[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its g...[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.展开更多
[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surf...[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.展开更多
Objective:To determine tick infestation of domestic ruminants and their infection to ovine theileriosis in northern Iran.Methods:About 425 domestic ruminants in Ghaemshahr city in northern Iran were inspected for tick...Objective:To determine tick infestation of domestic ruminants and their infection to ovine theileriosis in northern Iran.Methods:About 425 domestic ruminants in Ghaemshahr city in northern Iran were inspected for tick infestations.Twenty tick specimens(13 females and 7 males) of Rhipicephalus sanguineus(R.sanguineus).the most common lick in the study area, were tested by PCR amplification against 18s rRNA genome of Theileria spp using specie specific primers and then the PCR products were sequenced for species identification In comparison with data base available in GenBank.Results:About 323 ticks were collected from 102 animals(88 sheep,12 goats and 2 cattle).The prevalence of ticks infesting animals was R.sanguineus(82.35%), Rhipieeplialus bursa(R.bursa)(0.3%),Ixodes ricinus(I.ricinus)(15.2%),Boophilus annulatus (B.annulalus)(1.2%).Haemaphxsalis punctata(H.punctata)(0.3%) and Haemaphysalis numidiana(H.numidianu)(0.6%).Eleven(55%) tick specimens were PCR positive against genome of Theileria ovis(T.avis).Sequence analysis of the PCR products confirmed presence of T. oris in one R.sanguinus.Conclusions:This is the first report of tick infection to T.oris in Iran. Due to dominant prevalence of R.sanguineus as well as its infection to T.oris,it is postulated this tick is the main vector of ovine theileriosis in northern Iran.展开更多
Objective:To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea(MOE)and acetonic extract of Acacia laeta(AAL)against Babesia and Theileria parasites in vitro and evaluate the chemotherapeut...Objective:To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea(MOE)and acetonic extract of Acacia laeta(AAL)against Babesia and Theileria parasites in vitro and evaluate the chemotherapeutic effects of these extracts against Babesia(B.)microti in vivo.Methods:Fluorescence assay using SYBR Green 1 nucleic acid stain was used to detect inhibitory effects of the two extracts as well as the combination effects of the two extracts with diminazene aceturate and atovaquone on four Babesia species and Theileria equi in vitro while for in vivo experiments,8-weekold female BALB/c mice were injected intraperitoneally with 1× 107 B.microti-iRBCs and treated orally at a dose of 150 mg/kg of both extracts.Results:The half maximal inhibitory concentration(IC50)values of AAL against B.bovis,B.bigemina,B.divergens,B.caballi,and Theileria equi were lower than those of MOE extracts.Toxicity assay on Madin-Darby bovine kidney,mouse embryonic fibroblast(NIH/3T3),and human foreskin fibroblast cell lines showed that MOE and AAL affected only the viability of Madin-Darby bovine kidney cell line with half maximal effective concentrations(EC50)of(794.7±41.9)and(873.9±17.5)μg/mL,respectively.The oral treatments of MOE and AAL at 150 mg/kg inhibited the growth of B.microti in mice by 80.4% and 64.4%,respectively.The MOE and diminazene aceturate combination showed a higher chemotherapeutic effect than that of monotherapy.Conclusions:MOE and AAL have the potential to be an alternative remedy for treating piroplasmosis.Furthermore,the combination therapy of MOE + DA was more potent against B.microti infection in mice than their monotherapies.展开更多
Theileria luwenshuni and Theileria uilenbergi are important tick-borne pathogens and cause substantial losses to the sheep industry in China. The improvement in detection techniques has allowed the identification of m...Theileria luwenshuni and Theileria uilenbergi are important tick-borne pathogens and cause substantial losses to the sheep industry in China. The improvement in detection techniques has allowed the identification of multi-homing parasitism in Theileria parasites. Herein we evaluated the experimental infectivity of T. luwenshuni and T. uilenbergi in Chinese Kunming mice by screening blood samples of experimentally inoculated mice by microscopic examination（ME） and PCR. T. luwenshuni infected Chinese Kunming mice and 20 mice inoculated with this parasite were positive by ME and PCR. In addition, T. uilenbergi infected mice and 20 mice inoculated with this species were positive by ME and PCR. However, the number of red blood cells and the levels of hemoglobin of 40 infected mice had no obvious changes in the course of infection. Our results demonstrated the multi-homing parasitism of T. luwenshuni and T. uilenbergi, which were believed to be parasites of sheep and goats. This study was the first to demonstrate the infection of T. luwenshuni and T. uilenbergi in Kunming mice.展开更多
