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1.
Medicine (Baltimore) ; 100(33): e26767, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34414933

RESUMO

OBJECTIVE: The aim of this study was to investigate the effect of Ma Yinglong Shexiang Hemorrhoids Cream combined with pearl powder on pain and complications in patients with severe pressure ulcers. METHODS: One hundred seventeen patients with severe pressure ulcers hospitalized and treated in our hospital (January 2019--December 2019) were divided into Ma Yinglong Musk Hemorrhoid Cream Group (MY Group), Pearl Powder Group (PP Group), and combination with Ma Yinglong Musk Hemorrhoid Cream and Pearl Powder Group (MP group), 39 patients in each group. There was no significant difference in the general data of patients in MY group, PP group, and MP group. By analyzing the differences in clinical efficacy, secondary effects, scar incidence, pain, and clinical indicators of patients in the MY group, PP group, and MP group, the effects of Mayinglong Shexiang Hemorrhoid Cream combined with pearl powder in the treatment of pain and complications in patients with severe pressure ulcers were explored. RESULTS: After treatment, compared with the MY group and the PP group, the MP group had a higher clinical efficacy than the MY group and the PP group. Compared with MY group and PP group, the healing time, dressing change times, and dressing change time of MP group were better than MY group (P < .05). After treatment, the VAS score and incidence of secondary effects of the MP group was significantly lower than that of the MY group and PP group (P < .05). The incidence and area of scar formation in the MP group were lower than those in the MY group and the PP group (P < .05). CONCLUSION: Compared with Ma Yinglong Musk Hemorrhoid Cream or Pearl Powder, combination of Ma Yinglong Musk Hemorrhoid Cream and Pearl Powder are more effective in treating severe pressure ulcer patients, and can significantly reduce the pain in the affected area and reduce the occurrence of complications.


Assuntos
Medicina Tradicional Chinesa , Manejo da Dor/métodos , Dor/tratamento farmacológico , Dor/etiologia , Lesão por Pressão/complicações , Adulto , Feminino , Hemorroidas/tratamento farmacológico , Humanos , Masculino , Pomadas , Medição da Dor , Pós , Índice de Gravidade de Doença
2.
Genome ; 64(9): 869-877, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33617379

RESUMO

Molecular identification of acaroid mites is difficult because of the scarcity of molecular data in GenBank. Here, acaroid mites collected from ground flour dust in Xi'an, China, were preliminarily morphologically classified/grouped. Universal primers were then designed to amplify and screen suitable DNA barcodes for identifying these mites. Sixty mite samples were morphologically classified into six groups. Groups 1-2 were identified to Dermatophagoides farinae and Tyrophagus putrescentiae, while Groups 3-6 were not identified to the species level. ITS2 exhibited higher efficiency in molecular identification in comparison with COI, 12S, and 16S. Groups 1-6 were identified as D. farinae, T. putrescentiae, Suidasia nesbitti, Chortoglyphus arcuatus, Lepidoglyphus destructor, and Gohieria sp., respectively. The phylogenetic results were consistent with the morphological classification. Group 6 was further identified as G. fusca according to the morphology of the reproductive foramen. We conclude that the use of ITS2 and the availability of universal primers provide an ideal DNA barcode for molecular identification of acaroid mites. The use of multiple target genetic markers in conjunction with morphological approaches will improve the accuracy of Acaridida identification.

