Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily

A reverse line blot hybridisation (RLB) of 21 oligonucleotides with polymerase chain reaction (PCR) amplified regions of 16S rRNA (Ehrlichia/Anaplasma group) or 18S rRNA (Babesia/Theileria group) genes of haemoparasites detected Theileria annulata, T. buffeli/orientalis, Babesia bovis, B. bigemina, B. divergens, Ehrlichia bovis, Anaplasma marginale, A. centrale and unknown species within the Rickettsia tribe.A very high prevalence of mixed infections was detected, which indicated that animals infected with Babesia spp. were also infected with Theileria spp. and/or Anaplasma spp.The tick distribution appeared to be seasonal with Hyalomma marginatum as the most frequently observed tick and Boophilus annulatus and Ixodes ricinus as the least frequently observed ticks. Other species identified in the 818 ticks collected during the five sampling periods between April 1998 and November 1999 included H. lusitanicum, Rhipicephalus sanguineus group, R. bursa, Dermacentor marginatus, Haemaphysalis punctata, B. annulatus and I. ricinus.     

Genotyping of Babesia bigemina from cattle from a non-endemic area (Switzerland)

In August 2002, bovine anaplasmosis and concurrent infections with Mycoplasma sp. and piroplasms were reported in a cattle herd in an alpine region of Switzerland. The piroplasms were identified by PCR/sequencing of part of the 18S rRNA gene as Babesia bigemina and Theileria of the buffeli/sergenti/orientalis-complex, which have never been diagnosed in Switzerland before. The B. bigemina isolate was genetically characterised at two loci and compared with isolates from Italy, Spain, Turkey, Kenya and Mexico. Analysis of the internal transcribed spacer 2 (ITS2) of the rRNA genes revealed high polymorphism not only among the isolates but even within the isolates, and the presence of two types of the ITS2 in every isolate was confirmed. A dendrogram based on ITS2 sequences showed that the Swiss isolate was most closely related to a Spanish isolate but no sequences of the isolate from Switzerland were identical to any of the other isolates. The isolate from Italy was not positioned in the same cluster as the Swiss and the Spanish isolate. This had been anticipated as the nearest known endemic area of B. bigemina in Central Italy. Sequence analysis of the rhoptry-associated protein-1c gene (rap1c) confirmed the similarity of the Swiss and Spanish isolate. Hence, our molecular analyses of the Swiss B. bigemina isolate did not unequivocally track its geographical origin and the way of introduction remains obscure.     

东方巴贝斯虫的18S rRNA基因的扩增及系统发育研究

利用真核生物18S rRNA基因的PCR通用引物对寄生于中国水牛的巴贝斯虫（已命名为东方巴贝斯虫-BnbPsia orientalis）基因组DNA进行扩增，得到其18S rRNA全基因片段，测序后blast分析表明该虫种属巴贝斯虫无疑。将该基因1700bp长片段序列与GenBank中15种已知巴贝斯虫的相应序列进行比较分析，建立系统发育树。结果表明，东方巴贝斯虫与南非未定种的巴贝斯虫亲缘关系最近，与羊巴贝斯虫亲缘关系较近，与牛巴贝斯虫、双芽巴贝斯虫的亲缘关系较远。这一结果说明水牛东方巴贝斯虫是一独立种。     

Molecular and phylogenetic characterization of Theileria spp. parasites in autochthonous bovines (Mirandesa breed) in Portugal

A survey was conducted during the months of April-June 2003 in the northeast Portugal (Bragan?a district) in order to characterize the hemoparasite population of an autochthonous Mirandesa breed of Bos taurus. The polymerase chain reaction (PCR) analysis of the bovine blood revealed that 3 out of 116 animals were infected with Theileria and/or Babesia parasites, while reverse line blot hybridisation (RLB) analysis showed that these animals were infected with Theileria buffeli/orientalis. Cloning and sequencing confirmed the RLB results. Database sequence searches combined with phylogenetic analysis of the partial 18S ribosomal RNA gene sequences obtained enabled us to place the parasites in question as members of the T. buffeli/orientalis group, confirming the PCR/RLB diagnosis.     

