Prevalence and genotyping of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality / 中国血吸虫病防治杂志

Objective To investigate the prevalence and genotypes of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality. Methods A total of 145 fresh fecal samples were collected from pet dogs and cats in a pet hospital in Shanghai Municipality during the period from November 2021 to June 2022, including 99 dog fecal samples and 46 cat fecal samples. The small subunit ribosomal ribonucleic acid (SSU rRNA) gene of Cryptosporidium and the triose phosphate isomerase (TPI) gene of G. lamblia were amplified using nested PCR assay, and the positive amplification products were sequenced from both directions. The sequence assembly was performed using the software Clustal X 2.1, and sequence alignment was conducted using BLAST. A phylogenetic tree was created with the Neighbor-Joining method using MEGA 11.0 to identify parasite species or genotype. Results The overall prevalence of Cryptosporidium and G. lamblia was 20.00% (29/145) in 145 pet dog and cat fecal samples, with the prevalence of 0.69% (1/145) and 19.31% (28/145) in Cryptosporidium and G. lamblia, respectively. G. lamblia was only detected in dog fecal samples, with prevalence of 18.18% (18/99), while the detection rates of Cryptosporidium and G. lamblia were 2.17% (1/46) and 21.74% (10/46) in cat fecal samples. Nucleotide sequence analysis showed that one Cryptosporidium positive sample was characterized as C. felis, and 28 G. lamblia positive samples were all characterized as Giardia assemblage A, which showed 100% sequence homology with human isolates of Giardia. Phylogenetic analysis revealed that the sequences obtained in this study belonged to the same branch with the reported Giardia assemblage A. Conclusions Cryptosporidium and G. lamblia infection was prevalent in pet dogs and cats from the study pet hospital in Shanghai Municipality, and there is a zoonotic risk for the species and genotype. Intensified surveillance of Cryptosporidium and Giardia infection is recommended in pets and their owners, and improved management of pet keeping is required.

Retrospective analysis of multiple factors combination for early identification of pulmonary tuberculosis complicated with pulmonary embolism / 中华传染病杂志

Objective:To analyze the independent diagnostic indicators and their diagnostic values for pulmonary tuberculosis complicated with pulmonary embolism.Methods:A total of 34 cases of pulmonary tuberculosis complicated with pulmonary embolism treated in Huzhou Central Hospital from March 2014 to September 2021 were enrolled. And 136 patients with simple pulmonary tuberculosis who were hospitalized during the same period were collected with a ratio of 1∶4 according to the principle of age and gender matching. The general conditions, clinical symptoms, comorbidities and laboratory indicators of the patients were retrospectively analyzed. The univariate analysis was performed using independent samples t test, Mann-Whitney U test and chi-square test. Binary logistic regression was used to analyze the related diagnostic factors for pulmonary embolism in pulmonary tuberculosis patients, and the combined factors were constructed by transforming the model equation, and the receiver operating characteristic (ROC) curve was used to determine the optimal cut-off value and evaluate its diagnostic value. Results:The univariate analysis showed that patients with pulmonary tuberculosis complicated with pulmonary embolism had higher ratio of chest tightness (67.6%(23/34) vs 22.1%(30/136)), syncope (23.5%(8/34) vs 0.7%(1/136)), fever (55.9%(19/34) vs 36.0%(49/136)), hemostatic drug use (100.0%(34/34) vs 13.2%(18/136)), history of venous thrombosis (8.8%(3/34) vs 0.7%(1/136)), atrial fibrillation (11.8%(4/34) vs 2.2%(3/136)) and D-dimer levels (4.090 0(1.035 0, 10.790 0) mg/L vs 0.850 0(0.432 5, 2.145 0) mg/L) than those of simple pulmonary tuberculosis patients, and the differences were all statistically significant ( χ2=26.35, 28.19, 4.47, 96.44, 7.75, 6.30 and Z=-4.65, respectively; all P<0.050). The arterial partial pressure of oxygen (PaO 2)(61.90(52.95, 73.00) mmHg vs 82.00 (75.00, 87.00) mmHg, 1 mmHg=0.133 kPa) and albumin ((28.83±4.98) g/L vs (32.76±5.65) g/L) of patients with pulmonary tuberculosis complicated with pulmonary embolism were lower than those of simple pulmonary tuberculosis patients, and the differences were both statistically significant ( Z=-5.21 and t=3.71, respectively, both P<0.001). Binary regression analysis showed that chest tightness (odds ratio ( OR)=3.494, 95%confidence interval ( CI) 1.208 to 10.100, P=0.021), D-dimer ( OR=1.285, 95% CI 1.079 to 1.530, P=0.005) and PaO 2( OR=0.931, 95% CI 0.895 to 0.970, P=0.001) were the independent diagnostic indicators for pulmonary embolism in pulmonary tuberculosis patients. The areas under the ROC curve of chest tightness, D-dimer, PaO 2, and the combination of the three indicators (the combination factor) were 0.728, 0.758, 0.834, and 0.890, respectively. The optimal cut-off value of the combination factor was -3.1, with the sensitivity of 0.824 and the specificity of 0.824. Conclusions:Chest tightness, increased D-dimer and decreased PaO 2 are independent diagnostic indicators for pulmonary embolism in pulmonary tuberculosis patients. It is recommended to perform pulmonary artery computed tomography angiography promptly when the combination factor is higher than -3.1 to determine whether the patient is complicated by pulmonary embolism.

