Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

Microstructure characterization of different types of chlamydospores in Arthrobotrys flagrans.

The morphological and structural differences of different types of chlamydospore of Arthrobotrys flagrans, a nematophagous fungus, were studied under light microscope and electron microscope to provide a reference for the biological control of parasitic nematodiasis. In this study, A. flagrans isolate F088 dormant chlamydospore and nondormant chlamydospore were selected as the research objects. The structural differences of these spores were observed by optical microscopy through lactol cotton blue, Trypan blue, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. FunXite -1, 4',6-diamidino-2-phenylindole, and calcofluor white staining were used to observe the metabolic activity, cell wall, and nucleus differences of the two types of spores under fluorescence microscope. Ultrastructure of the two kinds of spores was observed using scanning electron microscope (SEM) and transmission electron microscope (TEM). Since lacto phenol cotton blue, trypan blue staining cannot distinguish dormant spores from dead spores, MTT assay was performed. Fluorescence microscopy observation showed that the cytoplasmic metabolic activity of nondormant spores was stronger than that of dormant spores. The nucleus of dormant spores was bright blue, and their fluorescence was stronger than that of nondormant spores. The cell wall of nondormant spores produced stronger yellow-green fluorescence than that of dormant spores. Ultrastructural observation showed that there were globular protuberances on the surface of the two types of spores but with no significant difference between them. The inner wall of dormant spore possesses a thick zona pellucida with high electron density which was significantly thicker than that of nondormant spores, and their cytoplasm is also changed. In this study, the microstructure characteristics of dormant and nondormant chlamydospores of A. flagrans fungi were preliminarily clarified, suggesting that the state of cell wall and intracellular materials were changed after spores entered to dormancy.

Immunogenicity and efficacy of serogroup A and D bacterins against Pasteurella multocida in mice.

Introduction: Pasteurella multocida is a widespread respiratory pathogen in pigs, causing swine pneumonia and atrophic rhinitis, and the capsular serogroups A and D are the main epidemic serogroups in infected animals. This study investigated the protective effects of serogroup A and D bacterins against current circulating P. multocida strains, to better understand the immunity generated by bacterins. Method: 13 serogroup A (seven A: L3 and six A: L6 strains) and 13 serogroup D (all D: L6 strains) P. multocida strains were isolated, and used as inactivated whole cell antigen to prepare P. multocida bacterins. Mice were immunized with these bacterins at 21-day interval and intraperitoneally challenged with the homologous and heterologous P. multocida strains, respectively. The antibody titer levels and immunization protective efficacy of vaccines were evaluated. Results: All of the bacterins tested induced high titer levels of immunoglobulin G antibodies against the parental bacterial antigen in mice. Vaccination with the six A: L6 bacterins provided no protection against the parent strain, but some strains did provide heterologous protection against A: L3 strains. Vaccination with the seven A: L3 bacterins provided 50%-100% protection against the parent strain, but none gave heterologous protection against the A:L6 strains. Immunization with the thirteen D: L6 bacterins offered 60%-100% protection against the parent strain, and almost all D: L6 strains gave cross-protection. Discussion: This study found that the cross-protectivity of serogroup A strains was poor, while serogroup D strains was effective, which provided some insights for P. multocida vaccine development.

Anti-osteosarcoma effect of antiserum against cross antigen TPD52 between osteosarcoma and Trichinella spiralis.

BACKGROUND: Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. METHODS: To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. RESULTS: Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 µg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. CONCLUSIONS: Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage.

ROS-mediated NLRP3 inflammasome activation participates in the response against Neospora caninum infection.

