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1.
J Parasitol ; 106(1): 71-81, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31995717

RESUMO

An unusual coccidian parasite was described previously from the prostate of a male Antechinus flavipes (family: Dasyuridae; common name: yellow-footed antechinus). Morphometrics and a partial nuclear 18S small subunit rDNA (18S rDNA) sequence were used to assign this parasite to the genus Eimeria; it was named Eimeria taggarti. We generated full nuclear 18S rDNA and mitochondrial genome sequences from this parasite and used the newly completed 18S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) sequences to perform a more in-depth phylogenetic analysis. The parasite clustered closely with Choleoeimeria spp. and Acroeimeria spp. infecting herptiles in a well-supported clade that was the sister lineage to the Eimeriidae sensu stricto. The mitochondrial genome of this parasite contained 2 inverted segments compared to mitochondrial genomes from parasites in the Eimeriidae sensu stricto (i.e., Stieda body-possessing coccidia with 4 dizoic sporocysts); this mitochondrial genome arrangement was shared with the only Choleoeimeria species for which sequence data were available publicly. Examination of histological preparations and TEM images uncovered bivalvate sporocysts and otherwise confirmed previously described morphological features of the parasite. Based on our phylogenetic analyses and histological observations, we propose the generic reclassification of E. taggarti to Choleoeimeria taggarti n. comb.


Assuntos
Coccidiose/veterinária , Eimeriidae/genética , Genoma Mitocondrial/genética , Marsupiais/parasitologia , Próstata/parasitologia , Animais , Coccidiose/parasitologia , DNA de Protozoário/química , DNA Ribossômico/química , Eimeriidae/classificação , Eimeriidae/isolamento & purificação , Eimeriidae/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/genética , Masculino , Anotação de Sequência Molecular , Oocistos/ultraestrutura , Filogenia , Reação em Cadeia da Polimerase , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Alinhamento de Sequência
2.
Exp Parasitol ; 209: 107814, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31816280

RESUMO

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates.


Assuntos
Técnicas de Genotipagem/normas , Giardia lamblia/classificação , Giardíase/parasitologia , Reação em Cadeia da Polimerase/métodos , Animais , Criança , Pré-Escolar , Cuba , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , DNA Espaçador Ribossômico/química , Fezes/parasitologia , Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Glutamato Desidrogenase/genética , Humanos , Polimorfismo de Fragmento de Restrição , Triose-Fosfato Isomerase/genética
3.
Exp Parasitol ; 209: 107824, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31870927

RESUMO

Giardiasis and cryptosporidiosis are recognized by the WHO as important emerging diseases of the 21st century. Symptoms are similar and include diarrhoea and vomiting, which may be severe, even life-threatening, for the immunocompromised and children under five years of age. Between 2013 and 2017, the Institute for Public Health in Serbia recorded 10 waterborne epidemics that manifested as gastrointestinal disease. Routine testing for enteropathogenic bacteria and viruses did not identify the aetiological agents of these outbreaks. As water is not examined for the presence of protozoa in Serbia, we performed a pilot study to analyse samples from four major rivers and their tributaries using a newly implemented methodology for detection of Giardia and Cryptosporidium, based on the ISO 15553:2006 standard. Using immunofluorescence microscopy, Giardia was detected in 10 out of the 31 samples, Cryptosporidium in five, while two samples were positive for both. Presence of G. duodenalis gDNA was confirmed by amplification of the ß-giardin gene in eight samples, of which one and two, respectively, were identified by RFLP as potentially zoonotic assemblages A and B. The results suggest that surface water in Serbia may be a potential source of infection and call for more in-depth studies using sophisticated molecular tools.


Assuntos
Cryptosporidium/isolamento & purificação , Giardia/isolamento & purificação , Rios/parasitologia , Animais , Cryptosporidium/genética , Proteínas do Citoesqueleto/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Técnicas de Genotipagem , Giardia/classificação , Giardia/genética , Humanos , Complexo Mediador/genética , Microscopia de Fluorescência , Projetos Piloto , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/genética , RNA Ribossômico/genética , Sérvia
4.
PLoS Negl Trop Dis ; 13(12): e0007900, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830038

RESUMO

Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples.


