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1.
Methods Enzymol ; 658: 83-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517961

RESUMO

Unicellular parasite Trypanosoma brucei maintains an elaborate mitochondrial mRNA processing pathway including 3'-5' exonucleolytic trimming of primary precursors, 5' and 3' modifications, and, in most cases, massive U-insertion/deletion editing. Whereas the role of editing in restoring protein coding sequence is apparent, recent developments suggest that terminal modifications are equally critical for generating a stable translationally competent messenger. The enzymatic activities responsible for 5' pyrophosphate hydrolysis, 3' adenylation and uridylation, and 3'-5' decay are positively and negatively regulated by pentatricopeptide repeat-containing (PPR) proteins. These sequence-specific RNA binding factors typically contain arrays of 35-amino acid repeats each of which recognizes a single nucleotide. Here, we introduce a combinatorial CTS affinity tag, which underlies a suite of methods for PPR proteins purification, in vivo RNA binding sites mapping and sub-cellular localization studies. These approaches should be applicable to most trypanosomal RNA binding proteins.


Assuntos
Trypanosoma brucei brucei , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Edição de RNA , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
2.
Ticks Tick Borne Dis ; 12(6): 101817, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34560427

RESUMO

Piroplasmosis is an economically important tick-borne disease worldwide. However, little is known about the presence of Babesia spp. and Theileria spp. in ticks in Eastern and Southern Kazakhstan (ESK). During 2016 - 2019, adult ticks (at 26 sampling sites in 16 districts of 5 oblasts in ESK) were collected. Tick species were identified according to morphological and molecular characteristics. Two fragments (487 bp and 438 bp) of 18S ribosomal RNA (18S rRNA) were used to determine piroplasm species in representative 698 ticks. The genotype characteristics of Babesia caballi and Theileria equi were further analyzed by longer 18S rRNA gene fragments. A total of 6107 adult ticks (4558 parasitizing ticks and 1549 off-host ticks), including 4665 hard ticks and 1442 soft ticks, were collected from their natural hosts (cattle, horses, sheep, camels, shepherd dogs and hedgehogs) and the surrounding environment, respectively. Among the hard tick species, Dermacentor marginatus (62.59%, 2920/4665) was the most abundant, followed by Hyalomma asiaticum (19.36%, 903/4665) and Hyalomma detritum (9.95%, 464/4665). All soft ticks were identified as Argas persicus. 16S ribosomal DNA (16S rDNA) phylogenic analysis showed that several tick species in Kazakhstan, as exemplified by Haemaphysalis erinacei and D. marginatus, clustered together with conspecific ticks reported from China. Five species of piroplasms, i.e. Babesia occultans, Babesia caballi, Theileria ovis, Theileria annulata and Theileria equi, were detected in 698 representative ticks. Genotype E of T. equi in Almaty, and genotype A of B. caballi in Almaty and South Kazakhstan were identified.


Assuntos
Argasidae/parasitologia , Babesia/isolamento & purificação , Ixodidae/parasitologia , Theileria/isolamento & purificação , Animais , Babesia/classificação , Babesia/genética , Genótipo , Cazaquistão , RNA de Protozoário/análise , RNA Ribossômico 16S/análise , RNA Ribossômico 18S/análise , Especificidade da Espécie , Theileria/classificação , Theileria/genética
3.
Nat Commun ; 12(1): 4851, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381047

RESUMO

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.


Assuntos
Quimiocina CXCL10/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Regiões 3' não Traduzidas , Quimiocina CXCL10/genética , Proteína DEAD-box 58/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Malária Falciparum/imunologia , Monócitos/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Biossíntese de Proteínas , RNA de Protozoário/metabolismo , Receptores Imunológicos/metabolismo , Ribossomos/metabolismo , Células THP-1
4.
Transfusion ; 61(10): 2969-2980, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34368968

