Piroplasma infection in dogs in northern Spain

Babesiosis is a world-wide zoonosis caused by tick-borne hematozoan parasites of the genus Babesia. Canine Babesidae have historically been classified as "large Babesia" (Babesia canis) and "small Babesia" (Babesia gibsoni) based on the size of their intraerythrocytic forms. Genetic sequencing technology using the polymerase chain reaction (PCR) has allowed further subdivision. B. gibsoni has three strains: "Asia", "California" and a recently identified small babesial-like parasite, Theileria annae. This newly recognised piroplasm appears to be hyperendemic in northwest Spain. In order to provide some insight into the situation, all the cases diagnosed in our laboratory (NW of Spain) during 2003 were evaluated. Our study (62 samples) shows the existence of a piroplasm morphologically different from B. canis and similar to B. gibsoni, which is genetically related to T. annae. Severe regenerative anemia and thrombocytopenia are almost constant characteristics of infection with T. annae in dogs. In many cases azotemia is found. Abnormally high serum concentrations of urea and creatinin, together with elevated concentrations of inorganic phosphorus, hypoalbuminemia, hypercholesterolemia, proteinuria, high protein/creatinin and presence of hyaline and granular casts in the microscopic examination of urine sediment suggest a glomerular component to the disease. We conclude that observational research and clinical trials should be conducted in order to improve our understanding of this emerging disease in order to provide some insight into the best therapeutic practices.     

Ixodes hexagonus is the main candidate as vector of Theileria annae in northwest Spain

Babesia canis and Babesia gibsoni have, until recently, been considered the only piroplasms that parasitise dogs. However, recent reports indicate that "small" Babesia infections in Spanish dogs are surprisingly frequent and molecular phylogenetic analysis indicates that the infecting agent is closely related to Babesia microti. Because the 18SrDNA sequence was not completely identical to that of B. microti, the new name "Theileria annae" was assigned to the canine agent. No information is available regarding the possible vector of the new piroplasm, T. annae. As part of an effort to identify the tick that may transmit T. annae in northwest Spain we asked veterinary surgeons practising in the region to collect and send to our laboratory ticks from dogs visiting their clinics. Seven hundred and twenty ticks collected from dogs of unknown clinical status during 1998 and 636 ticks collected between November 2001 and March 2002 from 38 dogs infected with T. annae and 131 uninfected dogs were identified. Results from the first study indicated that among the Ixodidae, Ixodes hexagonus clearly predominates over Ixodes ricinus (26.11% versus 6.67%). This observation was consistent with results of the second study, in which I. hexagonus was detected in all infected dogs and 71.8% of non-infected dogs and I. ricinus was not detected in either the infected or non-infected dogs. Results from the 2001-2002 study also indicate that the presence of Dermacentor reticulatus adult females is significantly less frequent among infected than non-infected dogs (OR=0.44; 95% CI: 0.21-0.92). On the other hand, I. hexagonus adult females and males are 6.75 and 4.24 times more likely to be detected among infected than non-infected dogs, respectively, with the association being, in both cases, statistically significant (95% CI: 1.97-23.12 and 1.92-9.36, respectively). I. hexagonus emerges as the main candidate as vector of T. annae because it feeds on dogs more frequently than other ticks and because B. microti is transmitted by Ixodes ticks, both in North America and Europe. In the absence of definitive confirmation of this hypothesis, our observations suggest that I. hexagonus might serve the same role as does Ixodes scapularis (=Ixodes dammini), the vector of B. microti in eastern North America.     

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.     

PCR-RFLP for the detection and differentiation of the canine piroplasm species and its use with filter paper-based technologies

Canine piroplasmosis is an emerging disease worldwide, with multiple species of piroplasm now recognised to infect dogs. A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection and differentiation of each of the piroplasm species currently known to infect dogs on the basis of the 18S ribosomal RNA gene. The assay can potentially amplify and discriminate between Theileria annae, Theileria equi, Babesia conradae, Babesia gibsoni, Babesia sp. (Coco) and each of the Babesia canis subspecies. Non-canine piroplasm species can also potentially be detected using the described assay, however amplification of Neospora caninum was also observed. The PCR was found to have a high detection limit, capable of detecting a 2.7x10(-7)% parasitaemia or the equivalent of 1.2 molecules of target DNA when using DNA extracted from whole EDTA blood and detected a parasitaemia of 2.7x10(-5)% using blood applied to both Flinders Technology Associates (FTA) cards and IsoCodetrade mark Stix. The application of blood samples to filter paper may greatly assist in piroplasm identification in regions of the world where local technologies for molecular characterisation are limited. The assay reported here has the potential to be standardised for routine screening of dogs for piroplasmosis.     

