[Objective] The study was to screen the Chinese medicines with good resistance to Neospora caninum. [Method] Healthy Kunming mice pretreated with methylprednisolone for the purpose of immunity decrease were randomly d...[Objective] The study was to screen the Chinese medicines with good resistance to Neospora caninum. [Method] Healthy Kunming mice pretreated with methylprednisolone for the purpose of immunity decrease were randomly divided into 15 groups, and each mouse was intraperitoneally inoculated with 1.0 ×104 N. caninum. Four hours later, the mice were gavaged with various Chinese medicines. Seven days post-administration, eyeball blood sampling was conducted and the serum was used for antibody level detection with ELISA method. [Result] Four among the 15 Chinese medicines showed lower antibody positive rates. [Conclusion] Scutellaria baicalensis, Stemona sessilifolia, Gastrodia elata and Coptis chinensis could enhance the immunity level of mice infected by N. caninum.展开更多
Objective:To determine Neospora caninum(N.caninum)as a cause of bovine abortion in dairy cows by ELISA,PCR and Pathological methods in Tabriz,Northwest of Iran.Methods:For study of outbreak of neosporosis,blood sample...Objective:To determine Neospora caninum(N.caninum)as a cause of bovine abortion in dairy cows by ELISA,PCR and Pathological methods in Tabriz,Northwest of Iran.Methods:For study of outbreak of neosporosis,blood samples were collected from 76 Holstein aborted dairy cows in Tabriz(Northwest Iran).Antibodies to N.caninum were assayed by using a commercially ELISA kit(1DEXX,USA).IgG against N.caninum were found in 14(18.4%)cases,Aborted fetuses of these seropositive dams were proposed for histopathological and molecular investigations.Brains,spinal cords and placentas of the fetuses were fixed in 10%buffered formalin for histopathology.Also 5-10 g of brain tissue was sampled for DNA extraction.In 6 out of 14(42.8%)fetuses,brain tissue was positive in PCR.All dams of these fetuses were serologically seropositive in ELISA test,Results:Histopathologically,the lesions consistent with N.caninum were observed in brains,spinal cords and placenta of all fetuses.Lesions in CNS included severe congestion.perivascular and perineuronal edema,status spongiosis,perivascular cuffing,focal gliosis,nourophagy and focal necrosis.There were some Neospora-like cysts in brain.In placentas,severe congestion,perivascular infiltration of mononuclear cells,vascular thrombosis,focal placentitis and necrotic foci in cotyledons were noticed.Conclusions:The results of present study agreed the results of similar studies about serological,histopathological and molecular results of other studies about neosporosis and it seems lo support the outbreak of N.caninum-associated abortion in dairy cows in Tabriz(Northwest Iran).展开更多
Objective:To estimate the extent to which abortion in dairy cows was associated with of Neospom caninum(N.caninum) and to determine the risk factors of neosporosis in dairy farms from 9 provinces in Iran.Methods:Polym...Objective:To estimate the extent to which abortion in dairy cows was associated with of Neospom caninum(N.caninum) and to determine the risk factors of neosporosis in dairy farms from 9 provinces in Iran.Methods:Polymerase chain reaction(PCR) test was used to detect Neospora infection in the brain of 395 bovine aborted fetuses from 9 provinces of Iran.In addition,the brains of aborted fetuses were taken for histopathological examination.To identify the risk factors associated with neosporosis,data analysis was performed by SAS.Results:N.caninum was detected in 179(45%) out of 395 fetal brain samples of bovine aborted fetuses using PCR.Among the PCR-positive brain samples,only 56 samples were suited for histopathological examination.The characteristic lesions of Neospora infection including non-suppurative encephalitis were found in 16(28%) of PCR-positive samples.The risk factors including season,parity of