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 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.     

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.     

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.     

Changes of splenic lymphocyte subpopulation in mice inoculated with Babesia microti and Babesia rodhaini.

Changes of splenic lymphocyte subpopulation after Babesia microti and Babesia rodhaini inoculation in mice were examined by flow cytometric analysis. The B. microti inoculated mice showed a longer period of time from inoculation to the onset of increase or decrease parasitaemia (%), packed cell volume, total spleen cell numbers and surface immunoglobulin positive splenic cell numbers than respective periods in B. rodhaini inoculated mice. The Thy-1 positive cell numbers in B. microti inoculated mice and B. rodhaini inoculated mice pre-immunized with homologous parasites were significantly higher than that of B. rodhaini inoculated mice. The ratio of L3T4 positive cell/Lyt-2 positive cell after inoculation with B. microti was quite similar to that in B. rodhaini mice pre-immunized. However, the ratio in B. rodhaini inoculated mice revealed a lack of an increasing phase. These results suggested that the T-cell dependent early immune response, especially suppressor activity, was closely related to the difference in the course of infection between the non-lethal B. microti and the lethal B. rodhaini infection in mice.     

Serological evidence for Babesia canis infection of horses and an endemic focus of B. caballi in Hungary

In order to evaluate the seroconversion of horses to Babesia caballi and B. canis in Hungary, blood samples were collected from 371 animals on 23 different locations of the country. The presence of antibodies to B. caballi was screened with a competitive ELISA. All 29 positive samples came from one region (the Hortobágy). The prevalence of infection did not show correlation with sexes, and reached 100% in the age group of 2-5 years. Babesia canis-specific antibodies were demonstrated by IFAT in 6.74% of animals kept in 7 regions. The titres were low or medium level (1:40 to 1:160), indicating that the horses had previously been exposed to this piroplasm, but their infection must have been limited. The highest seropositivity rate was observed in the age group of 3-4 years, and males (stallions and geldings) were significantly more frequently infected than females. However, neither B. caballi nor B. canis could be identified in the peripheral blood samples of infected horses by PCR. Since most of the B. caballi-positive horses remained negative in the B. canis IFAT, whereas seroconversion solely to B. canis was detected in several regions of the country, serological cross-reaction between the two species can be discounted. This is the first serological evidence of horses being naturally infected with B. canis, supporting the view that piroplasms are less host specific than previously thought.     

Serodiagnosis of Babesia gibsoni infection in dogs by an improved enzyme-linked immunosorbent assay with recombinant truncated P50

The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-length), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliable reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.     

Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia

Canine babesiosis, caused by intra-erythrocytic Babesia, is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Tunisia. Detection and analysis of Babesia species from naturally infected dogs and ticks recovered from dogs were attempted by reverse line blot hybridization and nucleotide sequence analysis based on 18S rRNA gene. Out of 180 blood samples collected from domestic dogs in 4 villages situated in different bioclimatic zones, 12 were positive for Babesia canis vogeli. In addition, a total of 160 Rhipicephalus sanguineus were analysed; only one male was infected by B. canis vogeli. This is the first report on the detection of DNA belonging to B. canis vogeli in domestic dogs and in R. sanguineus in Tunisia.     

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.     

Bovine babesiosis: anti-erythrocyte antibodies purification from the sera of naturally infected cattle

Babesia bigemina and Babesia bovis are intra-erythrocytic protozoan parasites transmitted by ticks to cattle in which they induce babesiosis, a disease that resembles human malaria. Anemia, caused by the destruction of non-infected erythrocytes, is a critical feature of the disease. Anti-erythrocyte antibodies could be one of the explanations for such destruction. These antibodies are found in the sera of dogs and mice respectively infected with B. gibsoni and B. rodhaini. However, data concerning the presence of anti-erythrocyte antibodies in the sera of infected cattle are not conclusive. In the present study, we made an attempt to detect anti-erythrocyte antibodies from the sera of cattle naturally infected with B. bigemina. Erythrocytes from a non-infected calf were used in ELISA reaction for the detection of antibodies from samples. Results confirmed the presence of anti-erythrocytes antibodies in higher amounts in the serum of infected cattle. In order to correlate this increment with the parasite, anti-erythrocyte antibodies from the sera from infected calves were purified, coupled to a Sepharose-4B column and than used for anti-idiotypic antibodies purification. These antibodies were found to react with the parasites, suggesting a correlation between both anti-parasite and anti-erythrocyte antibodies.     

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%.     

A survey for infection with Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi, and Babesia canis in feral and client-owned dogs in the Turks and Caicos Islands, British West Indies

The frequency of infection with Dirofilaria immitis and Babesia canis and seropositivity to Ehrlichia canis and Borrelia burgdorferi in feral and client-owned dogs was determined. Feral dogs were 14.8 and 11.2 times more likely to be seropositive to D. immitis and E. canis, respectively, than were client-owned dogs. None of the dogs tested positive for B. burgdorferi or B. canis.     

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.     

The prevention of transmission of Babesia canis canis by Dermacentor reticulatus ticks to dogs using a novel combination of fipronil, amitraz and (S)-methoprene

Four groups of seven dogs were treated topically with a novel combination of fipronil, amitraz and (S)-methoprene in a spot-on formulation (CERTIFECT?, Merial Limited, GA, USA) on 28, 21, 14 and 7 days prior to tick infestation, respectively and acaricidal efficacy and transmission blocking compared with an untreated control group (seven dogs). All dogs were infested with adult Dermacentor reticulatus ticks harbouring Babesia canis canis. Babesia canis canis was transmitted by D. reticulatus to all seven untreated control dogs, confirmed following demonstration of clinical signs, by the detection of B. canis parasites in thin blood smears and B. canis canis PCR-RLB DNA assay on blood and the development of B. canis canis antibody titres by 14-21 days after tick infestation. The majority of treated dogs remained sero-negative for 42 days after infestation. Therefore, the treatment of dogs with CERTIFECT applied up to 28 days prior to infestation with D. reticulatus harbouring B. canis canis, successfully prevented the development of clinical signs of canine babesiosis.     

Immunity against Babesia rossi infection in dogs vaccinated with antigens from culture supernatants

Soluble parasite antigens (SPA) from different Babesia species have been shown earlier to induce protective immunity when used as vaccine. However, initial attempts to produce such vaccine against Babesia rossi infection using SPA from B. rossi culture supernatants were not or only partially successful. Here we show that when dogs were vaccinated with a vaccine comprising SPA from B. rossi combined with SPA from Babesia canis protective immunity against experimental challenge infection was induced. Immunity was reflected in reduced clinical signs that resolved spontaneously, and reduction of parasitaemia and SPA in the blood. Not a single infected erythrocyte could be found in blood smears of dogs that had been repeatedly boosted (three vaccinations in total). In contrast, three out of four control dogs required chemotherapeutic treatment to prevent death. The fourth control dog showed a transient parasitaemia that resolved spontaneously. Vaccination did not prevent the development of a transient anaemia. It is concluded that a vaccine containing a mixture of SPA obtained from in vitro culture supernatants of B. rossi and B. canis induces protection in dogs against heterologous challenge infection with B. canis (as shown before) or B. rossi.     

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.     

Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland

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.     

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.     

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.