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1.
PLoS One ; 15(10): e0240062, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031471

RESUMO

The eukaryotic blood parasite genus Trypanosoma includes several important pathogens of humans and livestock, but has been understudied in wildlife broadly. The trypanosomes that infect birds are in particular need of increased attention, as these parasites are abundant and globally distributed, yet few studies have addressed their evolutionary origins and diversity using modern molecular and analytical approaches. Of specific interest are the deep evolutionary relationships of the avian trypanosomes relative to the trypanosome species that are pathogenic in humans, as well as their species level diversity in regions where they have been understudied such as North America. Here, we address these unresolved areas of study using phylogenomic data for two species of avian trypanosomes that were isolated as "bycatch" from host transcriptome assemblies, as well as a large 18S DNA barcode sequence dataset that includes 143 novel avian Trypanosoma 18S sequences from North America. Using a phylogenomic approach, we find that the avian trypanosomes are nested within a clade of primarily mammalian trypanosomes that includes the human pathogen Trypanosoma cruzi, and are paraphyletic with respect to the ruminant trypanosome Trypanosoma theileri. DNA barcode sequences showed that T. avium and an unidentified small, non-striated trypanosome that was morphologically similar to T. everetti are each represented by highly abundant and divergent 18S haplotypes in North America. Community-level sampling revealed that additional species-level Trypanosoma lineages exist in this region. We compared the newly sequenced DNA barcodes from North America to a global database, and found that avian Trypanosoma 18S haplotypes generally exhibited a marked lack of host specificity with at least one T. avium haplotype having an intercontinental distribution. This highly abundant T. avium haplotype appears to have a remarkably high dispersal ability and cosmopolitan capacity to evade avian host immune defenses, which warrant further study.


Assuntos
Aves/genética , Transcriptoma , Trypanosoma/genética , Animais , Teorema de Bayes , Evolução Biológica , Aves/parasitologia , Mapeamento de Sequências Contíguas , Código de Barras de DNA Taxonômico , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Bases de Dados Factuais , Haplótipos , Humanos , América do Norte , Filogenia , RNA Ribossômico 18S/química , RNA Ribossômico 18S/classificação , RNA Ribossômico 18S/metabolismo , Trypanosoma/classificação , Trypanosoma/patogenicidade , Trypanosoma cruzi/classificação
2.
Exp Parasitol ; 217: 107956, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32659234

RESUMO

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.


Assuntos
Ciclídeos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/fisiologia , Doenças dos Peixes/parasitologia , Actinas/química , Actinas/genética , Animais , Sequência de Bases , Evolução Biológica , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , Cryptosporidium/genética , Cryptosporidium/ultraestrutura , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças dos Peixes/epidemiologia , Pesqueiros , Genótipo , Funções Verossimilhança , Microscopia Eletrônica de Transmissão/veterinária , Filogenia , Reação em Cadeia da Polimerase/veterinária , Prevalência , RNA Ribossômico 18S/química , Washington/epidemiologia , Austrália Ocidental/epidemiologia
3.
Exp Parasitol ; 216: 107941, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32622940

RESUMO

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Adulto , Idoso , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Medula Óssea/parasitologia , Biologia Computacional , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças do Cão/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Imunoglobulina G/sangue , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas de Protozoários/química , Sensibilidade e Especificidade , Alinhamento de Sequência , Testes Sorológicos , Baço/parasitologia , Adulto Jovem
4.
J Parasitol ; 106(3): 395-399, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32556163

RESUMO

The objective of the present study was to determine the characterization of Toxoplasma gondii in cats, rats, and chickens in the border areas of Yunnan Province. A total of 259 samples was collected from 10 border areas in Yunnan Province including 94 cats, 58 rats, and 107 chickens. Samples were screened by a nested polymerase chain reaction (PCR) assay and the positive products were analyzed by multilocus PCR-restriction fragment length polymorphism (RFLP) to determine the genotypes. Toxoplasma gondii deoxyribonucleic acid (DNA) was detected from 15.96% of 94 cats, 15.52% of 58 rats, and 6.54% of 107 chickens, respectively, and the average infection rate is 11.97%. Using the multilocus PCR-RFLP, we found that the genotype of T. gondii in cats and rats was ToxoDB#9. Because of low DNA concentration, no genotype was determined from chickens. These results fill the gaps of knowledge in the prevalence and genotype of T. gondii in the border areas of Yunnan Province and have implications for the better control of T. gondii infection in humans and animals.