BACKGROUND: Neospora caninum is an obligate intracellular protozoan that causes neosporosis, N. caninum infection is a major cause of abortion in cattle worldwide. Currently, specific treatment for neosporosis is not available. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a cytoplasmic protein complex that plays an important role in host defense against N. caninum infection, but the underlying mechanisms are poorly understood. METHODS: The reactive oxygen species (ROS) inhibitor and the ROS inducer, wild-type (WT) and NLRP3-deficient peritoneal macrophages or mice were used to investigate the role of ROS in NLRP3 inflammasome activation and controlling parasite burdens. ROS production, cell death and cell viability, production of inflammasome-mediated IL-1ß or IL-18, cleavage of caspase-1 and NLRP3 expression, as well as parasite burdens were detected. RESULTS: In vitro, N. caninum induced ROS generation in a dose-dependent manner in peritoneal macrophages. The pretreatment of ROS inhibitor N-acetyl-L-cysteine (NAC) significantly attenuated N. caninum-induced ROS production, LDH release, IL-1ß secretion and NLRP3 expression, whereas N. caninum proliferation was notably increased. In contrary, the ROS inducer pyrogallol (PG) significantly enhanced ROS production and NLRP3 inflammasome activity and decreased the parasite burden in N. caninum-infected peritoneal macrophages. NADPH-dependent ROS-mediated NLRP3 inflammasome activation induced by N. caninum can also be confirmed by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). However, the NAC or DPI pre-treatment or PG treatment did not significantly alter N. caninum-induced inflammasome activities and parasite proliferation in Nlrp3-/- peritoneal macrophages. In vivo, IL-18 releases in serum and parasite burdens in peritoneal exudate cells were significantly increased in PG-treated WT mice after infection with N. caninum; however, IL-18 productions and parasite burdens were not changed in PG-treated Nlrp3-/- mice. Furthermore, PG treatment in WT mice infected with N. caninum significantly decreased the mortality, weight loss and parasite burdens in tissues and histopathological lesions. CONCLUSIONS: Neospora caninum-induced NADPH-dependent ROS generation plays an important role in NLRP3 inflammasome activation and controlling parasites. The ROS inducer PG can control N. caninum infection mainly by promoting NLRP3 inflammasome activation. ROS-mediated NLRP3 inflammasome axis can be a potential therapeutic target for neosporosis.

Newly excysted juveniles of Fasciola gigantica trigger the release of water buffalo neutrophil extracellular traps in vitro.

Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes and are among the first line of immune system defense. PMNs can form neutrophil extracellular traps (NETs) in response to some pathogens. The release of NETs plays an important role in trapping and killing invading parasites. However, the effects of NETs on parasitic trematode infections remain unclear. In the present study, water buffalo NET formation, triggered by the newly excysted juveniles (NEJs) of Fasciola gigantica, was visualized by scanning electron microscopy. The major components of the structure of NETs were characterized by immunofluorescence. Viability of flukes incubated with water buffalo PMNs were examined under light microscopy. The results revealed that F. gigantic juveniles triggered PMN-mediated NETs. These NETs were confirmed to comprise the classic characteristics of NETs: DNA, histones, myeloperoxidase and neutrophil elastase. Although NETs were formed in response to viable larvae, the larvae were not killed in vitro. These results suggest that NET formation may serve as a mechanism to hamper the migration of large larvae to facilitate immune cells to kill them. This study demonstrates, for the first time, that parasitic trematode juveniles can trigger NET formation.

Identification of a TRBD zinc finger-interacting protein in Giardia duodenalis and its regulation of telomerase.

BACKGROUND: Giardia duodenalis causes giardiasis, with diarrhea as the primary symptom. The trophozoite proliferation of this zoonotic parasite is mainly affected by telomerase, although the mechanism of telomerase regulation has not been thoroughly analyzed. METHODS: This study was performed to identify the telomerase RNA-binding domain (TRBD)-interacting protein in G. duodenalis and its regulation of telomerase. Interaction between TRBD and interacting proteins was verified via pulldown assays and co-immunoprecipitation (co-IP) techniques, and the subcellular localization of the protein interactions was determined in vivo via split SNAP-tag labeling. The hammerhead ribozyme was designed to deplete the mRNA of TRBD-interacting proteins. RESULTS: Using TRBD as bait, we identified zinc-finger domain (ZFD)-containing proteins and verified it via pulldown and co-IP experiments. Protein-protein interaction occurred in the nuclei of 293T cells and both nuclei of G. duodenalis. The hammerhead ribozyme depleted ZFD mRNA levels, which reduced the reproduction rate of G. duodenalis, telomerase activity and telomere length. CONCLUSIONS: Our findings suggest that ZFD may regulate telomere function in G. duodenalis nuclei.

Morphological variability, molecular phylogeny, and biological characteristics of the nematophagous fungus Duddingtonia flagrans.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

A novel recombinase polymerase amplification (RPA) assay for the rapid isothermal detection of Neospora caninum in aborted bovine fetuses.