一种牛源扇头蜱及其携带梨形虫种类鉴定

为了鉴定从汉中市牛体表采集到的蜱种类及其携带病原梨形虫(Piroplasma)的种类,在形态学初步观察的基础上,用PCR技术基于线粒体16SrDNA对蜱种类进行了分子鉴定,并基于梨形虫18S rRNA基因分别检测蜱体内携带巴贝斯虫属(Babesia)、泰勒虫属(Theileria)等病原情况.结果显示,所采集到的67只...     

Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.     

New advances in molecular epizootiology of canine hematic protozoa from Venezuela, Thailand and Spain

The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied. In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77 per cent of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied. In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30 per cent of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found. In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira 1 from Brazil.     

Semi-nested PCR detection of Babesia orientalis in its natural hosts Rhipicephalus haemaphysaloides and buffalo

Babesiosis has recently been recognized as an emerging infectious disease of buffalo in China. In order to investigate the epidemiology and enzootic potential of this parasite in Hubei province, we sought to develop a semi-nested PCR to detect Babesia orientalis in buffalo and the potential tick vector-Rhipicephalus haemaphysaloides by amplifying a specific 257bp fragment of B. orientalis 18S rRNA gene. The practical limit of detection showed that it had high sensitivity and an approximate parasitemia of 0.00000012% was detected by the PCR system. The blood samples of 121 asymptomatic buffaloes collected from four babesia endemic counties and that of 71 asymptomatic buffaloes collected from three babesia free counties in Hubei province of China were examined for the presence of B. orientalis using both Wright-Giemsa stained blood smear and semi-nested PCR. Microscopic examination revealed that 5/121 animals were positive, whereas 24/121 animals were positive by the semi-nested PCR assay. Of 378 ticks (R. haemaphysaloides) collected from buffaloes and examined by the semi-nested PCR, 35 were positive. The results showed that the semi-nested PCR was a useful method to investigate the epidemiology of buffalo babesiosis (B. orientalis), which is widely distributed in Hubei province, China.     

Molecular phylogenetic studies on <Emphasis Type="Italic">Theileria</Emphasis> spp. isolates (China) based on small subunit ribosomal RNA gene sequences

Six Theileria spp. from cattle, buffalo and black goat were identified in the Hubei province of China. In order to study the taxonomic status of these parasites, phylogenetic analysis of 18S rRNA genes were carried out. The 18S rRNA genes from each isolate were amplified by the polymerase chain reaction and the approximate 1.75 kb products were cloned and sequenced. Phylogenetic analysis of these gene sequences revealed that the five parasites from buffalo and cattle belonged to the Theileria sergenti/buffeli/orientalis group. The parasite from the Chinese goat (Macheng-Hubei, DQ286802) was closely related to Theileria luwenshuni isolated from sheep in the north of China. This represent the first report on the use of molecular phylogeny to classify Theileria spp. obtained in the Hubei province, showing that Theileria spp. from ruminants found in Hubei province belongs to the benign group of Theileria spp.     

Molecular phylogenetic studies on an unnamed bovine Babesia sp. based on small subunit ribosomal RNA gene sequences

The 18S small subunit ribosomal RNA (18S rRNA) gene of an unnamed Babesia species (designated B. U sp.) was sequenced and analyzed in an attempt to distinguish it from other Babesia species in China. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was ligated to the pGEM-T Easy vector for sequencing. It was found that the length of the 18S rRNA gene of all B. U sp. Kashi 1 and B. U sp. Kashi 2 was 1699 bp and 1689 bp. Two phylogenetic trees were, respectively, inferred based on 18S rRNA sequence of the Chinese bovine Babesia isolates and all of Babesia species available in GenBank. The first tree showed that B. U sp. was situated in the branch between B. major Yili and B. bovis Shannxian, and the second tree revealed that B. U sp. was confined to the same group as B. caballi. The percent identity of B. U sp. with other Chinese Babesia species was between 74.2 and 91.8, while the percent identity between two B. U sp. isolates was 99.7. These results demonstrated that this B. U sp. is different from other Babesia species, but that two B. U sp. isolates obtained with nymphal and adultal Hyalomma anatolicum anatolicum tick belong to the same species.     