RESUMO

BACKGROUND: Human babesiosis is a zoonotic infection caused by an intraerythrocytic parasite. The highest incidence of babesiosis is in the United States, although cases have been reported in other parts of the world. Due to concerns of transfusion-transmitted babesiosis, the US Food and Drug Administration (FDA) recommended year-round regional testing for Babesia by nucleic acid testing or use of an FDA-approved device for pathogen reduction. A new molecular test, cobas Babesia (Roche Molecular Systems, Inc.), was evaluated for the detection of the four species that cause human disease, Babesia microti, Babesia duncani, Babesia divergens, and Babesia venatorum. STUDY DESIGN AND METHODS: Analytical performance was evaluated followed by clinical studies on whole blood samples from US blood donations collected in a special tube containing a chaotropic reagent that lyses the red cells and preserves nucleic acid. Sensitivity and specificity of the test in individual samples (individual donation testing [IDT]) and in pools of six donations were determined. RESULTS: Based on analytical studies, the claimed limit of detection of cobas Babesia for B. microti is 6.1 infected red blood cells (iRBC)/mL (95% confidence interval [CI]: 5.0, 7.9); B. duncani was 50.2 iRBC/mL (95% CI: 44.2, 58.8); B. divergens was 26.1 (95% CI: 22.3, 31.8); and B. venatorum was 40.0 iRBC/mL (95% CI: 34.1, 48.7). The clinical specificity for IDT was 99.999% (95% CI: 99.996, 100) and 100% (95% CI: 99.987, 100) for pools of six donations. CONCLUSION: cobas Babesia enables donor screening for Babesia species with high sensitivity and specificity.


Assuntos
Babesia/isolamento & purificação , Babesiose/sangue , Doadores de Sangue , DNA de Protozoário/sangue , RNA de Protozoário/sangue , Babesia/genética , Babesia microti/genética , Babesia microti/isolamento & purificação , Babesiose/diagnóstico , Babesiose/microbiologia , DNA de Protozoário/genética , Testes Diagnósticos de Rotina , Seleção do Doador , Humanos , RNA de Protozoário/genética , Sensibilidade e Especificidade , Estados Unidos
5.
Ticks Tick Borne Dis ; 12(6): 101807, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34416567

RESUMO

Babesia spp. are tick-borne haemoparasites that infect a wide range of domestic and wild mammals. Free-ranging ungulates are considered to be important reservoir hosts of Babesia parasites. The European bison (Bison bonasus) is a large and rare ungulate species, reintroduced into the forests of Central Europe after an absence of several decades. Owing to their protected status, studies of tick-borne pathogens in European bison have so far been rare and fragmented. The aim of this study was to investigate the presence of Babesia infection in free-ranging and captive herds of European bison and their ticks. Tissue samples obtained from 37 European bison individuals and 242 ticks belonging to two species, Ixodes ricinus and Dermacentor reticulatus, collected from bison were subjected to PCR analysis of the 18S rRNA gene followed by sequencing. Babesia spp. were detected in 8% of the samples from European bison and in 11% of the ticks. Sequence analysis of partial 18S rRNA gene indicated the presence of B. divergens and B. capreoli in European bison, while B. divergens, B. microti and B. venatorum were detected in ixodid ticks. To the best of authors' knowledge, this is the first molecular detection and characterization of Babesia spp. in European bison and their ticks.


Assuntos
Babesia/isolamento & purificação , Babesiose/parasitologia , Bison , Ixodidae/parasitologia , Animais , Babesia/classificação , Feminino , Ixodidae/crescimento & desenvolvimento , Masculino , Ninfa/crescimento & desenvolvimento , Filogenia , RNA de Protozoário/análise , RNA Ribossômico 18S/análise
6.
Ticks Tick Borne Dis ; 12(6): 101810, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34416568

RESUMO

To investigate the presence of Theileria equi in an endemic area of equine piroplasmosis 42 horses (Equus caballus) from Corrientes City, Argentina were sampled. Eighty-one percent (34 blood samples) of the analyzed horses were tested positive to the presence of piroplasmid 18S rDNA. All these samples could be identified as T. equi by amplifying the specific EMA-1 (merozoite antigen 1) gene of this species. Phylogenetic analysis of an obtained 18S rDNA complete sequence from one strain resulted in the identification of this sample as T. equi sensu stricto (genotype A). This study presents the first molecular detection and characterization of T. equi by the complete 18S rDNA sequence in Argentina. Based on these results further studies should be carried out to investigate the distribution and heterogeneity of presented genotypes of T. equi in Argentina, which is essential for the diagnosis, prevention and treatment of equine piroplasmosis.


