Theileria ovis (Piroplasmida: Theileriidae) Detected in Melophagus ovinus (Diptera: Hippoboscoidea) and Ornithodoros lahorensis (Ixodida: Argasidae) Removed From Sheep in Xinjiang, China

2019 ◽  
Vol 57 (2) ◽  
pp. 631-635 ◽  
Author(s):  
Li Zhao ◽  
Jinling Wang ◽  
Yulin Ding ◽  
Kairui Li ◽  
Bo He ◽  
...  
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Economic Losses ◽  
Rrna Gene ◽  
Protozoan Parasites ◽  
Autonomous Region ◽  
Apicomplexan Parasites ◽  
Wide Range ◽  
Melophagus Ovinus ◽  
Theileria Spp

Abstract Theileria spp. are tick-transmitted, intracellular apicomplexan protozoan parasites that infect a wide range of animals and, as such, can cause significant economic losses. The aim of the present study was to detect and analyze apicomplexan parasites from two different ectoparasites that were collected from Xinjiang Uygur Autonomous Region, China. The PCR-based detection of 18S rRNA indicated that Ornithodoros lahorensis specimens from Kashgar, Xinjiang, and Aksu were positive for Theileria spp., as were Melophagus ovinus specimens from Aksu. Meanwhile, phylogenetic analysis, based on the 18S rRNA gene sequences, revealed that the four amplified Theileria sequences could be attributed to T. ovis. To the best of our knowledge, this is the first study to report the detection of T. ovis DNA in M. ovinus and the first molecular identification study to confirm the detection of T. ovis in O. lahorensis in China. Accordingly, the present study extends the known distribution of T. ovis.

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

Parasitology ◽  
2013 ◽  
Vol 141 (5) ◽  
pp. 646-651 ◽  
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.

scholarly journals Apicomplexa primers amplify Proteromonas (Stramenopiles, Slopalinida, Proteromonadidae) in tissue and blood samples from lizards

2012 ◽  
Vol 57 (4) ◽  
Author(s):  
João Maia ◽  
Elena Gómez-Díaz ◽  
D. Harris
Keyword(s):  
Cross Validation ◽  
Molecular Data ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Molecular Screening ◽  
Protozoan Parasites ◽  
Apicomplexan Parasites ◽  
Wide Range ◽  
Prevalence Estimates ◽  
Vertebrate Hosts

AbstractMicroscopy has traditionally been the most common method in parasitological studies, but in recent years molecular screening has become increasingly frequent to detect protozoan parasites in a wide range of vertebrate hosts and vectors. During routine molecular screening of apicomplexan parasites in reptiles using the 18S rRNA gene, we have amplified and sequenced Proteromonas parasites from three lizard hosts (less than 1% prevalence). We conducted phylogenetic analysis to confirm the taxonomic position and infer their relationships with other stramenopiles. Although our phylogeny is limited due to scarcity of molecular data on these protists, our results confirm they are closely related to Proteromonas lacertae. Our findings show that unexpected parasites can be amplified from host samples (blood and tissue) using general procedures to detect hemoparasites, and stress that positive PCR amplifications alone should not be considered as definitive proof of infection by particular parasites. Further validation by sequence confirmation and thorough phylogenetic assessment will not only avoid false positives and biased prevalence estimates but also provide valuable information on the biodiversity and phylogenetic relationships of other parasitic organisms. More generally, our results illustrate the perils of general diagnosis protocols in parasitological studies and the need of cross-validation procedures.

scholarly journals Emergence of Babesia Conradae Infection in Coyote-hunting Greyhounds in Oklahoma, USA

2021 ◽  
Author(s):  
Erin M Stayton ◽  
Megan Lineberry ◽  
Jennifer Thomas ◽  
Tina Bass ◽  
Kelly Allen ◽  
...  
Keyword(s):  
18S Rrna ◽  
Clinical Signs ◽  
18S Rrna Gene ◽  
Post Treatment ◽  
Rrna Gene ◽  
Babesia Gibsoni ◽  
Wide Range ◽  
Pcr Products ◽  
Life Threatening ◽  
Tick Vectors

