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Objective To compare the differences of bacterial distribution of intestinal flora in Microtus fortis living under laboratory feeding and wild survival conditions. Methods The 16S rDNA-V4-V5 region of bacteria in the ileocecal contents from Microtus fortis raised in lab and captured in wild were measured by high-throughput sequencing. The number of operational taxonomic units(OTUs)were sorted and calculated,and the species abundance and distribution and difference were analyzed. Results The rarefaction curves indicated that adequate sampling was achieved. At the phylum level,the distribution of intestinal flora between two groups was similar. The experimental group had a unique phylum, Lentisphaerae. The wild type group had 3 unique phylums,Fusobacteria,Thaumarchaeota and an unclassified phylum. At the genus level, the kind of intestinal flora in the wild type group was more abundant than the experimental group. Ruminococcus is the largest differential genus. Conclusions The microbial community structure and differences of Microtus fortis living under different conditions are obtained. It may further enrich the basic biology data of Microtus fortis.
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Objective To explore the biological functions of E77.43, a gene segment of Microtus fortis, in treating Schistoso-ma japonicum infection. Methods Recombinant retroviral vectors of pRevTRE-E77.43 was constructed, and recombinant retro-viral vectors were transfected into PA317 cells, and the stable cell lines were obtained by hygromycin screening, followed by the packaging, concentration and purification of recombinant retrovirus. The virus was transferred to the mice infected by S. japoni-cum via intravenous or intraperitoneal injection, through which the express of target gene and the treatment function in vivo were observed. Results The experiment showed the recombinant virus injected mice could efficiently express E77.43 on the 7th day after the injection which lasted for forty-five days thereafter. A significant reduction in adult worms (31.0％) and a high reduction (35.0％) in liver eggs were induced by pRevTRE-E77.43, while the reduction in adult worms and that in liver eggs was 1.2％and 0.9％induced by pRevTRE respectively (t=3.524, 9.485, both P<0.01). Conclusion pRevTRE-E77.43 could be used for the treatment of S. japonicum infection, indicating that E77.43 may involve in the natural resistance of M. fortis to S. japonicum infec-tion.
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Objective To isolate and culture the spontaneous ascites cells from Microtus fortis under artificial conditions, so as to investigate the molecular mechanism at the cell level. Methods The cells were isolated from spontaneous ascites of M. fortis artificially bred for 90 d,and were cultured and observed under a microscope. The differences of ascites cells among nor?mal,spontaneous ascites and schistosomiasis infected samples of M. fortis were compared. The lesion of tissue was observed si?multaneously. Results There were no obvious organ tissue lesions in M. fortis with spontaneous ascites,and the number and types of cells in peritoneal fluid were irregular and significantly changed. With the extension of culture time ,the colonies ap?peared and there were a large number of vacuole?like cells in the cultured medium and sequentially presenting proliferation ,de?formation,disintegration and the fiber?like changes and could be passaged 3-4 d only. Conclusion The cells from M. fortis with spontaneous ascites are similar to its abdominal cavity cells after infection of Schistosoma japonica.
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Objective To separate and purify intrahepatic macrophages from Microtus fortis Mf and identify its phagocy?tosis. Methods The intrahepatic macrophages from Mf were separated and purified by perfusion collagenase digestion and density gradient centrifugation. The function of the cells was identified by FACS analysis and ink phagocytosis activity. Results The macrophage cells from the liver of Mf were obtained. These cells were bright and circular and grew adhering to the wall. The proportion of the living cells was 95%. The binding rate of these cells from Mf with anti?mouse CD14 antibody Clone Sa2?8 was about 50%of the rate of macrophage from C57BL/6 mice with this monoclonal antibody. The result of ink?phagocytosis ex?periment of macrophage cells from the liver of Mf was positive. Conclusion The method above mentioned is useful to separate and purify macrophage from the liver of Mf. The study builds the foundation for further research on macrophages of Mf against Schistosoma japonicum.
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The principle, basis, necessity and significance of formulating the local standard of Microtu fortis as a laboratory animal were described in this paper, and the standard was compared with the relationship between this standard of Microtu fortis as laboratory animal and the existing laws, regulations of other standards of laboratory animals.The specific procedures and the degree of adoption of domestic standards and advanced foreign standards were introduced.Furthermore, the proposal and the reasons of recommendatory standards were presented.
