| Sparganosis is a serious food borne parasitic zoonosis caused by infection with sparganum. Sparganosis is sporadically distributed worldwide. Sparganosis poses a serious threat to human health. The larvae usually lodge in the subcutaneous tissues, sometimes invades the abdominal cavity, eye, and central nervous system causing blindness, epilepsy, paralysis, and even death. Traditionally, the species identification of Spirometra is by morphological observation, but the absence of distinguishing morphological characteristics in the larval stage does not allow the species to be easily identified. However an accurate identification of parasites and their characterization at different taxonomic levels has important implications for the prevention and control of parasitic diseases. The antigenic polypeptide from sparganum (antigenic polypeptide, SAP, GenBank No. AB019222.1) may be the protective antigen, the antigenic polypeptide plays an important role in evasion of the immune response.It might be a potential candidate for sparganosis vaccine.In this study, all spargana were collected from naturally infected frogs caught from a field site in six geographical locations (Luohe, Kaifeng, Xinxiang, Tongxu and Fugou) of Henan Province. Specific pathogen-free domestic cats were orally inoculated with plerocercoids from six locations.Choose three target genes (i.e., coxl, cox3and pnad4), Genetic variance analysis of six geographical locations. We expect to clone and express the protein SAP gene of sparganum with the technique for gene engineering. Mice were immunized by the purified recombinant protein and the serum against the recombinant protein was collected. SDS-PAGE and Western blot could be taken to research the antigenicity and immunogenicity of the recombinant protein. Indirect immunofluorescence assay (IFA) analyse the distribution of SAP protein in the worm. SAP-ELISA and ES-ELISA were used to assay anti-sparganum IgG antibodies in sera from mice infected. Materials and Methods1. Collection and examination of sparganum:All spargana were collected from naturally infected frogs caught from a field site in six geographical locations (Luohe, Kaifeng, Xinxiang, Tongxu and Fugou) of Henan Province. Six3-month-old specific pathogen-free domestic cats were orally inoculated with10plerocercoids from six locations. Faecal samples were microscopically examined by sedimentation during10-25days post infection (dpi), and cats were euthanized and examined for adult worms in small intestine at28dpi.2. Amplification and analysis of target genes of larvae and adult worms:Three target genes (i.e., coxl, cox3and pnad4) were amplified by polymerase chain reaction (PCR), and PCR products were purified and sequenced in both directions at the Genwiz Company (Beijing, China). Sequences of the three genes were initially aligned using the default settings in Clustal X v2.0and adjusted in Se-A1v2.0all. Uncorrected pairwise divergence was estimated by program MEGA v5.0.3. Phylogenetic analyses:The concatenated sequence of coxl, cox3and pnad4was analyzed by using three methods of maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) performed in PAUP*4b10, PhyML v3.0and MrBayes v3.1, respectively.4. Molecular cloning and expression of the SAP:The protein products were cloned into the expression vector pMAL-c2x with BamH I and HindⅢ sites. Then the recombinant plasmid was transformed into E. coli BL21(DE3). The expression of the recombinant protein was induced with1PTG. SDS-PAGE was used for the detection of the expressed products and the soluble analysis.5. Identification of the pMAL-c2X-SAP recombinant protein:The recombinant protein was purified by affinity chromatography through Amylose resin column. The recombinant protein was detected by Western blot with sera from the mice infected with sparganum. pMAL-c2X-SAP immune serum was collected from BALB/c mice injected subcutaneously with the recombinant protein. ES antigens and crude extract antigens from sparganum were detected by Western blot with pMAL-c2X-SAP immune serum.The different development stage of Spirometra mansoni was tested by IFA using the anti-pMAL-c2X-SAP serum to determine whether the protein SAP was located on the surface of the parasite or inner organs. SAP-ELISA and ES-ELISA were established using SAP and ES proteins of sparganum and other parasites, respectively and their sensitivity and specificity were evaluated. Results1. Morphological identification of eggs and adult worms:Spirometra tapeworm eggs were found in the faeces of cats experimentally infected with spargana at11dpi, and adult worms,26-45cm long, were recovered from the small intestine at28dpi. The egg with a thin shell is ellipsoidal, light brown in color, measuring52-76μm×31-44μm in size, containing one ovum and many yolk cells. The egg is tapered at two ends, one of which has an operculum. Adult worms were morphologically identified as S. mansoni according to the following features:scolex with2longitudinal grooves, mature and gravid proglottids with conspicuous uterus at the center of segments, spiral-shaped uterus, and there are three openings at the center of proglottid, including male-genital pore, female-genital pore, and uterine pore.2. Genetic variance analysis of collected isolates:All amplifications were successful, with fragments417bp,390bp, and578bp for cox1, cox3and pnad4respectively. And the length of corresponding sequence was identical between larvae and adult worms. The base composition of coxl, cox3and pnad4was generally AT rich with a mean of63.5%,68.3%and67%, respectively. The genetic divergence of coxl, coxi and pnad4sequences of Henan sparganum isolates ranged0-4.4%,0-0.8%and0-0.5%, respectively.3. Phylogenetic analysis of Henan sparganum isolates:The substitution models were TN93+I for coxl, HKY+G for cox3and HKY+I for pnad4. The combined analysis of cox1+cox3+pnad4revealed that one clade including all Henan sparganum isolates and other Spirometra isolates was supported by all three phylogenetic inference methods (MP, ML and BI) with high support values (100/99/1.0), and this clade had sibling relationship with the clade containing all Diphyllobothrium tapeworms.4. Molecular cloning and expression of the SAP:After being induced with1mM IPTG, the recombinant bacteria (BL21with pMAL-c2X-SAP) expressed a68.7kDa fusion protein and the size was in agreement with the predicted combined size of maltose binding proteinase(MBP) from the vector (40kDa) and the interest proteins(28.7kDa). The recombinant plasmid pMAL-c2X-SAP has been successfully constructed. The results showed that the expressed recombinant protein existed in a soluble form.5. Identification of the pMAL-c2X-SAP recombinant protein:Western blot showed that the recombinant pMAL-c2X-SAP can be recognized with the infection serum and anti-pMAL-c2X-SAP serum, but it was not immunostained with normal serum. And a component of ES antigens and crude extract antigens of approximately28.7kDa was recognized with the anti-pMAL-c2X-SAP serum. The immunofluorescence result showed that, the anti-pMAL-c2X-SAP serum reacted with the SAP localized on the sparganum. Not expressed in adult stage.The bright green fluorescence mainly distribute at the cuticle and the internal organization. The sensitivity of SAP-ELISA and ES-ELISA in detecting the serum samples of mouse with sparganosis was83.3%(25/30) and100%(30/30),respectively. However, the mouse sera collected from mice infected with T. gondii, T. spiralis, S. japanicum and before infection showed negative response by both SAP-ELISA and ES-ELISA.Conclusions1. Morphological and molecular analyses all showed that the sparganum isolates in Henan province represented Spirometra mansoni. The genetic variability among Spirometra sparganum isolates could be revealed by sequences of three mitochondrial DNA genes. For the three mtDNA genes, genetic variation of coxl was higher than cox3and pnad4, and cox3was higher than pnad4.2. The recombinant plasmid pMAL-c2X-SAP which has been successfully constructed. Western blot and IFA showed that the expressed recombinant protein existed in a soluble form. the SAP localized on the sparganum cuticle and the internal organization.3. SAP-ELISA which was used to detect the specific anti-sparganum IgG antibodies in mouse sera infected with sparganum, had good sensitivity and specificity, and SAP could be used for serological diagnosis for sparganosis. |
PDF Full Text Request