Assuntos
Doenças do Gato/epidemiologia , Galinhas/parasitologia , Doenças das Aves Domésticas/epidemiologia , Ratos/parasitologia , Doenças dos Roedores/epidemiologia , Toxoplasmose Animal/epidemiologia , Animais , Encéfalo/parasitologia , Doenças do Gato/parasitologia , Gatos , China/epidemiologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Marcadores Genéticos , Técnicas de Genotipagem/veterinária , Tipagem de Sequências Multilocus/veterinária , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Doenças das Aves Domésticas/parasitologia , Prevalência , Doenças dos Roedores/parasitologia , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose Animal/parasitologia
5.
Nature ; 582(7810): 104-108, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32427965

RESUMO

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.


Assuntos
Apoptose/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Parasitos/imunologia , Plasmodium falciparum/citologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Aotidae/imunologia , Aotidae/parasitologia , Caspases/metabolismo , Criança , Estudos de Coortes , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Ativação Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Quênia , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Parasitos/citologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Tanzânia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/imunologia , Vacúolos/imunologia
6.
Exp Parasitol ; 212: 107872, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32165145

RESUMO

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.


Assuntos
Doenças das Cabras/parasitologia , Doenças dos Ovinos/parasitologia , Theileria/isolamento & purificação , Theileriose/parasitologia , Distribuição por Idade , Análise de Variância , Animais , Coinfecção/epidemiologia , Coinfecção/parasitologia , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Ribossômico/isolamento & purificação , Feminino , Genótipo , Doenças das Cabras/epidemiologia , Cabras , Hibridização Genética , Funções Verossimilhança , Modelos Lineares , Masculino , Análise Multivariada , Sondas de Oligonucleotídeos , Paquistão/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Prevalência , RNA Ribossômico 18S/genética , Fatores de Risco , Análise de Sequência de DNA , Distribuição por Sexo , Ovinos , Doenças dos Ovinos/epidemiologia , Theileria/classificação , Theileria/genética , Theileriose/epidemiologia
7.
Malar J ; 19(1): 57, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32014000

RESUMO

BACKGROUND: The epidemiological control of malaria has been hampered by the appearance of parasite resistance to anti-malarial drugs and by the resistance of mosquito vectors to control measures. This has also been associated with weak transmission control, mostly due to poor control of asymptomatic patients associated with host-vector transmission. This highlights the importance of studying the parasite's sexual forms (gametocytes) which are involved in this phase of the parasite's life-cycle. Some African and Asian strains of Plasmodium falciparum have been fully characterized regarding sexual forms' production; however, few Latin-American strains have been so characterized. This study was aimed at characterizing the Colombian FCB2 strain as a gametocyte producer able to infect mosquitoes. METHODS: Gametocyte production was induced in in vitro cultured P. falciparum FCB2 and 3D7 strains. Pfap2g and Pfs25 gene expression was detected in FCB2 strain gametocyte culture by RT-PCR. Comparative analysis of gametocytes obtained from both strains was made (counts and morphological changes). In vitro zygote formation from FCB2 gametocytes was induced by incubating a gametocyte culture sample at 27 °C for 20 min. A controlled Anopheles albimanus infection was made using an artificial feed system with cultured FCB2 gametocytes (14-15 days old). Mosquito midgut dissection was then carried out for analyzing oocysts. RESULTS: The FCB2 strain expressed Pfap2g, Pfs16, Pfg27/25 and Pfs25 sexual differentiation-related genes after in vitro sexual differentiation induction, producing gametocytes that conserved the expected morphological features. The amount of FCB2 gametocytes produced was similar to that from the 3D7 strain. FCB2 gametocytes were differentiated into zygotes and ookinetes after an in vitro low-temperature stimulus and infected An. albimanus mosquitoes, developing to oocyst stage. CONCLUSIONS: Even with the history of long-term FCB2 strain in vitro culture maintenance, it has retained its sexual differentiation ability. The gametocytes produced here preserved these parasite forms' usual characteristics and An. albimanus infection capability, thus enabling its use as a tool for studying sexual form biology, An. albimanus infection comparative analysis and anti-malarial drug and vaccine development.