Assuntos
Genótipo , Leishmania/classificação , Leishmania/genética , Leishmaniose Visceral/parasitologia , Manejo de Espécimes/métodos , Sequenciamento Completo do Genoma/métodos , Adolescente , Criança , Pré-Escolar , DNA de Protozoário/química , DNA de Protozoário/genética , Humanos , Lactente , Leishmania/isolamento & purificação , Nepal
5.
Parasitol Res ; 118(12): 3469-3478, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31691003

RESUMO

Blastocystis is a prevalent parasite that has a wide distribution. In order to design HRM real-time PCR, primers were selected from SSU rRNA gene to amplify specific fragment with different melting temperatures for each subtype of Blastocystis. Subsequently, HRM real-time PCR was performed and melting curve analysis was done by Rotor-Gene Q software. The results of HRM real-time PCR was then compared with sequence results of "barcoding region" of SSU rRNA gene of Blastocystis. To evaluate sensitivity of test, 10-fold serial dilutions of the parasite were prepared from ~ 106 to 1 parasite per mL of stool sample and were investigated by HRM real-time PCR. In order to determine specificity of method, HRM real-time PCR was done for some microorganisms and Blastocystis-negative stool samples. In silico analysis showed that all seventeen subtypes of Blastocystis were distinguish. In vitro analysis revealed that the test discriminated subtypes with specific melting temperatures.


Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/diagnóstico , Primers do DNA/química , Primers do DNA/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Testes Diagnósticos de Rotina , Fezes/parasitologia , Humanos , Sensibilidade e Especificidade
6.
Exp Parasitol ; 207: 107776, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31628895

RESUMO

The availability of high quality genomic DNA in sufficient amounts to perform Next Generation Sequencing (NGS) experiments is challenging for pathogens that cannot be cultivated in vitro, as is the case for many parasites. Therefore, Whole Genome Amplification (WGA) of genomic DNA is used to overcome this limitation. In this study, we evaluated the effect of WGA using the intestinal flagellated protozoan Giardia duodenalis as a model, due to its genome compactness (12 Mb), the presence of two diploid nuclei with variable levels of allelic sequence heterogeneity (ASH), and the availability of reference genomes. We selected one isolate (ZX15) belonging to the same genetic group of the reference isolate WB, namely Assemblage A, sub-Assemblage AI. Genomic DNA from the ZX15 isolate (GEN dataset) and that obtained by WGA of 1 ng of the same genomic DNA (WGA dataset) were sequenced on a HiSeq Illumina platform. Trimmed reads from the GEN and WGA experiments were mapped against the WB reference genome, showing the presence of a very small number of mutations (846 and 752, respectively). The difference in the number of mutations is largely accounted by local variation in coverage and not by bias introduced by WGA. No significant difference were observed in the distribution of mutations in coding and non-coding regions, in the proportion of heterozygous mutations (ASH), or in the transition/transversion ratio of Single Nucleotide Variants within coding sequences. We conclude that the quantitative and qualitative impact of WGA on the identification of mutations is limited, and that this technique can be used to conduct comparative genomics studies.


Assuntos
DNA de Protozoário/genética , Giardia lamblia/genética , Giardíase/parasitologia , Pré-Escolar , Biologia Computacional , República Tcheca , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Feminino , Estudo de Associação Genômica Ampla , Variação Estrutural do Genoma , Humanos , Mutação , Técnicas de Amplificação de Ácido Nucleico , Fases de Leitura Aberta/genética
7.
Korean J Parasitol ; 57(4): 369-377, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31533403

RESUMO

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Marcadores Genéticos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Artemisininas/administração & dosagem , Artemisininas/uso terapêutico , Sequência de Bases , DNA de Protozoário/química , Combinação de Medicamentos , Resistência a Medicamentos/genética , Genes MDR/genética , Humanos , Repetição Kelch/genética , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Mutação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase , Tailândia
8.
PLoS One ; 14(7): e0218982, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31276473

