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1.
Vet Parasitol ; : 109160, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522393

RESUMO

The aim of this study was to ascertain the characteristics of a Trichinella spiralis cathepsin X (TsCX) and its role on larval invasion of intestinal epithelial cells (IECs). The full-length of TsCX cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of RT-PCR, IFA and Western blot revealed that TsCX was expressed at T. spiralis muscle larvae (ML), intestinal infective larvae, adult worm and newborn larvae, and it was located in whole worm section. The results of Far western and confocal microscopy demonstrated that there was a specific binding of rTsCX and IEC, and the binding site was located within the IEC cytoplasm. rTsCX promoted T. spiralis larval invasion of mouse IECs while anti-rTsCX antibody inhibited larval invasion into the IECs. Silencing TsCX by specific siRNA reduced the TsCX expression and larval invasive capacity. These results indicated that TsCX specifically binds to IECs and promotes larval invasion of intestinal epithelia, and it might be a potential target of vaccines against enteral stages of T. spiralis.

2.
Vet Res ; 51(1): 78, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32539772

RESUMO

A Trichinella spiralis aminopeptidase (TsAP) has been identified in intestinal infectious larvae (IIL) and adult worms (AW), but its biological function in the T. spiralis life cycle is unknown. The aim of this study was to characterize TsAP and ascertain its functions in the invasion, development and fecundity of T. spiralis. Recombinant TsAP (rTsAP) was expressed and purified. rTsAP has strong immunogenicity. qPCR and western blotting show that TsAP was transcribed and expressed at all T. spiralis lifecycle stages, but the expression level of TsAP mRNA and proteins at IIL and AW stages was obviously higher than those in muscle larvae (ML) and newborn larvae (NBL). The IFT results reveal that TsAP was principally located at the cuticle and the intrauterine embryos of this nematode. rTsAP had the enzymatic activity of natural aminopeptidase to hydrolyze the substrate Leu-pNA with an optimal temperature of 50 °C and optimal pH of 8.0. rTsAP promoted the larval penetration into intestinal epithelial cells, whereas anti-rTsAP antibodies suppressed the larval intrusion; the promotion and suppression was dose-dependently related to rTsAP or anti-rTsAP antibodies. TsAP protein expression level and enzymatic activity were reduced by 50.90 and 49.72% through silencing of the TsAP gene by specific siRNA 842. Intestinal AW and muscle larval burdens, worm length and female reproductive capacity were significantly declined in mice infected with siRNA-transfected ML compared to the control siRNA and PBS group. These results indicate that TsAP participates in the invasion, development and fecundity of T. spiralis and it might be a candidate target for anti-Trichinella vaccines.

3.
Acta Trop ; 211: 105592, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32565198

RESUMO

Elastase-1 is one member of serine protease family, distributes in organisms widely and plays a crucial role in the invasion and development of Trichinella spiralis. In order to identify the binding of T. spiralis elastase-1 (TsEla) with host's intestinal epithelial cells (IECs) and its role in Trichinella larval intrusion, TsEla gene was cloned and expressed in our previous study. The recombinant TsEla (rTsEla) has the enzymatic activity to degrade specific peptide substrate. A specific binding between rTsEla and IECs was detected by Far Western blot and ELISA. In an in vitro invasion assay, rTsEla promoted the larval intrusion, whereas anti-rTsEla serum inhibited the larval penetration. The larval intrusion was also suppressed after the silencing of TsEla by siRNA. Silencing of TsEla gene by siRNA-291 meditated RNA interference suppressed TsEla protein expression, reduced the worm infectivity, development and reproductive capacity. These results indicated that TsEla plays an important role in the T. spiralis intrusion of host's intestinal epithelia, and it could be a prospective vaccine molecular target against T. spiralis infection.