Babesia--a historical overview

The history of the genus Babesia is briefly outlined. The classical differences with the main other genus of non-pigment-forming hemoparasites, Theileria, are the absence of extra-erythrocytic multiplication (schizogony) in Babesia and the cycle in the vector tick, which includes transovarial transmission in Babesia but only transstadial transmission in Theileria. Also, the multiplication in the red cell of Babesia, by budding, most often results in two daughter cells (merozoites), while that of Theileria gives four merozoites, often as a Maltese cross. In particular this means that what is still commonly called Babesia microti is not a Babesia and that it would be just as logical to speak of human theileriosis as of babesiosis. The small piroplasm of horses, long known as Babesia equi, is already commonly designated as Theileria equi. However, on molecular grounds, it may be necessary to create a new genus for these parasites. The Babesia species of domestic animals are briefly discussed and presented in a table.     

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.     

Presence of Mycoplasma haemofelis,Mycoplasma haemominutum and piroplasmids in cats from southern Europe: a molecular study

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).     

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%).     

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.     

Identification of Babesia species infecting dogs using reverse line blot hybridization for six canine piroplasms,and evaluation of co-infection by other vector-borne pathogens

Canine infection by vector-borne hemoparasites is frequent in tropical and sub-tropical areas where exposure to hematophageous ectoparasites is intensive. A reverse line blot (RLB) assay was designed to improve the simultaneous detection of all named canine piroplasm species combined with other vector-borne pathogens of dogs including Ehrlichia canis, Hepatozoon canis and Leishmania infantum common in the Mediterranean basin. Blood samples of 110 dogs from Spain (n = 21), Portugal (n = 14) and Israel (n = 75) were analyzed. The study evaluated 2 groups of dogs, 49 dogs with piroplasm infection detected by blood smear microscopy from Portugal, Spain and Israel, and 61 dogs surveyed from rural areas in Israel, for which infection status with vector-borne pathogens was unknown. Among the dogs previously diagnosed with piroplasmosis, infection with Babesia canis, Babesia vogeli, Babesia gibsoni and Theileria annae was detected in the Iberian dogs while only B. vogeli was found in Israeli dogs. These differences are attributed to the absence of tick vectors for some piroplasm species such as Dermacentor reticulatus in Israel. Eleven (79%) of the Babesia-positive dogs from Portugal were co-infected with other pathogens including L. infantum, H. canis and E. canis. Eight of 61 (13%) rural Israeli dogs were co-infected with two or more pathogens including B. vogeli, L. infantum, E. canis, and H. canis. Triple infections were demonstrated in 2 dogs. The RLB detection limit for Babesia was 50-fold lower than that of PCR. This study presents a RLB to simultaneously detect and separate the major vector-borne dog pathogens in southern Europe and the Middle East.     

Serum protein response and renal failure in canine Babesia annae infection

Babesia annae piroplasms have recently been recognised as a cause of infection and disease among dogs in Europe. The pathogenesis and clinical implications of this emerging disease remain poorly understood. We conducted this study to describe the electrophoretic profiles associated with the infection and to determine if B. annae associated azotaemia is caused by renal failure. We examined by microscopy 2,979 canine blood samples submitted to a diagnostic laboratory in NW Spain between September 2001 and April 2002. Small ring-shaped piroplasms were detected in blood smears of 87 samples and the identity of 58 of these presumptive cases were confirmed by PCR. This group of 58 infected dogs and a reference group of 15 healthy non-infected dogs were our study population. For all the dogs, serum protein response to -albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin- was measured by capillary electrophoresis. The response of infected and non-infected dogs was compared and within infected dogs, the response of those with azotaemia (19) was compared with that of non-azotaemic dogs (39). Infected dogs presented a significant elevation of total proteins and all the different globulin fractions, and significantly lower levels of albumin compared to non-infected dogs. Among infected dogs, those presenting azotaemia had significantly lower concentrations of total proteins, albumin, beta and gamma globulins, and significantly higher values of alpha-2 globulin. Specific gravity was below the threshold of 1,025 for all dogs with azotaemia for which a urine sample was available (7) suggesting that azotaemia, in these dogs was of renal origin. Azotaemic dogs had higher concentrations of cholesterol and triglycerides, probably as a result of a liver compensatory response to the loss of proteins. We conclude that serum protein response in B. annae infected dogs corresponds to the pattern of a haemolytic syndrome with intense inflammatory reaction and that the azotaemia associated to the infection is very likely of renal origin.     

Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection

The present study provides the first epidemiological data regarding infection by Theileria and Babesia piroplasms in cattle in Minorca. More than 94% of the studied animals were positive for the presence of Theileria sp., and of those, 41.3% were positive for the presence of Theileria annulata. These results indicate that the prevalence of Mediterranean theileriosis caused by T. annulata is very high in Minorcan dairy farms and that other Theileria sp. are also present in the area. The prevalence of infection was similar throughout the study indicating an endemic situation in this island. The use of PCR resulted in significantly higher efficacy of detection of Theileria sp. compared to microscopical observation (MO) of blood smears and allowed the specific discrimination between pathogenic and non-pathogenic theilerias which cannot be accomplished by traditional diagnosis by MO. Babesia infection in the area was mainly due to Babesia bigemina (6.0% of the studied animals were infected), while one animal (0.75%) was found to be infected by Babesia bovis. It was observed that 31% of animals infected with B. bigemina had a concurrent infection of T. annulata. PCR also resulted in a significantly higher efficacy of detection of Babesia sp. compared to MO when infection levels were higher, towards the end of the study period. The results clearly demonstrate that parasitic infection by piroplasms, especially Theileria sp. is common and endemic in the island of Minorca and that PCR is the optimal approach for the detection and discrimination of these important parasites.     

Prevalence of antibodies against Babesia canis in dogs in an endemic area

A survey of 287 dogs for antibodies against Babesia canis in dogs in an endemic area, using ELISA, produced a prevalence of 43 per cent. Antibodies occurred in dogs of all age groups, the prevalence being significantly lower in dogs aged 1 to 6 months than in older dogs. There were no differences between indigenous Nigerian dogs and exotic (foreign) dogs; and between the sexes in the prevalence of antibodies. Antibodies were more prevalent in dogs with B. canis parasitaemia and in those with a higher risk of infection. Also antibodies were detected in some puppies born to seropositive bitches. The ELISA test failed to detect antibodies in 36.1 per cent of dogs with B. canis parasitaemia.     

Detection and identification of equine Theileria and Babesia species by reverse line blotting: epidemiological survey and phylogenetic analysis

Specific oligonucleotide probes were designed to develop a new and highly sensitive reverse line blot assay to detect and identify simultaneously different Theileria and Babesia species in horses. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against different generic and species-specific probes. The survey was conducted over 243 samples of equine blood divided into three different groups: group 1, 24 horses presented as possible clinical piroplasmosis; group 2, 181 clinically healthy free-ranging horses exposed to ticks; group 3, 38 riding horses with unrelated pathologies and low or no contact with ticks. The study demonstrated a high piroplasm prevalence in the first two groups of animals. Two Theileria genotypes sharing 96.8% similarity between their 18S rRNA gene sequences and two Babesia genotypes sharing 97.4% similarity, were identified. The biologic meaning of such genotypes is discussed in terms of their phylogenetic relationships and potential pathogenicity.     

Treatment of theileriosis with parvaquone in Zambia

Parvaquone was used to treat 126 cattle with theileriosis. Theileria species schizonts were present in their lymph node biopsy smears and the majority of the animals had clinical signs of theileriosis. One hundred and fifteen treated and one untreated cattle survived the infection while 11 treated and 12 untreated animals died of the disease. Despite serological evidence of a parasite challenge during the subsequent rainy season, recovered cattle did not develop clinical signs of theileriosis but untreated cattle in the area continued to die from the disease. An intermittent low piroplasm parasitaemia (less than 1 per cent) was observed in recovered cattle for up to 14 months after detailed monitoring of cattle in the trial; this could be evidence for a carrier status for the Theileria species or strains involved.     