Objective:To use two diagnostic antigens belonging to the frequently associated in Theileria domain,Theileria equi(T.equi)protein 82(Te 82)and T.equi 104 k Da microneme-rhoptry antigen precursor(Te 43),to diagnose T.e...Objective:To use two diagnostic antigens belonging to the frequently associated in Theileria domain,Theileria equi(T.equi)protein 82(Te 82)and T.equi 104 k Da microneme-rhoptry antigen precursor(Te 43),to diagnose T.equi infection in horses as compared with equi merozoite antigen-2(EMA-2).Methods:In the current study,we applied a cocktail-ELISA containing two antigens(EMA-2+Te 82)to diagnose T.equi infection either in experimentally infected horses or in field infection.Results:Our findings have revealed that a cocktail formula of EMA-2+Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T.equi infection in horses as compared with Te 82 or Te 43 alone.Conclusions:The ELISA technique using a cocktail formula of EMA-2+Te 82 offers a practical and sensitive diagnostic tool for diagnosing T.equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.展开更多
The authors studied the impact of tropical theileriosis onset on milk yield decrease in 10 local bred cows in Skikda(Northern Algeria) during 2015 summer season.The milk yield decrease estimated weekly during two mont...The authors studied the impact of tropical theileriosis onset on milk yield decrease in 10 local bred cows in Skikda(Northern Algeria) during 2015 summer season.The milk yield decrease estimated weekly during two months was 2.76 L/day/cow corresponding to31.92% of the total milk yield.This decrease corresponds to 110.5 Algerian Dinars(1.02US$)/day/diseased cow.The relative variation of milk yield showed a dramatic decrease from 82.72% to 0.76% at Day 21 then became constant.Further studies are needed to improve these estimations of financial losses due to bovine tropical theileriosis in Algeria.展开更多
Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measureme...Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.展开更多
环介导等温扩增技术(LAMP)是一种广泛应用的恒温下快捷检测技术,但其极易发生气溶胶污染导致假阳性结果,将LAMP与簇状规则间隔短回文重复序列(CRISPR)系统结合可以有效避免假阳性结果。为建立针对绵羊泰勒虫18S r RNA基因的LAMP-CRISPR/...环介导等温扩增技术(LAMP)是一种广泛应用的恒温下快捷检测技术,但其极易发生气溶胶污染导致假阳性结果,将LAMP与簇状规则间隔短回文重复序列(CRISPR)系统结合可以有效避免假阳性结果。为建立针对绵羊泰勒虫18S r RNA基因的LAMP-CRISPR/Cas12a检测方法,本研究根据绵羊泰勒虫18S rRNA基因设计LAMP的特异性引物,构建靶基因的质粒标准品作为模板,优化反应条件后建立LAMP方法。进一步将LAMP产物加入CRISPR/Cas12a检测体系作为模板,优化CRISPR/Cas12a检测体系,获得最佳反应体系。结果显示,优化后LAMP方法的最佳反应温度为65℃,最佳反应时间30 min,内外引物浓度比≥6:1时最佳,LAMP-CRISPR/Cas12a的最佳反应体系为Cas12a蛋白与CRISPR RNA(crRNA)浓度比为1:2,探针浓度为2.5μmol/L。利用建立的LAMP-CRISPR/Cas12a方法检测绵羊泰勒虫、东方泰勒虫、中华泰勒虫、马泰勒虫、驽巴贝斯虫、绵羊无形体、弓形虫的DNA,结果显示仅绵羊泰勒虫为阳性结果,其他病原均为阴性;将构建的质粒标准品10倍倍比稀释(10^(0)~10^(7)倍)后作为模板,利用该方法检测,结果显示该方法的检测限为4.04拷贝/μL;于同一时间和不同时间利用该方法检测重组质粒标准品pMD19-T-T.ovis(4.04×10^(3)拷贝/μL~4.04×10^(1)拷贝/μL),分析其重复性,结果显示该方法批内变异系数小于5%,批间变异系数小于10%,具有良好的重复性。利用建立的LAMP-CRISPR/Cas12a方法与套式PCR检测临床绵羊血液样品,结果显示二者的阳性符合率达100%(14/14)、阴性符合率96.43%(54/56),总符合率97.14%(68/70)。本实验基于绵羊泰勒虫18S rRNA基因首次建立了特异性、敏感性及重复性均良好的LAMP-CRISPR/Cas12a检测方法,为绵羊泰勒虫病的监测和防控奠定了技术基础。展开更多
文摘[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.
基金Supported by Science and Technology Development Program of Jilin Province (20050703-4)~~
文摘[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.
基金supported by Tehran Universitv of Medical Sciences
文摘Objective:To determine tick infestation of domestic ruminants and their infection to ovine theileriosis in northern Iran.Methods:About 425 domestic ruminants in Ghaemshahr city in northern Iran were inspected for tick infestations.Twenty tick specimens(13 females and 7 males) of Rhipicephalus sanguineus(R.sanguineus).the most common lick in the study area, were tested by PCR amplification against 18s rRNA genome of Theileria spp using specie specific primers and then the PCR products were sequenced for species identification In comparison with data base available in GenBank.Results:About 323 ticks were collected from 102 animals(88 sheep,12 goats and 2 cattle).The prevalence of ticks infesting animals was R.sanguineus(82.35%), Rhipieeplialus bursa(R.bursa)(0.3%),Ixodes ricinus(I.ricinus)(15.2%),Boophilus annulatus (B.annulalus)(1.2%).Haemaphxsalis punctata(H.punctata)(0.3%) and Haemaphysalis numidiana(H.numidianu)(0.6%).Eleven(55%) tick specimens were PCR positive against genome of Theileria ovis(T.avis).Sequence analysis of the PCR products confirmed presence of T. oris in one R.sanguinus.Conclusions:This is the first report of tick infection to T.oris in Iran. Due to dominant prevalence of R.sanguineus as well as its infection to T.oris,it is postulated this tick is the main vector of ovine theileriosis in northern Iran.
