Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats

In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.     

羊泰勒虫PCR检测方法的建立和初步应用

利用羊泰勒虫18SrRNA基因的序列特点,设计合成种特异性引物,建立羊泰勒虫PCR检测方法,该方法能特异性扩增398bp的羊泰勒虫18SrRNA基因片段,而对羊巴贝斯虫、羊无浆体、牛环形泰勒虫和牛伊氏锥虫的基因组DNA没有扩增带出现。对羊泰勒虫基因组DNA的最小检测量为0.12fgDNA。通过检测124份临床样品,24份为羊泰勒虫感染阳性,其余为阴性。结果表明,建立的PCR检测方法具有极高的敏感性和特异性,可用于羊泰勒虫病和临床健康带虫羊的诊断。     

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: II. In vitro studies.

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.     

Prevalence and distribution of tropical theileriosis in eastern Turkey

This study was carried out to determine the prevalence and distribution of tropical theileriosis in cattle in eastern Turkey by microscopical, serological and molecular methods. A total of 1561 whole blood, 1505 serum and 1483 blood smear samples were collected from cattle of various breeds and ages in 11 towns of Eastern Turkey. Theileria annulata piroplasm DNA extracted from cattle blood was amplified by polymerase chain reaction (PCR) using species-specific primers. Serum antibodies against T. annulata were investigated by indirect fluorescence antibody test (IFAT). Blood smears were examined for Theileria piroplasms by microscopical examination (ME). In the examination of DNA extracted from 1561 blood samples, an amplicon with the size of 721bp was obtained in 37.8% (590/1561) of these samples. Serum antibodies against T. annulata and piroplasm of Theileria spp. were detected in 34.9% (526/1505) and 19.7% (293/1483) of the samples, respectively. The differences between ME and PCR results and between ME and IFAT results were statistically significant (P < 0.05). In contrast, there was no significant difference between the PCR and IFAT results. A total of 179 ticks (136 female; 43 male) belonging to Hyalomma spp. were collected from cattle from three towns. Ticks were identified to be Hyalomma anatolicum anatolicum on the basis of morphological features.     

Molecular identification, genetic diversity and distribution of Theileria and Babesia species infecting small ruminants

Detection and identification of Theileria and Babesia species in 920 apparently healthy small ruminants in eastern Turkey, as well as parasite genetic diversity, was investigated using a specifically designed reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified and hybridized to a membrane onto which catchall and species-specific oligonucleotide probes were covalently linked. Three Theileria and one Babesia genotype were identified. Comparison of the Theileria genotypes revealed 93.6-96.2% similarity among their 18S rRNA genes. Two Theileria shared 100% and 99.7% similarity with the previously described sequences of T. ovis and Theileria sp. OT3, respectively. A third Theileria genotype was found to be clearly different from previously described Theileria species. The genotype was provisionally designated as Theileria sp. MK. The Babesia genotype shared 100% similarity with Babesia ovis. The survey indicated a high prevalence of piroplasm infections in small ruminants (38.36%). Theileria spp. prevalence was 36.08%. Prevalence of B. ovis was 5.43%. The most abundant Theileria species identified was T. ovis (34.56%) followed by Theileia sp. MK (1.30%) and Theileria sp. OT3 (0.43%).     

A molecular survey of bovine Theileria parasites among apparently healthy cattle and with a note on the distribution of ticks in eastern Turkey

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.     

PCR检测新疆南疆部分地方品种绵羊的嗜血支原体和泰勒虫感染情况

为了解新疆南疆部分地方品种绵羊嗜血支原体和泰勒虫的感染情况,采用PCR法检测新疆南疆5个地方品种绵羊共100份血液DNA样本.结果表明:新疆南疆部分地方品种绵羊泰勒虫和嗜血支原体感染率分别为35.0％(35/100)和3.0％(3/100).仅和田羊和多浪羊上检测到嗜血支原体感染,感染率分别为8.0％(2/25)和5....     

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: I. In vivo responses.

