First evidence and molecular characterization of Babesia vogeli in naturally infected dogs and Rhipicephalus sanguineus ticks in southern France

Babesiosis is an emerging tick-borne disease of animals and humans caused by intraerythrocytic protozoa of the genera Babesia and Theileria. In France canine babesiosis has a high prevalence with Babesia canis thought to be the main aetiological agent of the disease. Babesia vogeli has already been reported to occur in Europe and in other countries around the Mediterranean Sea. The tick Rhipicephalus sanguineus, the main known vector of B. vogeli, occurs in southern France. However, only one case of a B. vogeli infected dog has been reported to date in France. To gain further insight into the prevalence of Babesia and Theileria infections in dogs and ticks of the R. sanguineus complex, a study was conducted in a veterinary practice in the south of France from January to September 2010. Twelve bloods from dogs and 36 R. sanguineus ticks were analyzed using PCR and sequencing. For the analysis of ticks, a new primer was designed to specifically amplify the B. vogeli 18S rRNA gene. Four dogs (33.3%) and 8 ticks (22.2%) were found to be infected with B. vogeli. This approach has thus revealed for the first time a cluster of cases of canine babesiosis caused by B. vogeli in France and highlights the need to systematically screen for pathogens potentially responsible for canine babesiosis at the species level using suitable molecular tools.     

Molecular and serological detection of Ehrlichia canis and Babesia vogeli in dogs in Colombia

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.     

Confirmation of occurrence of Babesia canis vogeli in domestic dogs in South Africa

The prevalence of Babesia infections in domestic dogs in South Africa was studied using reverse line blot hybridization and 18S sequence analysis. Babesia canis vogeli was confirmed for the first time in domestic dogs in South Africa. Out of a total of 297 blood samples collected from domestic dogs in Bloemfontein, East London, Johannesburg, Durban and from the Onderstepoort Veterinary Academic Hospital, 31 were positive for Babesia canis rossi, whereas B. c. vogeli was detected in 13 dogs. None of the dogs carried both parasites. The detection of B. c. vogeli has implications with regard to prevalence and varied clinical manifestation of canine babesiosis in South Africa.     

Canine babesiosis in Slovenia: molecular evidence of Babesia canis canis and Babesia canis vogeli

Canine babesiosis, caused by intraerythrocytic Babesia spp., is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Slovenia. Therefore, 238 dogs admitted to the Small animal clinic in Ljubljana from the years 2000 to 2002 were tested for the presence of babesial parasites in the blood. Based on clinical, microscopic and molecular investigations, 14 dogs (5.9%) were determined as being infected with babesiae. Clinical signs relating to acute haemolysis, fever, anorexia, depression and haematological abnormalities such as anaemia and thrombocytopenia were noticed in most of the 14 infected dogs. The morphology of the parasites was indicative of Babesia canis infection. Two subspecies were detected, namely B. canis canis (11 dogs, 4.6%) and B. canis vogeli (3 dogs, 1.3%) using PCR and subsequent sequence analysis of portions of nns rRNA gene. In addition, based on nucleotide sequence analysis, the 11 isolates of B. c. canis could be subdivided into three groups, whereas the three B. c. vogeli isolates were genetically identical. The results of this study demonstrate the presence of canine babesiosis due to B. c. canis and B. c. vogeli in Slovenia.     

Babesia canis canis and Babesia canis vogeli infections in dogs from northern Portugal

Canine babesiosis represents an important veterinary medical problem. This study describes the molecular characterization of babesial parasites detected in eight clinically suspected dogs from northern Portugal, affected by lethargy, muscle tremors, weight loss, pale mucous membranes, hyperthermia or red-coloured urine. Microscopic examination of peripheral blood smears showed large intraerythrocytic piroplasms morphologically compatible with Babesia canis in all eight animals. DNA was extracted from blood on filter paper, and a Babesia spp. infection confirmed by polymerase chain reaction (PCR) amplification of a 408bp fragment of the 18S rRNA gene. Analysis of PCR-derived sequences revealed that seven dogs were infected with B. canis canis and one with B. canis vogeli. This is the first molecular identification report of both the species B. canis and the subspecies B. canis canis and B. canis vogeli in dogs from Portugal.     