Assuntos
Anopheles/parasitologia , Malária Falciparum/parasitologia , Mosquitos Vetores/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Colômbia/epidemiologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Eritrócitos/parasitologia , Feminino , Gametogênese , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Análise de Sequência de DNA , Espectrofotometria
8.
Parasit Vectors ; 13(1): 27, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937360

RESUMO

BACKGROUND: Henneguya Thélohan, 1892 (Myxobolidae) is one of the most species-rich genera of myxosporean parasites infecting fish. Although common in nature, there are few reports of these parasites causing important disease in aquaculture. In this paper, we describe a new species of Henneguya infecting Pagrus major (Temminck & Schlegel), a fish host introduced to the Mediterranean Sea from Japan in the late 1980s. RESULTS: Large plasmodia of the parasite were found in the bulbus arteriosus and in the ventricle of the infected fish. Spores were found mainly in the kidney and heart and were accompanied by melanized macrophages or vascular intimal proliferation mixed with a mild non-suppurative response, respectively. Comparisons of morphometric data for spore and polar capsule length and width, suggest a unique combination of features in the newly described species. Molecular analysis, based on 18S rDNA sequence of the parasite, followed by phylogenetic analysis, indicated that the parasite described here is a novel species of Henneguya, clustered with the marine congeneric species. CONCLUSIONS: Henneguya aegea n. sp. infects in aquaculture P. major, a host introduced as eggs to the Mediterranean from Japan. Despite the high host specificity of the myxobolid parasites, H. aegea n. sp. seems to be able to use P. major as a host and propagate successfully, causing morbidity and mortality. This could result in spillback of the new species from high density cultured non-native P. major to native fish hosts.


Assuntos
Doenças dos Peixes/parasitologia , Myxozoa/patogenicidade , Doenças Parasitárias em Animais/parasitologia , Perciformes/parasitologia , Animais , Aquicultura , Vasos Sanguíneos/parasitologia , Vasos Sanguíneos/patologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Diagnóstico Diferencial , Doenças dos Peixes/patologia , Grécia , Átrios do Coração/parasitologia , Ventrículos do Coração/parasitologia , Rim/parasitologia , Rim/patologia , Fígado/parasitologia , Fígado/patologia , Mar Mediterrâneo , Microscopia Eletrônica de Varredura/veterinária , Myxozoa/classificação , Myxozoa/genética , Myxozoa/ultraestrutura , Doenças Parasitárias em Animais/patologia , Filogenia , Esporos de Protozoários/genética , Esporos de Protozoários/ultraestrutura
9.
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
10.
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
11.
Avian Pathol ; 49(1): 47-55, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31486682

RESUMO

Outbreaks of avian trichomonosis are being reported worldwide; meanwhile, the genetic and virulence variations are under investigation. In this study, the occurrence and genetic variability of oral or faecal trichomonads among various avian species were investigated. Samples obtained from either the oropharyngeal cavity, crop/oesophagus, droppings/cloaca, or conjunctival swabs of avian species were inspected for flagellates. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples was performed to investigate the genetic diversity of the isolates. Investigation of 737 birds revealed an infection rate of 15.7% in the upper gastrointestinal tract, 7.3% in the faecal samples, and 0.7% involvement of the conjunctiva. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples, identified genotypes A and B of Trichomonas gallinae and genogroups A-C and E of Tetratrichomonas gallinarum. A novel ITS genotype of intestinal trichomonads was also detected in hooded crow (Corvus cornix) and common mynah (Acridotheres tristis). In the present study, in addition to Columbiformes and Falconiformes, trichomonads were detected in Passeriformes and Galliformes with the involvement of organs other than the gastrointestinal tract. Genotype A T. gallinae was detected in domestic pigeons (Columba livia domestica), a laughing dove (Spilopelia senegalensis), a common kestrel (Falco tinnunculus), a budgerigar (Melopsittacus undulates), and a canary (Serinus canaria). Distinct genotype B was detected in a common mynah and a budgerigar. Genogroups A-C of T. gallinarum were also demonstrated in Galliformes and Anseriformes. Furthermore, two novel trichomonad ITS genotypes were detected in hooded crows and a common mynah warranting detailed multi-locus molecular analysis.RESEARCH HIGHLIGHTSITS diversity of trichomonads was shown in various avian species.Diversity of the parasites' target organ and clinical manifestations was demonstrated.Two novel ITS genotype trichomonads from common mynah and hooded crow were identified.


Assuntos
Doenças das Aves/parasitologia , Infecções Protozoárias em Animais/parasitologia , Trichomonadida/genética , Animais , Anseriformes/parasitologia , Doenças das Aves/epidemiologia , Canários/parasitologia , Columbiformes/parasitologia , Corvos/parasitologia , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , DNA Espaçador Ribossômico/química , Falconiformes/parasitologia , Galliformes/parasitologia , Genótipo , Humanos , Irã (Geográfico)/epidemiologia , Melopsittacus/parasitologia , Tipagem de Sequências Multilocus/veterinária , Passeriformes/parasitologia , Filogenia , Prevalência , Infecções Protozoárias em Animais/epidemiologia , Psittaciformes/parasitologia , RNA Ribossômico 5,8S/genética , Estorninhos/parasitologia , Trichomonadida/classificação , Trichomonas/genética
12.
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
13.
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
14.
J Parasitol ; 105(6): 913-917, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31815596