Assuntos
Doenças dos Cavalos/parasitologia , Theileria/isolamento & purificação , Theileriose/parasitologia , Animais , Argentina , Cavalos , Filogenia , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Theileria/classificação
7.
Ticks Tick Borne Dis ; 12(5): 101776, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34271342

RESUMO

The present investigation was aimed to study the presence of Babesia caballi clades upon phylogenetic analysis of all available V4 hypervariable 18S rRNA gene sequences in GenBank in addition to the intra- and interclade genetic diversity in B. caballi and the distribution of parasite clades in different countries. Out of altogether 155 small-subunit ribosomal RNA gene sequences of B. caballi available in the database, only 92 sequences with a complete V4 hypervariable region (>293 bp) were used in multiple sequence alignment. The phylogenetic tree placed all the sequences into two distinct clades with high bootstrap values which are designated as B. caballi clades A and B. Clade A was further divided into two subclades A1 and A2 with 98% bootstrap support. On the contrary, clade B contained multiple small subclades which either lacked bootstrap support or did not have enough bootstrap support to further group them into subclades. All the sequences of B. caballi were 91.5-100% identical with each other. Clade B manifested a comparatively higher genetic diversity (95.2-100% identity) amongst sequences as opposed to clade A (97.3-100% identity). Moreover, it indicated 91.5-93.5%, 92.9-94.6% and 91.5-94.6% nucleotide identity with B. caballi subclades A1, A2, and clade A, respectively. Significant nucleotide variations were observed in one region, between nucleotide positions 126-178, in some of the sequences. A total of 21 molecular signature residues were identified in the V4 hypervariable region. The alignment report of the V4 hypervariable region of 18S rRNA gene of clades A and B exhibited nucleotide variation at nine and 24 places, respectively. The distribution map of all the clades of B. caballi is also reported. The number of 18S rRNA gene sequences employed in the study is relatively high compared to previous studies. Therefore, a fair comparison of definite genetic variations between isolates/sequences from different countries was carried out.


Assuntos
Babesia/fisiologia , Variação Genética , Filogenia , Babesia/classificação , Babesia/genética , Sequência de Bases , Geografia , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Alinhamento de Sequência/veterinária
8.
Ticks Tick Borne Dis ; 12(5): 101785, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34274574

RESUMO

We performed a cross-sectional epidemiological study with 456 household dogs from urban and rural areas in two different regions situated at different altitudes in the state of Rio de Janeiro. The PCR technique using 18S rRNA as target revealed prevalence of 7.9% of dogs positive for piroplasmids. These samples were sequenced, and all the sequences were 99.9% to 100% similar to Babesia vogeli sequences from other countries. The spatial distribution of positive cases was analysed using kernel interpolation in the QGIS software, and the spatial correlation indicators among positive dogs, altitude, and presence of ticks were obtained by calculating the local Moran index using the GeoDa software. The spatial correlation between positive cases and altitude was clear based on both visual and statistical observations. Logistic regression applying the Wald method with a cutoff point of 0.1 revealed that dogs from a region with altitude <600 m had a 2.29-fold chance of B. vogeli infection (OR = 2.29; p-value = 0.04; CI: 1.03-5.07), while the rainy season was 2.45 times more associated with B. vogeli infection (OR = 2.45; p-value = 0.01; CI: 1.20-5.01), and dogs infested with Rhipicephalus sanguineus sensu lato had a 2.47 times higher chance of being infected (OR = 2.47; p-value = 0.02; CI: 1.13-5.38). Entropy analysis of the alignment between B. vogeli 18S rRNA (> 1.600 bp) sequences revealed that the most variable region corresponds to the hypervariable V4 region. Genetic homogeneity was observed among the B. vogeli 18S rRNA sequences, with distance values ranging from 0 to 0.007 and a mean value of 0.001. The evolutionary distance (0.003) was greater between the sequences from the municipalities of Barra do Pirai (low altitude) and Teresopolis (high altitude). This study expands the molecular epidemiologic knowledge of B. vogeli and shows points of variability in the B. vogeli 18S rRNA. The results indicate the potential use of spatial analysis tools to improve screening for positive cases, enabling more in-depth studies to strengthen understanding of tick infection prevention in dogs.