4.
Vet Parasitol ; : 109128, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32402492

RESUMO

The purpose of this study was to determine the biological function of a Trichinella spiralis glutathione S-transferase (TsGST) in larval invasion and development by RNA interference (RNAi). The TsGST-specific siRNA 366 was transfected into T. spiralis muscle larvae (ML) via electroporation. At 1 day following transfection, the larval TsGST mRNA and protein expressions were reduced by 40.09 and 65.22 % (P < 0.05), respectively. The enzymatic activity of natural TsGST in siRNA-transfected ML was also suppressed by 45% compared with PBS group (P < 0.05). Silencing of the TsGST significantly inhibited the ability of larvae to invade intestinal epithelium cells (IECs) and isolated intestine. After challenge with siRNA-366-treated ML, the infected mice exhibited a 62.82% reduction of intestinal adult worms, and 65.03 % reduction of muscle larvae compared to the PBS group. Besides, the length of adults, newborn larvae and muscle larvae was significantly shorter than that of control siRNA and PBS group; the female fecundity of siRNA 366 group was lower than those of control siRNA and PBS group (P <  0.05). The results revealed that the specific RNAi significantly reduced the expression and enzymatic activity of TsGST, inhibited the larval invasive and developmental capacity, and impaired the female fecundity. The results further confirmed that TsGST plays a crucial role in the T. spiralis life cycle and it might be a potential molecular target for anti-Trichinella vaccines.

5.
PLoS Negl Trop Dis ; 14(4): e0008269, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339171

RESUMO

BACKGROUND: T. spiralis aspartic protease has been identified in excretion/secretion (ES) proteins, but its roles in larval invasion are unclear. The aim of this study was to characterize T. spiralis aspartic protease-2 (TsASP2) and assess its roles in T. spiralis invasion into intestinal epithelial cells (IECs) using RNAi. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant TsASP2 (rTsASP2) was expressed and purified. The native TsASP2 of 43 kDa was recognized by anti-rTsASP2 serum in all worm stages except newborn larvae (NBL), and qPCR indicated that TsASP2 transcription was highest at the stage of intestinal infective larvae (IIL). IFA results confirmed that TsASP2 was located in the hindgut, midgut and muscle cells of muscle larvae (ML) and IIL and intrauterine embryos of the female adult worm (AW), but not in NBL. rTsASP2 cleaved several host proteins (human hemoglobin (Hb), mouse Hb, collagen and IgM). The proteolytic activity of rTsASP2 was host-specific, as it hydrolyzed mouse Hb more efficiently than human Hb. The enzymatic activity of rTsASP2 was significantly inhibited by pepstatin A. The expression levels of TsASP2 mRNA and protein were significantly suppressed by RNAi with 5 µM TsASP2-specific siRNA. Native aspartic protease activity in ML crude proteins was reduced to 54.82% after transfection with siRNA. Larval invasion of IECs was promoted by rTsASP2 and inhibited by anti-rTsASP2 serum and siRNA. Furthermore, cell monolayer damage due to larval invasion was obviously alleviated when siRNA-treated larvae were used. The adult worm burden, length of adult worms and female fecundity were clearly reduced in mice challenged using siRNA-treated ML relative to the PBS group. CONCLUSIONS: rTsASP2 possesses the enzymatic activity of native aspartic protease and facilitates T. spiralis invasion of host IECs.


Assuntos
Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Endocitose , Células Epiteliais/parasitologia , Trichinella spiralis/enzimologia , Trichinella spiralis/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hemoglobinas/metabolismo , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Carga Parasitária , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Trichinella spiralis/genética , Triquinelose/parasitologia
6.
Res Vet Sci ; 130: 110-117, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32171999