3.
Acta Parasitol ; 66(2): 605-614, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33392956

RESUMO

PURPOSE: Pulex irritans are vectors of various zoonotic pathogens. However, molecular studies on P. irritans and flea-borne diseases are limited due to the lack of molecular data. This study aimed to conduct transcriptome sequencing, functional annotation, and pathogen analysis of P. irritans. METHODS: Fleas collected from a dog were identified morphologically and molecularly. RNA was extracted for transcriptome sequencing and functional annotation. Open reading frames (ORFs) of unigenes were confirmed by employing bioinformatics strategies, and maximum likelihood (ML) trees were reconstructed based on the highly expressed genes of ejaculation globulin-specific 3-like protein, salivary protein, and actin for phylogenetic relationship analysis. RESULTS: The obtained mitochondrial 16S rRNA gene sequences showed 99.71% of similarity with P. irritans obtained from GenBank database. Transcriptome sequencing generated 74,412 unigenes, of which 53,211 were functionally annotated. A total of 195 unigenes were assigned to fleas, of which 69 contained complete ORFs. Phylogenetic trees of both ejaculatory globulin and salivary protein genes demonstrated that P. irritans first clustered with Pulicidae sp., indicating the reliability of transcriptome data. It is noteworthy that 1070 unigenes were assigned to Hymenolepis microstoma and Dipylidium caninum, of which 62 contained complete ORFs. The phylogenetic tree of the actin gene showed that the unigenes had closer relationships with Echinococcus sp., suggesting the role of P. irritans as intermediate hosts of tapeworms. CONCLUSION: The results of this study provide the possibility for functional exploration of important genes and lay foundations for the prevention and control of P. irritans and flea-borne diseases.


Assuntos
Sifonápteros , Transcriptoma , Animais , Cães , Perfilação da Expressão Gênica , Masculino , Filogenia , RNA Ribossômico 16S , Reprodutibilidade dos Testes , Sifonápteros/genética
4.
Exp Parasitol ; 218: 108003, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980317

RESUMO

Dermatophagoides farinae, an important pathogen, has multiple allergens. However, their expression under physiological conditions are not understood. Our previous RNA-seq showed that allergens of D. farinae were up-regulated under temperature stress, implying that they may be involved in stress response. Here, we performed a comprehensive study. qRT-PCR detection indicated that 26 of the 34 allergens showed differential expression. Der f1 had the most abundant basic expression quantity. Der f 28.0201 (HSP70) and Der f3 had the same regulation pattern in 9 highly expressed transcripts, which only up-regulated at 41 °C and 43 °C, but Der f 28.0201 showed stronger regulation than Der f 3 (19.88-fold vs 6.02-fold). Whereas Der f 1, 2, 7, 21, 22, 27, and 30 were up-regulated under both heat and cold stress, and Der f 27 showed the strongest regulation ability among them. Der f 27 showed more significant up-regulation than Der f 28.0201 under heat stress (23.59-fold vs 19.88-fold), and Der f27 had more obvious up-regulation under cold than heat stress (30.70-fold vs 23.59-fold). The expression of Der f 27, 28.0201 and 1, and D. farinae survival rates significantly decreased following RNAi, indicating the upregulation of these allergens under temperature stress conferred thermo-tolerance or cold-tolerance to D. farinae. In this study, we described for the first time that these allergens have temperature-stress response functions. This new scientific discovery has important clinical value for revealing the more frequent and serious allergic diseases caused by D. farinae during the change of seasons.


Assuntos
Antígenos de Dermatophagoides/fisiologia , Resposta ao Choque Frio/fisiologia , Dermatophagoides farinae/fisiologia , Resposta ao Choque Térmico/fisiologia , Estresse Fisiológico/fisiologia , Animais , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/metabolismo , Antígenos de Dermatophagoides/farmacologia , Sequência de Bases , Dermatophagoides farinae/genética , Feminino , Inativação Gênica , Anotação de Sequência Molecular , RNA/química , RNA/isolamento & purificação , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/química , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Estresse Fisiológico/genética , Regulação para Cima
5.
Parasit Vectors ; 13(1): 251, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404192

