Epidemiological survey of Babesia gibsoni infection in dogs in eastern Japan

To determine the distribution of Babesia gibsoni infection in dogs in the eastern part of Japan, an epidemiological survey of dogs suspected of having B. gibsoni infection was attempted using the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Thirty-five of 115 such dogs (30.4%) were positive by PCR and/or ELISA. The 35 positive dogs consisted of 28 Tosa dogs, 4 American Pit Bull Terriers, and 3 mongrel dogs in Aomori, Fukushima, Ibaraki, Gunma, Chiba, Tokyo, Kanagawa, and Nagano Prefectures. The positive dogs had a significantly lower rate of tick exposure and a higher rate of bites by other dogs. Twenty-two of 35 B. gibsoni-positive dogs were infected with hemoplasma, and the rate of infection was significantly higher than that of B. gibsoni-negative dogs.     

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.     

Incidence of canine Babesia gibsoni infection and subclinical infection among Tosa dogs in Aomori Prefecture, Japan

To identify the incidence of Babesia gibsoni (B. gibsoni) in Aomori Prefecture, northeastern Japan, dogs with acute B. gibsoni infection were investigated at the Animal Teaching Hospital, Kitasato University, between April 2002 and March 2003. Eighteen dogs with acute B. gibsoni infection were recognized; they were all male dogs of the fighting dog breed Tosa. Their platelet counts were below normal and their packed cell volumes (PCVs) were at various levels. We collected blood samples from 141 Tosa dogs from Aomori Prefecture and used polymerase chain reaction assay to investigate the incidence of subclinical B. gibsoni infection. We also looked into the serological abnormalities associated with thrombocytopenia or anemia in subclinical infection. Forty-one of 87 dogs (47.1%) with histories of dog fighting, and one dog of 54 without a history of dog fighting were positive for B. gibsoni; that is, 42 of 141 dogs (29.8%) showed a positive result. The mean platelet counts of dogs with subclinical infection were significantly lower and levels of anti-platelet IgG were significantly higher than levels for dogs without infection. Anti-erythrocyte membrane IgG levels were significantly higher in dogs with subclinical infections, although mean PCVs were not significantly different. Tosa dogs from Aomori Prefecture, Japan, were highly infected with B. gibsoni subclinically and this pathogen might be successfully transmitted during dog fighting. Dogs with subclinical infections were at risk of chronic thrombocytopenia, which may be due to autoimmune mechanisms.     

Blood, Bull Terriers and Babesiosis: further evidence for direct transmission of Babesia gibsoni in dogs

This study reports on the epidemiology of Babesia gibsoni in American Pit Bull Terriers living in a region of western Victoria in southern Australia. Both American Pit Bull Terriers (n = 100) and other dog breeds (n = 51) were screened for B gibsoni using immunofluorescent antibody testing (IFAT) and/or polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). A questionnaire was also completed by each dog owner, ascertaining the husbandry and habits of the dogs sampled. Fourteen dogs were positive for B gibsoni using IFAT and/or PCR-RFLP and all were American Pit Bull Terriers. Dogs that were male and/or had been bitten by or were biters of other American Pit Bull Terriers were more likely to be B gibsoni positive, thus suggesting that blood-to-blood transmission contributes to the spread of this disease between dogs.     

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.     

Hemolytic anemia caused by Babesia gibsoni infection in dogs.

Babesia gibsoni caused severe hemolytic anemia in 11 dogs from southern California. The most common clinical signs of B gibsoni infection were lethargy, anorexia, anemia, and thrombocytopenia. Acute infection with B gibsoni may be misdiagnosed as autoimmune hemolytic anemia. Diagnosis was most reliably determined by identification of the intraerythrocytic parasites on Giemsa-stained blood smears. The pathogenicity of B gibsoni, difficulties in diagnosis, the parasite's resistance to treatment with available drugs, and frequent interstate movement of dogs indicate that this disease may be a serious threat to dogs throughout the United States.     