RESUMO

Cathepsin B is one member of cysteine protease family and widely distributed in organisms, it plays an important function in parasite penetrating, migrating, molting and immune escaping. The aim of this work was to investigate whether exist interaction between a Trichinella spiralis cathepsin B (TsCB) and mouse intestinal epithelium cells (IECs), and its influence in the process of larva cell invasion. The results of ELISA, indirect immunofluorescence assay (IIFA), confocal microscopy and Far western blotting showed that there was a strong specific binding of rTsCB and IEC proteins, and the binding positions were located in cytoplasm and nuclei of IECs. The results of the in vitro larva penetration test revealed that rTsCB facilitated the larva invasion of IECs, whereas anti-rTsCB antibodies impeded partially the larva intrusion of enterocytes, this promotive or inhibitory roles were dose-dependent of rTsCB or anti-rTsCB antibodies. Silencing TsCB by siRNA mediated RNA interference reduced the TsCB expression in T. spiralis larvae, and markedly inhibited the larva penetration of enterocytes. The results indicated that TsCB binding to IECs promoted larva penetration of host's enteral epithelia, and it is a promising molecular target against intestinal invasive stages of T. spiralis.

7.
Vet Res ; 51(1): 43, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32169101

RESUMO

Trichinella spiralis is an important foodborne parasitic nematode that represents an enormous threat to the food safety of pork meat. The development of a preventive vaccine is valuable for the prevention and control of Trichinella infection in domestic pigs to ensure pork safety. Elastase is a trypsin-like serine protease that hydrolyzes the host's diverse tissue components and participates in parasite penetration, and it might be a novel vaccine target molecule. The aim of this study was to assess the protective immunity produced by vaccination with a novel Trichinella spiralis elastase-1 (TsE) in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsE elicited a systemic humoral response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and significant local enteral mucosal sIgA responses. Anti-rTsE IgG recognized the native TsE at the cuticle, stichosome of intestinal infective larvae and adult worm (AW), and intrauterine embryos of female AW. The rTsE vaccination also produced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10) after spleen, mesenteric lymph node and Peyer's patch cells from immunized mice were stimulated with rTsE. The immunized mice exhibited a 52.19% reduction in enteral AW and a 64.06% reduction in muscle larvae after challenge infection. The immune response triggered by rTsE vaccination protected enteral mucosa from larval intrusion, suppressed larval development and reduced female fecundity. The results indicate that TsE may represent a novel target molecule for anti-T. spiralis vaccines.

8.
Acta Parasitol ; 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144548

RESUMO

PURPOSE: Human cases of thelaziasis caused by Thelazia callipaeda have increased in China in recent years. Although this species is of medical importance, our knowledge about the epidemiology of thelaziasis is still fragmentary. This study first reports a case of thelaziasis in central China. Then, the epidemiology of thelaziasis in China in the past 100 years (1917-2018) is reviewed. METHODS: A 5-year-old girl experienced discomfort in her left eye. Four thread-like worms were seen in the nasal upper eyelid of the left eye. The symptoms disappeared after these parasites were removed. In addition, we reviewed studies of Chinese human thelaziasis cited in articles or book chapters in all languages from inception to 31 Dec 2019. RESULTS: China is the nation with the most reports of thelaziasis (653 cases) in the world. More human cases were reported in central and eastern China than in other areas, and the majority of cases were from rural areas in poor socioeconomic settings. CONCLUSION: Special attention should be paid to this neglected disease in China. The use of a One Health approach is imperative for preventing eyeworm infections in humans.

9.
PLoS Negl Trop Dis ; 14(2): e0008019, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32101542

RESUMO

BACKGROUND: In China, frogs play an understudied role in the spread of human sparganosis (caused by the larval form of Spirometra). However, our knowledge about the prevalence of sparganum infection in frogs remains fragmented, and the taxonomic identification of the parasite is still controversial. METHODOLOGY/PRINCIPAL FINDINGS: The prevalence of sparganum infection in wild frogs was surveyed at 145 geographical locations from 28 of the 34 provinces/autonomous regions/municipalities in China for six years. The collected sparganum isolates from the different locations were subjected to molecular identification by a multiplex PCR assay and then were analysed with clustering analysis. In the survey, sparganum infection was found in 8 out of 13 of the collected frog species, and the most frequently infected species was Pelophylax nigromaculatus (the infection rate was up to 14.07%). Infected frogs were found in 80 of the 145 surveyed locations. The sparganum infection rates in the wild frogs in several regions of China were still high (above 10%), especially in South and Southwest China. A total of 72 spargana were selected for molecular identification, and the clustering analysis showed that sequences from the Chinese isolates were very similar to those identified as from Spirometra erinaceieuropaei. However, the taxonomy of the genus remains confused and further analysis is required. CONCLUSIONS: Eating wild frogs is associated with considerable health risks in China. Several traditional Chinese folk remedies may increase the risk of infection. The sparganum isolates in China are most likely from S. erinaceieuropaei, but new studies, especially comprehensive morphological analyses, are needed in the future.