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.     

Molecular studies on Babesia,Theileria and Hepatozoon in southern Europe. Part II. Phylogenetic analysis and evolutionary history

Following a study on molecular epizootiology of Hepatozoon canis and piroplasmids (Babesia spp. and Theileria spp.) in southern Europe, newly obtained sequences of 18s rRNA gene were used for phylogenetic analysis. Partial sequences were analysed in isolates showing high degree of homology (>99%) with previous GenBank entries: H. canis, B. canis vogeli, B. equi (two isolates, Spain1 and Spain2), T. annulata and Theileria sp. The complete gene sequences were used for B. ovis and B. bovis, that showed lower homology (<95%) with rapport to previously reported species or isolates. A first set of phylogenetic trees constructed with partial 18s rRNA sequences showed that most European isolates clustered unambiguously with previously described species, so that minor sequence dissimilarities found are due probably to strain variations.The second set of phylogenetic trees was made using the complete 18s rRNA sequences of 44 species from GenBank and the newly sequenced B. ovis and B. bovis. The analysis revealed for the first time a division of piroplasmids in five clades: (1) B. microti group, with B. rodhaini, B. felis, B. leo, B. microti and T. annae (proposed name for the group, without taxonomic value: Archaeopiroplasmids), (2) Western USA Theilerid-like group (proposed name: Prototheilerids), (3) Theileria group, containing all Theileria species from Bovinae (proposed name: Theilerids), (4) A first group of Babesia species including B. canis and B. gibsoni from canids together with B. divergens and B. odocoilei (proposed name: Babesids), (5) A second group composed mainly by Babesia species from ungulates: B. caballi, B. bigemina, B. ovis, B. bovis and Babesia sp. from cow (proposed name: Ungulibabesids). The bootstrap support obtained with several analytical procedures for this new dicotomy of Babesiidae was always very high. Taking into account the present phylogenetic analysis and additional paleogeographic, parasitological and zoological evidences, two hypothesis on the origin and evolution of piroplasmids groups are presented.     

New data on epizootiology and genetics of piroplasms based on sequences of small ribosomal subunit and cytochrome b genes

As a continuation of our studies on molecular epizootiology of piroplasmosis in Spain and other countries, we present in this contribution the finding of new hosts for some piroplasms, as well as information on their 18S rRNA gene sequences. Genetic data were complemented with sequences of apocytochrome b gene (whenever possible). The following conclusions were drawn from these molecular studies: Theileria annulata is capable of infecting dogs, since it was diagnosed in a symptomatic animal. According to cytochrome b sequences, isolates from cows and dog present slight differences. The same isolates showed, however, identical sequence in the 18S rRNA gene. This exemplifies well the usefulness of the mitochondrial gene for examining infra-specific variation. Babesia bovis is an occasional parasite of equines, since it was detected in two symptomatic horses. We found evidence of genetic polymorphism occurring in the 18S rRNA gene of Spanish T. equi-like and B. ovis isolates. B. bennetti from Spanish seagull is loosely related to B. ovis, and might represent a genetically distinct branch of babesids. A partial sequence of a cytochrome b pseudogene was obtained for the first time in Babesia canis rossi from South Africa. The pseudogene is distantly related to B. bigemina cytochrome b gene.These new findings confirm the ability of some piroplasms to infect multiple hosts, as well as the existence of a relatively wide genetic polymorphisms with respect to the cytochrome b gene. On the other hand, the existence of mtDNA-like pseudogenes of possible nuclear location in piroplasms is interesting due to their possible impact on molecular phylogeny studies.     