dam,history of bovine virus diarrhea and infectious bovine rhinotracheitis infection in herd,cow's milk production,herd size and fetal appearance did not show association with the infection.This study showed that Neospora caused abortion was significantly more in the second trimester of pregnancy than other periods.In addition,a significant association was observed between Neospora infection and stillbirth.Conclusions:The results showed N.caninum infection was detected in high percentage of aborted fetuses.In addition,at least one fourth of abortions caused by Neospora infection.These results indicate increasing number of abortions associated with the protozoa more than reported before in Iran.展开更多
Abstract [Objective] The study was aimed to screen some Chinese medicines with good inhibitory effects on Neospora [Method] The crude extracts of the tested Chinese medicines were added to the Vero cells that were in...Abstract [Objective] The study was aimed to screen some Chinese medicines with good inhibitory effects on Neospora [Method] The crude extracts of the tested Chinese medicines were added to the Vero cells that were infected by Neospora canium. Then, cell growth was ob- served. The death rate and relative suppression rate of Neospora canium were determined by trypan blue staining. [ Result] The extracts of Scutel- laria baicalensis, Stemona japonica, Rhizoma gastrodiae, Coptis chinensis, Areca catechu and Brucea javanica had better inhibitory and killing effects on Neospora canium in vitro. [ Conclusion ] The study serves the treatment of neosporosis in clinic.展开更多
The aim of the study was to estimate the prevalence of antibodies to Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) in pigs in Grenada, West Indies. T. gondii is a serious zoonosis affecting the unbor...The aim of the study was to estimate the prevalence of antibodies to Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) in pigs in Grenada, West Indies. T. gondii is a serious zoonosis affecting the unborn fetus and immunocompromized individuals. N. caninum is a similar coccidian parasite, which is not zoonotic, but is the cause of abortion and neonatal mortality in livestock similar to T. gondii. An earlier study conducted in Grenada and using a modified agglutination test (MAT) revealed seropositivity to T. gondii in pigs. No information is available on N. caninum infection of pigs in the Caribbean islands including Grenada. Serum samples from 185 pigs in Grenada, West Indies were tested for antibodies to T. gondii and N. caninum using an indirect enzyme linked immunosorbent assay (ELISA). Antibodies to T. gondii were found in 24.3% of pigs (95% confidence interval (CI): 18.12% to 30.48%) as all the tested pigs were negative for antibodies to N. caninum. Although, seroprevalence for T. gondii was higher in females (25.75%) than in males (20.70%), this result was statistically insignificant (p = 0.57). The results were similar to a previous study in Grenada confirming the continuity of infection in pigs. Human Toxoplasmosis is transmitted mainly through ingestion of tissue cysts in contaminated raw or undercooked meat or sporulated oocysts in soil, water or vegetables. Education of farmers and the Grenadian community on epidemiology of these parasites is warranted to prevent infection in pigs and in humans. This is the first report on the seroprevalence of N. caninum in pigs in the Caribbean region.展开更多