Clinical characteristics of 10 cases of brucellosis in Huzhou City, Zhejiang Province / 中华地方病学杂志

Objective:To analyze the clinical characteristics of brucellosis in Huzhou City, Zhejiang Province, so as to provide basis for clinicians to improve their understanding and diagnosis and treatment of brucellosis.Methods:The data of 10 cases of brucellosis in Huzhou Central Hospital from 2014 to 2019 were collected, and the epidemiological characteristics, clinical manifestations, laboratory examination results, imaging examination results, etiological examination results, diagnosis and treatment process and prognosis were summarized and analyzed by retrospective analysis method.Results:Among the 10 patients with brucellosis, 6 were males and 4 were females, aged from 32 to 71 years old, and 9 patients had a history of contact with goat, and one case was considered as food borne infection. The main clinical manifestations were fever (10 cases), sweating (2 cases), joint pain (7 cases). In imaging examination, 5 cases with splemomegaly, 1 cases with lymphadenopathy, 3 patients presented with spondylitis (including 1 case with paravertebral abscess) and 2 cases with prostatitis; Brucella was cultured in 2 patients and Brucella antibodies were positive in 10 patients. The treatment drugs included doxycycline, rifampicin and levofloxacin in two or three combinations. Eight patients were followed up for more than 1 month to more than 5 years, and the prognosis was good. Conclusions:Brucellosis lacks specificity in clinical features. Most patients with brucellosis are associated with osteoarthritis or urinary system diseases. Physicians should be vigilant, combine epidemiological features, conduct laboratory tests in time, and diagnose as soon as possible.

Cyclospora cayetanensis infections among diarrheal outpatients in Shanghai: a retrospective case study / 医学前沿

Cyclospora cayetanensis is a foodborne and waterborne pathogen that causes endemic and epidemic human diarrhea worldwide. A few epidemiological studies regarding C. cayetanensis infections in China have been conducted. During 2013, a total of 291 stool specimens were collected from patients with diarrhea at a hospital in urban Shanghai. C. cayetanensis was not detected in any of the stool specimens by traditional microscopy, whereas five stool specimens (1.72%, 5/291) were positive by PCR. These positive cases confirmed by molecular technology were all in the adult group (mean age 27.8 years; 2.94%, 5/170) with watery diarrhea. Marked infection occurred in the rainy season of May and July. Sequence and phylogenetic analyses of the partial 18S rRNA genes of C. cayetanensis isolated showed intra-species diversity of this parasite. This study showed, for the first time, that C. cayetanensis is a pathogen in outpatients with diarrhea in Shanghai, albeit at a low level. However, the transmission dynamics of this parasite in these patients remain uncertain.