At least two genetically distinct large Babesia species infective to sheep and goats in China

A fatal disease of sheep and goats in the northern part of China has been reported to be due to Babesia ovis. However, some characteristics of the causative agent in recent reports are not in accordance with the original attributes ascribed to this parasite. Therefore, the 18S small subunit ribosomal RNA (18S rRNA) genes of a number of Babesia isolates in China were sequenced and compared with that of other Babesia and Theileria species in an attempt to clarify their taxonomic position. In the present study, seven Babesia isolates were collected from distinct areas of northern China, and the 18S rRNA genes were amplified and sequenced. The phylogenetic trees were inferred based on 18S rRNA gene sequences of the Chinese ovine Babesia isolates and some of ovine Babesia and Theileria species available in GenBank. In the phylogenetic tree, Babesia sp. isolates from Madang, Tianzhu, Lintan, Ningxian, Hebei and Liaoning all grouped with B. motasi with 88.2-99.9% identity, while Babesia sp. Xinjiang grouped in a separate clade between B. ovis and B. crassa with 79.7-81.2% identity. The results indicated that there are at least two distinct Babesia species groups-B. motasi and Babesia sp. Xinjiang, the latter was distinctly different from other ovine Babesia isolates from China with less than 86.6% identity.     

Concurrent infection with Theileria buffeli caused depression of parasitaemia in Babesia bovis and Anaplasma centrale infections in splenectomised calves but not in B bigemina infections

In five experiments undertaken in splenectomised calves it was found that Theileria buffeli infections depressed the parasitaemias of superimposed Babesia bovis and Anaplasma centrale infections, but not B bigemina infections. The course of A centrale infections did not appear to be affected by a concurrent Eperythrozoon teganodes infection.     

Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method

Babesiosis and Theileriosis are tick-borne diseases widespread in tropical and sub-tropical regions with high economic impact worldwide. In Portugal there are at least 4 tick vectors known to be competent for the transmission of Babesia and Theileria sp. identified: Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Ixodes ricinus and Haemaphysalis punctata. All these potential Babesia and Theileria tick vectors are widely distributed in Portugal, although they are predominant in the Southern region. In this study, 1104 cattle blood samples were randomly collected from Central and Southern regions of Portugal and analyzed by PCR-reverse line blotting (RLB) for the detection of Babesia and Theileria sp. Testing indicated that 74.7% of the bovines tested were positive for either Babesia and/or Theileria sp. In addition, five different apicomplexan species, namely, Theileria buffeli, Theileria annulata, Babesia divergens, Babesia bovis, and Babesia bigemina were detected by RLB among the bovines tested. T. buffeli was the most frequently found species, being present in 69.9% of the positive samples either as single infections (52.4%), or as mixed infections (17.5%). The Babesia specie most frequently found was B. divergens, detected in 4.2% of the infected bovines. Overall, infected bovines were found in all regions tested; however the highest number of infected bovines was observed in évora district (96.2%) and in cattle from Limousin breeds (81.7%). The results indicate widespread Babesia and Theileria infections in Portuguese bovines, suggesting the need for improved control of ticks and tick-borne diseases.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

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.     

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 serological survey of bovine babesiosis in northern and eastern Zimbabwe

The geographical distribution of Babesia bovis and Babesia bigemina antibodies in communal herds in northern and eastern Zimbabwe was determined using the ELISA technique. The animals in different herds in the study region had different levels of natural exposure to B. bovis (mean 32%, range 0-79%) and B. bigemina (mean 52%, range 5-92%) infections. The majority of herds (90%) were endemically unstable for B. bigemina and 62% were unstable for B. bovis. Natural region 5 and Manicaland province had the highest seroprevalence of B. bovis infection, while natural region 5 and Masvingo province had the highest seroprevalence of B. bigemina infection.