Microneme proteins (MICs) are important for Apicomplexan parasite invasion due to their adhesion to host cells. Several studies have indicated that Neospora caninum MIC3 and MIC8 are important adhesion factors and pot...Microneme proteins (MICs) are important for Apicomplexan parasite invasion due to their adhesion to host cells. Several studies have indicated that Neospora caninum MIC3 and MIC8 are important adhesion factors and potential vaccine candidates against neosporosis. In this study, we evaluated the protective efficacy of recombinant proteins and DNA vaccines of NcMIC3 and NcMIC8. BALB/c mice were immunized with rNcMIC3, rNcMIC8, pcDNA3.1-NcMIC3 and pcDNA3.1-NcMIC8 respectively, and challenged with N. caninum tachyzoites. The immune responses were evaluated through cytokine, antibody measurements and the parasite burden in the mice brain tissues. Serological analysis showed that recombinant protein vaccines induced higher levels of immunoglobulin G (IgG) than other groups. The percentage of IgGl and IgG2a in the recombinant protein groups was higher than the other groups, and with a predominance of IgGl over IgG2a, suggesting that recombinant protein vaccines elicited a Th2-type immune response, while DNA vaccines mainly produce a Th 1-type immune response. In addition, mice immunized with rNcMIC3 and rNcMIC8 a had lower parasite burden in brain tissue compared with the other groups. These results demonstrate that rNcMIC3 and rNcMIC8 could induce humoral and Th2-type immune response, leading to a considerable level of resistance against neosporosis.展开更多
The Neospora caninum immune mapped protein 1(Nc IMP1) was identified as a membrane protein,and a previous study indicated that Nc IMP1 could be a promising vaccine candidate against neosporosis. In this study, the imm...The Neospora caninum immune mapped protein 1(Nc IMP1) was identified as a membrane protein,and a previous study indicated that Nc IMP1 could be a promising vaccine candidate against neosporosis. In this study, the immune response and protection efficacy of Nc IMP1 were evaluated. The coding sequence of Nc IMP1 was inserted into the eukaryotic expression vector pc DNA3.1(+), resulting in the recombination plasmid pc DNAIMP1, which was used for the intramuscular immunization of BALB/c mice. After immunization, the immune response was evaluated using a lymphoproliferative assay and cytokine and antibody measurements. Quantification of the cerebral parasite burden of mice challenged with 2106 N. caninum was performed 14 days after the last immunization. The results showed that the mice immunized with pc DNA-IMP1 developed a high level of specific antibody responses against recombinant Nc IMP1,with a mixed Ig G1/Ig G2 a response and a predominance of Ig G2 a production. The cellular immune response was associated with the production of IFN-γ, IL-2, IL-4 and IL-10 cytokines. The experiment was terminated 30 days p.i.,and the cerebral parasite burden in each mouse was assessed by quantitative PCR. The parasite burden was significantly reduced in the pc DNA-IMP1-vaccinated mice. These data suggest that IMP1 is a promising vaccine candidate against neosporosis.展开更多
为建立一种快速检测新孢子虫的环介导等温扩增方法(LAMP),本研究根据GenBank中登录的新孢子虫Nc-5基因序列(X84238.1)设计2对特异性引物,通过PCR扩增将新孢子虫Nc-5基因片段并克隆至pMD18-T载体中,构建重组质粒pMD18-T-Nc-5,并经PCR和...为建立一种快速检测新孢子虫的环介导等温扩增方法(LAMP),本研究根据GenBank中登录的新孢子虫Nc-5基因序列(X84238.1)设计2对特异性引物,通过PCR扩增将新孢子虫Nc-5基因片段并克隆至pMD18-T载体中,构建重组质粒pMD18-T-Nc-5,并经PCR和测序鉴定正确后作为质粒标准品。通过优化该方法的引物浓度、反应温度,并利用LAMP浊度仪测定扩增目的基因时出现白色焦磷酸镁的浊度值实时监控反应进程,初步建立了检测新孢子虫的LAMP方法,反应结束后通过加入SYBR Green I观察荧光对结果可视化判定。反应条件优化结果显示,引物F3/B3和FIP/BIP的终浓度分别为10 pmol/μL、20 pmol/μL,63℃扩增40 min效率最佳。利用该方法检测新孢子虫、弓形虫、瑟氏泰勒虫、环形泰勒虫、双芽巴贝斯虫、伊氏锥虫核酸,并在反应结束后加入SYBR Green I观察荧光以评估该方法的特异性。结果显示仅新孢子虫核酸扩增为阳性,其余病原核酸均为阴性结果。荧光观察结果显示,新孢子虫反应管呈翠绿色荧光(阳性),其他反应管呈橙黄色荧光(阴性);对重组质粒标准品10倍倍比稀释后作为模板(即4.0×10^(7)拷贝/μL~4.0×100拷贝/μL)进行LAMP的敏感性试验,结果显示该方法对质粒标准品的检测限为4.0×10^(1)拷贝/μL,且在反应结束后加入SYBR Green I,4.0×10^(7)拷贝/μL~4.0×10^(1)拷贝/μL质粒标准品的反应管均呈翠绿色荧光(阳性),4.0×100拷贝/μL反应管呈橙黄色荧光(阴性),敏感性与《新孢子虫病检疫技术规范》(SN/T 3499)中荧光定量PCR方法的敏感性一致;分别以同一时间和不同时间提取的3个不同稀释度的质粒标准品进行批内和批间重复性试验,结果显示批内和批间重复性试验的变异系数均小于5%。综上表明该方法特异性强、敏感性高、重复性好。对895份临床疑似感染新孢子虫的动物肝脏、脑组织样品同时采用建立的LAMP方法及《新孢子虫病检疫技术规范》(SN/T 3499)中的荧光定量PCR方法检测,结果显示LAMP的阳性检测率为41.45%(371/895),阴性率为58.55%(524/895),与SN/T 3499中的荧光定量PCR检测结果一致。本研究建立的新孢子虫LAMP方法快速、敏感、特异性强,且能够实时监测反应过程并能可视化判定结果,为新孢子虫现场快速检测提供了新的技术手段。展开更多