RESUMO

In epidemiological surveys and surveillance the application of molecular tools is essential in detecting submicroscopic malaria. A genus-specific conventional cytochrome b (cytb) PCR has shown high sensitivity in field studies, detecting 70% submicroscopic malaria. The main objective of this study was to assess the conversion from conventional to real-time PCR testing both SYBR and probe protocols, and including quantitative (q) PCR. The protocols were assessed applying well-defined clinical patient material consisting of 33 positive and 80 negative samples. Sequencing of positive PCR products was performed. In addition, a sensitivity comparison of real-time PCR methods was done by including five relevant assays investigating the effect of amplification target and platform. Sensitivity was further examined using field material consisting of 111 P.falciparum positive samples from Tanzanian children (< 5 years), as well as using related patient data to assess the application of q-PCR with focus on low-level parasitaemia. Both the cytb SYBR and probe PCR protocols showed as high sensitivity and specificity as their conventional counterpart, except missing one P. malariae sample. The SYBR protocol was more sensitive and specific than using probe. Overall, choice of amplification target applied is relevant for achieving ultra-sensitivity, and using intercalating fluorescence dye rather than labelled hydrolysis probes is favourable. Application of q-PCR analysis in field projects is important for the awareness and understanding of low-level parasitaemia. For use in clinical diagnosis and epidemiological studies the highly sensitive and user-friendly cytb SYBR q-PCR method is a relevant tool. The genus-specific method has the advantage that species identification by sequencing can be performed as an alternative to species-specific PCR.


Assuntos
Citocromos b/genética , Malária/diagnóstico , Parasitemia/diagnóstico , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pré-Escolar , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , Genes de Protozoários/genética , Humanos , Lactente , Recém-Nascido , Malária/parasitologia , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Parasitemia/parasitologia , Plasmodium falciparum/fisiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Exp Parasitol ; 204: 107727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31344389

RESUMO

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Citocinese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Trypanosoma rangeli/citologia , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Citocinese/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Citometria de Fluxo , Imunofluorescência , Hidroxiureia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Fatores de Tempo , Trypanosoma rangeli/efeitos dos fármacos , Trypanosoma rangeli/enzimologia , Trypanosoma rangeli/genética , Tripanossomíase/parasitologia
10.
PLoS Negl Trop Dis ; 13(6): e0007456, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31216270

RESUMO

In French Guiana, cutaneous leishmaniasis is highly endemic, whereas no autochthonous case of visceral leishmaniasis have been reported so far. However, due to its proximity to Brazil which is highly endemic for visceral leishmaniasis, and the high transboundary population flow, an epidemiological challenge could arise at any time. As an overseas department and region and the largest outermost region of the European Union, epidemiological surveillance of visceral leishmaniasis is of great importance. Our study aimed to investigate the presence of Leishmania spp. in domestic (dogs) and sylvatic (bats) animals from French Guiana. Over the 2008-2018 period, samples from 349 animals were collected. They included blood from 179 autochthonous dogs and 59 bats, spleen samples from 33 bats and, blood from 78 military working dogs (MWD) collected before their departure from continental France and at the end of their four-month stay in French Guiana. Samples were screened using real-time polymerase chain reaction (qPCR) assays targeting Leishmania DNA followed by sequencing of 18S rRNA, kDNA and ITS2 genes. L. infantum was detected in 2.3% (8/349) of animals with 1.7% (3/179) of autochthonous dogs, 5.1% (4/78) of MWD returning from French Guiana, whereas they were negative before their departure. One of them dates back to 2012. All these dogs were positive for serological tests. In addition, L. infantum DNA was detectable in one bat spleen sample, belonging to Carollia perspicillata species. We report here for the first time an infection with L. infantum in dogs and bat from French Guiana. Our results suggest the existence of potential reservoir and transmission cycle for visceral leishmaniasis, at least since 2012, which was unknown in this territory until now. Further studies are needed to determine how these animals were infected and which vectors are involved in the transmission in this area.