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Schistosomiasis is an endemic parasite disease and praziquantel is the only drug currently in use to control this disease. Experimental and epidemiological evidence strongly suggests that Microtus fortis ( Mf ) is a naturally resistant vertebrate host of Schistosoma japonicum . In the present study, we found that Mf serum albumin ( Mf -albumin) and the conditioned medium of pcDNA3.1- Mf -albumin caused 46.2% and 38.7% schistosomula death rates in 96 h, respectively, which were significantly higher than that of the negative control (p < 0.05). We also found that mice injected with Mf -albumin had a 43.5% reduction in worm burden and a 48.1% reduction in liver eggs per gram (p < 0.05) in comparison to the control animals. To characterise the mechanisms involved in clearance, schistosomula were incubated with fluorescein isothiocyanate-labelled Mf -albumin and fluorescent enrichment effects were found in the gut lumen of schistosomula after 48 h of incubation. Next, digestive tract excretions from schistosomula were collected and the sensitivity of Mf -albumin to digestive tract excretions was evaluated. The results indicated that schistosomula digestive tract excretions showed indigestibility of Mf -albumin. The death of schistosomula could be partially attributed to the lack of digestion of Mf -albumin by digestive tract excretions during the development of the schistosomula stage. Therefore, these data indicate the potential of Mf -albumin as one of the major selective forces for schistosomiasis.
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Animals , Arvicolinae/parasitology , Schistosoma japonicum/drug effects , Serum Albumin/pharmacology , Chromatography, Affinity , Serum Albumin/isolation & purificationABSTRACT
Objective To investigate the constitution,density changes and carrier rate about Yersinia pestis of rodents in plague foci,and to provide the scientific evidence for plague prevention.Methods According to the program of national monitoring plague,two survey procedures,namely quadrat of single-ha for 24 h and 5 m mouse jam,were used to monitor the host animals; culture and identification of Yersinia pestis in liver or spleen of the experimental animals was carried out by using self-made medium in the north of Beiyuanzi village in Dingbian town Shaanxi province.Results One hundred twelve rodents were captured using the first procedures and the rodent average density was 8.62 ind./hm2 and six species of rodents were found namely Meriones unguiculatus ( 100 individuals),Microtusfortis(5 individuals),Ochotona daurica(3 individuals),Meriones meridianus (2 individuals),Mus musculus Linnaeus (1 individual) and Cricetulus barabensis (1 individual).One hundred seventy-three field mouses were captured using the second procedures including Mus musculus Linnaeus (136 individuals),Cricetulus barabensis (36 individuals),and Microtus fortis ( 1 individual ).Among them,Microtus fortis was found in the salt marshes in the southern edge of Ordos Plateau steppe in plague area of Dingbian county.Yersinia pestis was not identified in all animals.Conclusions Microtus fortis is found in natural foci of plague in Shaanxi province for the first time,and a new geographic region was found.Its epidemiological significance needs further study.
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Objective To obtain the full-length cDNA sequences of CYP2E1,CYP2D5,ECHS1,which may be related with non-alcoholic fatty liver disease,from Microtus fortis.Methods To construct Microtus fortis liver cDNA plasmid library using SMART technique,to get the purposed colonies through screening libraries by PCR,and to obtain their full-length cDNA sequences by sequencing with pBluescript II SK universal primers M13R.Results Three full-length cDNA sequences of Microtus fortis,CYP2E1,CYP2D5 and ECHS1 were obtained.The CYP2E1 cDNA was 1685 bp in length and contained a 1482 bp open reading frame(ORF) encoding a 494 amino acids.The CYP2D5 cDNA was 1690 bp in length,and contained a 1514 bp ORF encoding 504 amino acids.The ECHS1 cDNA was 1013 bp in length,and containsed an 873 bp ORF encoding 290 amino acids.Sequence analysis revealed that the identity of the three cDNA sequences and deduced amino acids among Microtus fortis,Homo sapiens,Mus musculus and Rattus norvegicus was high.Conclusion The full-length cDNA sequences of CYP2E1,CYP2D5,ECHS1 were obtained from Microtus forti,liver cDNA library.and the gene sequences have been deposited in GenBank (GQ507485,GQ507486,GQ845171),which may lay the foundation for researchies of pathogenesis of non-alcoholic fatty liver disease in Microtus fortis models.
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Objective To study whether Microtus fortis can be infected with schistosome in wild. Methods Two villages (Banghu Village of Yueyang County and Nangang Village of Yuanjiang City) were selected as the study pilots. M. fortis were captured from both outside and inside embankment of the 2 villages. The liver, portal vein and mesentery vein of the captured M. fortis were examined for schistosome eggs, adult worms and schistosomula. Results A total of 1 440 M. fortis were captured, and after examined there were no eggs, adult worms and schistosomula of schistosome found. Conclusion M. fortis can not be infected with schistosome in wild environment.