文摘[Objective] The study was to screen the Chinese medicines with good resistance to Neospora caninum. [Method] Healthy Kunming mice pretreated with methylprednisolone for the purpose of immunity decrease were randomly divided into 15 groups, and each mouse was intraperitoneally inoculated with 1.0 ×104 N. caninum. Four hours later, the mice were gavaged with various Chinese medicines. Seven days post-administration, eyeball blood sampling was conducted and the serum was used for antibody level detection with ELISA method. [Result] Four among the 15 Chinese medicines showed lower antibody positive rates. [Conclusion] Scutellaria baicalensis, Stemona sessilifolia, Gastrodia elata and Coptis chinensis could enhance the immunity level of mice infected by N. caninum.
文摘Objective:To determine Neospora caninum(N.caninum)as a cause of bovine abortion in dairy cows by ELISA,PCR and Pathological methods in Tabriz,Northwest of Iran.Methods:For study of outbreak of neosporosis,blood samples were collected from 76 Holstein aborted dairy cows in Tabriz(Northwest Iran).Antibodies to N.caninum were assayed by using a commercially ELISA kit(1DEXX,USA).IgG against N.caninum were found in 14(18.4%)cases,Aborted fetuses of these seropositive dams were proposed for histopathological and molecular investigations.Brains,spinal cords and placentas of the fetuses were fixed in 10%buffered formalin for histopathology.Also 5-10 g of brain tissue was sampled for DNA extraction.In 6 out of 14(42.8%)fetuses,brain tissue was positive in PCR.All dams of these fetuses were serologically seropositive in ELISA test,Results:Histopathologically,the lesions consistent with N.caninum were observed in brains,spinal cords and placenta of all fetuses.Lesions in CNS included severe congestion.perivascular and perineuronal edema,status spongiosis,perivascular cuffing,focal gliosis,nourophagy and focal necrosis.There were some Neospora-like cysts in brain.In placentas,severe congestion,perivascular infiltration of mononuclear cells,vascular thrombosis,focal placentitis and necrotic foci in cotyledons were noticed.Conclusions:The results of present study agreed the results of similar studies about serological,histopathological and molecular results of other studies about neosporosis and it seems lo support the outbreak of N.caninum-associated abortion in dairy cows in Tabriz(Northwest Iran).
基金Supported by research fund of Ferdowsi University of Mashhad.Mashhad.Iran.(Grant No.3/25975)
文摘Objective:To estimate the extent to which abortion in dairy cows was associated with of Neospom caninum(N.caninum) and to determine the risk factors of neosporosis in dairy farms from 9 provinces in Iran.Methods:Polymerase chain reaction(PCR) test was used to detect Neospora infection in the brain of 395 bovine aborted fetuses from 9 provinces of Iran.In addition,the brains of aborted fetuses were taken for histopathological examination.To identify the risk factors associated with neosporosis,data analysis was performed by SAS.Results:N.caninum was detected in 179(45%) out of 395 fetal brain samples of bovine aborted fetuses using PCR.Among the PCR-positive brain samples,only 56 samples were suited for histopathological examination.The characteristic lesions of Neospora infection including non-suppurative encephalitis were found in 16(28%) of PCR-positive samples.The risk factors including season,parity of dam,history of bovine virus diarrhea and infectious bovine rhinotracheitis infection in herd,cow's milk production,herd size and fetal appearance did not show association with the infection.This study showed that Neospora caused abortion was significantly more in the second trimester of pregnancy than other periods.In addition,a significant association was observed between Neospora infection and stillbirth.Conclusions:The results showed N.caninum infection was detected in high percentage of aborted fetuses.In addition,at least one fourth of abortions caused by Neospora infection.These results indicate increasing number of abortions associated with the protozoa more than reported before in Iran.