Babesia gibsoni infection among dogs in the southeastern United States

OBJECTIVE: To identify subclinical Babesia gibsoni infection in American Pit Bull Terriers from the southeastern United States and to determine the genetic sequence of parasite DNA isolated from these dogs. DESIGN: Case series. ANIMALS: 33 American Pit Bull Terriers and 87 dogs of various other breeds. PROCEDURE: Blood smears were examined for microscopic evidence of the parasite, and DNA was extracted from blood samples and used in a polymerase chain reaction (PCR) assay designed to amplify the small subunit ribosomal RNA gene sequence of B. gibsoni. Amplification products of the expected size were sequenced, and sequences were compared with published sequences for B. gibsoni isolates. Hematocrit, platelet count, mean platelet volume, WBC count, and eosinophil count were compared between dogs with positive PCR assay results and dogs with negative results. RESULTS: Results of the PCR assay were positive for 18 of the 33 (55%) American Pit Bull Terriers, including all 10 dogs with microscopic evidence of parasitemia. Only 1 of these dogs was clinically ill at the time blood samples were collected. Results of microscopic evaluation of blood smears and of the PCR assay were negative for the 87 other dogs. Hematocrit and platelet count were significantly lower in dogs with positive PCR assay results than in dogs with negative results. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that American Pit Bull Terriers in the southeastern United States may be subclinically infected with B. gibsoni. However, subclinical infection was not identified in dogs of other breeds from the same geographic area.     

Comparison of partial ribosomal DNA sequences of Babesia gibsoni occurring in Miyazaki prefecture, Japan.

Nucleotide sequences of ribosomal DNA (rDNA) of Babesia (B.) gibsoni occurring in Miyazaki, western Japan, were examined using blood samples obtained from seven dogs suffering from natural canine babesiosis. DNA isolated from these blood samples was subjected to the polymerase chain reaction (PCR). The nucleotide sequences of the PCR products were determined and compared with other rDNA sequences of B. gibsoni isolated from Asia, Europe and U.S.A. Although homology values between our isolates and those isolated from Europe and U.S.A. were both 84.0%, respectively, our isolates were identical to the Asian types. In conclusion, B. gibsoni occurring in Miyazaki was revealed to have the genotype Asia 1 or Asia 2 from a comparison of the partial rDNA sequences.     

Clinical usefulness of antibodies against Babesia gibsoni detected by ELISA with recombinant P50

The clinical usefulness of antibodies against Babesia gibsoni detected by ELISA with recombinant P50 was examined in dogs in an area where B. gibsoni infection was endemic. Only 8 among 14 dogs with acute type B. gibsoni infection without a previous history of infection were positive. This high percentage of false-negative results is thought to be a weak point of ELISA as a diagnostic tool. However, 14 other anemic dogs with a confirmed history of B. gibsoni infection were all positive, thus confirming the higher sensitivity of ELISA in detecting a history of infection.     

Clindamycin in the treatment of Babesia gibsoni infections in dogs

This report examines the effectiveness of clindamycin for the treatment of babesiosis in dogs (n=10) experimentally infected with Babesia gibsoni (B. gibsoni). Clindamycin (25 mg/kg body weight, per os, q 12 hours for 14 days) gradually reduced parasitemia levels and induced morphological changes that indicated degeneration of parasites (e.g., segmentation; size reduction; localization in the cell limbic and/or torn state of the nucleus; and swelling, decrease, or disappearance of the cytoplasm) in the majority of dogs. Clindamycin treatment reduced the clinical symptoms characteristic of Babesia infection, including anemia, anorexia, and listlessness. Clindamycin might be useful as a medicine for treatment of B. gibsoni infection.     

Erythrocytic antioxidant defense, lipid peroxides level and blood iron, zinc and copper concentrations in dogs naturally infected with Babesia gibsoni

Babesiosis is a common tick borne disease of dogs in tropical and subtropical regions of the world caused by different species of Babesia. The present study aimed to examine erythrocyte lipid peroxide and erythrocytic antioxidant levels in dogs with clinical babesiosis, caused by Babesia gibsoni, and impact of the disease on blood iron, zinc and copper levels. The study was conducted on 10 naturally occurring cases of canine babesiosis with the history of tick infestation, erratic pyrexia, and prolonged illness. Microscopic examination of Giemsa stained peripheral blood smears confirmed B. gibsoni infection in the erythrocytes. Six apparently healthy dogs of different age, sex and breeds, brought for either health checkup or vaccination were used for comparison. Levels of erythrocytic antioxidant enzymes were significantly (P<0.01) higher in sick dogs than those of cytologically negative dogs (catalase: 0.192+/-0.024 units/mg Hb vs 0.074+/-0.004 units/mg Hb; superoxide dismutase: 0.014+/-0.0009 units/mg Hb vs 0.006+/-0.0008 units/mg Hb and lipid peroxide: 6.01+/-0.30 nmol MDA/mg Hb vs 1.89+/-0.10 nmol MDA/mg Hb). The levels of blood micronutrients were significantly low in these dogs (iron: 89.87+/-8.12 microg/g vs 126.44+/-14.65 microg/g; zinc: 3.67+/-1.85 microg/g vs 5.62+/-1.83 microg/g and copper: 0.55+/-0.63 microg/g vs 0.65+/-0.04 microg/g). The study demonstrated oxidative damage in dogs naturally infected with B. gibsoni. Low level of blood iron, zinc and copper seems to have an additional role in the genesis of anaemia and oxidative stress.     