In vitro pro ⁃ angiogenic activity of Echinococcus granulosus hydatid cysts from experimentally infected mice / 中国血吸虫病防治杂志

@#To preliminarily study the pro⁃angiogenic activity of Echinococcus granulosus hydatid cysts against hu⁃ man umbilical vein endothelial cells in vitro and the transcriptional level of potential pro⁃angiogenic factors. Methods The hydatid cysts and protoscolex derived from experimentally infected mice were collected and cultured in vitro，then the human umbilical vein endothelial cells were stimulated by the supernatant and cyst fluid respectively，and the angiogenesis was observed and analyzed through a microscope and the angiogenesis mode of the software NIH Image J. Meanwhile，the mouse homologous proteins of matrix metalloproteinase⁃9（MMP⁃9）and high mobility group box B1（HMGB1）were identified in E. granulosus genome through sequence alignment，and their transcriptional levels in the cyst wall and protoscolex were analyzed. Results The culture supernatant of hydatid cysts significantly promoted human umbilical vein endothelial cells into tubes（F = 73.03，P < 0.001），the transcriptions of MMP⁃9 and HMGB1 were detected in the cyst wall and protoscolex，and the transcriptional level of MMP⁃9 was higher in protoscolex（t = -11.65，P < 0.001），while the level of HMGB1 was higher in hydatid cysts（t = 6.43，P = 0.003）. Conclusion Some parasite⁃derived pro⁃angiogenic molecules may exist in the supernatant of E. granulosus hydatid cysts，while further researches are required into their exact mechanisms.

Epidemiology status of some new emerging Cryptosporidium species and genotypes in humans / 中国人兽共患病学报

Cryptosporidium spp.are protozoan parasites that infect the epithelial cells of the gstrointestinal tract of hosts.In humans,cryptosporidiosis is usually a self-limiting infection in immunocompetent individuals,but severe diarrhea and dissemination to extra-intestinal sites can occur in high-risk individuals,such as the very young,the elderly,immunedeficiency individuals,particularly in HIV-positive patients.So far,molecular epidemiological data have confirmed the presence of 30 species and over 40 genotypes with genus Cryptosporidium,with 21 species and genotypes being found in humans.The majority of human cryptosporidiosis cases are responsible for C.hominis and C.parvum.Human cases caused by C.meleagridis,C.ubiquitum,C.felis and C.canis have been increasing.Besides that,with data accumulation of molecular epidemiology of human cryptosporidiosis,some more Cryptosporidium species and genotypes were newly identified in humans.This paper mainly reviews epidemiology status of these new emerging Cryptosporidium species and genotypes in humans.

Observation on destroyed architecture of splenic lymphoid follicles in mice infected with Schistosoma japonicum by immunohistochemistry / 中国血吸虫病防治杂志

Objective To observe the destroyed architecture of splenic lymphoid follicles in mice infected with Schistosoma japonicum by immunohistochemistry. Methods The mice infected with S. japonicum(20 cercariae/mouse)for 8 weeks were sacrificed,and the splenic samples were paraffin embedded and sliced. The sections were first stained by hematoxylin and eosin to observe the massive structure of splenic lymphoid follicles,and then B cells,follicular dendritic cells(FDC)and germinal center cells were labeled with anti-B220,anti-CD21 or anti-Ki67 antibodies respectively by immunohistochemistry to observe the distribution of the specific cells of lymphoid follicles. Results The results of HE staining showed that the structure of lym-phoid follicles in spleens of infected mice was blurred,the number and area of follicles were significantly reduced compared to those of the normal mice. The immunohistochemical staining showed that the splenic T/B lymphocyte segregation ,FDC network and germinal centers of the infected mice all disappeared. Conclusion The structure of splenic lymphoid follicles in the mice infected with S. japonicum is obviously damaged.