Babesia canis canis and Babesia canis vogeli clinicopathological findings and DNA detection by means of PCR-RFLP in blood from Italian dogs suspected of tick-borne disease

The aims of this study were to determine the presence of Babesia spp. in blood samples from Italian dogs with clinical signs compatible with tick-borne diseases by means of PCR-restriction fragment length polymorphism (RFLP) and describe the clinicopathological findings of dogs with Babesia infection. We evaluated the majority of canine babesiosis cases by means of clinical history, physical examination, hematological, biochemical, serum electrophoresis, urinalysis and hemostatic tests. Forty-five out of 164 canine blood samples studied were positive to Babesia PCR-RFLP with the following results: Babesia canis canis (n=34) and Babesia canis vogeli (n=11). The majority of B. c. canis infections were detected in Northern Italy (29.1%; 30/103). B. c. vogeli cases were detected mainly in Central and Southern Italy (16.3%; 10/61). Only one B. c. vogeli was detected in Northern Italy (0.9%; 1/103). Three positive samples to B. c. canis and four positive samples to B. c. vogeli were selected for sequencing of a fragment of the 18S rRNA gene (410bp) for further molecular characterization. The sequence obtained from all seven dogs was 99/100% homologous to sequences from B. c. canis and B. c. vogeli, respectively, present in GenBank. Sixty-two percent of dogs infected with B. c. canis had recently travelled on a hunting trip to East European countries. The main acute clinical signs were dehydration, apathy, anorexia and fever. The majority of dogs infected with B. c. canis presented at initial clinical examination mild to severe thrombocytopenia, hyperfibrinogenemia, mild to moderate normocytic-normochromic non-regenerative anemia, hemolysis and neutropenia. The urinalysis showed hemoglobinuria in 13/19 dogs suggesting intravascular hemolysis. Dogs with B. c. canis infection had high levels of C-reactive protein. Hypoalbuminemia was present in 17/26 dogs. The 11 cases of B. c. vogeli infection did not present a homogenous clinicopathological pattern. B. c. vogeli infections were observed in young dogs causing hemolytic anemia and in adult/old does that frequently presented predisposing factors such as splenectomy or immunocompromised conditions. In conclusion, this study demonstrates the presence of B. c. canis and B. c. vogeli in Italian sick dogs and differences in clinicopathological pattern in these two species of B. canis.     

Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs

The morphologically small Babesia species isolated from naturally infected dogs in Europe, Japan, and US are described as Babesia gibsoni despite the fact that molecular techniques show that they should be assigned to two or three separate taxons. The morphologically large Babesia isolated from dogs in Europe, Africa, and US were generally classified as B. canis until it was proposed to distinguish three related, albeit genetically distinct subspecies of this genus, namely B. canis canis, B. canis rossi, and B. canis vogeli. The insight into the molecular taxonomy of canine piroplasms is, however, limited because only partial small subunit ribosomal RNA (ssrRNA) sequence data exist for two species from the B. canis group. In this work, we molecularly characterised natural Babesia infections in 11 dogs from Croatia, France, Italy, and Poland. These infections were diagnosed as caused by B. canis canis and B. canis vogeli based on the analysis of the complete sequence of the ssrRNA genes. Phylogenetic analysis confirmed that the large Babesia species of dogs belong the to the Babesia sensu stricto clade, which includes species characterised by transovarial transmission in the tick vectors and by exclusive development inside the mammalian host erythrocytes. The new data facilitate the reliable molecular diagnosis of the subspecies of B. canis.     