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%.     

The Effect of Diminazene Aceturate on Spienectomized Dogs with Babesia gibsoni Infection

The effect of diminazene aceturate on splenectomized and nonspienectomized dogs with Babesia gibsoni (B. gibsoni) infection was investigated. In splenectomized dogs, the fissional and multiplicational stages of B. gibsoni were observed in peripheral blood films, and hemoglobinuria was frequently observed. These findings were different from previous reports and were not changed by administration of diminazene aceturate. It is clear that the intramuscular administration of diminazene aceturate at the dose of 3 mg/kg body weight for 3 days is not effective against B. gibsoni infection in splenectomized dogs.     

Efficacy of atovaquone against Babesia gibsoni in vivo and in vitro

The therapeutic efficacy of atovaquone against Babesia gibsoni was examined in three dogs experimentally infected with B. gibsoni isolated from naturally infected dogs in Aomori Prefecture, Japan. Once parasitemia reached 10%, atovaquone was administered orally (30 mg/kg twice daily for 7 days). Within 2 days of atovaquone treatment, the parasite disappeared from blood smears without any clinical side effects. Anemia and thrombocytopenia were significantly improved in all the dogs. However, a polymerase chain reaction assay revealed that a B. gibsoni marker gene was intermittently present in peripheral blood after atovaquone therapy, indicating that the organism had not been eliminated, and parasites reappeared in blood smears 33 days after the last treatment. To investigate the change in sensitivity against atovaquone, an in vitro sensitivity test was performed using peripheral blood obtained from an untreated dog that was infected with the original parasite isolate, and from two of the experimentally infected and atovaquone-treated animals (blood was collected at the time of the post-treatment recurrence of the B. gibsoni infection). Atovaquone was added to the culture medium to final concentrations of 0.1, 1, 10, 100, and 1000 nM. For the untreated parasites, complete growth inhibition occurred at 1000 nM of atovaquone, whereas the recurrent parasites were inhibited by only 39.52 +/- 8.34% and 31.31 +/- 8.14% at this concentration after 48 h of incubation. Thus, the recurring parasites were less sensitive to atovaquone than the untreated originally isolated parasites.     

The therapeutic efficacy of two antibabesial strategies against Babesia gibsoni

Various combination strategies for treating Babesia gibsoni have been described. However, relapses after administering some combinations of antibabesial drugs and the presence of drug-resistant B. gibsoni still pose significant challenges to veterinarians. To compare the efficacy of a combination of clindamycin, diminazene, and imidocarb (CDI) to that of a combination of atovaquone and azithromycin (AA) for the treatment of B. gibsoni and to correlate drug efficacy with B. gibsoni mutations, 30 client-owned dogs with natural B. gibsoni infections were collected in the study. 17 dogs were treated with AA, and 13 dogs were treated with CDI combination. Hematological parameters were recorded on the day that the dogs were presented for treatment and during treatment. To detect the parasitic DNA, the B. gibsoni 18S rRNA gene was amplified, and to analyze the mutations, the cytochrome b (CYTb) gene was sequenced. The therapy duration for all of the dogs that recovered was 23.3±7.8 days in the AA group and 41.7±12.4 days in the CDI group. Nine of the 17 dogs in the AA group and 11 of the 13 dogs in the CDI group completely recovered. Seven dogs in the AA group and 2 dogs in the CDI group relapsed after treatment. The M121I mutation in the B. gibsoni CYTb gene was detected in all of the samples that were collected from AA-relapsed and AA-nonremission dogs. The dogs in the CDI group exhibited higher recovery rates and lower relapse rates during treatment for B. gibsoni infection. In addition, the detected M121I mutation was associated with AA treatment. The CDI combination is a promising alternative treatment strategy for B. gibsoni.     