文摘Abstract [Objective] The study was aimed to screen some Chinese medicines with good inhibitory effects on Neospora [Method] The crude extracts of the tested Chinese medicines were added to the Vero cells that were infected by Neospora canium. Then, cell growth was ob- served. The death rate and relative suppression rate of Neospora canium were determined by trypan blue staining. [ Result] The extracts of Scutel- laria baicalensis, Stemona japonica, Rhizoma gastrodiae, Coptis chinensis, Areca catechu and Brucea javanica had better inhibitory and killing effects on Neospora canium in vitro. [ Conclusion ] The study serves the treatment of neosporosis in clinic.
文摘The aim of the study was to estimate the prevalence of antibodies to Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) in pigs in Grenada, West Indies. T. gondii is a serious zoonosis affecting the unborn fetus and immunocompromized individuals. N. caninum is a similar coccidian parasite, which is not zoonotic, but is the cause of abortion and neonatal mortality in livestock similar to T. gondii. An earlier study conducted in Grenada and using a modified agglutination test (MAT) revealed seropositivity to T. gondii in pigs. No information is available on N. caninum infection of pigs in the Caribbean islands including Grenada. Serum samples from 185 pigs in Grenada, West Indies were tested for antibodies to T. gondii and N. caninum using an indirect enzyme linked immunosorbent assay (ELISA). Antibodies to T. gondii were found in 24.3% of pigs (95% confidence interval (CI): 18.12% to 30.48%) as all the tested pigs were negative for antibodies to N. caninum. Although, seroprevalence for T. gondii was higher in females (25.75%) than in males (20.70%), this result was statistically insignificant (p = 0.57). The results were similar to a previous study in Grenada confirming the continuity of infection in pigs. Human Toxoplasmosis is transmitted mainly through ingestion of tissue cysts in contaminated raw or undercooked meat or sporulated oocysts in soil, water or vegetables. Education of farmers and the Grenadian community on epidemiology of these parasites is warranted to prevent infection in pigs and in humans. This is the first report on the seroprevalence of N. caninum in pigs in the Caribbean region.
基金provided by the National Key Research and Development Program of China(2017YFD0501200)the National Key Basic Research Program(973 program)of China(2015CB150300)the National Natural Science Foundation of China(31772730)
文摘Microneme proteins (MICs) are important for Apicomplexan parasite invasion due to their adhesion to host cells. Several studies have indicated that Neospora caninum MIC3 and MIC8 are important adhesion factors and potential vaccine candidates against neosporosis. In this study, we evaluated the protective efficacy of recombinant proteins and DNA vaccines of NcMIC3 and NcMIC8. BALB/c mice were immunized with rNcMIC3, rNcMIC8, pcDNA3.1-NcMIC3 and pcDNA3.1-NcMIC8 respectively, and challenged with N. caninum tachyzoites. The immune responses were evaluated through cytokine, antibody measurements and the parasite burden in the mice brain tissues. Serological analysis showed that recombinant protein vaccines induced higher levels of immunoglobulin G (IgG) than other groups. The percentage of IgGl and IgG2a in the recombinant protein groups was higher than the other groups, and with a predominance of IgGl over IgG2a, suggesting that recombinant protein vaccines elicited a Th2-type immune response, while DNA vaccines mainly produce a Th 1-type immune response. In addition, mice immunized with rNcMIC3 and rNcMIC8 a had lower parasite burden in brain tissue compared with the other groups. These results demonstrate that rNcMIC3 and rNcMIC8 could induce humoral and Th2-type immune response, leading to a considerable level of resistance against neosporosis.