Assuntos
Infecções por Cestoides/veterinária , Ranidae/parasitologia , Spirometra/classificação , Animais , Infecções por Cestoides/epidemiologia , Infecções por Cestoides/parasitologia , China/epidemiologia , Interações Hospedeiro-Parasita
10.
Parasit Vectors ; 13(1): 97, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093735

RESUMO

BACKGROUND: Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. METHODS: The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. RESULTS: The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P > 0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P > 0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. CONCLUSIONS: rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.

11.
Res Vet Sci ; 128: 1-8, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31706217

RESUMO

The aim of this work was to identify the molecular characteristics of a chymotrypsin-like enzyme from Trichinella spiralis (Tschy) and its facilitation of larval penetration into enteral epithelial cells (EECs). The complete Tschy cDNA sequence was cloned and expressed in Escherichia coli BL21. RT-PCR, IIFA and western blotting showed that Tschy was expressed at the T. spiralis muscle larvae (ML), intestinal infective L1 larvae (IL1), adult worms (AW) and embryo stages and was primarily located in the stichosome of this parasite. The results of ELISA, IIFA and Far-western assays showed that there was a specific binding between rTschy and EECs, and the binding was dependent on the dose of both rTschy and EEC proteins. Confocal microscopy demonstrated that the binding was located in the EEC cytoplasm. rTschy facilitated T. spiralis larval penetration of EECs, and anti-rTschy antibodies impeded the larval intrusion of EECs. These results demonstrate that Tschy facilitated the larval intrusion of the host's enteral epithelium and could be a candidate molecular target for vaccine against the enteral invasive phase of T. spiralis.


Assuntos
Quimotripsina/genética , Expressão Gênica , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita/fisiologia , Trichinella spiralis/fisiologia , Animais , Quimotripsina/metabolismo , Embrião não Mamífero/enzimologia , Embrião não Mamífero/fisiologia , Células Epiteliais/parasitologia , Escherichia coli/genética , Proteínas de Helminto/metabolismo , Intestino Delgado/parasitologia , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Microrganismos Geneticamente Modificados/genética , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Vacinas/análise
12.
Parasit Vectors ; 12(1): 581, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31829230

RESUMO

BACKGROUND: Trichinella spiralis is a major zoonotic tissue-dwelling nematode, which is a public health concern and a serious hazard to animal food safety. It is necessary to exploit an anti-Trichinella vaccine to interrupt the transmission of Trichinella infection among animals and from animals to humans. The purpose of the present study was to characterize the novel T. spiralis cathepsin B (TsCB) and to evaluate the immune protection elicited by immunization with recombinant TsCB (rTsCB). METHODS: The complete cDNA sequences of the TsCB gene were cloned, expressed and purified. The antigenicity of rTsCB was investigated by western blot analysis and ELISA. Transcription and expression of TsCB at various T. spiralis life-cycle stages were analyzed by RT-PCR and indirect immunofluorescent assay (IIFA). The mice were subcutaneously immunized with rTsCB, and serum level of TsCB-specific IgG (IgG1 and IgG2a) and IgE antibodies were assayed by ELISA. Immune protection elicited by vaccination with rTsCB was investigated. RESULTS: The TsCB was transcribed and expressed in four T. spiralis life-cycle stages (adult worm, AW; newborn larvae, NBL; muscle larvae, ML; and intestinal infective L1 larvae), it was primarily located in the cuticle and stichosome of the parasitic nematode. Vaccination of mice with rTsCB produced a prominent antibody response (high level of specific IgG and IgE) and immune protection, as demonstrated by a 52.81% AW burden reduction of intestines at six days post-infection (dpi) and a 50.90% ML burden reduction of muscles at 35 dpi after oral larva challenge. The TsCB-specific antibody response elicited by immunization with rTsCB also impeded intestinal worm growth and decreased the female fecundity. CONCLUSIONS: TsCB might be considered as a novel potential molecular target to develop vaccines against T. spiralis infection.


