首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 31 毫秒
1.
  相似文献   

2.
The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.  相似文献   

3.
绵羊肺炎支原体（Mycoplasma ovipneumoniae,MO）是绵羊传染性胸膜肺炎（Contagious ovine pleuropneumo-nia）的主要致病菌。热休克蛋白Hsp70也叫（DnaK）,具有分子伴侣和免疫的作用。本试验以MO Y98基因组为模板,通过比对15种支原体Hsp70基因的序列并设计简并引物,分别利用同源克隆和染色体步移—Tail-PCR技术克隆到了四段Hsp70（DnaK）基因片断并进行了核苷酸序列测定。利用序列拼接软件对克隆的四段序列进行序列拼接,通过ORF Finder软件分析出了MO Y98的Hsp70的开放式阅读框架,预测分析该基因是由1 815bp组成,编码604个氨基酸。本试验于国内外首次克隆到了MO的Hsp70基因序列,为MO的抗原研究以及Hsp70的功能研究等奠定了基础。  相似文献   

4.
A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.  相似文献   

5.
Heat shock protein 90 (Hsp90), a molecular chaperone, is ubiquitous and involved in numerous cellular processes. To contribute to the relatively small collection of vertebrate Hsp90 sequences in the gene data bank, we cloned and sequenced horse (Equus caballus) Hsp90 alpha and beta cDNAs. This enabled identification of horse-specific primers for development of a convenient PCR-based method that could monitor horse stress tolerance. We analyzed the sequence data comparatively and phylogenetically with other Hsp90 cDNA sequences, and identified vertebrate-specific and isoform-specific conserved regions to facilitate future molecular investigations of Hsp90 functions. We found 4 highly conserved regions to vertebrate Hsp90 exclusively and 27 amino acids conserved among but differing between Hsp90 alpha and Hsp90 beta sequences. Protein-based phylogenetic trees revealed high conservation between mammal species within Hsp90 alpha and beta clusters. Comparison of nucleotide and amino acid substitution levels suggests that horse Hsp90 beta has undergone strong purifying selection, while rat Hsp90 beta and hamster Hsp90 alpha have been positively selected. Surprisingly, fish Hsp90 alpha genes clearly clustered with Hsp90 beta genes, and no distinct placement of fish Hsp90 alpha protein was found. The Hsp90 alpha isoform is apparently the result of beta gene duplication. Our results highlight the importance of organism- and isoform-specific Hsp90 functional analyses in describing the role of Hsp90 in cells.  相似文献   

6.
Various combination strategies for treating Babesia gibsoni have been described. However, relapses after administering some combinations of antibabesial drugs and the presence of drug-resistant B. gibsoni still pose significant challenges to veterinarians. To compare the efficacy of a combination of clindamycin, diminazene, and imidocarb (CDI) to that of a combination of atovaquone and azithromycin (AA) for the treatment of B. gibsoni and to correlate drug efficacy with B. gibsoni mutations, 30 client-owned dogs with natural B. gibsoni infections were collected in the study. 17 dogs were treated with AA, and 13 dogs were treated with CDI combination. Hematological parameters were recorded on the day that the dogs were presented for treatment and during treatment. To detect the parasitic DNA, the B. gibsoni 18S rRNA gene was amplified, and to analyze the mutations, the cytochrome b (CYTb) gene was sequenced. The therapy duration for all of the dogs that recovered was 23.3±7.8 days in the AA group and 41.7±12.4 days in the CDI group. Nine of the 17 dogs in the AA group and 11 of the 13 dogs in the CDI group completely recovered. Seven dogs in the AA group and 2 dogs in the CDI group relapsed after treatment. The M121I mutation in the B. gibsoni CYTb gene was detected in all of the samples that were collected from AA-relapsed and AA-nonremission dogs. The dogs in the CDI group exhibited higher recovery rates and lower relapse rates during treatment for B. gibsoni infection. In addition, the detected M121I mutation was associated with AA treatment. The CDI combination is a promising alternative treatment strategy for B. gibsoni.  相似文献   