RESUMO

Hemosporidians are a monophyletic group of protozoan parasites infecting all terrestrial vertebrate orders. Although Plasmodium is the most studied genus within the Haemosporidia, this research effort is heavily biased toward mammal and bird hosts. We screened 205 specimens of at least 18 reptile species from Brazil using a partial mitochondrial cytochrome b gene marker. Positive samples were sequenced and included in a phylogenetic assessment. Four positive PCR products matched others identified as Plasmodium using BLAST from 3 different host species, Ameiva ameiva, Tropidurus hispidus, and Hemidactylus mabouia. Recovery of similar haplotypes in the native T. hispidus and exotic H. mabouia (99.9%) indicate potential host-switching.


Assuntos
Malária/veterinária , Plasmodium/genética , Répteis/parasitologia , Animais , Teorema de Bayes , Brasil/epidemiologia , Citocromos b/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Variação Genética , Haplótipos , Funções Verossimilhança , Lagartos/parasitologia , Malária/epidemiologia , Malária/parasitologia , Programas de Rastreamento/métodos , Programas de Rastreamento/veterinária , Filogenia , Filogeografia , Plasmodium/classificação , Plasmodium/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Prevalência , Alinhamento de Sequência/veterinária
15.
J Parasitol ; 105(6): 890-892, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31738124

RESUMO

Toxoplasma gondii infections are prevalent in most warm-blooded animals worldwide. During the 2018 November hunting season in Pennsylvania, fresh (unfixed, not frozen) samples obtained from 99 harvested elk (Cervus canadensis) were tested for T. gondii infection. Antibodies to T. gondii were detected in 69 of 99 (69.7%) elk tested by the modified agglutination test (MAT, 1:25 cut-off). Tongues and hearts from 16 elk with high MAT titers (>1:200) were bioassayed for T. gondii by inoculation in outbred Swiss Webster (SW) and interferon-gamma gene knockout (KO) mice. Viable T. gondii was isolated from tongues of 2 elk with MAT titers of 1:200 and 1:3,200. Toxoplasma gondii from both isolates were successfully propagated in cell culture. 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 both isolates belonged to ToxoDB PCR-RFLP genotype #5 that is widely prevalent in wildlife in the United States. Our results suggest that elk may clear T. gondii organisms from their tissues.


Assuntos
Cervos/parasitologia , Toxoplasma/imunologia , Toxoplasmose Animal/epidemiologia , Testes de Aglutinação/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Bioensaio/veterinária , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Feminino , Técnicas de Genotipagem/veterinária , Coração/parasitologia , Masculino , Camundongos , Camundongos Knockout , Pennsylvania/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Estudos Soroepidemiológicos , Língua/parasitologia , Toxoplasma/genética , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/transmissão
16.
Parasit Vectors ; 12(1): 543, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31730024

RESUMO

BACKGROUND: Cryptosporidium spp. and Giardia duodenalis are major intestinal pathogens that can cause diarrheal diseases in humans, especially children. Enterocytozoon bieneusi is another parasite which can cause gastrointestinal tract disorders, with diarrhea being the main clinical symptom. However, few genetic studies of these parasites in pediatric inpatients in China have been published. METHODS: To assess the genetic characteristics and epidemiological status of these parasites, a total of 2284 fecal samples were collected from children in the pediatric departments of three hospitals in Zhengzhou, central China, and screened for these protozoans with PCR, based on the small subunit ribosomal RNA (SSU rRNA) genes of Cryptosporidium spp. and G. duodenalis and the internal transcribed spacer (ITS) of E. bieneusi. RESULTS: Six (0.26%), 14 (0.61%), and 27 (1.18%) of the samples were positive for Cryptosporidium spp., G. duodenalis and E. bieneusi, respectively. Of the 12 successfully sequenced G. duodenalis isolates, four were identified as assemblage A and eight as assemblage B. In subtype and multilocus genotype (MLG) analyses, C. parvum IIdA19G1 (n = 4) and two novel G. duodenalis MLGs belonging to subassemblage AII (n = 3) and BIV (n = 5) were successfully identified. The E. bieneusi isolates included genotypes D (n = 17), J (n = 2), PigEBITS7 (n = 1), BEB6 (n = 1), and CM8 (n = 1). This is the first report of C. parvum subtype IIdA19G1 in HIV-negative children and E. bieneusi genotype CM8 in humans. CONCLUSIONS: The dominance of zoonotic C. parvum subtype IIdA19G1 indicates that this parasite is turning into zoonotic origin from human-to-human transmission. The phylogenetic analysis also revealed the zoonotic origins and anthroponotic transmission potential of G. duodenalis and E. bieneusi, suggesting more efforts must be made to minimize the threat these pathogens pose to public health.


