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1.
During a year from 1999 to 2000, a total of 247 blood samples were collected from 214 cattle and 33 water buffaloes in 16 distinct geographical locations of Thailand and analyzed by allele-specific PCR amplification of major piroplasm surface protein (MPSP) genes of benign Theileria parasites. Four allelic MPSP gene types were determined namely C-type, I-type, B-type and Thai-type, which were originally designated from Japanese Theileria orientalis (Chitose, Ikeda), Australian T. buffeli (Warwick) and Thai T. sp. (Kamphaeng Saen), respectively. Only two allelic MPSP gene types were successively amplified from 204 (82.6%) blood samples. Among positive cases, 138 (67.6%) and 17 (8.3%) samples contained either Thai-type or C-type parasites, respectively, while 49 (24%) samples contained both types. However, nucleotide sequences of MPSP genes of Thai T. sp. amplified by C-type specific primers revealed higher (96.3%) similarity to Indonesian T. sp. rather than (87.8% similarity) to Japanese T. orientalis (Chitose) designated as C-type.  相似文献   

2.
A survey of Theileria parasites in cattle in eastern Turkey was carried out using specific polymerase chain reaction. A total of 252 blood samples were collected from clinically healthy cattle between June and July 2004. Of 252 blood samples examined, 41 (16%) were positive for piroplasms by microscopy, whereas 114 (45%) were positive for the presence of at least one species of Theileria by PCR. The percentages of positive animals for Theileria annulata and benign Theileria species (Theileria sergenti/buffeli/orientalis) were 39% (99/252) and 7% (18/252), respectively. By allele-specific PCR examination of 18 field isolates which were positive for benign Theileria parasites, 8 samples were only amplified by B-type specific primers and 10 samples were amplified by both of the B and C-type specific primers, indicating a mixed infection with B and C-type of the parasite. None of the field isolates was amplified by I-type specific primers. Three samples were co-infected with T. annulata and benign Theileria parasites. Two of them which were infected with B-type parasite were also infected with T. annulata, the other sample which was infected both of B and C-type parasites was also infected with T. annulata. A total of 724 ixodid ticks were collected from the cattle. Hyalomma anatolicum anatolicum was the dominant species with 32% (230/724) in the region. H. a. excavatum, Boophylus annulatus and Rhipicephalus bursa represented 25% (183/724), 19% (140/724) and 15% (112/724) of the total number of ticks, respectively. R. sanguineus was the minor species and represented 8% (59/724) of the tick population.  相似文献   

3.
Benign Theileria species of cattle are found in most parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a maker for epidemiological and phylogenetical studies of benign Theileria species. Parasites with Ikeda- or Chitose-type MPSP genes are dominant in Japan, but we report here mixed infection cases of Theileria parasites with an additional MPSP type parasite infecting cattle in Abashiri District, Hokkaido. The MPSP gene sequence found in the additional type was closely related to MPSP genes of Theileria parasites found in Southeast Asian countries, including Thailand (Narathiwat) and Indonesia (Java). Theileria parasites from the blood sample were also distinguishable from the Ikeda or Chitose type parasites by the small subunit (SSU) rRNA gene sequence analysis, and they are grouped into the SSU rRNA types C/D found in Korea, North America, and Spain. The present finding of mixed infections of cattle with three different types of Theileria makes epidemiological feature of bovine theileriosis in Japan more complex. We have designed a set of primers specific to this MPSP type in order to conduct further epidemiological study.  相似文献   

4.
Benign Theileria parasites of cattle distributed in the Okinawa prefecture were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. Using universal or allele-specific primer sets, parasite DNA was amplified in 31 out of 48 blood samples obtained from beef cattle. Among the positive cases, mixed infections involving various combinations of I-, C-, and B-type parasites were detected in 24 (77.4%) samples. Phylogenetic analysis based on the MPSP gene sequences revealed that parasites with the MPSP types 1-5 and 7, exist within the Okinawa prefecture.  相似文献   

5.
Complete major piroplasm surface protein (MPSP) gene sequences of benign Theileria parasites were isolated from ticks of grazing cattle in Korea. A total of 556 tick samples were collected in five provinces: Chungbuk, Jeonbuk, Jeonnam, Gyeongbuk, and Jeju during 2010-2011. Fifteen samples from Chungbuk and Jeonnam were positive for the Theileria MPSP gene by PCR amplification using a specific primer set. A phylogenetic tree was constructed with the amplified gene sequences and 26 additional sequences published in GenBank. The benign Theileria parasites were classified into eight types, those isolated from Korean cattle ticks belonged to Types 1 (Ikeda), 2 (Chitose), 4, and 8. Types 2 and 4 were the most common types, with the rate of 40%, followed by Types 1 and 8 (with the rate of 13% and 7%, respectively). Nucleotide sequence identities of 23 theilerial MPSP sequences (15 MPSP gene sequences amplified and 8 sequences published) ranged from 67.3 to 99.8%. Multiple alignments of the deduced amino acid sequences also showed that each type was characterized by specific amino acids: 7 for Type 1, 9 for Type 2, 4 for Type 4, and 3 for Type 8.  相似文献   