Molecular characteristics and RNA interference efficiency of Schistosoma ja-ponicum Sj79 gene / 中国血吸虫病防治杂志

Objective To study the structural features and characteristics of a novel gene Schistosoma japonicum 79(Sj79), and observe its effect of RNA interference(RNAi),so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. Methods The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA(mRNA)during the different develop?mental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The tran?scriptional levels of Sj79 after RNAi were detected by real time PCR. Results The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity,and its transcriptional level in mature female worms was the highest. After soaking for 3 d,the Sj79 mRNA level[(41.0 ± 12.3)%]in the siRNA?1 group with low dosage(20 nmol/L) was lower than that in the siRNA?NC group[(103.2 ± 14.4)%],the difference was statistically significant(t=3.28,P<0.05). When with high dosage(200 nmol/L ),both the Sj79 mRNA levels in the siRNA?1 group[(15.8 ± 10.9)%]and siRNA?2 group [(11.1 ± 8.8)%]were significantly lower than that in the siRNA?NC group[(100.1 ± 6.3)%](t=13.44,27.84,both P<0.01). After soaking for 7 d,only the Sj79 mRNA levels in the siRNA?1group[(43.4 ± 4.5)%]and siRNA?2 group[(62.5 ± 5.4)%]with low dosage were lower than that in the siRNA?NC group[(100.4 ± 5.2)%],and the differences had statistical sig?nificance(t=8.33,5.07,both P<0.01). Conclusion Through this study,we have improved the mRNA sequence and genom?ic information of Sj79 gene,and understood its structural features,as well as selected out two effect fragments siRNA?1and siR? NA?2 which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schisto?some in vitro.

Role of zinc finger protein 1 in rat liver fibrosis and as related to TGFb expression / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To determine the role of zinc finger protein 1 (ZEB 1) in liver fibrosis and in regards to expression of the tumor growth factor-beta (TGFb) signaling factor using a rat model system.</p><p><b>METHODS</b>Sprague-Dawley rats were randomly divided into a normal (control) group, liver fibrosis (model) group and a liver fibrosis + therapy (ZEB1 intervention) group. The model group and the ZEB1 intervention group were given intraperitoneal injections of dimethylnitrosamine (DMN) for the first 3 days of each week over a 7-week period; starting at week 5, the ZEB 1 intervention group was started on a routine of every other day tail vein injections of recombinant ZEB1. During this 7-week period, the control group was given intraperitoneal injections of 0.9% NaC1 alone on the DMN schedule. Liver tissues were collected for pathological examination (with hematoxylin-eosin and Masson staining) and for detection of TGFb1 and ZEB 1 expression (by RT-PCR and western blotting). Measurement data were compared between groups using the single-factor analysis of variance test, followed by the least significant difference LSD test. Count data were analyzed by Fisher's exact test.</p><p><b>RESULTS</b>The model group's liver tissues showed degeneration and necrosis, as well as obvious fibrous septa accompanied by pseudo lobules. The ZEB 1 intervention group's liver tissues showed a significantly higher degree of fibrosis (x²=21.63, P=0), with more coarse fiber cords. The expression of ZEB1 and TGFb1 was significantly higher in the model group than in the control group (both P less than 0.05). However, the ZEB 1 intervention group showed the highest levels of ZEB 1 and TGFb1 expression (vs. model group, P less than 0.05).</p><p><b>CONCLUSION</b>ZEB 1 may promote the development of liver fibrosis in rats through a mechanism involving the TGFb/Smad signaling pathway.</p>

Study on Nogo-B participating in transforming growth factor-β1/Smad2 signaling pathway in mice models of hepatic fibrosis / 中华传染病杂志