Assuntos
Quirópteros , Reservatórios de Doenças , Cães , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Estruturas Animais/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , DNA de Protozoário/química , DNA de Protozoário/genética , Feminino , Guiana Francesa/epidemiologia , Humanos , Leishmania infantum/genética , Leishmaniose Visceral/epidemiologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
11.
Malar J ; 18(1): 192, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185976

RESUMO

BACKGROUND: Mutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia. This problem can be overcome by collecting large volumes of venous blood. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance. METHODS: Methods for k13 amplification from standard Whatman 3MM DBS (stored for 14 days at 28 °C with 80% relative humidity) were optimized by systematically testing different extraction conditions. Conditions that improved parasite DNA recovery as assessed by quantitative polymerase chain reaction (PCR) of 18S rDNA were then tested for their impact on k13 PCR amplification. RESULTS: The optimized protocol for amplification of k13 from DBS is markedly more sensitive than standard methods using commercial kits. Using this method, k13 was successfully amplified from laboratory-created DBS samples with parasite densities as low as 500 parasites/mL. Importantly, the method recovers both DNA and RNA, making it compatible with RNA-based ultrasensitive techniques currently in use. CONCLUSIONS: The optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.


Assuntos
Artemisininas/farmacologia , Sangue/parasitologia , DNA de Protozoário/isolamento & purificação , Resistência a Medicamentos , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antimaláricos/farmacologia , Ásia Sudeste , DNA de Protozoário/química , DNA de Protozoário/genética , Dessecação/métodos , Humanos , Plasmodium falciparum/efeitos dos fármacos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Manejo de Espécimes/métodos
12.
J Parasitol ; 105(3): 442-445, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31192761

RESUMO

Larval tapeworms of Echinococcus granulosus have been viewed as the etiological agent for the zoonotic disease cystic echinococcosis, but the species is a complex readily divided into several species and genotypes. Cystic echinococcosis is an important public health issue. Here, the case of liver hydatid cyst in a donkey and molecular characterization of the cyst is presented. The fluid-filled hydatid cyst materials were obtained from the liver of a necropsied donkey. Genomic DNA was extracted and PCR amplification of mitochondrial 12S rRNA gene as well as partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (COI) gene were performed. All cysts were fertile. Traditional polymerase chain reaction (PCR) amplification of 12S rRNA and COI yielded bands (254 and 446 base pairs, respectively) for all 3 cyst samples. However, partial COI gene sequences were identical to those reported for Echinococcus equinus (formerly E. granulosus genotype G4). Thus E. equinus is still transmitting among the equids in Turkey but the mitochondrial 12S rRNA gene primers may not be sufficient for the molecular characterization of members of the E. granulosus species/genotype complex. Molecular diagnosis must be confirmed by partial COI sequence analysis.


Assuntos
Equinococose Hepática/veterinária , Echinococcus/genética , Echinococcus/isolamento & purificação , Equidae/parasitologia , Animais , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Equinococose Hepática/parasitologia , Echinococcus/classificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Fígado/parasitologia , Masculino , Filogenia
13.
Korean J Parasitol ; 57(2): 197-200, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31104414

RESUMO

Cryptosporidium is a common intestinal protozoan that can lead to diarrhea in humans and dogs. The predominant species of infection are C. hominis and C. parvum in humans, and C. canis in dogs. However, C. canis can infect immunocompromised humans. Considering the close contact with humans, dogs have the potential to be reservoirs for human cryptosporidiosis. Breeding kennels are the major supply source of puppies for pet shops. The present study is to determine the molecular prevalence and characteristics of Cryptosporidium spp. found in breeding kennel dogs. A total of 314 fecal samples were collected from young and adult dogs kept in 5 breeding kennels. A polymerase chain reaction targeting the small subunit rRNA gene was employed for the detection of Cryptosporidium spp. To determine the species, the DNA sequences were compared to GenBank data. Overall, 21.0% of the fecal samples were positive for Cryptosporidium spp. infection. Cryptosporidium spp. was detected in all 5 facilities. A sequencing analysis demonstrated that all isolates shared 99-100% similarity with C. canis. The results suggest that Cryptosporidium spp. infection is present at a high-level in breeding kennel dogs. However, because dominant species in this survey was C. canis, the importance of breeding kennel dogs as reservoirs for Cryptosporidium spp. transmission to humans is likely to be low in Japan.