基金supported by the Japan Society for the Promotion of Science(JSPS)(KAKEN Grant Number:18H02337)
文摘Objective:To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea(MOE)and acetonic extract of Acacia laeta(AAL)against Babesia and Theileria parasites in vitro and evaluate the chemotherapeutic effects of these extracts against Babesia(B.)microti in vivo.Methods:Fluorescence assay using SYBR Green 1 nucleic acid stain was used to detect inhibitory effects of the two extracts as well as the combination effects of the two extracts with diminazene aceturate and atovaquone on four Babesia species and Theileria equi in vitro while for in vivo experiments,8-weekold female BALB/c mice were injected intraperitoneally with 1× 107 B.microti-iRBCs and treated orally at a dose of 150 mg/kg of both extracts.Results:The half maximal inhibitory concentration(IC50)values of AAL against B.bovis,B.bigemina,B.divergens,B.caballi,and Theileria equi were lower than those of MOE extracts.Toxicity assay on Madin-Darby bovine kidney,mouse embryonic fibroblast(NIH/3T3),and human foreskin fibroblast cell lines showed that MOE and AAL affected only the viability of Madin-Darby bovine kidney cell line with half maximal effective concentrations(EC50)of(794.7±41.9)and(873.9±17.5)μg/mL,respectively.The oral treatments of MOE and AAL at 150 mg/kg inhibited the growth of B.microti in mice by 80.4% and 64.4%,respectively.The MOE and diminazene aceturate combination showed a higher chemotherapeutic effect than that of monotherapy.Conclusions:MOE and AAL have the potential to be an alternative remedy for treating piroplasmosis.Furthermore,the combination therapy of MOE + DA was more potent against B.microti infection in mice than their monotherapies.
基金financially supported by the National Key Research and Development Program of China (2017YFD0501200, 2016YFC1202000, 2016YFC1202002)the earmarked fund for China Agriculture Research System (CARS-37)+5 种基金the National Natural Science Foundation of China (31272556, 31402189, 31372432)the Agricultural Science and Technology Innovation Program, China (2014ZL010)the National Basic Research Program of China (2015CB150300)the Special Funds for Agroscientific Research in the Public Research, China (201303035)the Gansu International Collaboration Special Project, China (1504WKCA056)the Jiangsu Co-Innovation Center Programme for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,State Key Laboratory of Veterinary Etiological Biology Project, China
文摘Theileria luwenshuni and Theileria uilenbergi are important tick-borne pathogens and cause substantial losses to the sheep industry in China. The improvement in detection techniques has allowed the identification of multi-homing parasitism in Theileria parasites. Herein we evaluated the experimental infectivity of T. luwenshuni and T. uilenbergi in Chinese Kunming mice by screening blood samples of experimentally inoculated mice by microscopic examination（ME） and PCR. T. luwenshuni infected Chinese Kunming mice and 20 mice inoculated with this parasite were positive by ME and PCR. In addition, T. uilenbergi infected mice and 20 mice inoculated with this species were positive by ME and PCR. However, the number of red blood cells and the levels of hemoglobin of 40 infected mice had no obvious changes in the course of infection. Our results demonstrated the multi-homing parasitism of T. luwenshuni and T. uilenbergi, which were believed to be parasites of sheep and goats. This study was the first to demonstrate the infection of T. luwenshuni and T. uilenbergi in Kunming mice.
基金supported by the Ministry of Higher Education Egypt
文摘Objective:To use two diagnostic antigens belonging to the frequently associated in Theileria domain,Theileria equi(T.equi)protein 82(Te 82)and T.equi 104 k Da microneme-rhoptry antigen precursor(Te 43),to diagnose T.equi infection in horses as compared with equi merozoite antigen-2(EMA-2).Methods:In the current study,we applied a cocktail-ELISA containing two antigens(EMA-2+Te 82)to diagnose T.equi infection either in experimentally infected horses or in field infection.Results:Our findings have revealed that a cocktail formula of EMA-2+Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T.equi infection in horses as compared with Te 82 or Te 43 alone.Conclusions:The ELISA technique using a cocktail formula of EMA-2+Te 82 offers a practical and sensitive diagnostic tool for diagnosing T.equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.
文摘The authors studied the impact of tropical theileriosis onset on milk yield decrease in 10 local bred cows in Skikda(Northern Algeria) during 2015 summer season.The milk yield decrease estimated weekly during two months was 2.76 L/day/cow corresponding to31.92% of the total milk yield.This decrease corresponds to 110.5 Algerian Dinars(1.02US$)/day/diseased cow.The relative variation of milk yield showed a dramatic decrease from 82.72% to 0.76% at Day 21 then became constant.Further studies are needed to improve these estimations of financial losses due to bovine tropical theileriosis in Algeria.
文摘Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.