Transfusion-associated Babesia gibsoni infection in a dog

A 2.5-year-old spayed female German Shepherd Dog was referred for evaluation of progressive anemia, lethargy, and weight loss. Seventeen days earlier, the dog had received a whole blood transfusion to manage hemorrhage after ovariohysterectomy. Mild fever, splenomegaly, and thrombocytopenia were also identified. Von Willebrand disease and Babesia gibsoni infection were diagnosed. Because of the serologic cross-reactivity of B gibsoni and B canis in the immunofluorescent antibody assay for IgG antibodies against these organisms, polymerase chain reaction amplification of parasite DNA was required to identify the infecting Babesia sp. The source of the B gibsoni infection was traced to an apparently healthy American Pit Bull Terrier blood donor. Despite resolution of clinical signs in the dog of this report, a series of antiparasitic treatments failed to eliminate the B gibsoni infection. Screening of potential blood donor dogs for Babesia spp is becoming increasingly important in the United States.     

Sequence conservation in the rRNA first internal transcribed spacer region of Babesia gibsoni genotype Asia isolates

Babesia gibsoni genotype Asia is a small, tick-transmitted intraerythrocytic protozoan that parasitizes dogs. Reports suggest that it is increasingly diagnosed in the United States. The clinical outcome of infection with this piroplasm is often variable, leading us to hypothesize that the different clinical outcomes resulting from B. gibsoni genotype Asia infection are due to genetically distinguishable strains that differ in virulence. As a first step to assess the genetic variability of B. gibsoni isolates originating from the southeastern United States, we sequenced the rRNA first internal transcribed spacer region of recent isolates from Georgia and Alabama, and compared these sequences with isolates originating from Japan and Australia. All isolates examined proved to be genetically identical at the first internal transcribed spacer region, although this region differed distinctly from other Babesia species and closely related apicomplexan species. Although negating our hypothesis, this information gives us insight into the recent evolutionary history and spread of B. gibsoni genotype Asia in dogs in the U.S. Our research suggests that the gradual rise in prevalence of canine babesiosis due to B. gibsoni genotype Asia in the United States may be a result of clonal expansion of a single strain within a susceptible host population.     

Is the detection of anti-Rhipicephalus sanguineus (Rs24p) antibodies a valuable epidemiological tool of tick infestation in dogs?

Antibodies against the 24 kDa Rhipicephalus sanguineus (Rs24p) protein were detected by ELISA to evaluate the relationship between antibodies and tick infestation. The mean titer of 3 dogs that underwent 2 experimental infestations with adult ticks was transiently increased after the second infestation. There was a significant difference in mean titers between positive control dogs naturally infested with ticks and tick-naive dogs. These results suggested that anti-Rs24p antibodies detected by ELISA are a marker of tick exposure. There was no significant difference in mean titers between tick-naive dogs and seropositive dogs to Ehrlichia canis. Some dogs positive for E. canis antibodies showed, however, higher titers than most tick-naive dogs. R. sanguineus may be related to the E. canis infection in Japan.     

Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA.

A real-time fluorogenic polymerase chain reaction (PCR) assay based on SYBR green that allows for sensitive, reproducible, and accurate quantification of Babesia gibsoni (Asian genotype). DNA from peripheral blood of infected dogs was developed. Standard curves were created by plotting the input amount of a standard template, constructed with plasmid DNA containing 182 base pairs (bp) of the p18 gene, against threshold cycle numbers. The curves showed a wide dynamic range (1,000,000-fold input) and high correlation values (>0.99). The PCR amplification efficacy of the standard template was similar to that of intact genomic DNA obtained from peripheral blood with B. gibsoni infection. The detection limit of the assay was 9 parasites/microl of blood with B. gibsoni infection. The intra-assay and interassay coefficients of variation of the threshold cycles ranged from 0.70% to 1.89% and from 1.18% to 1.92%, respectively. This assay system was found to be reproducible and accurate for the quantification of parasite DNA in experimentally infected dogs and far more sensitive than traditional microscopic examination.     

Anti-erythrocyte membrane antibodies detected in sera of dogs naturally infected with Babesia gibsoni.

Due to the potential for anti-erythrocyte membrane antibodies as possible enhancers of erythrocyte destruction, the presence of serum anti-erythrocyte membrane antibodies in 31 dogs with Babesia gibsoni infection admitted to a veterinary hospital was investigated by an enzyme linked immunosorbent assay (ELISA) and immunoblotting analyses. This infection resulted in an increase of anti-erythrocyte membrane antibodies in 84% (IgG) and 74% (IgM) of 31 infected dogs, respectively. This was confirmed by the similarity in the protein profiles of the erythrocyte membrane antigens immunoblotted with rabbit antiserum to dog erythrocyte membrane antigens and infected dog serum. These results suggest the production of anti-erythrocyte membrane antibodies was induced by B. gibsoni infection.