Assuntos
Criptosporidiose/epidemiologia , Criptosporidiose/patologia , Cryptosporidium/classificação , Cryptosporidium/isolamento & purificação , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Fezes/parasitologia , Animais , Análise por Conglomerados , Cryptosporidium/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Cães , Feminino , Japão/epidemiologia , Masculino , Epidemiologia Molecular , Filogenia , Prevalência , RNA Ribossômico 18S/genética , Análise de Sequência de DNA
14.
J Parasitol ; 105(3): 391-394, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31059382

RESUMO

Toxoplasmosis in wild turkeys (Meleagris gallopavo) is of epidemiological interest because turkeys feed from the ground, and detection of infection in turkeys indicates contamination by oocysts in the environment. During the 2018 spring hunting season in Pennsylvania, fresh (unfixed, not frozen) samples were obtained from 20 harvested wild turkeys and tested for Toxoplasma gondii infection. Hearts from all wild turkeys and skeletal muscle from 1 were bioassayed for T. gondii by inoculation in outbred Swiss Webster (SW) and interferon-gamma gene knockout (KO) mice. Antibodies to T. gondii were detected in 1:5 dilution of neat serum from 5 of 15 wild turkeys and in fluid from the heart of 1 of 4 wild turkeys with the modified agglutination test (MAT); neat serum was not available from 4 wild turkeys. Viable T. gondii was isolated from hearts of 5 wild turkeys, 1 with MAT of 1:10, 1 with MAT of 1:5, and 3 seronegative (MAT < 1:5). Toxoplasma gondii was isolated from both heart and skeletal muscle in the 1 wild turkey that had skeletal muscle submitted. The KO mice inoculated with tissue from all 5 infected wild turkeys died or were euthanatized when ill, 7-21 days post-inoculation (PI). Tachyzoites were detected in lungs of all KO mice, and the T. gondii strains were successfully propagated in cell culture. The SW mice inoculated with tissues of wild turkeys remained asymptomatic, and tissue cysts were seen in the brains of infected mice when euthanatized in good health at 46 days PI; 1 of the 2 SW mice inoculated with the heart of 1 turkey died on day 26, and tachyzoites were detected in its lung. Genetic typing on DNA extracted from culture-derived tachyzoites using the PCR restriction fragment length polymorphism with 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed that 4 isolates belonged to ToxoDB PCR-RFLP genotype #5 and 1 was genotype #216.


Assuntos
Doenças das Aves/parasitologia , Toxoplasma/genética , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Perus/parasitologia , Testes de Aglutinação/veterinária , Animais , Animais Selvagens , Anticorpos Antiprotozoários/sangue , Doenças Assintomáticas , Doenças das Aves/epidemiologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Técnicas de Genotipagem/veterinária , Coração/parasitologia , Camundongos , Camundongos Knockout , Músculo Esquelético/parasitologia , Pennsylvania/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Toxoplasma/imunologia , Toxoplasmose Animal/epidemiologia
15.
Exp Parasitol ; 202: 1-6, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31077732

RESUMO

Neospora caninum is an apicomplexan parasite distributed worldwide. Although a positive association between the presence of birds and abortions in cattle associated to N. caninum has been reported, the role of the birds in the epidemiologic cycle of the parasite is unknown. To the best knowledge, no experimental studies have evaluated N. caninum in the eared dove, Zenaida auriculata. Therefore, we aimed to determine whether Z. auriculat can act as intermediate host for N. caninum. Eighteen birds were divided into four groups, G1, G2, G3, and G4 (control); G1, G2 and G3 received 2 × 106 tachyzoites of NC-1 strain via different routes: subcutaneous, intramuscular, and intraperitoneal, respectively. G4 composed of three birds. Serum samples were collected weekly, and one bird each from G1, G2 and G3 was euthanized on the 7th and 14th day post-inoculation (dpi). The remaining birds were euthanized after the 28th dpi. Tissues from the doves were evaluated using histopathological analysis, PCR and dog bioassay to detect the parasite. Dogs were fed with tissues from the birds and monitored for 30 days. Serum samples were collected weekly from the dogs for serological analysis, and feces samples were collected daily until the end of the experiment for coproparasitological examinations. No dove showed clinical signs of the infection; however, all of them seroconverted after the inoculation, with stronger immunological response in the G3 birds. The lung tissue of one G3 bird showed positive PCR results; it was euthanized on the 7th dpi, and an inflammatory infiltrate was observed in the lung and kidney from this dove. The dogs did not shed oocysts or seroconverted. Our results indicate that the intraperitoneal route induced infection in the doves; however, the parasite may have been eliminated by the host, and the doves may be resistant to chronic infection.