Assuntos
Antígenos de Helmintos/imunologia , Catepsina B/imunologia , Trichinella spiralis/imunologia , Triquinelose/prevenção & controle , Vacinas Sintéticas/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/administração & dosagem , Catepsina B/administração & dosagem , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fertilidade , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Injeções Subcutâneas , Camundongos , Carga Parasitária , Resultado do Tratamento , Trichinella spiralis/crescimento & desenvolvimento , Trichinella spiralis/isolamento & purificação , Triquinelose/parasitologia , Triquinelose/patologia , Vacinas Sintéticas/administração & dosagem
13.
Vet Res ; 50(1): 106, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31806006

RESUMO

The binding and activation of host plasminogen (PLG) by worm surface enolases has been verified to participate in parasite invasion, but the role of this processes during Trichinella spiralis infection has not been clarified. Therefore, the expression and immunolocalization of a T. spiralis enolase (TsENO) and its binding activity with PLG were evaluated in this study. Based on the three-dimensional (3D) molecular model of TsENO, the protein interaction between TsENO and human PLG was analysed by the ZDOCK server. The interacting residues were identified after analysis of the protein-protein interface by bioinformatics techniques. The key interacting residues were confirmed by a series of experiments. The qPCR analysis results demonstrated that Ts-eno was transcribed throughout the whole life cycle of T. spiralis. The immunofluorescence assay (IFA) results confirmed that TsENO was distributed on the T. spiralis surface. The binding assays showed that recombinant TsENO (rTsENO) and native TsENO were able to bind PLG. Four lysine residues (90, 289, 291 and 300) of TsENO were considered to be active residues for PLG interaction. The quadruple mutant (Lys90Ala + Lys289Ala + Lys291Ala + Lys300Ala) TsENO, in which the key lysine residues were substituted with alanine (Ala) residues, exhibited a reduction in PLG binding of nearly 50% (45.37%). These results revealed that TsENO has strong binding activity with human PLG. The four lysine residues (90, 289, 291 and 300) of TsENO play an important role in PLG binding and could accelerate PLG activation and invasion of the host's intestinal wall by T. spiralis.


Assuntos
Proteínas de Helminto/genética , Fosfopiruvato Hidratase/genética , Plasminogênio/fisiologia , Trichinella spiralis/fisiologia , Triquinelose/imunologia , Animais , Feminino , Proteínas de Helminto/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fosfopiruvato Hidratase/metabolismo , Trichinella spiralis/genética , Triquinelose/parasitologia
14.
Sci Rep ; 9(1): 15703, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673002

RESUMO

Intestinal helminth infections elicit Th2-type immunity, which influences host immune responses to additional threats, such as allergens, metabolic disease, and other pathogens. Th2 immunity involves a shift of the CD4+ T-cell population from type-0 to type-2 (Th2) with increased abundance of interleukin (IL)-4 and IL-13. This study sought to investigate if existing gut-restricted intestinal helminth infections impact bacterial-induced acute airway neutrophil recruitment. C57BL/6 mice were divided into four groups: uninfected; helminth-Heligmosomoides polygyrus infected; Pseudomonas aeruginosa infected; and coinfected. Mice infected with H. polygyrus were incubated for 2 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage, blood, and lung samples were analyzed. Interestingly, infection with gut-restricted helminths resulted in immunological and structural changes in the lung. These changes include increased lung CD4+ T cells, increased Th2 cytokine expression, and airway goblet cell hyperplasia. Furthermore, coinfected mice exhibited significantly more airspace neutrophil infiltration at 6 hours following P. aeruginosa infection and exhibited an improved rate of survival compared with bacterial infected alone. These results suggest that chronic helminth infection of the intestines can influence and enhance acute airway neutrophil responses to P. aeruginosa infection.