Endocrine predictors of mortality in canine babesiosis caused by Babesia canis rossi

This prospective, cross-sectional, observational study was designed to determine the association between the hormones of the pituitary-adrenal and pituitary-thyroid axes and outcome in dogs with naturally occurring Babesia canis rossi babesiosis. Ninety-five dogs with canine babesiosis were studied and blood samples were obtained from the jugular vein in each dog prior to treatment at admission to hospital. Serum cortisol, adrenocorticotrophic hormone (ACTH), thyroxine, free thyroxine and thyrotropin (TSH) concentrations were measured. Diagnosis was confirmed by polymerase chain reaction and reverse line blot and dogs infected with Babesia canis vogeli or Ehrlichia canis were excluded. Three outcomes were defined: hospitalization with subsequent death (n=7); hospitalization followed by recovery (n=56); and treatment as an outpatient (n=32). Serum cortisol and ACTH concentrations were significantly higher in the dogs that died, compared to hospitalized dogs that survived and compared to dogs treated as outpatients. Serum T4 and free T4 concentrations were significantly lower in the dogs that died, compared to the hospitalized dogs that survived and compared to dogs treated as outpatients. Serum TSH concentrations were not significantly different between any of the groups. Mortality was significantly associated with high cortisol and high ACTH concentrations and with low T4 and fT4 concentrations in dogs suffering from B. canis rossi babesiosis.     

Detection and molecular characterization of a novel large Babesia species in a dog

Babesia canis has generally been considered the only large Babesia to infect dogs. Here we describe the molecular characterization of a large Babesia species that was detected in the blood and bone marrow of a dog with clinical and hematological abnormalities consistent with babesiosis. Analysis of the 18S rRNA genes revealed a unique sequence that shared 93.9% sequence identity with B. bigemina and 93.5% sequence identity with B. caballi, compared to 91.2-91.6% identity with B. canis canis, B. c. vogeli, and B. c. rossi. Cross-reactive antibodies against B. canis, B. gibsoni (Asian genotype), or B. gibsoni (California genotype) antigens were not detected in acute or convalescent serum samples. The dog was treated with imidocarb diproprionate, which resulted in the resolution of clinical signs, and subsequently Babesia DNA was not detectable by PCR in post-treatment samples. The organism described in this report represents a genetically unique large Babesia sp. and is the eighth genetically distinct piroplasm capable of infecting the domestic dog.     

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.     

Molecular survey of Babesia infection in dogs in Okinawa, Japan

A total of 80 free-roaming dogs on Okinawa Island, Japan, were examined for Babesia infection using the polymerase chain reaction (PCR) and sequence analysis. Of 80 samples, 12 were positive in a Babesia genus-specific PCR. Consequent species-specific PCR for B. canis and B. gibsoni revealed that 5 (6.3%) and 7 (8.8%) dogs were infected with B. canis and B. gibsoni, respectively. Sequence analysis of the PCR products revealed that the 18S rRNA gene sequence of B. canis detected from dogs in Okinawa was very close to B. canis vogeli with sequence similarity of 99.94%.     

Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada

To identify the tick-borne pathogens in dogs from Grenada, we conducted a serologic survey for Ehrlichia canis in 2004 (104 dogs) and a comprehensive serologic and molecular survey for a variety of tick-borne pathogens in 2006 (73 dogs). In 2004 and 2006, 44 and 32 dogs (42.3% and 43.8%) were seropositive for E. canis, respectively. In 2006, several tick-borne pathogens were identified by serology and PCR. DNA of E. canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, and Bartonella sp. were identified in 18 (24.7%), 14 (19.2%), 5 (7%), 5 (7%), and 1 (1.4%) dogs, respectively. Six (8.2%) dogs were seropositive for Bartonella vinsonii subsp. berkhoffii. All dogs were seronegative and PCR-negative for Rickettsia spp. Coinfection with two or three pathogens was observed in eight dogs. Partial 16S rRNA E. canis and A. platys sequences were identical to sequences in GenBank. Partial 18S rRNA gene sequences from the Grenadian H. canis were identical to each other and had one possible mismatch (ambiguous base) from H. canis detected from Spain and Brazil. Grenadian B. c. vogeli sequences were identical to B. c. vogeli from Brazil and Japan. All of the detected pathogens are transmitted, or suspected to be transmitted, by Rhipicephalus sanguineus. Results of this study indicate that dogs from Grenada are infected with multiple tick-borne pathogens; therefore, tick-borne diseases should be included as differentials for dogs exhibiting thrombocytopenia, leukopenia, fever, or lethargy. One pathogen, E. canis, is also of potential public health significance.     

