out to genotype the species and then obtained results were sequenced.Results. Three out of 100 samples were diagnosed as positive and overall prevalence ofCryptosporidiumspp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples andC. parvumpattern was identified. Finally PCR-RFLP findings were sequenced and presence ofC. parvumwas confirmed again.Conclusions. Our study showed rodents could be potential reservoir forC. parvum. So an integrated program for control and combat with them should be adopted and continued.

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A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.00060-20 ◽

2020 ◽

Vol 58 (6) ◽

Author(s):

Stefan Moritz Neuenschwander ◽

Miguel Angel Terrazos Miani ◽

Heiko Amlang ◽

Carmen Perroulaz ◽

Pascal Bittel ◽

...

Keyword(s):

Sanger Sequencing ◽

Sequence Data ◽

Automated Analysis ◽

Amplicon Sequencing ◽

Turnaround Time ◽

Clinical Samples ◽

Rrna Gene ◽

Nanopore Sequencing ◽

Taxonomic Identification ◽

Consensus Analysis

ABSTRACT Amplicon sequencing of the 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations, with the most significant being the inability to resolve mixed amplicons when closely related species are coamplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read next-generation sequencing (NGS) technologies could solve the mixed amplicon issue but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling a flexible number of samples per run and an adjustable sequencing time. Here, we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (long read consensus analysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN’s behavior when dealing with samples with known cross-contamination levels. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single-base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability, and availability of amplicon sequencing in diagnostic settings.

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Phylogenetic relationships of the family Campulidae (Trematoda) based on 18S rRNA sequences

Parasitology ◽

10.1017/s0031182098003126 ◽

1998 ◽

Vol 117 (4) ◽

pp. 383-391 ◽

Cited By ~ 33

Author(s):

M. FERNANDEZ ◽

D. T. J. LITTLEWOOD ◽

A. LATORRE ◽

J. A. RAGA ◽

D. ROLLINSON

Keyword(s):

18S Rrna ◽

Fasciola Hepatica ◽

18S Rrna Gene ◽

Host Switching ◽

Rrna Gene ◽

Intermediate Hosts ◽

Marine Gastropods ◽

Echinostoma Caproni ◽

The Family ◽

Rrna Sequences

Traditionally, the family Campulidae has been associated either with the family Fasciolidae, parasites of ruminants, or the Acanthocolpidae, parasites of fishes, based on morphological similarities. Since morphology does not seem to resolve clearly the problem of the relationships of campulids, we have used the sequences of the 18S rRNA gene of the campulids Zalophotrema hepaticum, Campula oblonga and Nasitrema globicephalae, the fasciolid Fasciola hepatica, the acanthocolpid Stephanostomum baccatum and the outgroup Schistosoma mansoni to infer a phylogeny. Maximum parsimony and neighbour-joining methods were applied. Both methods indicated that campulids are closer to acanthocolpids than fasciolids. In order to confirm this relationship, we generated a second phylogeny using all the partial sequences of the 18S published for trematodes: Lobatostoma manteri, Echinostoma caproni, Calicophoron calicophorum, Tetracerasta blepta, Gyliauchen sp. and Opistorchis viverrini, plus those mentioned above, and Dicrocoelium dendriticum. The aspidogastrean L. manteri was used as the outgroup. Results were identical to the first analysis. According to this and the most recent Digenean phylogeny, which considers campulids and acanthocolpids as sister groups, we suggest that a common origin for these 2 groups would imply a host-switching process. The life-cycle of acanthocolpids includes marine gastropods as first intermediate hosts, and fishes as second intermediate and definitive hosts. In this context, the hypothesis would be that trematodes whose cycle ended in fishes were able to switch to mammalian hosts.