RESUMO

BACKGROUND: The morphological and molecular identification of mites is challenging due to the large number of species, the microscopic size of the organisms, diverse phenotypes of the same species, similar morphology of different species and a shortage of molecular data. METHODS: Nine medically important mite species belonging to six families, i.e. Demodex folliculorum, D. brevis, D. canis, D. caprae, Sarcoptes scabiei canis, Psoroptes cuniculi, Dermatophagoides farinae, Cheyletus malaccensis and Ornithonyssus bacoti, were collected and subjected to DNA barcoding. Sequences of cox1, 16S and 12S mtDNA, as well as ITS, 18S and 28S rDNA from mites were retrieved from GenBank and used as candidate genes. Sequence alignment and analysis identified 28S rDNA as the suitable target gene. Subsequently, universal primers of divergent domains were designed for molecular identification of 125 mite samples. Finally, the universality of the divergent domains with high identification efficiency was evaluated in Acari to screen DNA barcodes for mites. RESULTS: Domains D5 (67.65%), D6 (62.71%) and D8 (77.59%) of the 28S rRNA gene had a significantly higher sequencing success rate, compared to domains D2 (19.20%), D3 (20.00%) and D7 (15.12%). The successful divergent domains all matched the closely-related species in GenBank with an identity of 74-100% and a coverage rate of 92-100%. Phylogenetic analysis also supported this result. Moreover, the three divergent domains had their own advantages. D5 had the lowest intraspecies divergence (0-1.26%), D6 had the maximum barcoding gap (10.54%) and the shortest sequence length (192-241 bp), and D8 had the longest indels (241 bp). Further universality analysis showed that the primers of the three divergent domains were suitable for identification across 225 species of 40 families in Acari. CONCLUSIONS: This study confirmed that domains D5, D6 and D8 of 28S rDNA are universal DNA barcodes for molecular classification and identification of mites. 28S rDNA, as a powerful supplement for cox1 mtDNA 5'-end 648-bp fragment, recommended by the International Barcode of Life (IBOL), will provide great potential in molecular identification of mites in future studies because of its universality.


Assuntos
Código de Barras de DNA Taxonômico , Ácaros/classificação , Ácaros/genética , Filogenia , RNA Ribossômico 28S/genética , Animais , Análise de Sequência de DNA
6.
MycoKeys ; 66: 135-157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32377155

RESUMO

New collections of six Squamarina species from type localities in China were studied. The comparison of morphological characteristics and secondary metabolites with those of the type specimens and phylogenetic analyses suggest that S. callichroa and S. pachyphylla belong to Rhizoplaca, S. semisterilis belongs to Lobothallia and S. chondroderma should be retained in Lecanora temporarily. Only two species, S. kansuensis and S. oleosa, remain in Squamarina. The new combinations Lobothallia semisterilis (H. Magn.) Y. Y. Zhang, Rhizoplaca callichroa (Zahlbr.) Y. Y. Zhang and R. pachyphylla (H. Magn.) Y. Y. Zhang are proposed. Detailed descriptions to aid the identification of these species, distributions and phylogenetic trees, based on multiple collections, are presented. The generic concept of Squamarina is recircumscribed in this study.

7.
J Med Entomol ; 57(4): 1049-1056, 2020 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-32215556

RESUMO

The microbiota of mites is closely related to their growth, development, and pathogenicity. Therefore, it is necessary to study the bacteria in mites. Here, for the first time, based on 16s rRNA V3-V4 region, the microbiota of 45 samples of nine species in six families of medically important mites were analyzed using Illumina MiSeq sequencing technique. The results showed that, at the phylum level, Proteobacteria (56.20-86.40%) were the dominant, followed by Firmicutes (6.41-19.43%), Bacteroidetes (5.56-13.38%) and Actinobacteria (1.93-28.07%). But at the genera the microbiota of mites are different, showing four characteristics: 1) The microbiota is related to the parasitic host. Demodex folliculorum (Acariforms: Demodicidae) and D. brevis (Acariforms: Demodicidae), both parasitizing humans, showed similar microbial composition, as did D. canis (Acariforms: Demodicidae) and Sarcoptes scabiei canis (Acariforms: Sarcoptidae) parasitizing dogs, but D. caprae (Acariforms: Demodicidae) parasitizing sheep showed unique microbial community; 2) The microbiota is related to mite's species. Dermatophagoides farinae and Cheyletus malaccensis (Acariforms: Cheyletidae), both collecting from flour, show respective microbial composition; 3) The microbiota is related to the life stage. There were differences in microbiota between adults and larvae of D. farinae, but no differences observed in Psoroptes cuniculi (Acariforms: Psoroptidae); and 4) The microbiota is related to the blood-feeding state. The microbiota of blood-fed Ornithonyssus bacoti (Parasitiformes: Macronyssidae) adults was significantly higher than that of unfed adults. This indicates that the microbiota of mites is affected by mite species, parasitic host, growth stage and habitat. Therefore, understanding these influencing factors will have a very important guiding significance for the prevention and control of mite-borne diseases.