Assuntos
Babesia/isolamento & purificação , Babesiose/epidemiologia , Altitude , Animais , Babesiose/parasitologia , Brasil/epidemiologia , Doenças do Cão , Cães , Feminino , Masculino , Epidemiologia Molecular , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Análise Espacial
9.
Parasit Vectors ; 14(1): 370, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34284816

RESUMO

BACKGROUND: Epidemiological surveys in Oman have revealed a high prevalence of the co-occurrence of the pathogenic Theileria lestoquardi and the non-pathogenic Theileria ovis among sheep in the Barka region, Oman. Our most recent data illustrated an interaction and reduced mortality risk in animals co-infected with T. lestoquardi and T. ovis, suggesting that the latter confers protection against pathogenicity of T. lestoquardi. The present study extends the above findings and examines disease outcomes; clinical markers, hematological parameters, and parasite density in mixed and single T. lestoquardi infections. METHODS: A total of 390 blood samples were collected from 16 sheep pens located in Barka, Oman between July and November 2019. Theileria spp. were detected and quantified using qPCR assay targeting 18S rRNA, and the extent of genetic diversity was estimated by a panel of T. lestoquardi specific micro- and mini-satellites. The association of some disease markers with the presence of Theileria spp. and genetic diversity was tested. RESULTS: Theileria spp. were detected in 75 (19.2%) sheep; of these 65 (86.7%) had mixed infections (T. lestoquardi plus T. ovis), 8 (10.6%) were infected with T. lestoquardi alone, and 2 (2.7%) with only T. ovis. Exotic breeds had a higher risk for Theileria spp. infection. The density (18S rRNA gene copies) of both parasites was higher in single infection against mixed infection, and there was a relatively lower density of T. lestoquardi in mixed infections. However, there was no difference in hematological indices between single T. lestoquardi and mixed infections. High genetic diversity was observed among T. lestoquardi in Barka, with no differences of T. lestoquardi in single and mixed infections. The extent of diversity seen in Barka was higher (He = 0.772) than that reported in Oman in 2019 (He = 0.582), with distinct T. lestoquardi genotypes. CONCLUSION: The lower density of T. lestoquardi as mixed infection with T. ovis compared to single infection supports the hypothesis that T. ovis confers protection against lethal T. lestoquardi infection. However, there were no differences in disease correlations (clinical markers, hematological parameters, and density of parasites) or the extent of diversity of T. lestoquardi between the two types of infection. The presence of distinct T. lestoquardi genotypes in Barka, compared to that reported earlier in Oman, likely reflects movement of carrier animals and highlights the need for further analysis of the parasite populations to inform novel approaches for controlling malignant ovine theileriosis.


Assuntos
Variação Genética , Doenças dos Ovinos/parasitologia , Ovinos/parasitologia , Theileria/genética , Theileriose/parasitologia , Animais , Coinfecção/veterinária , Estudos Transversais , Feminino , Genótipo , Masculino , Repetições de Microssatélites/genética , Omã/epidemiologia , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Doenças dos Ovinos/epidemiologia , Theileria/isolamento & purificação , Theileriose/epidemiologia
10.
Ticks Tick Borne Dis ; 12(4): 101729, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33984595