Detection of Anaplasma platys and Babesia canis vogeli and their impact on platelet numbers in free-roaming dogs associated with remote Aboriginal communities in Australia

OBJECTIVE: To detect Anaplasma platys and Babesia canis vogeli infection, using polymerase chain reaction (PCR)-based assays, in free-roaming dogs associated with eight Aboriginal communities in remote areas of Australia and to determine the impact of infection through the assessment of platelet numbers. PROCEDURES: Blood samples from 215 dogs were screened by PCR for A platys and B canis vogeli using established genus-specific DNA primers for the 16S and 18S rRNA genes respectively. Both A platys DNA and B canis vogeli DNA were confirmed from the screening PCR either by sequencing or by the use of species-specific primers. Peripheral blood films from 92 of the 215 dogs were used to estimate platelet numbers through an indirect method. RESULTS: Of 215 dogs, 69 (32%) were positive for A platys, 22 (10%) for B canis vogeli and 24 (11%) for both. The two organisms were detected singularly and as coinfection in all communities. For the 92 dogs in which peripheral blood films were examined, the mean estimated platelet counts for the non-infected dogs was 318 x 10(9)/L, those infected with A platys alone was 256 x 10(9)/L, those with B canis vogeli alone was 276 x 10(9)/L and those infected with both parasites was 169 x 10(9)/L. In young dogs, infection produced significantly decreased mean platelet counts when compared to uninfected dogs. Thrombocytopenia (< 200 x 10(9)/L) was detected in 18 (51%) dogs infected with A platys alone, 3 (33%) dogs infected with B canis vogeli alone, 13 (72%) dogs coinfected, and 8 (27%) uninfected dogs. CONCLUSIONS: A platys and B canis vogeli infection, either singularly or together, was widespread in free roaming dogs associated with remote Aboriginal communities in the Northern Territory and north-western New South Wales. Moreover, both A platys and B canis vogeli infections were associated with a reduction in mean platelet numbers in dog populations, particularly in young dogs. The fact that 51% of dogs infected with A platys alone and 72% dogs coinfected were thrombocytopenic compared to 27% of uninfected dogs suggests that the organism alone or in combination with B canis vogeli has the potential to cause thrombocytopenia and perhaps contribute to a clinical bleeding disorder in infected dogs.     

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.     

Evolution of clinical, haematological and biochemical findings in young dogs naturally infected by vector-borne pathogens

Longitudinal studies evaluating the evolution of clinical, haematological, biochemical findings in young dogs exposed for the first time to multiple vector-borne pathogens have not been reported. With the objective of assessing the evolution of clinical, haematological and biochemical findings, these parameters were serially monitored in naturally infected dogs throughout a 1-year follow-up period. Young dogs, infected by vector-borne pathogens based on cytology or polymerase chain reaction, were examined clinically and blood samples were obtained at seven different follow-up time points. Dogs were randomized to group A (17 dogs treated with a spot-on formulation of imidacloprid 10% and permethrin 50%) or to group B (17 dogs untreated). In addition, 10 4-month-old beagles were enrolled in each group and used as sentinel dogs. At baseline, Anaplasma platys was the most frequently detected pathogen, followed by Babesia vogeli, Bartonella spp., Ehrlichia canis and Hepatozoon canis. Co-infections with A. platys and B. vogeli, followed by E. canis and B. vogeli, A. platys and H. canis and A. platys and Bartonella spp. were also diagnosed. In dogs from group B, abnormal clinical signs were recorded at different time points throughout the study. No abnormal clinical signs were recorded in group A dogs. Thrombocytopenia was the most frequent haematological alteration recorded in A. platys-infected dogs, B. vogeli-infected dogs and in dogs co-infected with A. platys and B. vogeli or A. platys and Bartonella spp. Lymphocytosis was frequently detected among dogs infected with B. vogeli or co-infected with A. platys and B. vogeli. Beagles were often infected with a single pathogen rather than with multiple canine vector-borne pathogens. There was a significant association (p<0.01) between tick infestation and A. platys or B. vogeli, as single infections, and A. platys and B. vogeli or A. platys and Bartonella spp. co-infections. This study emphasizes the clinical difficulties associated with assigning a specific clinical sign or haematological abnormality to a particular canine vector-borne disease.     