6.
An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.  相似文献   

7.
Onuma  M.  Kubota  S.  Kakuda  T.  Sako  Y.  Asada  M.  Kabeya  H.  Sugimoto  C. 《Tropical animal health and production》1997,29(4):119S-123S

Bovine piroplasmosis caused byTheileria sergenti is a major cause of economic loss in grazing cattle in Japan. Infected calves show chronic anaemia with intraerythrocytic piroplasms and occasionally die in severe cases. We found that parasite stocks and isolates consist of genetically and antigenically mixed populations. To differentiate parasite populations bearing 3 allelic forms of p32/34, an immunodominant piroplasm surface protein, 3 sets of oligonucleotide primers were designed to amplify either of 3 alleles by polymerase chain reaction (PCR). By using this allele-specific PCR, we found that the majority ofT. sergenti-infected calves in Japan harbored mixed parasite populations bearing C and I type parasites. To controlTheileria infection, we produced 2 vaccine candidates: recombinant baculovirus p32 and synthetic peptide containing Lys-Glu-Lys (KEK) motif. Immunization with either recombinant p32 or synthetic peptide containing KEK sequences with Freund’s complete adjuvant resulted in low parasitemia and reduced the clinical symptoms compared to control calves. Interestingly, the parasite with the p32 allelic form corresponding to the one used as the immunogen was suppressed.

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8.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.  相似文献   

9.
A survey of Theileria parasite infection in cattle in Cambodia and Vietnam was carried out by using allele-specific polymerase chain reaction. A total of 137 blood samples from draught animals in Cambodia and 40 blood samples from dairy cattle in Vietnam were analyzed. In Cambodia, 69 out of 137(50.4%) samples were PCR-positive containing mainly the Thai and the C type parasites. In Vietnam, 11 (27.5%) samples were positive and all were of the Thai type parasite.  相似文献   

10.
为了探究表位疫苗是否可以应用于预防牛瑟氏泰勒虫(Theileria sergenti),根据MPSP (Major piroplasm surface protein)基因序列(FJ515689.1)设计合成了2对引物,以T.sergenti基因组DNA为模板,通过SOE-PCR扩增出长约361 bp的双表位基因融合片段,2个表位基因通过linker (Gly4Ser)3相连.将该片段连接pGEX-4T-2,构建pGEX-4T-E1-2重组表达载体,转化BL21,IPTG诱导表达,经SDS-PAGE电泳显示表达了约39 ku的融合蛋白.Westem blot分析表明该双表位重组蛋白可与T.sergenti阳性血清发生特异性反应,表明融合蛋白具有反应原性.  相似文献   

11.
为了解吉林省延边地区流行的羊泰勒虫种类,采用血涂片镜检和PCR方法对羊泰勒虫进行鉴定和遗传学分析。血涂片镜检观察发现,血涂片红细胞中存在圆形虫体。对羊泰勒虫MPSP基因生物信息学分析显示,MPSP编码的蛋白包含3个N-糖基化位点,14个潜在抗原表位,含有信号肽结构。建立系统进化树显示,分离到的3个虫株与Theileria luwenshunli(GQ281044)同源性为100%,亲缘关系较近,分离的羊泰勒虫为吕氏泰勒虫。由该试验结果可以得出,吉林省延边地区流行的羊泰勒虫为吕氏泰勒虫。  相似文献   

12.
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13.
Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79 x 10(-4)%.  相似文献   

14.