Assuntos
Cryptosporidium/classificação , Cryptosporidium/genética , Enterocytozoon/classificação , Enterocytozoon/genética , Fezes/parasitologia , Giardia lamblia/classificação , Giardia lamblia/genética , Adolescente , Criança , Criança Hospitalizada , Pré-Escolar , China , Análise por Conglomerados , Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Transmissão de Doença Infecciosa , Enterocytozoon/isolamento & purificação , Feminino , Variação Genética , Genótipo , Giardia lamblia/isolamento & purificação , Giardíase/parasitologia , Hospitais Pediátricos , Humanos , Lactente , Masculino , Microsporidiose/parasitologia , Filogenia , RNA Ribossômico 18S/genética , Análise de Sequência de DNA
17.
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
18.
Parasit Vectors ; 12(1): 548, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31753041

RESUMO

BACKGROUND: Avian malaria parasites are a highly diverse group that commonly infect birds and have deleterious effects on their hosts. Some parasite lineages are geographically widespread and infect many host species in many regions. Bird migration, natural dispersal, invasive species and human-mediated introductions into areas where competent insect vectors are present, are probably the main drivers of the current distribution of avian malaria parasites. METHODS: A total of 412 and 2588 wild house sparrows (Passer domesticus) were captured in 2012 and 2013 in two areas of the Iberian Peninsula (central and southern Spain, respectively). Genomic DNA was extracted from blood samples; parasite lineages were sequenced and identified by comparing with GenBank and/or MalAvi databases. RESULTS: Thirteen Plasmodium lineages were identified in house sparrows corresponding to three major clades. Five individuals were infected by the African Plasmodium lineage PAGRI02, which has been proposed to actively circulate only in Africa. CONCLUSIONS: Despite the low prevalence of PAGRI02 in sparrows in Spain, our results suggest that the area of transmission of this parasite is more widespread than previously thought and covers both Africa and Europe. Further studies of the global distribution of Plasmodium lineages infecting wild birds are required to identify the current transmission areas of these parasites. This is vital given the current scenario of global change that is providing new opportunities for avian malaria transmission into areas where parasites were previously absent.


Assuntos
Doenças das Aves/transmissão , Transmissão de Doença Infecciosa , Genótipo , Malária/veterinária , Plasmodium/classificação , Plasmodium/genética , Pardais , África , Animais , Doenças das Aves/parasitologia , DNA de Protozoário/química , DNA de Protozoário/genética , Malária/parasitologia , Malária/transmissão , Epidemiologia Molecular , Plasmodium/isolamento & purificação , Prevalência , Análise de Sequência de DNA , Espanha
19.
Mycologia ; 111(6): 981-997, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613722

RESUMO

The genus Siphoptychium is resurrected on the basis of comparative morphology and phylogeny of partial nuc 18S rDNA (18S) and translation elongation factor 1-alpha (EF1A) nucleotide sequences. The genus is characterized by the firm upper surface of the pseudoaethalium, accreted but easily separable sporothecae, a tubular or fibrous columella, and spores with a reticulate ornamentation consisting of 7-9 meshes across the diameter. In addition to the currently known single species S. casparyi (= Tubifera casparyi), two new members of Siphoptychium are described: S. violaceum from coniferous forests of Europe, east Asia, and southeast Asia, and S. reticulatum from temperate and subarctic regions of North America and alpine regions of Europe. A second genus, Thecotubifera, is described to accommodate Tubifera dictyoderma. The fruiting body of this species is transitional between a pseudoaethalium and a true aethalium. It is covered by a contiguous membranous cortex formed by the fused tips of the sporothecae, a feature typical for aethalia. However, the inner portions of sporothecae remain discernible, a feature more typical for pseudoaethalia. Columellae of Th. dictyoderma are formed by perforated plates, and the spores have a reticulate ornamentation consisting of 2-5 meshes across the diameter. For Th. dictyoderma, we could confirm records only for tropical regions and Japan, whereas all studied European specimens, including those mentioned in current monographs, represent species of Siphoptychium.


Assuntos
Mixomicetos/classificação , Mixomicetos/genética , Filogenia , Ásia , Análise por Conglomerados , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Europa (Continente) , Microscopia , Mixomicetos/citologia , Mixomicetos/isolamento & purificação , América do Norte , Fator 1 de Elongação de Peptídeos/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNA
20.
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
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