RESUMO

Canine vector-borne pathogens can act as zoonotic agents in humans; however, it poorly understood whether dogs play a role as reservoirs of vector-borne parasites in livestock animals. Here, we report the unexpected detection of 18S rRNA gene (rDNA) sequences of five ruminant Theileria species from the peripheral blood of dogs in Myanmar, in addition to those of two canine Babesia species. Using novel BTH primers capable of amplifying the 18S rDNA of Babesia, Theileria, and Hepatozoon spp., approximately 1,500 bp nested PCR products were detected in 19% (17/91) of local or imported dog breeds in different regions of Myanmar. Among the sequences of the 17 PCR products, ten were determined as Theileria 18S rDNA, including three as Theileria orientalis, three as Theileria buffeli, two as Theileria cf. velifera, one as Theileria luwenshuni, and one as Theileria sp. Most of these sequences showed higher identities with Theileria sequences determined in previous studies of cattle, water buffaloes, and goats in Myanmar. Six PCR products were identified as Babesia vogeli and one sample was determined as Babesia gibsoni. Furthermore, we obtained approximately 900 bp thrombospondin-related adhesive protein (TRAP) gene fragments from three dog blood DNA samples. Phylogenetic analysis of the TRAP gene showed that B. gibsoni parasites in Myanmar were considerably related to isolates from China, Korea, Japan, and Taiwan, but clearly separated from those from Bangladesh and India. These results provide new insights into a possible role of dogs in maintaining and spreading tick-borne pathogens among livestock and canine populations in Myanmar.


Assuntos
Babesia/isolamento & purificação , Babesiose/epidemiologia , Reservatórios de Doenças/veterinária , Doenças do Cão/epidemiologia , Theileria/isolamento & purificação , Theileriose/epidemiologia , Animais , Babesiose/parasitologia , Búfalos , Bovinos , Cães , Cabras , Mianmar/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Proteínas de Protozoários/análise , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Theileriose/parasitologia
11.
Parasitol Int ; 84: 102386, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34019996

RESUMO

An Isospora species, Isospora amphiboluri, originally described by Canon in 1967 and later by McAllister et al. (1995), was isolated from a central netted dragon (Ctenophorus nuchalis) housed at a wildlife rehabilitation centre in Perth, Western Australia. Sporulated oocysts of Isospora amphiboluri (n = 30) are spherical, 24.2 (26.5-23.0) µm in length and 23.9 (22.4-25.9) µm in width, with a shape index of 1.01. The bilayered oocyst wall is smooth and light-yellow in color. Polar granule, oocyst residuum and micropyle are absent. The sporocysts are lemon-shaped, 15.7 (15.2-18.0) × 10.2 (8.9-11.2) µm, with a shape index (length/width) of 1.53. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda half-moon-shaped. Each sporocyst contains four vermiform sporozoites arranged head to tail. The sporozoites are 11.7 (9.9-16.2) × 3.0 (2.4-3.5) µm, with a shape index (length/width) of 3.87. A sporocyst residuum is present. Sporozoites contain a central nucleus with a finely distributed granular residuum. Comparison of oocyst measurements and their features with other valid Isospora species from hosts in the Agamid family confirmed that this Isospora species is Isospora amphiboluri. Molecular characterization of I. amphiboluri at the 18S rRNA and MTCOI loci showed the highest similarity with I. amphiboluri from the central bearded dragon, 99.8% and 99.7% respectively. This is the first report of I. amphiboluri from a central netted dragon in Australia.


Assuntos
Interações Hospedeiro-Parasita , Isospora/isolamento & purificação , Isosporíase/veterinária , Lagartos , Animais , Animais de Zoológico , Complexo IV da Cadeia de Transporte de Elétrons/análise , Isospora/classificação , Isospora/citologia , Isospora/genética , Isosporíase/parasitologia , Masculino , Proteínas Mitocondriais/análise , Oocistos/classificação , Oocistos/citologia , Oocistos/isolamento & purificação , Filogenia , Proteínas de Protozoários/análise , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Esporozoítos/classificação , Esporozoítos/citologia , Esporozoítos/isolamento & purificação , Austrália Ocidental
12.
Mol Biochem Parasitol ; 243: 111373, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33961917