7.
We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection.  相似文献   

8.
Antibodies that recognized either Babesia gibsoni or canine red blood cell (RBC) 70-kilodalton (kDa) protein were detected in serum from acutely and chronically B. gibsoni-infected. In those sera, antibodies that reacted with recombinant B. gibsoni and canine heat shock protein 70 (rBgHsp70 and rcHsp70) were detected; therefore, B. gibsoni and canine RBC 70-kDa proteins seemed to be BgHsp70 and cHsp70, respectively. In infected dogs, the amounts of these antibodies increased after infection. Interestingly, polyclonal antibody raised against rBgHsp70 in two rabbits reacted not only with rBgHsp70 but also with rcHsp70 and native cHsp70 from canine RBCs. Because BgHsp70 showed high homology with cHsp70 (70.8%), anti-rBgHsp70 antibody might cross-react with cHsp70. Additionally, the localizations of both BgHsp70 and cHsp70 were observed by indirect fluorescence assay. As a result, cHsp70 was not found on the membrane surface of erythrocytes, suggesting that erythrocytes would not be targets of anti-cHsp70 antibody. Meanwhile, only exoerythrocytic parasites were stained by anti-rBgHsp70 antibody. This result showed that BgHsp70 would be expressed on the surface of parasites during the exoerythrocytic stage. These results indicated that BgHsp70 was a highly immunogenic protein in canine B. gibsoni infection, and that exoerythrocytic parasites might be targets of anti-BgHsp70 antibody.  相似文献   

9.
Nucleotide sequences of ribosomal DNA (rDNA) of Babesia (B.) gibsoni occurring in Miyazaki, western Japan, were examined using blood samples obtained from seven dogs suffering from natural canine babesiosis. DNA isolated from these blood samples was subjected to the polymerase chain reaction (PCR). The nucleotide sequences of the PCR products were determined and compared with other rDNA sequences of B. gibsoni isolated from Asia, Europe and U.S.A. Although homology values between our isolates and those isolated from Europe and U.S.A. were both 84.0%, respectively, our isolates were identical to the Asian types. In conclusion, B. gibsoni occurring in Miyazaki was revealed to have the genotype Asia 1 or Asia 2 from a comparison of the partial rDNA sequences.  相似文献   

10.
OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.  相似文献   

11.
An atovaquone (ATV)-resistant Babesia gibsoni was developed by in vitro exposure of uncloned wild type (WT) B. gibsoni to 800 nM ATV for 6 days. Sequence analysis of mitochondrial genes showed a single-nucleotide polymorphism (SNP) at cytb nt363 (G to T) that resulted in the substitution of methionine with isoleucine (M121I), which is one of the SNPs reported in a previous in vivo study. 363T or 363G allele-specific real-time polymerase chain reaction (PCR) revealed that an M121I variant was present in over 99% of the ATV-resistant population. As neither ATV resistance nor gene polymorphisms appeared in the B. gibsoni WT sibling clones, the expression of ATV resistance in this study was suspected to be because of selective multiplication of the B. gibsoni M121I variant. This ATV-resistant B. gibsoni displayed the same sensitivity as the WT B. gibsoni against 5 other drugs, including diminazene aceturate, azithromycin, doxycycline, clindamycin, and proguanil. This is the first report on the in vitro establishment of an ATV-resistant B. gibsoni with gene polymorphisms.  相似文献   

12.
OBJECTIVE: To identify subclinical Babesia gibsoni infection in American Pit Bull Terriers from the southeastern United States and to determine the genetic sequence of parasite DNA isolated from these dogs. DESIGN: Case series. ANIMALS: 33 American Pit Bull Terriers and 87 dogs of various other breeds. PROCEDURE: Blood smears were examined for microscopic evidence of the parasite, and DNA was extracted from blood samples and used in a polymerase chain reaction (PCR) assay designed to amplify the small subunit ribosomal RNA gene sequence of B. gibsoni. Amplification products of the expected size were sequenced, and sequences were compared with published sequences for B. gibsoni isolates. Hematocrit, platelet count, mean platelet volume, WBC count, and eosinophil count were compared between dogs with positive PCR assay results and dogs with negative results. RESULTS: Results of the PCR assay were positive for 18 of the 33 (55%) American Pit Bull Terriers, including all 10 dogs with microscopic evidence of parasitemia. Only 1 of these dogs was clinically ill at the time blood samples were collected. Results of microscopic evaluation of blood smears and of the PCR assay were negative for the 87 other dogs. Hematocrit and platelet count were significantly lower in dogs with positive PCR assay results than in dogs with negative results. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that American Pit Bull Terriers in the southeastern United States may be subclinically infected with B. gibsoni. However, subclinical infection was not identified in dogs of other breeds from the same geographic area.  相似文献   