Assuntos
Doenças das Aves/parasitologia , Coccidiose/veterinária , Columbidae/parasitologia , Neospora/isolamento & purificação , Animais , Anticorpos Antiprotozoários/análise , Bioensaio/métodos , Bioensaio/veterinária , Coccidiose/parasitologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Cães , Feminino , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Imunoglobulina G/análise , Imuno-Histoquímica/veterinária , Rim/patologia , Fígado/patologia , Pulmão/patologia , Masculino , Músculo Esquelético/patologia , Neospora/genética , Neospora/imunologia , Reação em Cadeia da Polimerase/veterinária
16.
Vet Parasitol Reg Stud Reports ; 16: 100283, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31027592

RESUMO

Among the protozoa of the genus Sarcocystis (Apicomplexa; Sarcocystidae), Sarcocystis bertrami (syn. Sarcocystis fayeri) is an obligate intracellular parasite of donkeys and horses with worldwide distribution. Here, we report the detection of S. bertrami in naturally infected donkeys from southern Italy and describe their structure by light microscopy (LM) and transmission electron microscopy (TEM). Protozoal cysts were detected both morphologically and molecularly in skeletal muscles of 28.57% (40/140) donkeys. Mature cysts of S. bertrami were found in skeletal muscle measuring 31-102 µm long and 19-83 µm wide with radially striated thick cyst wall. The high prevalence of infected donkeys suggests that dogs, the definitive hosts of S. bertrami, are contaminating environment with environmentally resistant sporocysts. Considering the increased consumption of raw donkey meat results also suggest a potential risk for human health.


Assuntos
Equidae/parasitologia , Músculo Esquelético/parasitologia , Sarcocystis/isolamento & purificação , Sarcocistose/veterinária , Língua/parasitologia , Matadouros , Animais , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Itália/epidemiologia , Carne/parasitologia , Carne/normas , Microscopia Eletrônica de Transmissão/veterinária , Reação em Cadeia da Polimerase/veterinária , Prevalência , Sarcocystis/genética , Sarcocystis/ultraestrutura , Sarcocistose/epidemiologia , Sarcocistose/parasitologia , Alinhamento de Sequência/veterinária , Língua/ultraestrutura
17.
Vet Parasitol Reg Stud Reports ; 16: 100281, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31027606

RESUMO

Trichomonosis is an important cause of mortality in multiple avian species; however, there have been relatively few reports of this disease in owls. Two barn owls (Tyto alba) and four barred owls (Strix varia) submitted for diagnostic examination had lesions consistent with trichomonosis including caseous necrosis and inflammation in the oropharynx. Microscopically, these lesions were often associated with trichomonads and molecular testing, if obtainable, confirmed the presence of Trichomonas gallinae, the species most commonly associated with trichomonosis in birds. The T. gallinae genotype in one barn owl and two barred owls was identified as ITS-OBT-Tg-1 by sequence analysis. Columbids are the primary hosts for T. gallinae, and columbid remains found within the nest box of the barn owls were the likely source of infection. This study is the first to formally describe the strains and genetic variation of T. gallinae samples from clinical cases of trichomonosis in barn and barred owls in the eastern USA.


Assuntos
Doenças das Aves/parasitologia , Estrigiformes/parasitologia , Tricomoníase/veterinária , Animais , Doenças das Aves/diagnóstico , Doenças das Aves/patologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Feminino , Genótipo , Masculino , Estudos Retrospectivos , Trichomonas/classificação , Trichomonas/genética , Tricomoníase/diagnóstico , Tricomoníase/parasitologia , Tricomoníase/patologia , Estados Unidos
18.
J Clin Pathol ; 72(7): 487-492, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30952829

RESUMO

AIMS: Helminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay. METHODS: We designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested. RESULTS: The PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples. CONCLUSIONS: The highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.