基金supported by National Key Basic Research Program of China(2015CB150300)Beijing Municipal Natural Science Foundation(6131001)
文摘The Neospora caninum immune mapped protein 1(Nc IMP1) was identified as a membrane protein,and a previous study indicated that Nc IMP1 could be a promising vaccine candidate against neosporosis. In this study, the immune response and protection efficacy of Nc IMP1 were evaluated. The coding sequence of Nc IMP1 was inserted into the eukaryotic expression vector pc DNA3.1(+), resulting in the recombination plasmid pc DNAIMP1, which was used for the intramuscular immunization of BALB/c mice. After immunization, the immune response was evaluated using a lymphoproliferative assay and cytokine and antibody measurements. Quantification of the cerebral parasite burden of mice challenged with 2106 N. caninum was performed 14 days after the last immunization. The results showed that the mice immunized with pc DNA-IMP1 developed a high level of specific antibody responses against recombinant Nc IMP1,with a mixed Ig G1/Ig G2 a response and a predominance of Ig G2 a production. The cellular immune response was associated with the production of IFN-γ, IL-2, IL-4 and IL-10 cytokines. The experiment was terminated 30 days p.i.,and the cerebral parasite burden in each mouse was assessed by quantitative PCR. The parasite burden was significantly reduced in the pc DNA-IMP1-vaccinated mice. These data suggest that IMP1 is a promising vaccine candidate against neosporosis.
文摘为建立一种快速检测新孢子虫的环介导等温扩增方法(LAMP),本研究根据GenBank中登录的新孢子虫Nc-5基因序列(X84238.1)设计2对特异性引物,通过PCR扩增将新孢子虫Nc-5基因片段并克隆至pMD18-T载体中,构建重组质粒pMD18-T-Nc-5,并经PCR和测序鉴定正确后作为质粒标准品。通过优化该方法的引物浓度、反应温度,并利用LAMP浊度仪测定扩增目的基因时出现白色焦磷酸镁的浊度值实时监控反应进程,初步建立了检测新孢子虫的LAMP方法,反应结束后通过加入SYBR Green I观察荧光对结果可视化判定。反应条件优化结果显示,引物F3/B3和FIP/BIP的终浓度分别为10 pmol/μL、20 pmol/μL,63℃扩增40 min效率最佳。利用该方法检测新孢子虫、弓形虫、瑟氏泰勒虫、环形泰勒虫、双芽巴贝斯虫、伊氏锥虫核酸,并在反应结束后加入SYBR Green I观察荧光以评估该方法的特异性。结果显示仅新孢子虫核酸扩增为阳性,其余病原核酸均为阴性结果。荧光观察结果显示,新孢子虫反应管呈翠绿色荧光(阳性),其他反应管呈橙黄色荧光(阴性);对重组质粒标准品10倍倍比稀释后作为模板(即4.0×10^(7)拷贝/μL~4.0×100拷贝/μL)进行LAMP的敏感性试验,结果显示该方法对质粒标准品的检测限为4.0×10^(1)拷贝/μL,且在反应结束后加入SYBR Green I,4.0×10^(7)拷贝/μL~4.0×10^(1)拷贝/μL质粒标准品的反应管均呈翠绿色荧光(阳性),4.0×100拷贝/μL反应管呈橙黄色荧光(阴性),敏感性与《新孢子虫病检疫技术规范》(SN/T 3499)中荧光定量PCR方法的敏感性一致;分别以同一时间和不同时间提取的3个不同稀释度的质粒标准品进行批内和批间重复性试验,结果显示批内和批间重复性试验的变异系数均小于5%。综上表明该方法特异性强、敏感性高、重复性好。对895份临床疑似感染新孢子虫的动物肝脏、脑组织样品同时采用建立的LAMP方法及《新孢子虫病检疫技术规范》(SN/T 3499)中的荧光定量PCR方法检测,结果显示LAMP的阳性检测率为41.45%(371/895),阴性率为58.55%(524/895),与SN/T 3499中的荧光定量PCR检测结果一致。本研究建立的新孢子虫LAMP方法快速、敏感、特异性强,且能够实时监测反应过程并能可视化判定结果,为新孢子虫现场快速检测提供了新的技术手段。