The development of a method to rapidly diagnose Neospora caninum infection is highly desirable. Recombinase polymerase amplification (RPA), combined with lateral flow (LF) strips, is a novel approach to rapidly amplify and visualize DNA. We have developed a prototype LF-RPA assay, using primers and a probe that targeted a specific sequence in the N. caninum NC-5 gene. The N. caninum-specific LF-RPA assay was first tested on purified DNA from oocysts and amplified N. caninum DNA to detectable levels in 10 min, at a constant temperature and without the need for an expensive thermocycler. The designed RPA primers and probe displayed 100% specificity for detecting N. caninum without any cross-reaction with DNA from nine related protozoan spp. (eg Toxoplasma gondii, Sarcocystis gigantean, Sarcocystis zuoi, Hammondia hammondi, Hammondia heydorni, Eimeria cylindrica, Plasmodium falciparum, Theileria annulata and Babesia bigemina). Although, LF-RPA assay detected amounts as low as 50 fg of N. caninum DNA, it was nearly 5-fold less sensitive than previously published qPCR and nested PCR assays. We tested the diagnostic performance of the LF-RPA assay for the detection of N. caninum DNA in aborted bovine fetal tissue samples, and compared the results with those obtained from nested PCR. Out of the 75 samples examined, 18 (24%) and 17 (22.6%) tested positive using LF-RPA and nested PCR, respectively. Our results indicate that LF-RPA is a suitable assay for the rapid and reliable detection of N. caninum.

Seroprevalence of and risk factors for Neospora caninum infection in yaks (Bos grunniens) in China.

The aim of the present study was to detect the seroprevalence of Neospora caninum infection in yaks in Gansu province, China. Serum samples from 974 white yaks and 610 black yaks were tested by a commercial competitive-inhibition ELISA for the detection of specific anti-N. caninum antibodies.; N. caninum antibodies were detected in 10.4% of yaks with higher prevalence in black (11.5% of 610) than white yak (8.6% of 974). Age, regions and sampling times were considered as risk factors associated with N. caninum infection. These results revealed the seroprevalence of N. caninum in white yaks for the first time in China.

Prevalence and Molecular Characterization of Intestinal Trichomonads in Pet Dogs in East China.

The trichomonad species Tritrichomonas foetus and Pentatrichomonas hominis were recently detected in the feces of dogs with diarrhea. However, little information is available on the prevalence and pathogenicity of these parasites in the canine population. Therefore, the aim of this study was to determine the prevalence and molecular characterization of trichomonads infecting pet dogs in Anhui and Zhejiang provinces, east China. In total, 315 pet dogs, with or without diarrhea, from 7 pet hospitals were included in this epidemiological survey. Microscopy and PCR detected P. hominis in 19.7% (62/315) and 31.4% (99/315) of fecal samples, respectively. T. foetus infection was detected in 0% (0/315) of samples with microscopy and in 0.6% (2/315) with PCR. The prevalence of P. hominis was significantly higher in young dogs (≤12 months) than in adult dogs (>12 months), and was significantly higher in diarrheic dogs (50.6%) than in non-diarrheic dogs (24.3%; P<0.05). Infection with T. foetus did not correlate with any risk factors evaluated in this study. A sequence analysis of the P. hominis PCR products showed minor allelic variations between our sequences and those of P. hominis strains from other hosts in different parts of the world. Type CC1 was the most common strain in dogs in east China. The internal transcribed spacer 1 (ITS1)-5.8S rRNA gene sequences from the 2 T. foetus isolates detected in this study displayed 100% identity and were homologous to the sequences of other strains isolated from domestic cats in other countries.

Intestinal microbes influence the survival, reproduction and protein profile of Trichinella spiralis in vitro.

The interactions between intestinal microbes and parasitic worms play an essential role in the development of the host immune system. However, the effects of gut microbes on Trichinella spiralis are unknown. The aim of this work was to explore microbe-induced alterations in the survival and reproduction of T. spiralis in vitro. To further identify the proteins and genes involved in the response of nematodes to microbes, quantitative proteomic analysis of T. spiralis was conducted by iTRAQ-coupled LCMS/MS technology and quantitative real-time-PCR was used to measure changes in mRNA expression. The results showed Lactobacillus acidophilus, and especially Lactobacillus bulgaricus, significantly enhanced the survival and reproductive rates of nematodes. Salmonella enterica, and especially Escherichia coli O157:H7 (EHEC), had opposite effects. Genetic responses were activated mainly by EHEC. A total of 514 proteins were identified and quantified, and carbohydrate metabolism-related proteins existed in a higher proportion. These findings indicated that some gut bacteria are friendly or harmful to humans and in addition they may have similar beneficial or detrimental effects on parasites. This may be due to the regulation of expression of specific genes and proteins. Our studies provide a basis for developing therapies against parasitic infections from knowledge generated by studying the gut microbes of mammals.