RESUMO

The Coccidia is the largest group of parasites within the Apicomplexa, a phylum of unicellular, obligate parasites characterized by the possession of an apical complex of organelles and structures in the asexual stages of their life cycles, as well as by a sexual reproductive phase that occurs enterically in host animals. Coccidian sexual reproduction involves morphologically distinct microgametes and macrogametes that combine to form a diploid zygote and, ultimately, following meiosis and mitosis, haploid, infectious sporozoites, inside sporocysts within an oocyst. Recent transcriptomic analyses have identified genes involved in coccidian sexual stage development and reproduction, including genes encoding for microgamete- and macrogamete-specific proteins with roles in gamete motility, fusion and fertilization, and in the formation of the resilient oocyst wall that allows coccidians to persist for long periods in the environment. Transcriptomics has also provided important clues about the regulation of gene expression in the transformation of parasites from one developmental stage to the next, a complex sequence of events that may involve transcription factors such as the apicomplexan Apetala2 (ApiAP2) family, alternative splicing, regulatory RNAs and MORC (a microrchida homologue and regulator of sexual stage development in Toxoplasma gondii). The molecular dissection of coccidian sexual development and reproduction by transcriptomic analyses may lead to the development of novel transmission-blocking strategies.


Assuntos
Coccídios/fisiologia , Regulação da Expressão Gênica , Proteínas de Protozoários/genética , Processamento Alternativo , Coccídios/isolamento & purificação , Coccídios/patogenicidade , Perfilação da Expressão Gênica , Estágios do Ciclo de Vida , Merozoítos/genética , MicroRNAs , Oocistos/genética , RNA Longo não Codificante , RNA de Protozoário , Análise de Célula Única/métodos
13.
Ticks Tick Borne Dis ; 12(4): 101713, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827035

RESUMO

Babesia bovis is a known causative agent of bovine babesiosis and is widely distributed across China. Rapid detection and accurate identification of B. bovis is essential for follow-up management and epidemiological investigations. In this study, a cross-priming amplification combined with vertical flow (CPA-VF) assay was developed. The detection limit of the CPA-VF assay targeting the 18S rRNA gene was 320 fg per reaction at 61 °C for 60 min. No cross-reactions were observed with other piroplasms infective to cattle. Furthermore, 36 blood samples from experimentally-infected animals were accurately assessed using the CPA-VF assay. The performance of the CPA-VF assay was compared with the results of conventional PCR for 219 blood samples from the field. Our results demonstrate that the CPA-VF assay is a practical and effective diagnostic tool for bovine babesiosis caused by B. bovis infection.


Assuntos
Babesia bovis/isolamento & purificação , Babesiose/diagnóstico , Doenças dos Bovinos/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/veterinária , Animais , Babesiose/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Apresentação Cruzada , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Sensibilidade e Especificidade
14.
Ticks Tick Borne Dis ; 12(4): 101710, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827036

RESUMO

Babesia species (Apicomplexa: Piroplasmorida) are tick-borne protozoan hemoparasites, which pose a significant threat to domestic animals, wildlife and humans. This study aimed to determine and characterize Babesia species in red foxes (Vulpes vulpes), Asian badgers (Meles leucurus) and their ticks. Blood, heart, liver, spleen, lung, kidney, large intestine and small intestine were collected from 19 wild carnivores (12 red foxes and 7 Asian badgers). All ticks were removed from these animals and identified according to morphological and molecular characteristics. The samples were tested for the presence of Babesia species using the 18S rRNA gene. Molecular analyses showed that the DNA of Babesia vogeli and Babesia vulpes was present in red fox organs/tissues and blood samples. A total of 54 hard ticks (38 Ixodes canisuga, 6 Haemaphysalis erinacei, 9 Ixodes kaiseri and 1 Dermacentor marginatus) were collected from red foxes and 12 (I. kaiseri) from Asian badgers. All ticks were adults. Among them, one I. kaiseri parasiting a red fox contained the DNA of B. vulpes while one I. canisuga was positive for Babesia sp. belonging to the clade "Babesia sensu stricto". Molecular and phylogenetic analyses indicated the presence of a novel genotype, Babesia sp. "badger China". Babesia sp. badger type A and type B from Asian badgers were different from those in European badgers. Co-infection with three Babesia genotypes was found in one Asian badger. This study provides the first data on Babesia infection in red foxes, Asian badgers and their ticks in China. Babesia vogeli was detected for the first time in red foxes in Asia. Co-infection and genetic diversity of Babesia genotypes in Asian badgers were also demonstrated.