15.
Front Genet ; 10: 1093, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31737057

RESUMO

In China, the nematode Trichinella spiralis is the main aetiological agent of human trichinellosis. We performed multi-locus microsatellite typing of T. spiralis isolates to improve the current knowledge of the evolution and population diversity. First, seven polymorphic microsatellite loci were used to infer the genetic diversity of T. spiralis collected in 10 endemic regions. Then, a Bayesian model-based STRUCTURE analysis, a clustering based on the neighbor-joining method, and a principal coordinate analysis (PCA) were performed to identify the genetic structure. Finally, the phylogenetic position of Chinese isolates was explored based on six mitochondrial and nuclear genetic markers (cox1, cytb, 5S ISR, ESV, ITS1, and 18S rDNA) using the maximum likelihood and Bayesian methods. In addition, the divergence time was estimated with multiple genes using an uncorrelated log-normal relaxed molecular-clock model. A total of 16 alleles were detected in 2,310 individuals (1,650 muscle larvae and 660 adult worms) using seven loci. The STRUCTURE analysis indicated that the T. spiralis isolates could be organized and derived from the admixture of two ancestral clusters, which was also substantiated through the clustering analysis based on the allelic data. PCA separated most samples from Tiandong, Guangxi (GX-td), and Linzhi, Tibet (Tibet-lz), from the remaining isolates. However, both maximum likelihood and Bayesian inference supported the close relationship between Xiangfan, Hubei (HB-xf), and GX-td. The molecular dating analysis suggested that the Chinese isolates started to diverge during the Late Pleistocene (0.69 Mya). Generally, T. spiralis was observed to harbor low genetic variation, and further investigation with deeper sampling is needed to elucidate the population structure.

16.
Vet Res ; 50(1): 70, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547875

RESUMO

Molting is a key step for body-size expansion and environmental adaptation of parasitic nematodes, and it is extremely important for Trichinella spiralis growth and development, but the molting mechanism is not fully understood. In this work, label-free LC-MS/MS was used to determine the proteome differences between T. spiralis muscle larvae (ML) at the encapsulated stage and intestinal infective larvae (IIL) at the molting stage. The results showed that a total of 2885 T. spiralis proteins were identified, 323 of which were differentially expressed. These proteins were involved in cuticle structural elements, regulation of cuticle synthesis, remodeling and degradation, and hormonal regulation of molting. These differential proteins were also involved in diverse intracellular pathways, such as fatty acid biosynthesis, arachidonic acid metabolism, and mucin type O-glycan biosynthesis. qPCR results showed that five T. spiralis genes (cuticle collagen 14, putative DOMON domain-containing protein, glutamine synthetase, cathepsin F and NADP-dependent isocitrate dehydrogenase) had significantly higher transcriptional levels in 10 h IIL than ML (P < 0.05), which were similar to their protein expression levels, suggesting that they might be T. spiralis molting-related genes. Identification and characterization of T. spiralis molting-related proteins will be helpful for developing vaccines and new drugs against the early enteral stage of T. spiralis.


Assuntos
Proteínas de Helminto/genética , Muda/genética , Doenças dos Suínos/parasitologia , Trichinella spiralis/fisiologia , Triquinelose/veterinária , Animais , Cromatografia Líquida/veterinária , Feminino , Proteínas de Helminto/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Camundongos , Proteômica , Organismos Livres de Patógenos Específicos , Sus scrofa , Suínos , Doenças dos Suínos/fisiopatologia , Espectrometria de Massas em Tandem/veterinária , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Triquinelose/parasitologia , Triquinelose/fisiopatologia
17.
Parasitol Res ; 118(7): 2247-2255, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31081529