Molecular survey of Babesia canis in dogs in Nigeria

An epidemiological study of Babesia canis in dogs in Nigeria was performed. Four hundred blood samples collected from dogs in Nigeria were investigated using nested PCR and sequence analysis. On nested PCR screening, nine samples (2.3%) produced a band corresponding to a 698-bp fragment indicative of B. canis infection. Sequence analysis of the PCR products identified eight samples (2.0%) as B. canis rossi and the ninth (0.3%) as B. canis vogeli. This is the first report of the prevalence of B. canis rossi and B. canis vogeli in dogs in Nigeria.     

Autochthonous cases of canine babesiosis in the canton Solothurn

Starting in November 2003 a series of five clinical cases of canine babesiosis was registered in the region of Oberg?sgen (canton Solothurn). All presented dogs showed increased body temperature, thrombocytopenia and hemoglobinuria, and none of the dogs had been abroad or visited endemic regions in the southern or western part of Switzerland so far. Babesia canis was detected in the blood smears of all five patients, but only three had detectable specific antibodies against this parasite. However, seroconversion was found in a second sample collected from the negative dogs at a later timepoint, confirming the diagnosis of canine babesiosis. The blood samples of two parasitized dogs were used for DNA-isolation and were tested with a Babesia-specific PCR, detecting the 18S rRNA-gene. Sequencing of the amplified products revealed a 100% identity with the sub-species B. canis canis. The ticks Rhipicephalus sanguineus and Dermacentor marginatus are potential vectors for B. canis. In the area where the infection with B. canis was suspected a total of 152 ticks was collected and characterized; one was a female R. sanguineus.Although babesia could not be detected in the latter tick and the final prooffor the complete life cycle is still lacking, it is very probable that B. canis has become autochthonous in the canton Solothurn.     

Geographic distribution of babesiosis among dogs in the United States and association with dog bites: 150 cases (2000-2003)

OBJECTIVE: To identify the geographic distribution of babesiosis among dogs in the United States and determine, for dogs other than American Pit Bull Terriers (APBTs), whether infection was associated with a recent dog bite. DESIGN: Retrospective study. ANIMALS: 150 dogs. PROCEDURE: Canine blood samples submitted to the North Carolina State University Vector-Borne Disease Diagnostic Laboratory between May 2000 and October 2003 for which results of a Babesia-specific polymerase chain reaction assay were positive were identified, and breed and geographic origin of dogs from which samples were obtained were recorded. History and hematologic abnormalities for dogs that were not APBTs were recorded, and possible associations with a recent dog bite were examined. RESULTS: Dogs positive for Babesia DNA were located in 29 states and 1 Canadian province (Ontario). Babesia gibsoni was the most commonly detected species, with B gibsoni DNA detected in blood samples from 131 of 144 (91%) dogs. Of the 131 dogs positive for B gibsoni DNA, 122 (93%) were APBTs. Of the 10 dogs positive for Babesia canis vogeli DNA, 6 were Greyhounds. In dogs other than APBTs, there was an association between having recently been bitten by another dog, particularly an APBT, and infection with B gibsoni. CONCLUSIONS AND CLINICAL RELEVANCE: Results document an expansion of the known geographic range for babesiosis among dogs in the United States. Testing for babesiosis should be pursued in dogs with clinicopathologic abnormalities consistent with immune-mediated hemolytic anemia or thrombocytopenia, particularly if there is a history of a recent dog bite.     

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay

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.