Abstract Background: Babesia species are intraerythrocytic Apicomplexan parasites that infect a wide range of vertebrate hosts. These pathogens are typically transmitted either by tick vectors or by direct blood-to-blood contact, and may cause life-threatening clinical disease such as thrombocytopenia, hemolytic anemia, and acute renal failure in canine hosts. While Babesia vogeli and Babesia gibsoni infections have both been reported in Oklahoma, reports of B. conradae infections have been limited to California. Methods: Whole blood samples were collected in EDTA tubes from all dogs in four separate kennels in Oklahoma. DNA was extracted from each blood sample and a nested PCR was performed using general Apicomplexan primers for the partial 18S rRNA gene. PCR products were electrophoresed in agarose matrix and appropriately sized amplicons were sequenced. Sequences were compared to reference 18S rRNA sequences available in GenBank, and samples with >98% homology to B. conradae (GenBank MK256976) were considered positive. B. conradae positive dogs were then treated with atovaquone (13.5 mg/kg TID) and azithromycin (10 mg/kg SID) for 10 days and retested at 30 and 60 days post treatment by PCR. Results: Fifteen of 40 dogs tested positive for B. conradae with 98–100% sequence homology to B. conradae from California. All positive cases were coyote-hunting Greyhounds. Treatment of clinically ill dogs with atovaquone and azithromycin resulted in complete clinical recovery in clinically ill dogs and all treated dogs had negative follow-up PCR at 30 and 60 days post treatment. Conclusions: Collectively, this study (i) documents the occurrence of B. conradae in Oklahoma, (ii) highlights this pathogen as a differential to be considered when clinical signs are present, and (iii) supports the use of atovaquone and azithromycin as effective treatment in these cases.

scholarly journals High-resolution molecular identification of smalltooth sawfish prey

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Taylor L. Hancock ◽  
Gregg R. Poulakis ◽  
Rachel M. Scharer ◽  
S. Gregory Tolley ◽  
Hidetoshi Urakawa
Keyword(s):  
18S Rrna ◽  
Stomach Content Analysis ◽  
18S Rrna Gene ◽  
Taxonomic Resolution ◽  
Molecular Technique ◽  
Rrna Gene ◽  
Fecal Samples ◽  
Predator Prey ◽  
Critically Endangered Species ◽  
Wide Range

AbstractThe foundation of food web analysis is a solid understanding of predator-prey associations. Traditional dietary studies of fishes have been by stomach content analysis. However, these methods are not applicable to Critically Endangered species such as the smalltooth sawfish (Pristis pectinata). Previous research using the combination of stable isotope signatures from fin clips and 18S rRNA gene sequencing of fecal samples identified the smalltooth sawfish as piscivorous at low taxonomic resolution. Here, we present a high taxonomic resolution molecular technique for identification of prey using opportunistically acquired fecal samples. To assess potential biases, primer sets of two mitochondrial genes, 12S and 16S rRNA, were used alongside 18S rRNA, which targets a wider spectrum of taxa. In total, 19 fish taxa from 7 orders and 11 families native to the Gulf of Mexico were successfully identified. The sawfish prey comprised diverse taxa, indicating that this species is a generalist piscivore. These findings and the molecular approach used will aid recovery planning for the smalltooth sawfish and have the potential to reveal previously unknown predator-prey associations from a wide range of taxa, especially rare and hard to sample species.

scholarly journals pr2-primers: an 18S rRNA primer database for protists

2021 ◽  
Author(s):  
Daniel Vaulot ◽  
Stefan Geisen ◽  
Frédéric Mahé ◽  
David Bass
Keyword(s):  
Web Application ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Taxonomic Distribution ◽  
System A ◽  
Microbial Eukaryotes ◽  
Wide Range ◽  
Amplicon Size ◽  
On Line

AbstractMetabarcoding of microbial eukaryotes (collectively known as protists) has developed tremendously in the last decade, almost uniquely relying on the 18S rRNA gene. As microbial eukaryotes are extremely diverse, many primers and primer pairs have been developed. To cover a relevant and representative fraction of the protist community in a given study system, a wise primer choice is needed as no primer pair can target all protists equally well. As such, a smart primer choice is very difficult even for experts and there are very few on-line resources available to list existing primers. We built a database listing 179 primers and 76 primer pairs that have been used for eukaryotic 18S rRNA metabarcoding. In silico performance of primer pairs was tested against two sequence databases: PR2 for eukaryotes and a subset of Silva for prokaryotes. This allowed to determine the taxonomic specificity of primer pairs, the location of mismatches as well as amplicon size. We developed a R-based web application that allows to browse the database, visualize the taxonomic distribution of the amplified sequences with the number of mismatches, and to test any user-defined primer set (https://app.pr2-primers.org). This tool will provide the basis for guided primer choices that will help a wide range of ecologists to implement protists as part of their investigations.