Assuntos
Cestoides/isolamento & purificação , Infecções por Cestoides/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Nematoides/isolamento & purificação , Infecções por Nematoides/diagnóstico , Trematódeos/isolamento & purificação , Infecções por Trematódeos/diagnóstico , Animais , Sequência de Bases , Cestoides/genética , Infecções por Cestoides/parasitologia , Estudos de Coortes , Primers do DNA/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Fezes/parasitologia , Humanos , Nematoides/genética , Infecções por Nematoides/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Alinhamento de Sequência , Trematódeos/genética , Infecções por Trematódeos/parasitologia
19.
J Parasitol ; 105(1): 186-194, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30817219

RESUMO

Equine piroplasmosis stands out among the diseases that affect Equidae in Brazil and the world. It is caused by the protozoa Theileria equi and Babesia caballi. The objective of the present study was to carry out the molecular characterization of T. equi using equine blood samples collected in the 5 geographic regions of Brazil. Samples from all over the country were tested for the presence of T. equi by real-time PCR. The 18S rRNA sequences (∼1,600 bp) obtained from 23 samples taken from naturally infected horses were characterized by sequencing and analyzed to identify the genotypes and the possible sites of genetic variability. Thirteen different T. equi 18S rRNA sequences were identified, and 2 different genotypes were demonstrated to be in circulation in Brazil. Alignment entropy analysis demonstrated the existence of three hypervariable regions (V2, V4, and V8) within the 18S rRNA sequence of T. equi. The V2 region is located between nucleotides 63 and 75, V4 is located between nucleotides 524 and 586, and V8 is located between nucleotides 1,208 and 1,226. The hypervariable region V4 demonstrated the greatest variation within the 18S rRNA sequence of T. equi. Phylogenetic analysis based on the 18S rRNA sequences revealed the formation of 3 distinct clades (A, B, and C). The Brazilian samples belonged to 2 clades (A and C). The present study describes the characterization and heterogeneity of the circulating T. equi 18S rRNA sequences in Brazil. The results confirm that the country is an endemic area for the disease, and they indicate that at least 2 distinct T. equi genotypes are naturally infecting equines in Brazil.


Assuntos
Variação Genética , Doenças dos Cavalos/parasitologia , RNA Ribossômico 18S/genética , Theileria/genética , Theileriose/parasitologia , Animais , Brasil , Sequência Consenso , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças Endêmicas/veterinária , Doenças dos Cavalos/sangue , Cavalos , Funções Verossimilhança , RNA de Protozoário/sangue , RNA de Protozoário/genética , RNA Ribossômico 18S/sangue , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Alinhamento de Sequência/veterinária , Theileria/classificação , Theileriose/sangue
20.
Parasit Vectors ; 12(1): 80, 2019 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744665

RESUMO

BACKGROUND: In Kenya, malaria remains a major public health menace equally affecting the semi-arid to arid ecologies. However, entomologic knowledge of malaria vectors in such areas remains poor. METHODS: Morphologically-identified wild-caught Anopheles funestus (s.l.) specimens trapped outdoors from the semi-arid to arid area of Kacheliba, West Pokot County, Kenya, were analysed by PCR and sequencing for species identification, malaria parasite infection and host blood-meal sources. RESULTS: Three hundred and thirty specimens were analysed to identify sibling species of the An. funestus group, none of which amplified using the available primers; two were infected with Plasmodium falciparum and Plasmodium ovale, separately, while 84% (n = 25) of the blood-fed specimens had fed on humans. Mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences of 55 specimens (Plasmodium-positive, blood-fed and Plasmodium-negative) did not match reference sequences, possibly suggesting a previously unreported species, resolving as two clades. CONCLUSIONS: Our findings indicate the existence of yet-to-be identified and described anopheline species with a potential as malaria vectors in Kenya.


Assuntos
Anopheles/classificação , Malária/transmissão , Mosquitos Vetores/classificação , Plasmodium falciparum/fisiologia , Animais , Anopheles/genética , Anopheles/parasitologia , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Ecologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Monitoramento Ambiental , Feminino , Humanos , Quênia/epidemiologia , Malária/epidemiologia , Malária/parasitologia , Mitocôndrias/enzimologia , Mosquitos Vetores/genética , Mosquitos Vetores/parasitologia , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
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