Assuntos
Babesia/isolamento & purificação , Babesiose/epidemiologia , Raposas , Ixodidae/parasitologia , Mustelidae , Animais , Babesia/classificação , Babesiose/parasitologia , China/epidemiologia , Filogenia , Prevalência , RNA de Protozoário/análise , RNA Ribossômico 18S/análise
15.
Sci Rep ; 11(1): 8274, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859278

RESUMO

Ciliated protozoans form dormant cysts for survival under adverse conditions. The molecular mechanisms regulating this process are critical for understanding how single-celled eukaryotes adapt to the environment. Despite the accumulated data on morphology and gene coding sequences, the molecular mechanism by which lncRNAs regulate ciliate encystment remains unknown. Here, we first detected and analyzed the lncRNA expression profile and coexpressed mRNAs in dormant cysts versus vegetative cells in the hypotrich ciliate Pseudourostyla cristata by high-throughput sequencing and qRT-PCR. A total of 853 differentially expressed lncRNAs were identified. Compared to vegetative cells, 439 and 414 lncRNAs were upregulated and downregulated, respectively, while 47 lncRNAs were specifically expressed in dormant cysts. A lncRNA-mRNA coexpression network was constructed, and the possible roles of lncRNAs were screened. Three of the identified lncRNAs, DN12058, DN20924 and DN30855, were found to play roles in fostering encystment via their coexpressed mRNAs. These lncRNAs can regulate a variety of physiological activities that are essential for encystment, including autophagy, protein degradation, the intracellular calcium concentration, microtubule-associated dynein and microtubule interactions, and cell proliferation inhibition. These findings provide the first insight into the potentially functional lncRNAs and their coexpressed mRNAs involved in the dormancy of ciliated protozoa and contribute new evidence for understanding the molecular mechanisms regulating encystment.


Assuntos
Cilióforos/genética , Cilióforos/fisiologia , Perfilação da Expressão Gênica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Transcriptoma/genética , Transcriptoma/fisiologia , Morte Celular Autofágica/genética , Cálcio/metabolismo , Proliferação de Células/genética , Dineínas , Proteínas Associadas aos Microtúbulos , Proteólise , Proteínas de Protozoários/metabolismo
16.
Parasitol Res ; 120(6): 2165-2174, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33893549

RESUMO

Neospora caninum is an important pathogen commonly causing spontaneous abortion in livestock. The parasite is known to remain in cysts in an inactive state; or it can undergo expansive development within an intermediate host. However, the mechanisms that trigger the proliferation of N. caninum have not been thoroughly elucidated. For various organisms, it has been demonstrated that microRNAs (miRNAs) can act as important endogenous regulatory factors in gene regulation during cell differentiation and development. However, miRNAs and their function have not been studied in N. caninum. In this study, small RNA libraries from N. caninum tachyzoites (NC-1 strain) were analyzed using a high-throughput RNA sequencing technology combined with systematic bioinformatics analysis. A considerable number of novel miRNAs from N. caninum NC-1 strain tachyzoites were identified. Of the 300 miRNAs found by bioinformatics analysis, 10 were conserved miRNAs belonging to 10 metazoan miRNA families, while 290 were novel miRNAs. The expression of 13 novel miRNAs was verified by real-time quantitative PCR (qRT-PCR). Data from this study provided and identified authentic miRNAs for the first time in N. caninum. The study also introduces a framework for further investigations of RNAi-dependent regulatory mechanisms of the parasite and provides data for further understanding of N. caninum development.


Assuntos
MicroRNAs/metabolismo , Neospora/genética , RNA de Protozoário/metabolismo , Animais , Chlorocebus aethiops , Coccidiose , Regulação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Neospora/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Células Vero
17.
Parasitol Res ; 120(6): 2219-2231, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33904983