In a series of experiments, sporozoite stabilates of a Theileria lestoquardi (Lahr) and a T. annulata (Ankara) stock prepared from Hyalomma anatolicum anatolicum ticks, were used to examine the infectivity of both parasite species for sheep and cattle and to study the development of cross-immunity between these parasite species. In the first experiment sheep and cattle were inoculated with T. lestoquardi sporozoites. Surviving animals and naive sheep and cattle were, in the second experiment, inoculated with T. annulata. In the third experiment, naive sheep and sheep previously infected with T. annulata, were inoculated with T. lestoquardi. The following responses to inoculations were monitored: clinical and haematological signs of infection, appearance of parasitic stages of the parasites in lymph node biopsies and in peripheral blood and serological response to T. lestoquardi and T. annulata schizont antigens. While T. lestoquardi readily infected sheep and caused severe disease, it did not infect cattle. On the other hand, T. annulata infected both cattle and sheep. However, whereas cattle became severely affected, infected sheep showed mild clinical symptoms only and piroplasms did not develop. Despite their different behaviour in the host species examined, cross-immunity studies suggested that the parasite species are very closely related. Experiments in sheep indicated that T. lestoquardi infection protected against subsequent T. annulata infection. On the other hand, recovery from T. annulata infection did not prevent infection by sporozoites of T. lestoquardi, resulting in the establishment of schizonts and their subsequent development into piroplasms, although it protected against the major clinical effects of T. lestoquardi infection.     

利用套式PCR检测牛羊乏质体

为同时检测和鉴定牛边缘乏质体、中央乏质体及绵羊乏质体,根据这3种病原体的msp4基因核苷酸序列,自行设计、合成了针对3种乏质体的2对通用引物,及分别针对三者的特异引物,通过PCR条件优化,建立了检测乏质体及分别鉴定3种乏质体的套式PCR方法,并与OIE推荐的msp5半套式PCR比较.结果显示:该方法对牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫、伊氏锥虫均未扩增出特异性片段.套式PCR检测乏质体DNA量为0.2 pg(相当于6个感染红细胞).检测l 119份来自6个不同地区的奶牛、肉牛、水牛及羊的临床样品,阳性106份,经鉴定边缘乏质体46份,中央乏质体15份,绵羊乏质体35份,混合感染中央乏质体和绵羊乏质体4份,混合感染边缘乏质体和绵羊乏质体3份,混合感染边缘乏质体和中央乏质体3份.首次在分子生物学水平证明中央乏质体存在于中国.同时,证明牛可以混合感染边缘乏质体和中央乏质体或绵羊乏质体,以及混合感染中央乏质体和绵羊乏质体.上述848份样品用OIE推荐的msp5半套式PCR同时检测,两者符合率为98.5％(835/848).检测结果表明,msp4套式PCR特异、敏感,可用于边缘乏质体、中央乏质体、绵羊乏质体的检测和鉴定.     

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Molecular studies on Babesia,Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects

Molecular epizootiology of piroplasmids (Babesia spp., Theileria spp.) and Hepatozoon canis was studied in mammals from southern Europe (mainly from Spain, but also from Portugal and France). Partial amplification and sequencing of the 18s rRNA gene was used for molecular diagnosis. In some particular cases (B. ovis and B. bovis) the complete 18s rRNA gene was sequenced. Blood samples were taken from domestic animals showing clinical symptoms: 10 dogs, 10 horses, 10 cows, 9 sheep and 1 goat. In addition, DNA samples were isolated from blood of 12 healthy dogs and from spleen of 10 wild red foxes (Vulpes vulpes). The results of the survey were the following: Piroplasmid infections: Approximately from 50 to 70% of wild or domestic mammals (symptomatic) were infected.Piroplasmids detected in ruminants were:COW: B. bovis, T. annulata and Theileria sp. (type C). Sheep and goat: B. ovis. Piroplasmids present in canids were: Babesia canis vogeli, Babesia canis canis, Theileria annae and B. equi. The only piroplasmid found in asymptomatic dogs was B. equi. Piroplasmids found in horse were: B. equi and B. canis canis.H. canis infections in canids: H. canis was absent of domestic dog samples, whereas all foxes studied were infected by this protozoa.Genetic analysis showed that most of piroplasmid and Hepatozoon isolates from southern Europe matched unambigously with previously described species, as demonstrated by the high level sequence identity between them, usually between 99 and 100%. Minor differences, usually detected in hypervariable regions of 18s rRNA gene are probably due to strain variations or rare genetic polymorphisms. A possible exception was B. bovis, which shows a relatively lower degree of homology (94%) with regard to other B. bovis isolates from several countries. The same is true for B. ovis, that showed a 94% identity with regard to Babesia sp. from South African cow and a 92% with rapport to B. bovis from Portugal.     

Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil

Intraerythrocytic protozoan species of the genera Theileria and Babesia are known to infect both wild and domestic animals, and both are transmitted by hard-ticks of the family Ixodidae. The prevalences of hemoprotozoa and ectoparasites in 15 free-living Mazama gouazoubira, two captive M. gouazoubira and four captive Blastocerus dichotomus from the State of Minas Gerais, Brazil, have been determined through the examination of blood smears and the use of nested polymerase chain reaction (nPCR). The cervid population was inspected for the presence of ticks and any specimens encountered were identified alive under the stereomicroscope. Blood samples were collected from all 21 animals, following which blood smears were prepared, subjected to quick Romanowsky staining and examined under the optical microscope. DNA was extracted with the aid of commercial kits from cervid blood samples and from tick salivary glands. The nPCR assay comprised two amplification reactions: the first was conducted using primers specific for a 1700 bp segment of the 18S rRNA gene of Babesia and Theileria species, whilst the second employed primers designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank. The ticks Amblyomma cajennense, Rhipicephalus microplus and Dermacentor nitens were identified in various of the cervids examined. Of the animals investigated, 71.4% (15/21) were infected with hemoprotozoa, including Theileria cervi (47.6%), Theileria sp. (14.3%), Babesia bovis (4.8%) and Babesia bigemina (4.8%). However, only one of the infected wild cervids exhibited accentuated anaemia (PCV=17%). This is first report concerning the occurrence of Theileria spp. in Brazilian cervids.     

Identification of a Mycoplasma ovis-like organism in a herd of farmed white-tailed deer (Odocoileus virginianus) in rural Indiana

Mycoplasma ovis is a hemoplasma parasite of sheep, goats, and reindeer; however, natural hemoplasma infection in white-tailed deer has not previously been reported. Subsequent to finding many coccoid, bacillary, and ring-shaped organisms, consistent with hemotropic mycoplasmas, on RBCs from a 72-day-old female white-tailed fawn, we sought to (1) identify the putative hemoplasma observed in blood from the fawn, (2) evaluate others in the herd for hemoplasma infection, and (3) identify clinicopathologic characteristics of hemoplasma-infected white-tailed deer. EDTA-anticoagulated whole blood was collected from the fawn and 8 apparently healthy does in the same herd. CBCs were performed on 7 nonclotted samples from the fawn and 6 does. DNA was extracted from all samples, followed by PCR amplification of bacterial (16S rDNA) and protozoal (18S rDNA) genes. The nearly complete 16S rDNA product from the fawn's sample was directly sequenced and compared with known sequences in the GenBank database. Samples from the fawn and 7 of 8 does were PCR-positive using hemoplasma-specific and M ovis-specific protocols. The fawn was PCR-negative for Anaplasma spp., Babesia spp., and Theileria spp. The 16S rDNA sequence from the fawn (GenBank accession number, FJ824847) was most closely related to M ovis (AF338268), having 98.5% sequence identity. The fawn had a mild nonregenerative anemia, a neutrophilic left-shift with toxic change, aspiration bronchopneumonia, and gastrointestinal disease. Hematologic values, including blood film evaluation, in infected does were unremarkable. The M ovis-like organism may have acted as either an opportunistic or primary pathogen in the fawn. The high occurrence of subclinical infections in the does suggests that white-tailed deer may act as wildlife reservoirs for M ovis.     

Prevalence of Theileria infections in goats and sheep in southeastern China

Theileriosis is an important tick-borne hemoprotozoan disease, which can cause severe economic loss in animal husbandry. In this paper, one hundred peripheral blood samples of goats and sheep from southeastern China were examined for the Theileria infection. A region of Theileria 18S rRNA gene was amplified by nested PCR in 26 samples. All the nested PCR amplicons were cloned and sequenced. Alignment analysis has shown that these sequences are highly homologus to each other with identities from 99.2% to 100%. Blast the sequences against NCBI database indicated 99% homology with Theileria sp. China 1 (Theileria luwenshuni). Phylogenetic tree has also shown that the newly identified Theileria are in the same clade with T. luwenshuni. The results have revealed a relatively high prevalence of Theileria in some areas of southeastern China and little genetic diversity in these infections.     

Detection of pathogenic leptospires in urine from naturally infected cattle by nested PCR

A nested polymerase chain reaction (PCR) using primers from the LipL32 sequence of Leptospira spp. was used to detect shedding of pathogenic leptospires in urine from naturally infected cattle. Amplicons (497bp) were obtained from 21 pathogenic reference serovars belonging to four species (L. interrogans, L. borgpetersenii, L. santarosai, L. kirschneri). DNA was amplified from 26/30 urine samples taken from cattle with suspected leptospirosis and from leptospires cultivated from 10 of these samples. The limit of detection of DNA in the clinical samples was 200pg and the nested PCR detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified using DNA from other common bacterial species from the bovine urogenital tract or urine, or from the non-pathogenic L. biflexa Andamana serovar. The nested PCR exhibited high specificity and sensitivity for detection of pathogenic serovars in urine from cattle.     