13.
Tian Z  Liu G  Zhang L  Yin H  Wang H  Xie J  Zhang P  Luo J 《Veterinary parasitology》2011,181(2-4):282-290
A Haemaphysalis longicornis heat shock protein 70 (HLHsp70) was identified from a cDNA library synthesized from tick eggs. The HLHsp70 cDNA is 2311 bp in length and encodes 661 amino acid residues with the predicted molecular weight of 72.5 kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmic reticulum (ER) retention signal "KDEL" that is common among ER-localized proteins. The HLHsp70 exhibits 90% amino acid identity to the putative Hsp70 of Ixodes scapularis, and 85% to Gallus gallus 78 kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were significantly higher than in other tested tissues in partially fed females. Although the expression level of the HLHsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. Further, the expression was positively correlated to the temperature (4-37°C, tested). Western blot analysis showed that the rabbit antiserum against the recombinant HLHsp70 protein (rHLHSP70) recognized bands of approximately 100, 72, and 28 kDa from egg lysates, as well as a 72kDa fragment in protein extracts from partially fed larvae. Immunization of rabbits with the rHLHSP70 did not result in a statistically significant reduction of female tick engorgement and oviposition. These results suggest that although HLHSP70 plays a role in the physiological activities of ticks, as a constitutive protein it was not suitable for selection as a candidate vaccine antigen against ticks.  相似文献   

14.
Only little is known about the heat shock proteins (Hsp) and Hsp-encoding genes of mycoplasmas. The aim of this study was to identify and sequence the hsp60 gene of Mycoplasma agalactiae, Mycoplasma arthritidis, Mycoplasma bovis, and Mycoplasma hyopneumoniae, and to investigate the immune response to Hsp60.Fragments of the hsp60 genes of M. agalactiae, M. arthritidis, M. bovis and M. hyopneumoniae representing almost the entire coding region were amplified by PCR. Two fragments of a hsp60 gene were cloned in Escherichia coli and the antibody response of pigs infected with M. hyopneumoniae against the recombinant Hsp60 fusion proteins was analysed. Within the mycoplasmas, the hsp60 genes showed sequence identities of nearly 100%, with the exception of the hsp60 gene of Mycoplasma genitalium, which was determined to be only 76.5-77.7% identical. Identities to Clostridium perfringens, Bacillus subtilis and E. coli were determined between approximately 50 and 60%. The predicted amino acid sequences of Hsp60 showed an identity of 90 to nearly 100% among mycoplasmas and 50-60% to the other bacteria indicated above. Two Hsp60 derived glutathione-S-transferase fusion proteins containing mycoplasma peptides of 28 and 35kDa were isolated. M. hyopneumoniae-ELISA positive porcine convalescent sera reacted strongly with the recombinant Hsp60 fusion proteins in Western immunoblotting indicating for the first time that mycoplasmal Hsp60 is immunogenic in natural infection.  相似文献   

15.
Molecular characterization of a Babesia gibsoni isolate from a Spanish dog   总被引：1，自引：0，他引：1  
Babesia gibsoni is a morphologically small Babesia species that infects dogs. Molecular techniques have shown that some small Babesia sp. recently described in canids are not related to the original B. gibsoni and they should be assigned to separate taxons. Although the 18s rRNA gene of true B. gibsoni isolates has been studied in the USA, Asia and Australia, no molecular data on the presence and genetic characteristics of B. gibsoni in Europe are available. Blood collected from a Babesia-symptomatic dog from Spain was used for DNA diagnosis by seminested PCR. DNA amplification was positive and the complete 18s rRNA gene of the dog isolate was sequenced, showing 98% homology with B. gibsoni (isolate Asia 1). Evidence from phylogenetic analysis indicated that: The Spanish isolate unambiguously belongs to the B. gibsoni group. The B. gibsoni complex might be diphyletic. In the absence of genetic data from African isolates of B. gibsoni, Asia seems to be the most likely geographical location of origin.  相似文献   

16.
A total of 80 free-roaming dogs on Okinawa Island, Japan, were examined for Babesia infection using the polymerase chain reaction (PCR) and sequence analysis. Of 80 samples, 12 were positive in a Babesia genus-specific PCR. Consequent species-specific PCR for B. canis and B. gibsoni revealed that 5 (6.3%) and 7 (8.8%) dogs were infected with B. canis and B. gibsoni, respectively. Sequence analysis of the PCR products revealed that the 18S rRNA gene sequence of B. canis detected from dogs in Okinawa was very close to B. canis vogeli with sequence similarity of 99.94%.  相似文献   