RESUMO

Blastocystis sp. is an intestinal protist parasite commonly found in the feces of humans and animals worldwide. Blastocystis exhibits extensive genetic diversity and has been identified in humans and a variety of animals including other mammals and birds. Blastocystis subtypes do not exhibit strict host specificity which raises the possibility of zoonotic transmission through either direct contact or fecal contamination of food or water. However, reports detailing the subtypes and prevalence of Blastocystis in avian species are limited. Therefore, this study investigated the presence of Blastocystis in chickens by molecular characterization of the small subunit rRNA (SSU rRNA) gene. Fecal samples from 130 chickens were collected from local markets in Uberlândia and Belo Horizonte in the state of Minas Gerais, Brazil. To detect and identify subtypes of Blastocystis, a next-generation amplicon sequencing protocol was used. Forty-four of the 130 (33.8%) chickens examined were positive for Blastocystis. Blastocystis subtypes ST6 (23/130; 17.7%), ST7 (43/130; 33.1%), ST10 (1/130; 0.8%), ST14 (5/130; 3.8%), ST25 (1/130; 0.8%), and a novel subtype (ST29) (2/130; 1.5%) were observed. A nanopore sequencing strategy was used to obtain the near full-length SSU rRNA gene nucleotide sequence and validate novel subtype ST29. Mixed infections containing multiple subtypes were common and identified in 63.6% of Blastocystis-positive chickens. All positive samples contained one or both potentially zoonotic subtypes ST6 and ST7. The prevalence of Blastocystis in chickens was high, and molecular characterization mostly identified subtypes previously found in humans. Thus, chickens may be a source of human infection and environmental contamination.


Assuntos
Infecções por Blastocystis/veterinária , Blastocystis/genética , Galinhas/parasitologia , Doenças das Aves Domésticas/parasitologia , Animais , Infecções por Blastocystis/parasitologia , Brasil , Fezes/parasitologia , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Especificidade de Hospedeiro , Humanos , Enteropatias Parasitárias/parasitologia , Enteropatias Parasitárias/veterinária , Filogenia , Prevalência , RNA de Protozoário , Zoonoses/parasitologia
18.
Malar J ; 20(1): 181, 2021 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-33838672

RESUMO

BACKGROUND: Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. METHODS: A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. RESULTS: The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. CONCLUSIONS: The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.


Assuntos
Testes Diagnósticos de Rotina/métodos , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Humanos , Hidrólise , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Sensibilidade e Especificidade
19.
Parasitology ; 148(7): 779-786, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33843504

RESUMO

Metchnikovellids are a deep-branching group of microsporidia, parasites of gregarines inhabiting the alimentary tract of polychaetes and some other invertebrates. The diversity and phylogeny of these hyperparasites remain poorly studied. Modern descriptions and molecular data are still lacking for many species. The results of a light microscopy study and molecular data for Metchnikovella spiralis Sokolova et al., 2014, a hyperparasite of the eugregarine Polyrhabdina sp., isolated from the polychaete Pygospio elegans, were obtained. The original description of M. spiralis was based primarily on the analysis of stained preparations and transmission electron microscopy images. Here, the species description was complemented with the results of in vivo observations and phylogenetic analysis based on the SSU rRNA gene. It was shown that in this species, free sporogony precedes sac-bound sporogony, as it occurs in the life cycle of most other metchnikovellids. Spore sacs are entwined with spirally wound cords, and possess only one polar plug. Phylogenetic analyses did not group M. spiralis with M. incurvata, another metchnikovellid from the same gregarine species, but placed it as a sister branch to Amphiacantha. The paraphyletic nature of the genus Metchnikovella was discussed. The taxonomic summary for M. spiralis was emended.


Assuntos
Apicomplexa/parasitologia , Interações Hospedeiro-Parasita , Microsporídios/classificação , Microsporídios/citologia , Poliquetos/parasitologia , Animais , Microsporídios/genética , Microsporídios/fisiologia , Filogenia , RNA de Protozoário/análise , RNA Ribossômico/análise
20.
J Zoo Wildl Med ; 52(1): 200-205, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33827177

RESUMO

Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and ß-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The ß-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the ß-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.


Assuntos
Babesia/genética , Babesiose/epidemiologia , Cervos/parasitologia , Theileria/genética , Theileriose/epidemiologia , Tubulina (Proteína)/isolamento & purificação , Animais , Animais Selvagens , Babesia/classificação , Babesia/isolamento & purificação , Babesiose/parasitologia , Japão/epidemiologia , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Theileria/classificação , Theileria/isolamento & purificação , Theileriose/parasitologia , Tubulina (Proteína)/classificação , Tubulina (Proteína)/genética
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