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1.
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   

2.
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.  相似文献   

3.
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.  相似文献   

4.
Clinical symptoms produced by Mycoplasma spp. and piroplasmids in cats are sometimes similar. Diagnosis of these pathogens is difficult by microscopic procedures and molecular methods have been used as an alternative. We present in this work, the development of new molecular procedures for diagnosis of the aforementioned organisms, together with a molecular characterization of isolates found in southern European cats.A single PCR-RFLP procedure was designed for diagnosis of Mycoplasma spp. and a seminested PCR-RFLP was designed for diagnosis of piroplasmids. The 16S or 18S rRNA genes of isolates found in clinical samples were partially sequenced in all positive cases.Mycoplasma spp. was detected in 9 (30%) out of 30 symptomatic cats from Spain. Sequencing indicated that 66.6% of these isolates can be ascribed to Mycoplasma haemofelis and only 33.3% to Mycoplasma haemominutum. Partial 16S rRNA sequences obtained in Spanish isolates were very similar to those previously published from the UK and the USA.The presence of piroplasmids (Babesia and Theileria spp.) was studied in 16 cats from Spain (n=13) and Portugal (n=3). Animals analyzed were 10 cats with immunosuppressive viral infection (either FeLV or FIV), 5 asymptomatic cats and 1 cat with Babesia-compatible symptoms. Asymptomatic cats were all PCR-negative. Partial sequencing of 18S rRNA gene demonstrated that the Babesia-symptomatic cat was infected with Babesia canis canis whereas 3 (30%) out of the 10 cats with immunosuppressive viral infection were coinfected with piroplasmids (1 with B. canis canis, 1 with Theileria annae, and 1 with B. canis canis and T. annae both).  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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%).  相似文献   

8.
DNA of two species of piroplasmids was detected in dromedaries during a survey of blood protozoans in Jordan between 2007 and 2009. Ten clinically healthy camels (10%) originating from three Jordanian districts were found, using a PCR assay, to harbor Theileria or Babesia species in their blood and no mix infection was determined. Analysis of the partial 18S rRNA gene sequences of these parasites allowed their unambiguous identification as equine piroplasmids Babesia caballi (n=6) and Theileria equi (n=4). In case of latter species, a novel genotype was found in horses. This first molecular-based species determination of piroplasmids from camels further contributes to the growing evidence of low host specificity of piroplasmids.  相似文献   

9.
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.  相似文献   

10.
Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.  相似文献   

11.
Luo J  Yin H  Liu Z  Yang D  Guan G  Liu A  Ma M  Dang S  Lu B  Sun C  Bai Q  Lu W  Chen P 《Veterinary parasitology》2005,133(1):1-6
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.  相似文献   

12.
Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.  相似文献   

13.
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.  相似文献   

14.
The presence and prevalence of tick-borne haemoparasites in water buffalo from the Hubei province, south China was investigated using the reverse line blot (RLB) hybridization assay and phylogenetic analysis of the parasite 18S rRNA gene. Theileria buffeli (19.1%) was the most frequently found species in all of the locations, followed by Babesia orientalis (8.9%), Babesia bovis (1.0%) and Babesia bigemina (0.7%). Only 12 (3.9%) of the samples had mixed infections. Eleven samples with single infections were selected for further characterization using 18S rRNA gene sequence analysis. Phylogenetic analysis showed that the eight T. buffeli 18S rRNA gene sequences obtained grouped into four clusters, of which three grouped with the known T. buffeli types B and D. The remaining five grouped separately from the previously describe T. buffeli types, constituting new T. buffeli types. The two B. bigemina 18S rRNA gene sequences obtained grouped closely with B. bigemina Kunming; this serves as the first report of B. bigemina in the Hubei province. The B. orientalis Daye 18S rRNA gene sequence obtained grouped closely with the previously reported B. orientalis Wuhan strain and with Babesia sp. Kashi 1 and Kashi 2.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

18.
Ehrlichiosis and babesiosis are tick-borne diseases, caused mainly by Ehrlichia canis and Babesia canis, respectively, with a worldwide occurrence in dogs, whose main vector is the brown-dog tick, Rhipicephalus sanguineus. The present work aimed to detect the presence of E. canis and Babesia sp. in 91 dog blood samples in Colombia, by molecular and serological techniques. We also performed sequence alignment to indicate the identity of the parasite species infecting these animals. The present work shows the first molecular detection of E. canis and B. vogeli in dogs from Colombia. Immunoglobulin-G (IgG) antibodies to E. canis and Babesia vogeli were found in 75 (82.4%) and 47 (51.6%) sampled dogs, respectively. Thirty-seven (40.6%) and 5 (5.5%) dogs were positive in PCR for E. canis and Babesia sp., respectively. After sequencing, amplicons showed 99% of identity with isolates of E. canis and B. vogeli. The phylogenetic trees based on 16S rRNA-Anaplasmataceae sequences and 18S rRNA-piroplasmid sequences supported the identity of the found E. canis and B. vogeli DNAs, respectively. The present work shows the first molecular detection of E. canis and B. vogeli in dogs in Colombia.  相似文献   

19.
Liu Z  Luo J  Bai Q  Ma M  Guan G  Guan G  Yin H 《Veterinary microbiology》2005,107(1-2):145-148
In this study, a phylogenetic tree was inferred through comparing five 16S rRNA gene sequences of four isolates of Anaplasma ovis and one of Anaplasma marginale in China with all nineteen 16S rRNA gene sequences deposited in GenBank (12 A. marginale, 3 A. ovis and 4 Anaplasma centrale derived from America, Uruguay, South Africa, Zimbabwe, Australia, Isreal and Japan). The analysis showed that all A. ovis isolated in China were separated into an A. ovis cluster, while the A. marginale in China was separated into an A. marginale cluster (see Fig. 1). This analysis demonstrated that there are at least two different Anaplasma species widespread among ruminants in North China.  相似文献   

20.
The Babesia gibsoni heat shock protein 70 gene (BGHsp70) was cloned by polymerase chain reaction (PCR) and sequenced. The length of the gene was 1938 bp and the predicted polypeptide was 646 amino acids long with a calculated molecular weight of 70,627. The amino acid sequences of BGHsp70 from 17 isolates were identical, though there were six types of polymorphisms among the corresponding nucleotide sequences. There was no intron in the BGHsp70 gene. Phylogenetic analysis of the amino acid sequence of Hsp70 showed that B. gibsoni was most closely related to B. bovis and lies within a phylogenetic cluster with Theileria. These results suggest that Hsp70 was well conserved among intraerythrocytic protozoa.  相似文献   

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