Pentatrichomonas hominis: prevalence and molecular characterization in humans, dogs, and monkeys in Northern China.

Pentatrichomonas hominis is an anaerobic amitochondrial flagellated protist that primarily colonizes the large intestines of a number of species, including cats, dogs, nonhuman primates, and humans. The prevalence of this parasite in dogs, monkeys, and humans is, however, poorly understood. In this study, a total of 362 fecal samples including 252 dogs, 60 monkeys, and 50 humans from northern China were collected for an epidemiological survey of P. hominis infection.The average prevalence of P. hominis infection determined by nested PCR was 27.38% (69/252), 4.00% (2/50), and 46.67% (28/60) in dogs, humans, and monkeys, respectively. The prevalence was significantly higher in 6-month-old dogs (41.53%) and children (7.69%) than in older dogs (14.39%) and adults (0%) (P < 0.05). Sequencing of amplicons revealed that four variable positions separated sequences into three types, called CC1-3. CC1 was the most prevalent in the study population. This study determined that P. hominis infection is common in dogs, monkeys, and humans, especially in children and young dogs. Given the infection prevalence, P. hominis may pose a risk of zoonotic and anthroponotic transmission.

[In vitro effect of osthole on ultrastructure of Giardia lamblia].

Giardia lamblia trophozoites were cultivated axenically in TYI-S-33 modified medium containing 1.345 mg/ml of osthole (24 h IC50). The parasites were observed by scanning and transmission electron microscopes after treated with osthole for 24 h. The surface of the trophozoites treated with osthole was rough. The surface of ventral sucker and median body had obvious lesions, the cell membrane was damaged and the content spilled out. There were a lot of vacuoles in the cytoplasm. And the nuclear was severely deformed with a serrated edge and marginated nuclear chromatin. The microtubules of sucker had partially disintegrated.

Pentatrichomonas hominis: first isolation from the feces of a dog with diarrhea in China.

A trichomonad-like parasite isolated from canine fecal samples in Changchun, China was successfully cultivated in vitro using RPMI1640 medium supplemented with 10% heat-inactivated calf serum and antibiotics. These were then subjected to scanning and transmission electron microscopy for ultrastructural study. This parasite has four anterior flagella of unequal length, one independent flagellum, and one recurrent flagellum. It exhibits an anterior nucleus, a Golgi complex, an axostyle, food vacuoles, and hydrogenosomes. These features are consistent with the ultrastructural characteristics of previously described Pentatrichomonas hominis. Polymerase chain reaction and sequence analysis of three genetic loci, including ITS1-5.8S rRNA-ITS2, 18S rRNA, and EF-1α, were also used to compare these samples with other trichomonad species. Molecular identification was also consistent with P. hominis. This is the first time that isolation of P. hominis has been isolated from dog in China, although several other strains of P. hominis have been isolated from human samples.

Eimeria maxima: efficacy of recombinant Mycobacterium bovis BCG expressing apical membrane antigen1 against homologous infection.

Coccidiosis is one of the most important protozoan diseases and inflicts severe economic losses on the poultry industry. The aim of this study was to evaluate the capacity of Bacillus Calmette-Guerin (BCG) to deliver apical membrane antigen1 (AMA1) of Eimeria maxima to stimulate specific cellular and humoral immune responses in chickens. Day-old birds were immunized twice with rBCG/pMV261-AMA1, rBCG/pMV361-AMA1, or BCG via oral, intranasal, and subcutaneous routes and then orally challenged with homologous E. maxima sporulated oocysts. Gain of body weight, fecal oocyst output, lesion scores, serum antibody responses, numbers of splenocyte CD4(+) and CD8(+) T cells, and gut cytokine transcript levels were assessed as measures of protective immunity. Challenge experiments demonstrated that rBCG vaccination via intranasal or subcutaneous routes could increase weight gain, decrease intestinal lesions, and reduce fecal oocyst shedding, and the subcutaneous and intranasal routes were superior to the oral route based on the immune effects. Furthermore, intranasal rBCG immunization could also lead to a significant increase in serum antibody, the percentage of CD4+ and CD8+ T lymphocyte cells, and the levels of IL-1ß, IFN-γ, IL-15, and IL-10 mRNAs compared with the control group. These results suggested that intranasal rBCG immunization could induce a strong humoral and cellular response directed against homologous E. maxima infection. This study provides data for the use of rBCG to develop a prophylactic vaccine against coccidiosis.