scholarly journals Molecular survey of Cryptosporidium spp. in calves from the state of Mato Grosso, Brazil

2020 ◽  
Vol 41 (5supl1) ◽  
pp. 2437-2444
Author(s):  
Thábata dos Anjos Pacheco ◽  
Felippe Danyel Cardoso Martins ◽  
Sayanne Luns Hatum de Almeida ◽  
Thiago Borges Fernandes Semedo ◽  
Michelle Igarashi Watanabe ◽  
...  
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
The State ◽  
Severe Illness ◽  
Rrna Gene ◽  
Mato Grosso ◽  
Fecal Samples ◽  
Cryptosporidium Spp ◽  
Gp60 Gene ◽  
Wide Range

Cryptosporidium spp. is a protozoan that infects a wide range of vertebrate hosts; it has been reported to be the cause of severe illness or death in livestock worldwide, which leads to decreased performance and production losses, especially in young animals. This study investigated the presence of Cryptosporidium in calves from beef farms in the state of Mato Grosso, midwestern Brazil. For this purpose, fecal samples from 237 animals aged ? 45 days, raised in 20 rural properties were subjected to DNA extraction and nested polymerase chain reaction (nPCR) targeting 18S ribosomal RNA (18S rRNA) gene followed by sequencing. Additionally, positive samples, previously identified as Cryptosporidium parvum by sequencing and phylogenetic analyses based on 18S rRNA gene, were subsequently analyzed focusing the amplification and sequencing using nPCR of a fragment of the 60 kDa glycoprotein (gp60) gene. Of the 237 fecal samples analyzed by PCR (18S rRNA), 50 (21.1%) fecal samples were positive for Cryptosporidium spp., while 14 (70%) of the 20 properties had at least one positive animal. The following Cryptosporidium species were detected: C. bovis, C. parvum, and C. ryanae. Thereafter, two potentially zoonotic subtypes (IIaA15G2R1 and IIaA16G3R1) of C. parvum were identified based on gp60 gene sequences. This study resulted in the detection of subtype IIaA16G3R1 for the first time in South America and showed a wide distribution of the protozoan in beef farms in the studied area of the State.

scholarly journals Surveillance of Giardia and Cryptosporidium in sewage from an urban area in Brazil

2019 ◽  
Vol 28 (2) ◽  
pp. 291-297 ◽  
Author(s):  
Felippe Danyel Cardoso Martins ◽  
Winni Alves Ladeia ◽  
Roberta dos Santos Toledo ◽  
João Luis Garcia ◽  
Italmar Teodorico Navarro ◽  
...  
Keyword(s):  
18S Rrna ◽  
Fragment Length Polymorphism ◽  
Sewage Water ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Protozoan Parasites ◽  
Treated Sewage ◽  
Multiple Species ◽  
Pcr Targeting

Abstract Cryptosporidium and Giardia are protozoan parasites that cause diarrhea in humans and animals. Molecular characterization of these pathogens in sewage may provide insight on their occurrence and prevalence in Brazil. This study aimed to investigate the presence of Giardia and Cryptosporidium in raw and treated sewage from Londrina, Paraná, Brazil. Samples were collected every two weeks during a year. Samples were concentrated, then DNA was extracted and subjected to a nested PCR targeting the Giardia 18S rRNA gene and the Cryptosporidium 18S rRNA gene. Species of Cryptosporidium were characterized by restriction fragment length polymorphism (RFLP). All raw sewage and 76% of the treated sewage were positive for Giardia; 84% of raw sewage samples and 8% of treated sewage were positive for Cryptosporidium. C. muris, C. hominis, C. baileyi, C. parvum and C. suis were detected in 100%, 19%, 9%, 9% and 4% of raw sewage, respectively. C. muris was the only species found in treated sewage. Multiple species of Cryptosporidium were present in 19.04% of the raw sewage. Treated sewage water can pose a threat to human health. The speciation of Cryptosporidium revealed the presence of non-common zoonotic species as C. suis and C. muris.

scholarly journals Cross-Genera PCR Amplification of DNA from Apicomplexan Parasites

2018 ◽  
Author(s):  
Philippe Gil de Mendonça
Keyword(s):  
Pathogenic Bacteria ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Apicomplexan Parasites ◽  
Diagnostic Samples ◽  
Successful Amplification ◽  
Babesia Spp ◽  
Theileria Spp