Assuntos
Microbiota , Ácaros/microbiologia , Animais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Masculino
8.
Int J Biol Macromol ; 144: 351-361, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31812740

RESUMO

Dermatophagoides farinae is a major exogenous allergen. Its ability to tolerate adverse external temperatures makes it responsible for widespread occurrence of allergies. Heat shock protein (HSP), a recognized temperature stress response gene, but its role in D. farinae remained unclear. Here, we performed a comprehensive study. First, we found that 25 °C was the optimal temperature, and all mites died at 48 or -20 °C for 1 h (LT100). Thus, 41 °C (LT15), 43 °C (LT25), 45 °C (LT45), and -10 °C (LT25) were selected as stress temperatures to perform de novo RNA-seq. Then, 17 main genes of the 47 differentially expressed HSP, were detected by qRT-PCR. Temperature and time gradient versus expression magnitude histogram revealed that HSP70, HSP83-1, HSP83-2, and HSP16-1 showed heat stress response only at 41-43 °C, while HSC71 and HSF played a regulatory role under both heat and cold stress, particularly HSF, with strong intensity, long duration, and quick upregulation at recovery for 10-20 min. Finally, gene expression and D. farinae survival rates significantly decreased following RNAi. These findings indicated that HSPs conferred thermo-tolerance or cold-tolerance to D. farinae. In conclusion, this was the first meaningful exploration that confirmed HSP and HSF playing an important role in temperature resistance of D. farinae.


Assuntos
Dermatophagoides farinae/fisiologia , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Alérgenos , Animais , Temperatura Baixa , Dermatophagoides farinae/genética , Dermatophagoides farinae/imunologia , Feminino , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Temperatura Alta , Interferência de RNA , Estresse Fisiológico , Temperatura
9.
Parasitol Res ; 118(12): 3223-3235, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31691855

RESUMO

Demodex are among the tiniest organisms in Acari and are important mammalian parasites. However, differences in pathogenicity between two human parasites, Demodex folliculorum and Demodex brevis, remain unknown. Related genetic studies are limited by RNA extraction difficulties and molecular data deficiencies. In this study, RNA extraction, de novo sequencing, functional annotation, and differential gene expression analyses were performed to compare D. folliculorum and D. brevis. This yielded 67.09 and 65.10 million clean reads, respectively, with similar annotations. Bioinformatics analyses and manual alignments identified 237 coding sequences comprising 48 genes from 29 families, including five important functional classes. Of these, 30 genes from 20 families related to metabolism, motion, detoxification and stress response, and allergic reaction were differentially expressed between the two species. Cathepsin type 1, serine protease inhibitor, arginine kinase, triosephosphate isomerase, muscle-specific protein 20-2, myosin alkaline light chain, troponin C, tropomyosin, and heat shock protein 90 were highly expressed in D. folliculorum, whereas cathepsin type 2, aspartic protease, serine protease, myosin heavy chain type 2, and alpha tubulin type 1C were highly expressed in D. brevis. Verified coding sequences were nearly consistent with unigene clusters. Further, absolute quantification results demonstrated that differentially expressed genes followed the predicted expression trend. Therefore, the first RNA sequencing and functional annotation analysis of two Demodex species was successful. Differential expression of important functional genes is likely implicated in pathogenicity disparities between these two species. Our study provides molecular data and technical support for further studies on human Demodex pathogenicity and functional genes.