RESUMO

In a previous study, immunoproteomics was used to identify a serine protease inhibitor (TsSPI) of T. spiralis excretory/secretory (ES) proteins that exhibited an inhibitory effect on trypsin enzymatic activity, but the precise role of TsSPI on worm infection and development in its host is not well understood. The objective of the present study was to use RNA interference to ascertain the function of TsSPI in larval invasion and growth. TsSPI-specific small interference RNAs (siRNAs) were delivered to muscle larvae (ML) to silence TsSPI expression by electroporation. Four days after electroporation, the ML transfected with 2 µM siRNA-653 exhibited a 75.75% decrease in TsSPI transcription and a 69.23% decrease in TsSPI expression compared with control ML. Although the silencing of TsSPI expression did not decrease worm viability, it significantly suppressed the larval invasion of intestinal epithelium cells (IEC) (P < 0.01), and the suppression was siRNA dose-dependent (r = 0.981). The infection of mice with siRNA-653-treated ML produced a 63.71% reduction of adult worms and a 72.38% reduction of muscle larvae. In addition, the length of the adults, newborn larvae, and ML and the fecundity of female T. spiralis from mice infected with siRNA-treated ML were obviously reduced relative to those in the control siRNA or PBS groups. These results indicated that the silencing of TsSPI by RNAi suppressed larval invasion and development and decreased female fecundity, further confirming that TsSPI plays a crucial role during the T. spiralis lifecycle and is a promising molecular target for anti-Trichinella vaccines.


Assuntos
Doenças Transmitidas por Alimentos/prevenção & controle , RNA Interferente Pequeno/administração & dosagem , Inibidores de Serino Proteinase/genética , Trichinella spiralis/genética , Triquinelose/prevenção & controle , Animais , Feminino , Fertilidade , Doenças Transmitidas por Alimentos/imunologia , Doenças Transmitidas por Alimentos/parasitologia , Humanos , Mucosa Intestinal/imunologia , Larva , Camundongos , Camundongos Endogâmicos BALB C , Músculos/parasitologia , Proteômica , Interferência de RNA , Inibidores de Serino Proteinase/metabolismo , Trichinella spiralis/crescimento & desenvolvimento , Trichinella spiralis/imunologia , Trichinella spiralis/patogenicidade , Triquinelose/imunologia , Triquinelose/parasitologia
18.
Exp Parasitol ; 201: 1-10, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004570

RESUMO

The aim of this study was to observe the intestinal mucosal/systemic responses triggered by intranasal vaccination using recombinant Trichinella spiralis serine protease (rTsSP) and its capacity to elicit immune protection against larva challenge in a murine model. rTsSP coupled with cholera toxin B subunit (CTB) was used to vaccinate mice via intranasal route. The results revealed that intranasal vaccination with rTsSP plus CTB elicited significantly intestinal local sIgA response and a TsSP-specific systemic antibody response in vaccinated mice. Furthermore, more goblet cells/acidic mucins and IgA-secreting cells were observed in jejunum from vaccinated mice. Anti-rTsSP immune serum strongly recognized the cuticle of various worm stages (muscle larva, intestinal infective larva and adult worm). The level of IFN-γ, IL-4 and IL-10 of rTsSP-vaccinated mice was significantly elevated relative to CTB and PBS control groups. The vaccinated mice exhibited a 71.10% adult reduction at 9 days pi and a 62.10% muscle larva reduction at 42 days pi following larva challenge. Additionally, vaccination with rTsSP also dampened intestinal T. spiralis development and decreased the female fecundity. Our results showed that intranasal vaccination using rTsSP adjuvanted with CTB triggered significantly local sIgA response and systemic concurrent Th1/Th2 response that induced an obvious protection against Trichinella infection.


Assuntos
Serina Proteases/imunologia , Trichinella spiralis/imunologia , Administração Intranasal , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/administração & dosagem , Antígenos de Helmintos/imunologia , Citocinas/análise , Duodeno/química , Duodeno/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Células Caliciformes/química , Soros Imunes/imunologia , Imunoglobulina A/sangue , Imunoglobulina A Secretora/análise , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Mesentério , Camundongos , Camundongos Endogâmicos BALB C , Mucinas/isolamento & purificação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Serina Proteases/administração & dosagem , Organismos Livres de Patógenos Específicos , Baço/citologia , Baço/imunologia , Trichinella spiralis/enzimologia
19.
Parasitology ; 146(7): 947-955, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30859932

RESUMO

The plerocercoid (sparganum) of Spirometra erinaceieuropaei is the main aetiological agent of human sparganosis. To improve the current knowledge on S. erinaceieuropaei evolution, we performed multi-locus microsatellite typing of sparganum isolates from China for the first time. All available expressed sequence tag (EST) sequences for the Spirometra were downloaded from the GenBank. The identification and localization of microsatellites in ESTs was accomplished by MISA. Based on the selected microsatellites, the genetic structure of 64 sparganum isolates collected from 11 geographical locations in southwest China were investigated through principal component analysis, STRUCTURE analysis and neighbour-joining clustering. A total of 522 non-redundant ESTs containing 915 simple sequence repeats were identified from 12 481 ESTs screened. Five primer pairs were finally selected. Using these loci, a total of 12 alleles were detected in 64 sparganum isolates. Little variability was observed within each of geographical population, especially among isolates derived from Kunming of Yunnan (YN-KM) province. Both STRUCTURE analysis and the clustering analysis supported that two genotypes existed among the sparganum isolates from southwest China. In conclusion, five microsatellite markers were successfully developed, and sparganum population was observed to harbour low genetic variation, further investigation with deeper sampling was needed to elucidate the population structure.


Assuntos
Etiquetas de Sequências Expressas , Genética Populacional , Repetições de Microssatélites , Plerocercoide/genética , Alelos , Animais , Anuros/parasitologia , China , Marcadores Genéticos , Variação Genética , Genótipo , Filogenia , Serpentes/parasitologia , Esparganose
20.
Vet Res ; 49(1): 119, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518422

RESUMO

Trichinellosis is one of the most serious foodborne parasitic zoonosis with worldwide distribution, and it is necessary to develop a vaccine to interrupt transmission from animals to humans. Trichinella spiralis adult-specific DNase II-1 (TsDNase II) were identified by immunoproteomics in surface or excretory/secretory proteins of adult worms (AW) and intestinal infective larvae (IIL). The aim of this study was to investigate the systemic, mucosal responses and immune protection elicited by oral vaccination with TsDNase II DNA vaccine delivered by attenuated Salmonella typhimurium strain⊿cyaSL1344. Oral vaccination with TsDNase II DNA vaccine triggered an obvious mucosal sIgA response and a systemic IgG response in mice, and IgG1 was predominant. Th1 (IFN-γ) and Th2 (IL-4, 10) cytokines were distinctly increased in the spleen and mesenteric lymph node (MLN) cells of vaccinated mice. An indirect immunofluorescent test revealed that native TsDNase II is present at the cuticle of this nematode after the 2nd molting, further confirming that TsDNase II is adult-specific and expressed at AW and pre-adult stages. Oral immunization of mice with TsDNase II exhibited a 53.85% reduction in AW and a 59.26% reduction in ML after larval challenge. The in vitro NBL production of adult females from TsDNase II-vaccinated mice was also reduced in comparison with pcDNA3.1 or the PBS control group (P < 0.01). Our results show that oral immunization of mice with TsDNase II produced an intestinal and systematic concurrent Th1/Th2 immune response, and a significant immune protection against challenge.


Assuntos
Endodesoxirribonucleases/uso terapêutico , Proteínas de Helminto/uso terapêutico , Imunidade nas Mucosas , Trichinella spiralis/imunologia , Triquinelose/prevenção & controle , Vacinação , Vacinas de DNA/imunologia , Administração Oral , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Salmonella typhimurium/genética , Vacinas Atenuadas/imunologia
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