17.
Using RT-PCR amplifications with mRNA from mitogen-stimulated feline peripheral blood mononuclear cells, cDNA of feline B7-1 (CD80) and B7-2 (CD86) were cloned. The cDNA were sequenced and putative translated protein sequences compared with known counterpart sequences. Hydrophilicity patterns of the feline CD80 and CD86 which were only 26.8% identical at the amino acid sequence were very distinct from each other, but similar to the putative human CD80 and CD86 proteins, respectively. The feline CD80 gene encoded a protein of 292 amino acids and the CD86 gene encoded a protein of 329 amino acids. Amino-terminal signal sequences, extracellular Ig V- and Ig C-like domains, transmembrane domains, and carboxyl cytoplasmic domains were identified in both molecules. Although the most conserved domain among the CD80 sequences was the Ig C-like domain, the most conserved domain among the CD86 sequences was the Ig V-like domain. Among the known sequences, the bovine CD80 and the porcine CD86 sequences available for comparisons were identified as most closely related to the feline CD80 (63.3%) and CD86 (67.5%), respectively. The mouse molecules were the least identical (43.6 and 43.6%, respectively) with the feline CD80 and CD86 proteins. The human CD80 and CD86 molecules were 56.3 and 57.0% identical with the feline molecules.  相似文献   

18.
The clinical usefulness of antibodies against Babesia gibsoni detected by ELISA with recombinant P50 was examined in dogs in an area where B. gibsoni infection was endemic. Only 8 among 14 dogs with acute type B. gibsoni infection without a previous history of infection were positive. This high percentage of false-negative results is thought to be a weak point of ELISA as a diagnostic tool. However, 14 other anemic dogs with a confirmed history of B. gibsoni infection were all positive, thus confirming the higher sensitivity of ELISA in detecting a history of infection.  相似文献   

19.
A cDNA encoding the Babesia bovis 12D3 antigen homologue was obtained by immunoscreening the expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1406 bp. Computer analysis suggested that the sequence contains an open reading frame of 1052 bp encoding an expected protein with a molecular weight of 36kDa. Based on homology analysis, this putative protein was designated as the B. gibsoni 12D3 antigen (Bg12D3). The Bg12D3 gene was expressed in the Escherichia coli BL21 strain, and the chronically infected dog serum reacted with the recombinant protein. The antiserum against the recombinant Bg12D3 protein can recognize a 38-kDa native protein, which is consistent with its expected size. Moreover, the purified recombinant proteins were used as the antigen to detect the antibody response in an experimentally infected dog by the enzyme-linked immunosorbent assay (ELISA). Our results indicated that the Bg12D3 protein was recognized by the host immune system and that it induced an antibody response in chronic B. gibsoni infection. These results allowed us to identify a new member of the 12D3 antigens and its characteristic immune response in canine B. gibsoni infection.  相似文献   

20.
利用抗体法（picoBlueTMImmunscreening Kit,应用B.gibsoni感染血清）从犬吉氏巴贝斯虫（B.gibsoni）裂殖子mRNA制备的吉氏巴贝斯虫cDNA文库中进行免疫筛选,选出目的基因相cDNA片段（阳性克隆）.测序验证后将该cDNA（基因）克隆至原核表达载体pGEX-4T-3,构建重组质粒,在大肠杆菌E.coli（DH5a）中以GST融合蛋白的形式表达（SDS-PAGE分析证明）;表达产物为130kDa的可溶性融合蛋白.免疫学分析（Western blot和ELISA）结果表明,犬吉氏巴贝斯虫重组GST-P130kDa融合蛋白（rBg GST-P130kDa）能与犬吉氏巴贝斯虫（B.gibsoni）感染血清起反应,且与犬巴贝斯虫（B.canis）无交叉反应.本试验利用犬吉氏巴贝斯虫重组BgGST-P130kDa融合蛋白（作为重组抗原）检测巴西（n=310）、日本（n=100）和中国（n=114,n=30）等国随机采集的自然感染狗血清,其结果为巴西狗血清阳性率为55%、日本为8%、中国为1～9%;其结果与间接荧光抗体法（IFAT）结果为一致（作为验证）.结果表明重组BgGST-P130kDa融合蛋白具有较强的免疫原性和特异性,可用于吉氏巴贝斯虫病的诊断,这为吉氏巴贝斯虫病的早期诊断和深入研究犬吉氏巴贝斯虫病的特异性重组抗原及重组疫苗奠定基础.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号