Assuntos
Ácaros/genética , Animais , Proteínas de Artrópodes/genética , Perfilação da Expressão Gênica , Humanos , Ácaros/classificação , Ácaros/patogenicidade , Especificidade da Espécie , Transcriptoma , Virulência/genética
10.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
11.
Acta Parasitol ; 64(4): 807-820, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31418165

RESUMO

PURPOSE: Haemaphysalis longicornis (Neumann) is a hematophagous tick widely distributed in northern China. It not only causes enormous economic loss to animal husbandry, but also as a vector and reservoir of various zoonotic pathogens, it spreads natural focal diseases, such as severe fever with thrombocytopenia syndrome, seriously threatening human health. Lack of transcriptomic and genomic data from H. longicornis limits the study of this important medical vector. METHODS: The engorged female H. longicornis from Gansu, China, was used for RNA extraction, de novo RNA-seq, functional annotation, and ORF prediction. RESULTS: As a result, 53.09 million clean reads (98.88%) with a GC content of 54.29% were obtained. A total of 65,916 Unigenes were assembled, of which 34.59% (23,330) were successfully annotated. Of these Unigenes, 22,587 (34.27%) were annotated to species by NCBI non-redundant protein (nr). Ixodes scapularis, Limulus polyphemus, Parasteatoda tepidariorum, Stegodyphus mimosarum, and Metaseiulus occidentalis were the top BLAST hit species, accounting for 47.23%, 9.58%, 4.11%, 3.50%, and 2.69%, respectively. A total of 29,182 ORFs were predicted, and 35 complete ORFs for functional genes were identified, including ORFs involved in digestion (14), stress responses (8), anticoagulation (3), reproduction (3), antimicrobial (2), drug resistance (2), movement (2), autophagy (1), and immunity (1), respectively. The Unigene ORFs encoding cathepsin and heat shock proteins were further analyzed phylogenetically. CONCLUSION: De novo RNA-seq and functional annotation of H. longicornis were successfully completed for the first time, providing a molecular data resource for further research on blood-sucking, pathogen transmission mechanisms, and effective prevention and control strategies.


Assuntos
Ixodidae/genética , Anotação de Sequência Molecular , RNA-Seq , Animais , Cães/parasitologia , Feminino , Fases de Leitura Aberta , RNA/genética
12.
Genome ; 62(5): 295-304, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30998112

RESUMO

Whether ribosomal genes can be used as DNA barcodes for molecular identification of Demodex (Acariformes: Demodicidae) is unclear. To examine this, Demodex folliculorum, D. brevis, D. canis, and D. caprae were collected for DNA extraction, rDNA fragments amplification, sequencing, and analysis. The V2 and V4 regions of SSU rDNA; D5, D6, and D8 regions of LSU rDNA; and ITS region were obtained from the four morphospecies. BLAST analysis showed that the obtained sequences matched those of Demodex or Aplonobia (Acariformes: Tetranychidae) in Raphignathae. Phylogenetic trees derived from V2, V4, D5, D6, and D8 regions, but not from ITS region, showed that the four species of Demodex clustered independently. Sequence divergence analysis further demonstrated that D5, D6, and D8 regions had obvious barcoding gap between intraspecific and interspecific divergences, with the gap of D5 (16.91%) larger than that of D6 (11.82%) and D8 (4.66%). The V2 and V4 regions did not have a barcoding gap, as the intraspecific and interspecific divergences partially overlapped. For the ITS region, intraspecific and interspecific divergences completely overlapped. These results suggest that the D5, D6, and D8 regions of LSU rDNA, especially D5, are suitable DNA barcodes for Demodex.


Assuntos
Código de Barras de DNA Taxonômico , Ácaros/classificação , Animais , DNA Intergênico/genética , DNA Ribossômico/genética , Ácaros/genética , Filogenia
13.
Gene ; 705: 82-89, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30981836

RESUMO

Dermatophagoides farinae are an important mite species that cause stored product deterioration and allergic diseases. They widely breed in human habitats because of their strong tolerance to extreme external temperatures. However, mechanisms underlying the stress response and tolerance of D. farinae are unclear. We hypothesized that heat shock protein 70 plays an important role in the heat stress response of D. farinae. In this study, we determined the survival rates of D. farinae at high temperatures (37 °C-45 °C) by performing temperature-gradient experiments in vitro and assessed the expression level of HSP70 by performing RT-qPCR. First, we confirmed that HSP70 regulated the heat stress response of D. farinae, with maximum heat stress regulation observed at 41 °C. Next, we confirmed the presence of a Dicer enzyme-mediated RNA interference (RNAi) pathway in D. farinae by searching the NCBI database and a Dicer site prediction website. Finally, we performed RNAi in D. farinae by using an immersion method with screened dsHSP70 fragments. Moreover, we performed concentration-gradient experiments to determine that 600 ng/µl was the minimal effective concentration of dsHSP70 for silencing HSP70. These results confirm that HSP70 regulates the heat stress response of D. farinae. The present study is the first to report the use of the non-invasive and highly sensitive immersion method for performing RNAi in D. farinae. The results of the present study provide a technical foundation for performing functional gene research and for developing molecular prevention and control strategies against medically important mites.