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ObjectiveTo explore the poss ib le mechanisms of killing effects of serum from Microtus fortis to Schistosoma japonicum schisto somula in vitro. MethodsSerum was separated into protein fraction and
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Objective To obtain novel vaccine candidate antigens against Schistosoma japonicum. Methods S. japonicum schistosomula cDNA library was screened by using sera of Microtus fortis that was naturally resistant to schistosomiasis. The positive clones were transformated into Escherichia coli BM25.8, E. coli clones containing the plasmid cultured in LB, and then selected for plasmid extraction, the plasmid DNA was digested by EcoRⅠand Hind Ⅲ, and analysed by agarose gel electrophoresis. The positive clones were also sequenced and the data were analysed through the internet Nucleotide BLAST software of NCBI and Expert Protein Analysis system of GeneRunner and HNN. Results Twelve positive clones were obtained after repeatedly immunoscreening the library and their sizes ranged from 300 bp to 1100 bp. Two novel genes (named as Sj-sMf1 and Sj-sMf2) with complete ORF were obtained. The deduced protein of Sj-sMf1 consisted of 93 amino acids while Sj-sMf2 consisted of 61 amino acids. Sj-sMf1 protein predicted containing one cAMP phosphorylation site and Casein kinase C phosphorylation site, respectively. Sj-sMf1 protein predicted containing one Casein kinase C phosphorylation site and two Protein kinase C phosphorylation sites. Conclusion Two novel genes predictably encoding unknown proteins are obtained from immunoscreening of Schistosoma japonicum schistosomula cDNA library by M. fortis sera.
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Objective To study the killing effects of fractional proteins from Microtus fortis (Mf) serum on schistosomula of Schistosoma japonicum. Methods Mf serum proteins were separated into albumin and globulin by means of salt out of ammonium sulfamate. The globulin was then separated into 4 big and 12 small fractional proteins through Sephacryl S-300 column chromatography and electrophoresis elution. The killing effects were observed in vitro in cultivation in which the purified fractional proteins and schistosomula of S. japonicum were incubated together for 48 h. Results The mortality rate of schistosomula acted by Mf globulin was 59.2% and when added with complements was 68.4%. The killing effects of the 2nd and 3rd big fractional proteins were the same as that of Mf globulin. Three small fractional proteins (3.2, 3.3, 3.4) showed the higher killing effects which made the mortality rate of schistosomula 45.1%, 57.6% and 67.2%, respectively. The fractional protein of 100-135 kDa also showed the same killing effect as that of Mf globulin. Globulin from BALB/c mice sera had no significant effect on schistosomula. There was no significant difference in the mortality rate of schistosomula acted by both albumins. Conclusions Mf globulin has significant killing effects on schistosomula of S.japonicum in vitro and 100-135 kDa fractional protein may be an important effective molecule.
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Objective To look for the genes of Schistosoma japonicum related to the Schistosoma-resistance of Microtus fortis.Methods The fresh sera of Microtus fortis were used to screen a T7 phage display cDNA library from worms of Schistosoma japonicum established in our lab.The positive clones were sequenced and functionally analysed through bioinformatics.Results The specific phages binding to the sera of Microtus fortis were enriched 857-fold after three rounds of biopanning,and 58 positive clones picked at random were sequenced and 10 ESTs were obtained.BLASTn results showed that 7 ESTs had 99%-100% similarity to the genes of Shistosoma japonicum reported in GenBank and 1 EST had 82% similarity to a zinc finger protein encoden gene from Pan troglodytes.The results of these ESTs function prediction indicated most of them were involved in the regulation of gene expresion of Schistosoma japonicum.Conclusions Several target genes of Schistosoma japonicum related to the Schistosoma-resistance of Microtus fortis are obtained and those would lay foundation to expatiate the native resistance mechnism of Microtus fortis to Schistosoma japonicum.
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Objective To construct a T7 phage display cDNA library from the lung of Microtus fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the double strain cDNA was given with EcoRⅠ and Hind Ⅲ adhering ends by ligation with the directional EcoRⅠ/Hind Ⅲ linkers and digestion with EcoRⅠ and Hind Ⅲ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoRⅠ and Hind Ⅲ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5?106 clones and the titer of the amplied library was 1.1?1012 pfu/ml. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion A T7 phage display cDNA library from the lung of Microtus fortis is successfully constructed.