Survey of Theileria lestoquardi antibodies among Sudanese sheep

The prevalence of Theileria lestoquardi antibodies in Sudanese sheep from nine geographical areas in Sudan was determined using indirect fluorescent antibody "IFA" test. Out of 315 samples examined, 51 (16.2%) were found positive and ranged between 23.4% in River Nile State and 10% in Kasala and Darfour Provinces with an overall prevalence of 16.2% indicating widespread distribution of the infection. We also report on presence of antibodies reactive to Theileria annulata in sheep sera.     

套式PCR扩增山羊吕氏泰勒虫18S rRNA基因

泰勒虫是一种危害动物的重要血液寄生性原虫,常寄生于牛、羊、骆驼和其他野生动物,在中国的东北、西北等地较为流行。采集安徽定远县某山羊场疑似焦虫病的山羊血样,首先采用血涂片法检查,再用Blood DNA&Tissue kit提取血液基因组,参照Kim和Wei的方法设计2对引物用于泰勒虫18S rRNA基因的扩增,并对该方法进行敏感性和特异性分析。结果显示:血涂片检查明显可见红细胞内有典型的虫体,大小为0.6~2μm,初步怀疑为泰勒虫(Theileria);PCR扩增可扩增出359 bp大小目的片段,与预期结果相一致;基因序列同源性表明,该泰勒虫分离株基因与已报道泰勒虫毒株(JQ926740.1)核苷酸同源性最高,可达99.72%;在遗传进化方面,所分离的泰勒虫和吕氏泰勒虫(Theileria luwenshuni)Nanjingdong(JQ926740.1)最接近,而且在一个分支上。本试验所建立的套式PCR具有良好的特异性,且敏感性较高,最低测出量是3.8 fg/μL。本次分离的羊血液原虫为吕氏泰勒虫。     

Detection of Babesia ovis by PCR in Rhipicephalus bursa collected from naturally infested sheep and goats

Polymerase chain reaction (PCR) was used to assess the presence and the frequency of Babesia ovis infection in the adult Rhipicephalus bursa and their hosts in Elazig province located in eastern Turkey. Tick and blood samples were collected from 32 sheep and 28 goats of four selected herds. A total of 226 R. bursa were randomly selected from the collected ticks and their salivary glands were dissected out in 0.85% saline under stereo microscope. DNA amplification method revealed that the frequency of B. ovis infections in the ticks and the small ruminants were 16.37% (37/226) and 6.66% (4/60), respectively. Three positive products, two of which were from the salivary glands of R. bursa and the other from sheep blood were purified from agarose gel and sequenced. The results showed that nucleotide sequences were identical to the previously reported nucleotide sequences of B. ovis. It is concluded that R. bursa might play an important role in the field as a natural vector of the parasite.     

新疆南疆部分地方品种羊无浆体的分子检测与鉴定

为了解新疆南疆部分地方品种羊的无浆体感染情况和分子特征,用PCR法检测新疆南疆5种地方品种绵羊共100份血液DNA样本,发现无浆体总感染率为67.0％(67/100).以多浪羊感染率最高,为100％(20/20),和田羊感染率最低,为44.0％(11/25);散养和圈养羊的无浆体感染率分别为74.0％(37/50)和6...     

马泰勒虫病PCR检测方法的建立和应用

为寻求一种快速、有效的马泰勒虫 (Theileria equi,T.equi) PCR检测方法。基于马泰勒虫18S rRNA基因序列,在其V4高变区设计特异性引物Te-18F、Te-18R,通过PCR技术获得了531 bp的核酸片段。用该引物对马泰勒虫、马泰勒虫和驽巴贝斯虫混合样本、驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫基因组模板进行特异性试验。同时对马泰勒虫基因组模板进行不同浓度稀释后扩增,以便于确定试验的敏感性。用本试验建立的方法与常规显微镜镜检方法对45份马属动物血样进行检测。特异性试验显示,在被检测的9个样本中,只有马泰勒虫及马泰勒虫和驽巴贝斯虫混合模板中扩增出了符合大小的特异核酸片段。驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫的扩增结果均为阴性。灵敏度试验结果表明,PCR对马泰勒虫的扩增效率可达到10^-13。对本试验建立的PCR检测马泰勒虫方法评估结果显示,PCR对马泰勒虫的检出率为17.78%(8/45),显微镜镜检结果只有8.89%(4/45)两者的符合率为100%。本试验建立的马泰勒虫PCR检测方法不失,为一种好的检测方法。