Assuntos
Dermatophagoides farinae/crescimento & desenvolvimento , Proteínas de Choque Térmico HSP70/genética , Interferência de RNA , Animais , Clonagem Molecular , Bases de Dados Genéticas , Dermatophagoides farinae/genética , Dermatophagoides farinae/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico , Ribonuclease III/metabolismo
14.
Acta Parasitol ; 64(2): 251-256, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30864098

RESUMO

BACKGROUND: Otodectes cynotis (Hering, 1838) is the pathogen of otodectic mange distributed worldwide. The mite mainly infests carnivores and, sometimes, humans. However, due to the lack of cDNA library, research on its pathogenesis has been challenging. METHODS: To solve this problem, the present study first sampled O. cynotis mites from an infested cat from Xi'an, China, for RNA extraction. Then, the full-length cDNA library was constructed using the SMART technique. Finally, positive clones > 500 bp and Hsc70-5 gene fragment specifically amplified from the cDNA library were sequenced and analyzed to verify the library's reliability. RESULTS: Results showed that RNA extracted from 300 mites had good quality with a concentration of 149 ng/µl and OD260/OD280 of 1.99. The library satisfied the quality standard of a good library with a titer of 5.02 × 105 PFU/ml and a combination rate of 97.61%. In addition, clone 4 and Hsc70-5 showed 98.38% and 99.72% identity with Ef1-α and Hsc70-5 gene sequences of O. cynotis in GenBank, respectively. CONCLUSION: The cDNA library of O. cynotis constructed here was successful and reliable, creating the basis for research on RNA sequencing and functional genes of O. cynotis.


Assuntos
DNA Complementar/genética , Biblioteca Gênica , Ácaros/genética , Animais , Sequência de Bases , Gatos/parasitologia , Infestações por Ácaros/parasitologia , Infestações por Ácaros/veterinária , Reprodutibilidade dos Testes
15.
Exp Parasitol ; 200: 67-72, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30904695

RESUMO

Demodex is a type of parasitic mite which could cause serious dermatoses in 11 orders of mammals. However, due to the tiny body with thick chitin hard to be ruptured as well as the difficulty in obtaining a large number of mites, the quantity and quality of extracted RNA could hardly satisfied for transcriptome sequencing. This has hampered the research on functional genes and molecular pathogenesis of Demodex for a long time. To solve the problems above, the present study established a new RNA extraction method in combination Azanno method with liquid nitrogen grinding using 16 human and canine Demodex mite samples. The RNA quality detection results of Agilent 2100 Bioanalyzer showed that 8 of 16 RNA samples met the requirements for trace RNA-Seq, with RIN of 5.0-6.5 and RNA quantity of 1.1-16.0 ng. RNA quality was affected by grinding process and parasitic position of Demodex. Enough grinding number (≥2000) in moderate time (≤20 min) was significant for mites' complete rupture and RNA degradation prevention. D. brevis (100%, 3/3) parasitizing in human sebaceous glands had significantly higher RNA qualification rate than D. folliculorum (57.14%, 4/7) parasitizing in human hair follicles. Yet D. canis parasitizing in dog had lower RNA qualification rate (16.67%, 1/6) as mites were embedded in skin tissues and blood clots. It should be pointed out that microplate reader had defects with a lower RNA qualification rate of 6.25% (1/16) unmatched with 2100 Bioanalyzer, reminding that it could be only used as reference in RNA quality evaluation.