Background: The discovery of an unexpected genetic sequence raised doubts about the specificity of a primer pair targeting Babesia spp. and Theileria spp. This study aimed to check the specificity of this primer pair. Methods: Conventional end-point PCR and real-time PCR protocols using primers targeting the 18S rRNA gene V4 hypervariable region of Babesia spp. and Theileria spp. were tested for potential cross-genera amplification using DNA from a palette of parasitic protists and pathogenic bacteria as a template. These investigations took place at the Ludwig Maximilian University of Munich (Germany) in 2010 as part of the EDEN project. Results: Successful amplification was obtained with DNA from five apicomplexan genera: Babesia, Theileria, Hepa­tozoon, Toxoplasma, and Hammondia. No amplicons were obtained when DNA from Leishmania infantum or bacte­ria within the genera Borrelia, Leptospira or Anaplasma was used as a template. Conclusion: This cross-genera amplification ability is useful for the quick exclusion of many parasite species from PCR negative diagnostic samples. Accurate species identification from PCR positive samples requires genetic se­quencing of the amplicon.

scholarly journals Molecular Detection of Tick-Borne Agents in Cats from Southeastern and Northern Brazil

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 106
Author(s):  
Marcos Rogério André ◽  
Ana Cláudia Calchi ◽  
Maria Eduarda Chiaradia Furquim ◽  
Isabela de Andrade ◽  
Paulo Vitor Cadina Arantes ◽  
...  
Keyword(s):  
18S Rrna ◽  
Clinical Signs ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Molecular Identity ◽  
Northern Brazil ◽  
Rrna Gene Sequencing ◽  
First Time ◽  
Theileria Spp

Even though the epidemiology of tick-borne agents (TBA) in dogs has been extensively investigated around the world, the occurrence, vectors involved, and molecular identity of these agents in cats remains elusive in many regions. Among TBA, Ehrlichia, Anaplasma, Babesia, Cytauxzoon, and Hepatozoon are responsible for diseases with non-specific clinical signs in cats, making essential the use of molecular techniques for accurate diagnosis and proper treatment. The present work aimed to investigate the occurrence and molecular identity of tick-borne agents (Ehrlichia, Anaplasma, Babesia/Theileria, Cytauxzoon, and Hepatozoon) in cats from southeastern (states of São Paulo (SP) and Minas Gerais (MG)) and northern (state of Rondônia (RO)) Brazil. For this purpose, 390 blood samples were collected from domiciled cats in MG (n = 155), SP (n = 151), and RO(n = 84) states, submitted to DNA extraction and PCR assays for Ehrlichia spp. (dsb gene), Anaplasma spp. (rrs gene), piroplasmids (18S rRNA gene), and Hepatozoon spp. (18S rRNA gene), sequencing, and phylogenetic inferences. The overall positivity for Anaplasma spp., Ehrlichia spp., Babesia/Theileria spp., Cytauxzoon spp., and Hepatozoon spp. were 7.4% (12.3% (MG) and 6.6% (SP)), 2% (4.5% (MG) and 0.6% (SP)), 0.7% (0.6% (MG), 0.6% (SP) and 1.2% (RO)), 27.2% (41.9% (MG), 24.5% (SP) and 4.8% (RO), and 0%, respectively. The phylogenetic analysis grouped the obtained sequences with ‘Candidatus Anaplasma amazonensis’, A. platys, B. vogeli, and Cytauxzoon sp. previously detected in wild felids from Brazil. qPCR specific for E. canis based on the dsb gene confirmed the molecular identity of the detected ehrlichial agent. The present study expanded the list and geographical distribution of hemoparasites in cats. ‘Candidatus Anaplasma amazonensis’, recently detected in sloths from northern Brazil, was described for the first time in cats. This is the first report of piroplasmids infecting cats in northern Brazil. Coinfection by Cytauxzoon and other TBA (Ehrlichia, Anaplasma, and B. vogeli) reported in the present study raises the need for veterinary practitioners’ awareness of cats parasitized by multiple TBA.

scholarly journals  Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

2012 ◽  
Vol 57 (No. 5) ◽  
pp. 224-232 ◽  
Author(s):  
M. Adamska ◽  
A. Leonska-Duniec ◽  
M. Sawczuk ◽  
A. Maciejewska ◽  
B. Skotarczak
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
High Concentration ◽  
Intestinal Protozoan ◽  
Wide Range ◽  
Stool Samples

Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst’s walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5–20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.  

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