牛中华泰勒虫新种(梨形虫亚目:泰勒虫科)2.分子分类学研究

分离自我国甘肃中部地区土种黄牛的一种形态特异的泰勒虫，采用传统分类学研究鉴定为新种，被定名为中华泰勒虫（Theileria sinensis sp.nov）。又利用对泰勒虫属特异的PCR引物（92/93）对该种基因组DNA扩增，结果表明该种属泰勒虫属原虫确定无疑，并对代表虫种进化和分类的18S rRNA基因序列进行了测定，对已测出该基因1067bp长片断序列与基因库中9种巴贝斯虫，7种已知牛泰勒虫的相应序列进行比较、分，建立起系统发生树。树图表明，瑟氏泰勒虫和水牛泰勒虫的亲缘关系非常近， 中华泰勒虫与这两个种的亲缘关系较近，与附膜泰勒虫、小泰勒虫、环形泰勒虫、斑羚泰勒虫、突变泰勒虫差异较大。     

A review of the small canine piroplasms from California: Babesia conradae in the literature

Small piroplasms as a cause of canine babesiosis in southern California were first documented in 1990. Initially these piroplasms were considered to be Babesia gibsoni, the only small Babesia parasite known to infect dogs at that time. In the following decade, the use of molecular analysis made it clear that small canine Babesia in fact are comprised of at least three distinct species, and the isolates from dogs in southern California were not B. gibsoni. Molecular, antigenic, and morphological characteristics of the southern California species of canine piroplasm supported naming it as a distinct species, Babesia conradae. The renaming of this species prompted this literature review of small canine piroplasms in California in order to clarify clinical, diagnostic, epidemiological, and molecular characteristics of B. conradae in comparison to other small canine piroplasms. Clinical symptoms of B. conradae are similar to those of B. gibsoni; however, B. conradae infections may be more pathogenic, resulting in higher parasitaemia and more pronounced anaemia when compared with B. gibsoni-infected dogs. The immunofluorescent antibody test is the most commonly used test to diagnose B. conradae. It is important to specify which small Babesia species to test for since there is little serological cross reactivity between the small canine Babesia antigens or cross-detection in the newer molecular tests. Molecular characterization of B. conradae, based principally on the 18S small subunit rRNA gene, and recently the second internal transcribed spacer region, demonstrate that B. conradae is most closely related to piroplasms recovered from humans and animals in the western United States.     

Theileria annae in a young Swedish dog

A severe regenerative anemia was detected in a 12-week-old mixed breed puppy in Sweden.A small protozoan parasite was observed in erythrocytes on a blood smear. It was initially suspected to be Babesia gibsoni based on its size and because B. gibsoni was previously recorded in Sweden.Surprisingly, specific polymerase chain reaction analysis identified the protozoan as Theileria annae. T. annae is endemic in Northwest Spain, is very uncommonly reported elsewhere and has never been recorded in Scandinavia. T. annae has been identified in dogs used for dog fighting, and it is thought to be transmitted by dog bites. This puppy was a mixed pit bull terrier. Pit bull terriers are sometimes used for dog fighting. T. annae has been reported to be transmitted vertically, and in light of the puppy’s age, this transmission was suspected in the present case.     

Blood parasites of dogs in Ibadan

Between March, 1974 and February, 1975, blood smears made from 500 of the dogs brought to the small animal clinic of the University of Ibadan and the state owned veterinary clinic in the same town were stained with Giemsa and examined for blood parasites. Forty-nine per cent of the dogs carried blood parasites, the commonest of which was Babesia cards. Others were B. gibsoni, Haemobartonella canis, Trypanosoma congolense, Eperythrozoon and Microfilaria. The parasitaemia due to these parasites in the dogs examined was mostly light, although a substantial number of dogs had medium and heavy parasitaemias. No significant difference was found in the susceptibility of the local and exotic breeds to infection with the parasites.     

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.