Assuntos
Ácaros/genética , RNA/isolamento & purificação , Transcriptoma , Animais , Cães , Folículo Piloso/parasitologia , Humanos , Ácaros/classificação , RNA/química , RNA/normas , RNA Ribossômico 18S/isolamento & purificação , Glândulas Sebáceas/parasitologia , Análise de Sequência de RNA , Trombose/parasitologia
16.
Exp Appl Acarol ; 75(2): 191-208, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29855753

RESUMO

Ornithonyssus bacoti (Hirst) (Acari: Macronyssidae) is a vector and reservoir of pathogens causing serious infectious diseases, such as epidemic hemorrhagic fever, endemic typhus, tularemia, and leptospirosis. Its genome and transcriptome data are lacking in public databases. In this study, total RNA was extracted from live O. bacoti to conduct RNA-seq, functional annotation, coding domain sequence (CDS) prediction and simple sequence repeats (SSRs) detection. The results showed that 65.8 million clean reads were generated and assembled into 72,185 unigenes, of which 49.4% were annotated by seven functional databases. 23,121 unigenes were annotated and assigned to 457 species by non-redundant protein sequence database. The BLAST top-two hit species were Metaseiulus occidentalis and Ixodes scapularis. The procedure detected 12,426 SSRs, of which tri- and di-nucleotides were the most abundant types and the representative motifs were AAT/ATT and AC/GT. 26,936 CDS were predicted with a mean length of 711 bp. 87 unigenes of 30 functional genes, which are usually involved in stress responses, drug resistance, movement, metabolism and allergy, were further identified by bioinformatics methods. The unigenes putatively encoding cytochrome P450 proteins were further analyzed phylogenetically. In conclusion, this study completed the RNA-seq and functional annotation of O. bacoti successfully, which provides reliable molecular data for its future studies of gene function and molecular markers.


Assuntos
Repetições de Microssatélites , Ácaros/genética , Transcriptoma , Animais , Biologia Computacional , Ácaros/metabolismo , Anotação de Sequência Molecular , Análise de Sequência de RNA
17.
Parasitol Res ; 116(12): 3285-3290, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29032499

RESUMO

There has been no widely accepted DNA barcode for species identification of Demodex. In this study, we attempted to solve this issue. First, mitochondrial cox1-5' and 12S gene fragments of Demodex folloculorum, D. brevis, D. canis, and D. caprae were amplified, cloned, and sequenced for the first time; intra/interspecific divergences were computed and phylogenetic trees were reconstructed. Then, divergence frequency distribution plots of those two gene fragments were drawn together with mtDNA cox1-middle region and 16S obtained in previous studies. Finally, their identification efficiency was evaluated by comparing barcoding gap. Results indicated that 12S had the higher identification efficiency. Specifically, for cox1-5' region of the four Demodex species, intraspecific divergences were less than 2.0%, and interspecific divergences were 21.1-31.0%; for 12S, intraspecific divergences were less than 1.4%, and interspecific divergences were 20.8-26.9%. The phylogenetic trees demonstrated that the four Demodex species clustered separately, and divergence frequency distribution plot showed that the largest intraspecific divergence of 12S (1.4%) was less than cox1-5' region (2.0%), cox1-middle region (3.1%), and 16S (2.8%). The barcoding gap of 12S was 19.4%, larger than cox1-5' region (19.1%), cox1-middle region (11.3%), and 16S (13.0%); the interspecific divergence span of 12S was 6.2%, smaller than cox1-5' region (10.0%), cox1-middle region (14.1%), and 16S (11.4%). Moreover, 12S has a moderate length (517 bp) for sequencing at once. Therefore, we proposed mtDNA 12S was more suitable than cox1 and 16S to be a DNA barcode for classification and identification of Demodex at lower category level.


Assuntos
Código de Barras de DNA Taxonômico , Genes Mitocondriais , Ácaros/classificação , Animais , Sequência de Bases , DNA Mitocondrial/genética , Doenças do Cão , Cães , Infestações por Ácaros/parasitologia , Infestações por Ácaros/veterinária , Mitocôndrias/genética , Filogenia , Análise de Sequência de DNA
18.
Acta Parasitol ; 62(2): 354-376, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28426424

RESUMO

The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.