Objective To study the relationship between Nogo-B and transforming growth factor-β1 (TGF-β1)/Smad2 signaling pathway in mice models of hepatic fibrosis.Methods Twenty four healthy male ICR mice were divided into two groups,with 6 in the control group and 18 in the model group.Mice in the model group were further divided into three subgroups according to different time points:subgroups of 4,8 and 12 weeks,with 6 mice in each subgroup.Hepatic fibrosis of mice was induced by subcutaneous injection of carbon tetrachloride (CCl4).The histopathologic changes of the liver were observed by optical microscope using hematoxylin-eosin and Masson trichrome stainings of the liver tissues.Expressions of Nogo-B,Smad2 and TGF-β1 mRNA and proteins in liver were detected by reverse transcription-polymerase chain reaction (RT-PCR),Western blot and immunohistochemistry assays,respectively.Means among groups were compared by univariate analysis of variance.Results The hepatic fibrosis models were successfully induced by CCl4 injection.The expressions of two subtypes of Nogo-B,Nogo-B1 and Nogo-B2 mRNA in normal livers were 0.140±0.050 and 0.104±0.023,but both significantly increased in the livers of mice in the 12 week model subgroup (1.054±0.040 and 0.500±0.057,F=431.41 and 135.46,respectively; both P＜0.01).The Nogo-B protein was mainly expressed in nonparenchymal cells of the liver,and was hardly expressed in hepatocytes.Linear correlation analysis showed that the expressions of Nogo-B mRNA and proteins were positively correlated with Smad2 and TGF-β1 mRNA and proteins (all P＜0.01),which were considered to participate in the signaling pathway of hepatic fibrosis.Conclusion Nogo-B might play a role in the development and progression of hepatic fibrosis by participating in TGF-β1/Smad2 signaling pathway.

The identification of the Cryptosporidium ubiquitum in pre-weaned Ovines from Aba Tibetan and Qiang autonomous prefecture in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Cryptosporidium spp. are prevalent globally and sheep are an important zoonotic reservoir. Little data regarding the rates of Cryptosporidium infections in ovines in China are available. This study assessed the prevalence of Cryptosporidium spp. in pre-weaned ovines from Aba Tibetan and Qiang Autonomous Prefecture in the Sichuan province of China.</p><p><b>METHODS</b>A total of 213 fecal samples were collected from pre-weaned ovines and were examined microscopically (following modified acid fast staining). In addition, 18S rRNA genetic sequences were amplified from fecal samples by nested PCR and phylogenetically analyzed.</p><p><b>RESULTS</b>The prevalence of Cryptosporidium in the collected samples was at 14.6% (31/213) and four isolates identified by PCR belonged to the Cryptosporidium cervine genotype (Cryptosporidium ubiquitum) demonstrating that this species was the primary sheep species found in sheep in China.</p><p><b>CONCLUSION</b>The present study suggested that the high incidence of Cryptosporidium in sheep poses a significant public health threat and that surveillance practices must be established to prevent zoonotic disease of humans.</p>

Immunoscreeening of Schistosoma japonicum schistosomula cDNA library with sera from Microtus fortis and bioinformatic analysis of novel genes / 中国血吸虫病防治杂志

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.

Cloning and expression of gene encoding myophilin-like protein of Schistosoma japonicum and study on the antigenicity of recombinant protein / 中国血吸虫病防治杂志

Objective To clone and express the gene encoding Schistosoma japonicum myophilin-like protein (SjcMLP) and to study the antigenicity of the recombinant protein. Methods The SjcMLP gene was amplified by PCR. The PCR product was cloned into T vector, and then subcloned into expression vector pQE30. The recombinant plasmid of pQE30-SjcMLP was transformed into E.coli M15, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analysed by SDS-PAGE. The recombinant protein (reSjcMLP)was purified with the Ni-NTA resin, and analysed with SDS-PAGE and Western blot. The titers of sera from C57BL/6 mice immunized subcutaneously with reSjcMLP were detected by ELISA. Results The results of SDS-PAGE and Western blot showed that the molecular weight of expressed fusion protein was around 24.8 kDa and was recognized by the sera from the mice infected with Schistosoma japonicum. The purified protein of reSjcMLP was coated for ELISA test and the IgG titers in the sera from the mice immunized with reSjcMLP were as high as 1∶12 800 reacted with. However, no significant difference was found in worm reduction rates between the immunized mice and control mice. Conclusions The fused recombinant protein of reSjcMLP is successfully ex-pressed and purified. The recombinant protein in this experiment fails to induce significant protection against the challenge infection in C57BL/6 mice.