Giardia intestinalis: effects of Pulsatilla chinensis extracts on trophozoites.

Pulsatilla chinensis is a medicinal root plant that has been used to treat a wide range of disease conditions. Our study determined the antiprotozoal activity of various P. chinensis extracts and fractions against Giardia intestinalis including their effects on parasite growth, cell viability, adherence, and morphology. Ethyl acetate extracts (IC50 = 257.081 µg/ml) were the most active to inhibit the growth of G. intestinalis followed by aqueous extract (PWE), saponins, and n-butanol extract. The PWE and ethyl acetate extract inhibited G. intestinalis trophozoites adherence after 3 h of incubation and killed almost 50 % of the parasite population in a time-dependent manner. Changes in morphology, presence of precipitates in the cytoplasm, dissolved cytoplasm with large vacuole, break of flagella and ventral disk, membrane blebs, and intracellular and nuclear clearance of the treated trophozoites were observed by scanning and transmission electron microscopy. We demonstrated that P. chinensis induced these changes in G. intestinalis morphology and consequently has potential therapeutic use against giardiasis.

Detection of Trypanosoma lewisi from wild rats in Southern China and its genetic diversity based on the ITS1 and ITS2 sequences.

Trypanosoma lewisi has widely been considered as a non-pathogenic rat trypanosome. However, more and more cases of humans infected with T. lewisi have been reported around the world, indicating that it can infect humans in some undetermined circumstances. Quick and sensitive diagnosis of infection by T. lewisi is important for both treatment of patients and epidemiological studies of this parasite. In this paper, three methods i.e. wet blood smear (diagnosis by microscopy), PCR and LAMP were used to detect T. lewisi from 238 wild rats (Rattus norvegicus) collected from the field in Huadu, Guangdong province, China. Infection rates of these samples detected by the 3 methods was 6.7% (16/238), 12.6% (30/238), and 18.9% (45/238), respectively. LAMP could detect all samples shown positive by microscopical observation of wet smear and by single PCR indicating good potential for application in the detection of T. lewisi. So far as we know, this is the first report of the LAMP method being used to detect T. lewisi in wild rats. The specific T. lewisi LAMP primers were able to amplify the target fragment from the genomic DNA of 19 T. lewisi strains isolated from Huadu, Guangdong province (n=16), Changchun, Jilin province of China (n=1) and from Thailand (n=2). Based on the analyses of ITS1 (internal transcribed spacer 1) and ITS2 sequences, these 19 strains show a very close genetic relationship with over 96-97% similarity to the other corresponding sequences of T. lewisi published in Genbank. Phylogenetic trees of the species in the subgenus Herpetosoma were constructed, based on the ITS1 and ITS2 sequences, and these results also indicate that they are closely related and in the same clade.

[Research progress on anti-tumor mechanism of Trichinella spiralis].

Trichinella spiralis has restrain effect on tumors. Different amount of T. spiralis can emerge different tumor inhibition effect T. spiralis infection can reduce tumor growth to various extents in mice bearing tumor cells at different times post infection. Each developmental stage of T. spiralis in the host may have anti-tumor effect T. spiralis may play anti-tumor roles by stimulating cell-mediated immune response, and/or possessing tumor-associated antigen and anti-tumor active substances of the parasite.

[Prokaryotic expression and identification of S-dsRNA gene from Cryptosporidium parvum virus].

OBJECTIVE: To clone and express S-dsRNA gene of Cryptosporidium parvum virus, and investigate the reactogenicity of the recombinant. METHODS: Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a(+)-S was transformed into E. coli BL21 (DE3) and induced with IPTG. The expression situation of recombinant protein was analyzed by SDS-PAGE. Its reactogenicity was examined by Western blotting analysis. RESULTS: pET-28a (+)-S was identified by PCR and double endonuclease digestion. SDS-PAGE result showed that the recombinant protein (M, 37,000) was expressed in the form of inclusion body. High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 degrees C for 4 h and reached up to 72.6% of the total protein. The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts. CONCLUSION: The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactogenicity.