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1.
Front Immunol ; 12: 664998, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995396

RESUMO

Inflammatory bowel disease (IBD) increases the risk of colorectal cancer, and it has the potential to diminish the quality of life. Recent clinical and experimental evidence demonstrate protective aspects of parasitic helminth infection against IBD. Reports have highlighted the potential use of helminths and their byproducts as potential treatment for IBD. In the current study, we studied the effect of a newborn larvae-specific serine protease from Trichinella spiralis (TsSp) on the host immune and inflammatory responses. A 49-kDa recombinant TsSp (rTsSp) was expressed in Escherichia coli BL21 (DE3) and purified. The cytotoxicity of rTsSp was analyzed. The immune protective effect of rTsSp was studied by using dextran sodium sulfate (DSS)-induced mouse colitis model. The result illustrated that rTsSp has no toxic effects on cells. We further demonstrated that administration of the rTsSp without the additional adjuvant before the induction of DSS-induced colitis reduced the severity of intestinal inflammation and the disease index; it suppressed macrophage infiltration, reduced TNF-α secretion, and induced IL-10 expression. Our findings suggest therapeutic potential of rTsSp on colitis by altering the effect of macrophages. Data also suggest immunotherapy with rTsSp holds promise for use as an additional strategy to positively modulate inflammatory processes involved in IBD.

2.
J Morphol ; 282(5): 733-745, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33644867

RESUMO

Sensory structures on the antennae and mouthparts of insects are associated with various activities, such as host location, feeding, attracting a mate, and identifying a suitable oviposition site. Athetis lepigone (Möschler) is an important polyphagous Eurasian pest with more than 30 species of host plants. The larvae target bud leaves, prop roots, and tender stems of many agricultural crops, but the feeding habits of the adults remain poorly known. Aiming to understand the feeding behavior of the species, we investigated the fine morphology of its antennae and proboscis using scanning electron microscopy. The antennae of both sexes are filiform, and bear eight types of sensilla: Böhm's bristles, sensilla squamiformia, trichodea, chaetica, basiconica, coeloconica, styloconica, and auricillica. Sensilla trichodea are the most abundant among these sensillum types. The proboscis consists of two elongated, interlocked maxillary galeae that enclose the food canal by dorsal and ventral legulae. The external galeal surface is covered with numerous triangular microtrichia on Zone 1 and abundant blunt microbumps on Zone 2. The surface of the food canal bears closely connected and smooth semicircular ridges, gradually tapering toward the proboscis tip. Three types of sensilla are noticeable on the proboscis: sensilla trichodea, basiconica, and styloconica. We briefly discuss the putative functional significance of the antennal and proboscis sensilla and, based on the specific structural modifications of the proboscis, predict a flower-visiting habit for A. lepigone.

3.
Acta Trop ; 216: 105825, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33421420

RESUMO

The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host's immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism.


Assuntos
Proteínas de Helminto/metabolismo , Peptídeo Hidrolases/metabolismo , Proteoma/análise , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Animais , Cromatografia Líquida , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita , Intestinos/parasitologia , Larva/genética , Larva/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/parasitologia , Peptídeo Hidrolases/genética , Reação em Cadeia da Polimerase , Proteômica/métodos , RNA de Protozoário , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Espectrometria de Massas em Tandem , Triquinelose/parasitologia
4.
Vet Res ; 52(1): 6, 2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33413587

RESUMO

Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg2+, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.


Assuntos
Pirofosfatase Inorgânica/fisiologia , Trichinella spiralis/crescimento & desenvolvimento , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Larva , Camundongos , Camundongos Endogâmicos BALB C , Muda/fisiologia , Mutagênese Sítio-Dirigida , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Trichinella spiralis/fisiologia , Triquinelose/parasitologia , Triquinelose/veterinária
5.
Res Vet Sci ; 134: 1-11, 2020 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-33276221

RESUMO

The aim of this study was to characterize the biological properties of a novel aspartic protease-1 from Trichinella spiralis (TsASP1) and evaluate its potential in inducing immune response. TsASP1 gene was cloned and expressed in Escherichia coli BL21 (DE3). On Western blotting analysis with anti-rTsASP1 serum, native TsASP1 was detected in various T. spiralis phases other than newborn larvae (NBL). qPCR results showed that TsASP1 transcription was the highest in intestinal infective larvae (IIL) and the lowest in the NBL stage. Immunofluorescence test result shows that native TsASP1 was principally localized in stichosome, muscle cells of muscle larvae (ML) and IIL, and surrounded intrauterine embryos in female adult worms (AW). After silencing TsASP1 gene of the ML by siRNA, the worm development was significantly inhibited, showed by shorter AW and more wrinkles and longitudinal crack on epicuticle of AW on scanning electron microscopy; the AW and ML burdens were reduced by 41.82 and 56.36% respectively, compared with the control siRNA or PBS group (P < 0.001). Immunization of mice with rTsASP1 elicited an evident antibody response (serum IgG, IgG1/IgG2a and enteral sIgA), and systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune response, demonstrated by a prominent elevation of IFN-γ and IL-4. The results suggested TsASP1 participated in T. spiralis development and survival in host, and immunization of mice with rTsASP1 induced systemic/intestinal local mucosal humoral and cellular immune response against Trichinella.

6.
Folia Parasitol (Praha) ; 672020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33082302

RESUMO

The elastase, which belongs to the serine protease family, hydrolyses various proteins and may be involved in the parasite invasion. In this study, complete sequence of elastase-1 (TsE) the nematode Trichinella spiralis (Owen, 1835) was cloned into the plasmid pcDNA3.1 as TsE DNA vaccine. After intramuscular vaccination, serum anti-Trichinella antibodies (IgG and subclass IgG1/IgG2a, and IgA), total and specific intestinal mucosal sIgA in mice vaccinated with pcDNA3.1/TsE were measured by ELISA. The results showed that vaccination with pcDNA3.1/TsE induced a systemic humoral immune response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and local intestinal mucosal immune responses (high levels of TsE-specific sIgA). Vaccination of mice with TsE DNA vaccine also triggered a systemic and local concomitant Th1/Th2 response, as demonstrated by significant elevation of Th1 (IFN-γ and IL-2) / Th2 (IL-4 and IL-10) cytokine levels after the spleen, mesenteric lymph node and Peyer's patch cells from vaccinated mice were stimulated with recombinant TsE (rTsE). The vaccination of mice with pcDNA3.1/TsE displayed a 17% reduction of intestinal adult worms and a 39% reduction of muscle larvae. Our results indicated that TsE DNA vaccine elicited a systemic concomitant Th1/Th2 response and an enteral local sIgA response, and produced a partial protection against infection with T. spiralis. The TsE may be regarded as a potential candidate vaccine target against Trichinella infection. The oral polyvalent vaccines should be developed to improve the protective efficacy of anti-Trichinella vaccines.

7.
Vet Res ; 51(1): 125, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32988413

RESUMO

The aim of this study was to investigate the biological characteristics and functions of a Trichinella spiralis serine proteinase (TsSerp) during larval invasion and development in the host. The full-length TsSerp cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of RT-PCR, IFA and western blotting analyses showed that TsSerp was a secretory protein that was highly expressed at the T. spiralis intestinal infective larva and muscle larva stages and primarily located at the cuticle, stichosome and intrauterine embryos of the parasite. rTsSerp promoted the larval invasion of intestinal epithelial cells (IECs) and the enteric mucosa, whereas an anti-rTsSerp antibody impeded larval invasion; the promotion and obstruction roles were dose-dependently related to rTsSerp and the anti-rTsSerp antibodies, respectively. Vaccination of mice with rTsSerp elicited a remarkable humoral immune response (high levels of serum IgG, IgG1/IgG2a, IgE and IgM), and it also triggered both systemic (spleen) and local intestinal mucosal mesenteric lymph node (MLN) cellular immune responses, as demonstrated by a significant elevation in Th1 cytokines (IFN-γ) and Th2 cytokines (IL-4) after the spleen and MLN cells from vaccinated mice were stimulated with rTsSerp. Anti-TsSerp antibodies participated in the killing and destruction of newborn larvae via ADCC. The mice vaccinated with rTsSerp exhibited a 48.7% reduction in intestinal adult worms and a 52.5% reduction in muscle larvae. These results indicated that TsSerp participates in T. spiralis invasion and development in the host and might be considered a potential candidate target antigen to develop oral polyvalent preventive vaccines against Trichinella infection.

8.
Vet Res ; 51(1): 111, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32891183

RESUMO

In our previous studies, a novel T. spiralis peptidase (TsP) was identified among the excretory/secretory (ES) proteins of T. spiralis intestinal infective larvae (IIL) and T. spiralis at the adult worm (AW) stage using immunoproteomics, but the biological function of TsP in the life cycle of T. spiralis is not clear. The objective of this study was to investigate the biological properties and functions of TsP in larval intrusion and protective immunity induced by immunization with rTsP. The complete TsP cDNA sequence was cloned and expressed. The results of RT-PCR, indirect immunofluorescence assay (IIFA) and western blotting revealed that TsP is a surface and secretory protein expressed in T. spiralis at different stages (muscle larvae, IIL, AWs and newborn larvae) that is principally localized at the epicuticle of the nematode. rTsP facilitated the larval intrusion of intestinal epithelial cells (IECs) and intestinal mucosa, whereas anti-rTsP antibodies suppressed larval intrusion; these facilitative and suppressive roles were dose-dependently related to rTsP or anti-rTsP antibodies. Immunization of mice with rTsP triggered an obvious humoral immune response (high levels of IgG, IgG1/IgG2a, and sIgA) and also elicited systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune responses, as demonstrated by an evident increase in the cytokines IFN-γ and IL-4. Immunization of mice with rTsP reduced the numbers of intestinal adult worms by 38.6% and muscle larvae by 41.93%. These results demonstrate that TsP plays a vital role in the intrusion, development and survival of T. spiralis in hosts and is a promising candidate target molecule for anti-Trichinella vaccines.

9.
Vaccines (Basel) ; 8(3)2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32764274

RESUMO

Trichinella spiralis is a major foodborne parasite and has a serious threat to meat safety. Development of anti-Trichinella vaccines is prospective to eliminate Trichinella infection in food animal. The aim of this study was to assess the biological properties of a novel T. spiralis trypsin (TsT) and its elicited immune protection against larval challenge. The cDNA sequence of TsT gene was cloned and expressed. Western blotting showed rTsT was identified by infection serum and anti-TsT serum. RT-PCR results revealed that TsT gene was transcribed at diverse T. spiralis lifecycle stages. The IIFT results showed that natural TsT was principally expressed at epicuticle of 5-6 day adult worms, indicating that TsT is a worm somatic antigen and adult-stage specific surface antigen. Vaccination of mice with rTsT triggered an evident humoral immune response (high levels of serum IgG, IgG1/IgG2a, and enteral sIgA), and it also induced the systemic and enteral local cellular immune response, demonstrated by an significantly elevation of cytokines IFN-γ and IL-4. The mice vaccinated with rTsT exhibited a 33.17% reduction of enteral adult worms and a 37.80% reduction of muscle larvae after larval challenge. The results showed that TsT might be considered as a candidate target antigen for anti-T. spiralis vaccines.

10.
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.

11.
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.

12.
Acta Trop ; 211: 105592, 2020 Nov.
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.

13.
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.

14.
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
15.
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.


Assuntos
Proteínas de Helminto/farmacologia , Imunidade Humoral , Elastase Pancreática/farmacologia , Trichinella spiralis/efeitos dos fármacos , Triquinelose/prevenção & controle , Vacinação/veterinária , Animais , Feminino , Fertilidade , Proteínas de Helminto/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Elastase Pancreática/administração & dosagem , Trichinella spiralis/fisiologia , Triquinelose/parasitologia
16.
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.


Assuntos
Catepsina B/genética , Enterócitos/parasitologia , Células Epiteliais/parasitologia , Proteínas de Helminto/genética , Mucosa Intestinal/parasitologia , Trichinella spiralis/fisiologia , Animais , Catepsina B/metabolismo , Feminino , Proteínas de Helminto/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência de DNA/veterinária , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Triquinelose/parasitologia
17.
Acta Parasitol ; 65(3): 783-786, 2020 Sep.
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.

18.
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.


Assuntos
Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Elastase Pancreática/química , Elastase Pancreática/imunologia , Trichinella spiralis/enzimologia , Triquinelose/diagnóstico , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Humanos , Estágios do Ciclo de Vida , Camundongos Endogâmicos BALB C , Elastase Pancreática/genética , Alinhamento de Sequência , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Trichinella spiralis/imunologia , Triquinelose/sangue , Triquinelose/imunologia , Triquinelose/parasitologia
19.
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
20.
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
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