Cloning and expression of the gene encoding hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chinese-strain hypoxanthine-guanine phosphoribosyltransferase(HGPRT)and its expression in Escherichia coli.MethodsA couple of primers were designed with the BamHI restriction endonuclease site introduced in forward primer and SalI in reverse primer.Total RNA was isolated from adult worms of S.japonicum Chinese-strain(Anhui-strain,Sjc-A)and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHI and SalI.The target DNA fragments were purified and cloned properly into pET28a.After identification by en-donucleases digestion,PCR and sequencing,the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E.coli BL21 and expressed in the presence of IPTG.Results pET28a-SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S.japonicum Chinese-strain(Hunan-strain,Sjc-H)and S.mansoni HGPRT,respectively.The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica.Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein(reSjcHGPRT)is also successfully purified.

Preparation of DNA from Cryptosporidium parvum Oocysts for PCR Detection / 中国寄生虫学与寄生虫病杂志

Objective To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. Methods After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C.parvum gene (L16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex-100 method was also tested by PCR. Results One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. Conclusion The three kinds of extraction can all be served as templates for PCR detection of C.parvum oocysts, while Chelex-100 method is simpler, quicker and more reliable for DNA extraction of the parasite.

Dendritic Cells and Th2 Response Induced by Schistosome Infection / 中国寄生虫学与寄生虫病杂志

Dendritic cells act as the major antigen presenting cells in the body and play a central role in intri-guing the adaptive immune response. Protective immunity against schistosome and immuno-pathological response in host caused by eggs are both closely associated with Th2 response. Further understanding on immune mechanism will contribute to the development of vaccines against schistosome infection, as well as the relief of the pathological lesion in schistosomiasis. This article discusses the central role of dendritic cells in the mechanism of Th2 response induced by schistosome (including eggs).

Cloning, Expression and Analysis of the Heat Shock Protein of Cryptosporidium andersoni / 中国寄生虫学与寄生虫病杂志

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

Isolation and Identification of an Isolate of Cow-origin Cryptosporidium sp / 中国寄生虫学与寄生虫病杂志

Objective To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. Methods Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheather sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene (SSU rRNA) and Cryptosporidium oocyst wall protein gene (COWP), respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. Results The results suggested that the size of oocysts was (7.4?0.32)?m by(5.4?0.21)?m and the ratio of length and width was 1.37?0.07 (n=20). BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of C.andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C.andersoni clade based on the sequence of SSU rRNA and COWP gene. Conclusion What isolated from naturally infected cow feces has been identified as C. andersoni.

Protective Immunity Induced by Recombinant Schistosoma japonicum Thioredoxin in Mice / 中国寄生虫学与寄生虫病杂志

Objective To investigate the protective immunity of the recombinant thioredoxin of Schistosoma japonicum(reSjcTrx)in mice. Methods Thirty 6-week old female C57BL/6 mice were randomly divided into 3 groups with 10 each: reSjcTrx with Montanide ISA720 adjuvant, adjuvant control, and infection control. Mice were vaccinated subcutaneously at week 0, 2, 4 with reSjcTrx emulsified in Montanide ISA720 adjuvant. The mice in adjuvant group was injected three times with Montanide ISA720 and saline only. Mice in infection control group were given no injection. Three weeks after final injection, each mouse was challenged with 30?1 cercariae of S. japonicum (Chinese strain). At the week six after challenge, all mice were sacrificed and perfused. The number of recovered worms and eggs from liver tissue of mice were counted. Sera were collected from mice before immunization, before challenge and before killing. The anti-SjcTrx antibodies in sera were detected with ELISA. Results ELISA showed a high level of specific IgG antibodies in mice immunized with the reSjcTrx. The worm reduction rate and egg reduction rate of reSjcTrx immunization group were 22.8% and 29.5% respectively, significantly higher than those of the control groups (P﹤0.05). SDS-PAGE and Western blotting revealed that the molecular weight of expressed protein was around Mr 14 000 and could be recognized by sera from rabbit infected with S.japonicum and from mice immunized with reSjcTrx. Conclusion The reSjcTrx induces certain protective immunity against schistosomiasis japonica in mice.