A total of 1000 clinically healthy small ruminants comprising 500 sheep and 500 goats from five districts within Riyadh Province in Saudi Arabia were investigated by routine Giemsa staining for hematozoan parasites. Out of these, 100 sheep and 95 goat samples were investigated by PCR using three pairs of hemoprotozoan-specific primers. Based on microscopic examination, 33.2% of sheep and 25.2% of goats were found infected with hemoprotozoan parasites, while PCR detected hematozoan infection in 46% of sheep and 33.7% of goats. Extensive molecular characterization of hematozoan infection using six pairs of species-specific primers revealed the dominance of Theileria ovis, rather than any other species, which is recorded for the first time in small ruminants in Saudi Arabia. Prevalence of T. ovis in sheep and goats was found to be the highest in Riyadh (32, 48%) followed by AL-Kharj (31, 35%), Ad-Dawadimi (31, 33%), AL-Majmaah (15, 27%), and Rumah (17, 23%). The highest parasite prevalence was recorded in the 3 years of age and >?4 years of age ruminants, while the lowest prevalence was recorded in <?1 year of age ruminants. No noticeable differences in parasite prevalence between male or female ruminants were recorded. Partial sequencing of 18S rRNA gene revealed the infection of the studied ruminants with four new isolates of T. ovis. Further characterization of the pathogenicity and the clinical effects of these T. ovis isolates in sheep and goats is highly needed. The current results can be helpful in protecting and improving livestock industry in the countries that depend on a high number of small ruminants.

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15.
In the studies previously reported, the tick-borne protozoan parasites Theileria lestoquardi and Theileria annulata were shown to differ in their capacity to infect sheep and cattle. In the studies presented here, these findings were further supported. In vitro infectivity of T. lestoquardi and T. annulata sporozoites for peripheral blood mononuclear cells of sheep and cattle were determined by analysis of cell cultures for cell proliferation, the detection of parasites in Giemsa-stained cytospin smears and the establishment of continuously growing schizont-infected cell lines. In the same way, the development of schizont-infected cells into continuously growing cell lines was studied with material isolated ex vivo from the sheep and cattle undergoing primary infections described elsewhere. Comparisons were also made between development of ex vivo cell lines from animals undergoing primary infections with those of the animals undergoing challenge infection with the other parasite species. Theileria species specific primers were used in a PCR to determine the identity of the parasites in the cell lines. These in vitro studies confirmed earlier observations that T. lestoquardi was unable to infect cattle, whereas infection of all sheep with T. annulata was proven. Moreover, earlier indications of the development of partial cross-immunity in sheep of T. annulata to T. lestoquardi and vice versa were strengthened. These findings may thus have consequences for the understanding of the epidemiology of T. lestoquardi infections of sheep. On the other hand. since piroplasms were not demonstrated in sheep infected with T. annulata, such sheep will not be infective to ticks and will consequently be unlikely to play a role in the maintenance and transmission of T. annulata to cattle.  相似文献   

16.
Piroplasms are tick-transmitted, intracellular, hemoprotozoan parasites that cause anorexia, fever, anemia, and icterus. Theileriosis is caused by Theileria sergenti and causes major economic losses in grazing cattle in Japan and Korea. In May 2003, we examined the antigenic diversity of the major piroplasm surface protein (MPSP) gene in 35 healthy Jeju black cattle that were born and raised at the National Institute of Subtropical Agriculture. On microscopic examination of Giemsa-stained blood smears, 9 of 35 cattle had intra-erythrocytic piroplasms. Hematological data were within normal range for all 35 cattle. Amplification of DNA from all blood samples using universal MPSP gene primers showed mixed infections with C, I, and B type Theileria spp. Type C was identified in 20 of 35 blood samples, and type B was identified in 17 samples. Allelic variation was seen in type B.  相似文献   

17.
A simple procedure was developed for detection of Theileria sergenti infection on the basis of hybridization of parasite DNA with a specific probe. A genomic DNA library of T. sergenti constructed in pUC-18 was screened to detect clones containing the parasite's DNA sequences by colony and Southern hybridizations. Two positive DNA inserts were purified from the recombinant plasmids and used as probes labelled with 32P or non-isotopic reagent, biotin-11-dUTP. 32P-radiolabelled and non-radioactive probes appear to be sensitive enough to detect 15 pg (equivalent to 1,200 parasites) and 125 pg (equivalent to 10,000 parasites) of purified T. sergenti DNA, and in diluted T. sergenti-infected red blood cells, they are able to detect 8,000 parasites and 16,000 parasites, respectively.  相似文献   

18.
Tissues from 4 aborted polar foxes (3 samples of brain and 4 samples of liver) were selected for Toxoplasma gondii PCR assay. Positive results of serological tests of mothers and immunofluorescence test (IFT) of fetal organ smears were the criteria of sample selection. Five sets of primers designed from B1 gene and ITS1 sequences of T. gondii were used for detection of the parasite in fetal fox tissues. All used primer sets successfully amplified T. gondii DNA in PCR from organs which were positive by IFT. Single tube nested PCR also showed positive result from a sample negative by IFT, but this product was not confirmed. The studies showed usefullness of PCR for routine diagnosis of toxoplasmosis in carnivores.  相似文献   

19.
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20.
Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).  相似文献   

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