Assuntos
DNA Complementar/genética , Biblioteca Gênica , Ácaros/genética , Animais , Sequência de Bases , DNA Mitocondrial/genética , Humanos , RNA/genética
19.
Parasitol Res ; 116(3): 945-951, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28063008

RESUMO

The role of bacteria is unclear in the facial skin lesions caused by Demodex. To shed some light on this issue, we conducted a case-control study comparing cases with facial dermatoses with controls with healthy skin using denaturing gradient gel electrophoresis (DGGE) technique. The bacterial diversity, composition, and principal component were analyzed for Demodex bacteria and the matched facial skin bacteria. The result of mite examination showed that all 33 cases were infected with Demodex folliculorum (D. f), whereas 16 out of the 30 controls were infected with D. f, and the remaining 14 controls were infected with Demodex brevis (D. b). The diversity analysis showed that only evenness index presented statistical difference between mite bacteria and matched skin bacteria in the cases. The composition analysis showed that the DGGE bands of cases and controls were assigned to 12 taxa of 4 phyla, including Proteobacteria (39.37-52.78%), Firmicutes (2.7-26.77%), Actinobacteria (0-5.71%), and Bacteroidetes (0-2.08%). In cases, the proportion of Staphylococcus in Firmicutes was significantly higher than that in D. f controls and D. b controls, while the proportion of Sphingomonas in Proteobacteria was significantly lower than that in D. f controls. The between-group analysis (BGA) showed that all the banding patterns clustered into three groups, namely, D. f cases, D. f controls, and D. b controls. Our study suggests that the bacteria in Demodex should come from the matched facial skin bacteria. Proteobacteria and Firmicutes are the two main taxa. The increase of Staphylococcus and decrease of Sphingomonas might be associated with the development of facial dermatoses.


Assuntos
Bactérias/isolamento & purificação , Dermatoses Faciais/microbiologia , Dermatoses Faciais/parasitologia , Infestações por Ácaros/parasitologia , Ácaros/microbiologia , Adulto , Animais , Bactérias/classificação , Bactérias/genética , Biodiversidade , Estudos de Casos e Controles , Eletroforese em Gel de Gradiente Desnaturante , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ácaros/fisiologia , Pele/microbiologia , Pele/parasitologia , Adulto Jovem
20.
Parasitol Res ; 115(7): 2661-70, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26997341

RESUMO

The transcriptomic data of Sarcoptes is still lacking in the public database due to the difficulty in extracting high-quality RNA from tiny mites with thick chitin. In this study, total RNA was extracted from live Sarcoptes mites for quality assessment, RNA-Seq, functional annotation, and coding region (CD) prediction and verification. The results showed that the sample JMQ-lngm was qualified for cDNA library construction. Firstly, Agilent 2100 detection showed that the RNA baseline was smooth and the 18S peak was single. Second, the Illumina platform generated 65.78M clean reads and 20,826 unigenes with 35.43M were assembled, occupying 62.98 % of the 56.26M genome. In total, 15,034 unigenes were annotated in seven functional databases. Finally, 13,122 CDs were detected in the 20,826 unigenes, of which 70 complete CDs were matched with Sarcoptes manually in non-redundant nucleotide (NT). Three CDs with indels ≥10 bp were verified. Those results indicated that peritrophin sequences of JMQ-lngm missed 35 bp during the assembly; the pressure-sensitive sodium channel sequences of all the six Sarcoptes scabiei canis isolates were confirmed to be 90 bp shorter than that of a Sarcoptes scabiei hominis isolate; three introns remained in PH chlorine ion channel gating sequences of JMQ-lngm. Moreover, the allergen gene prediction for JMQ-lngm indicated that 61 unigenes were matched with 19 allergen genes of Dermatophagoides, of which Der 1, Der 3, Der 8, and Der 10 had been confirmed in NT. In conclusion, this study successfully completed the RNA-Seq and functional annotation of S. s. canis for the first time, which provides molecular data for future studies on the identification and pathogenic genes of Sarcoptidae.


Assuntos
Sarcoptes scabiei/genética , Animais , Doenças do Cão/parasitologia , Cães , RNA , Escabiose/parasitologia , Escabiose/veterinária , Análise de Sequência de RNA
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