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Morphological and molecular characterization of Mermisnigrescens Dujardin, (Nematoda: Mermithidae) parasitizing the introduced European earwig (Dermaptera: Forficulidae) in New Zealand

Journal of Helminthology ◽

10.1017/s0022149x14000017 ◽

2014 ◽

Vol 89 (3) ◽

pp. 267-276 ◽

Cited By ~ 7

Author(s):

B. Presswell ◽

S. Evans ◽

R. Poulin ◽

F. Jorge

Keyword(s):

New Zealand ◽

18S Rrna ◽

Sequence Data ◽

18S Rrna Gene ◽

Parasitic Nematodes ◽

Rrna Gene ◽

28S Rrna ◽

Forficula Auricularia ◽

Adult Females ◽

European Earwig

AbstractParasitic nematodes of the family Mermithidae were found to be infecting the introduced European earwig Forficula auricularia (Dermaptera: Forficulidae) in Dunedin, South Island, New Zealand. Adult females were later collected from various garden plants while depositing eggs. These mermithid specimens were identified morphologically as Mermis nigrescens Dujardin, 1842. A genetic distance of 0.7% between these specimens and a M. nigrescens isolate from Canada (18S rRNA gene), suggests that they have diverged genetically, but there are currently no available comparable sequences for the European M. nigrescens. Two additional nuclear fragments were also amplified, the 28S rRNA and the ribosomal DNA first internal transcribed spacer (ITS1), providing a basis for future studies. Bearing in mind the morphological similarity with other reported M. nigrescens and the lack of sequence data from other parts of the world, we retain the name M.nigrescens, and suggest that the species may be found to represent a complex of cryptic species when more worldwide data are available. Herein, we present a brief description of the post-parasitic worms and adult females, along with an inferred phylogeny using 18S rRNA gene sequences.

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Hepatozoon canis and Leishmania spp. coinfection in dogs diagnosed with visceral leishmaniasis

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612016065 ◽

2016 ◽

Vol 25 (4) ◽

pp. 450-458 ◽

Cited By ~ 6

Author(s):

Fernanda Nazaré Morgado ◽

Amanda dos Santos Cavalcanti ◽

Luisa Helena de Miranda ◽

Lúcia Helena O’Dwyer ◽

Maria Regina Lucas da Silva ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Dna Isolation ◽

Parasite Burden ◽

Leishmania Species ◽

18S Rrna Gene ◽

Hepatozoon Canis ◽

Histopathological Analysis ◽

Rrna Gene ◽

Pcr Rflp ◽

Leishmania Spp

Abstract This study describes the occurrence of dogs naturally co-infected with Hepatozoon canis and two Leishmania species: L. infantum or L. braziliensis. Four dogs serologically diagnosed with Visceral Leishmaniasis were euthanized. Liver and spleen samples were collected for histopathological analysis and DNA isolation. H. canis meronts were observed in tissues from all four dogs. H. canis infection was confirmed by PCR followed by sequencing of a fragment of 18S rRNA gene. Leishmania detection and typing was confirmed by ITS1' PCR-RFLP and parasite burden was calculated using ssrRNA quantitative qPCR. A DPP - Dual Path platform test was performed. One out (Dog #2) of four animals was asymptomatic. Dogs #1 and #4 were infected by L. infantum and were DPP test positive. Dogs #2 and #3 were infected by L. braziliensis and were DPP test negative. Furthermore, visceral dissemination was observed in Dogs #2 and #3, since L. braziliensis was detected in liver and spleen samples. The visceral dissemination of L. braziliensis associated with systemic signs suggested that this co-infection could influence the parasite burden and disease progression.

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Legionella Species Diversity in an Acidic Biofilm Community in Yellowstone National Park

Applied and Environmental Microbiology ◽

10.1128/aem.71.1.507-511.2005 ◽

2005 ◽

Vol 71 (1) ◽

pp. 507-511 ◽

Cited By ~ 46

Author(s):

Kathy B. Sheehan ◽

Joan M. Henson ◽

Michael J. Ferris

Keyword(s):

Yellowstone National Park ◽

National Park ◽

Pcr Amplification ◽

18S Rrna Gene ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Thermal Gradients ◽

Legionella Species ◽

Legionella Micdadei

ABSTRACT Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30Ã¯Â¿Â½C) and higher (35 to 38Ã¯Â¿Â½C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.

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