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.     

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.     

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.     

Platelet size,platelet surface-associated IgG,and reticulated platelets in dogs with immune-mediated thrombocytopenia

Abstract: Immune-mediated thrombocytopenia (IMT) is a disorder in which bound IgG on the surface of platelets results in platelet removal and alterations in mean platelet volume. Using flow cytometry, alterations in platelet size, platelet surface-associated IgG (PSAIgG), and numbers of reticulated platelets were determined in 13 dogs with primary IMT and 4 dogs with secondary IMT induced by experimental infection with Babesia gibsoni . Effects of sample age on platelet parameters also were determined, using samples from 20 dogs with normal platelet counts analyzed within 4 hours and after 24, 48, and 72 hours of storage in EDTA. No significant changes in platelet count, platelet size, or reticulated platelet percentage were observed in samples assayed within 4 and 24 hours of blood collection; whereas PSAIgG values increased 3 to 7 fold in samples stored for 24–72 hours. Using reference values for freshly collected or 24-hour-old samples, 10 of 13 (77%) dogs with primary IMT and all B gibsoni-inf ected dogs had increased PSAIgG levels. In 12 (75%) of the 16 dogs with thrombocytopenia the percentage of reticulated platelets was increased; however, absolute numbers of reticulated platelets were within reference values. Moreover, PSAIgG level and the percentage of reticulated platelets were not always increased concurrently in dogs with primary and secondary IMT. Platelet microparticles were detected in all B gibsoni-infected dogs, 8 of 13 (62%) dogs with primary IMT, and transiently in a dog that responded to immunosuppressive treatment. The results of this study indicate that sample age and time of sampling during disease affect interpretation of platelet parameters in dogs with IMT.     

Epidemiological survey of Babesia gibsoni infection in dogs in Japan by enzyme-linked immunosorbent assay using B. gibsoni thrombospondin-related adhesive protein antigen

A nationwide epidemiological survey of Babesia gibsoni infection in non-fighting dogs was conducted using an improved ELISA with recombinant B. gibsoni thrombospondin-related adhesive protein (BgTRAP). A total of 1206 dogs from 27 prefectures were examined and 128 (10.6%) tested positive. In the eastern part of Japan, 39 dogs out of the 559 (7.0%) examined were positive, while 89 dogs out of 647 (13.8%) tested positive in the western part of Japan. Although the percentage of dogs that tested positive was significantly (p=0.0001) lower in the eastern part compared to the western part of Japan, overall these results indicate that B. gibsoni infection of dogs has a widespread geographic distribution throughout the country. A history of tick infestation was identified as a significant risk factor for B. gibsoni infection (p=0.0091), while sex (p=0.9411), age (p=0.0920) and breed (p=0.0549) of dogs were not statistically significant risk factors. These results indicate that tick infestation is the most dominant risk factor for B. gibsoni infection of non-fighting dogs in Japan and suggest that other B. gibsoni transmission routes, such as fighting and transplacental transmission, may be less important.     

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.     

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.     

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.     

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.     

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.     

A Possible treatment strategy and clinical factors to estimate the treatment response in Bebesia gibsoni infection

The effectiveness of combination therapy using clindamycin, metronidazole and doxycycline against canine babesiosis, and the usefulness of platelet count and the plasma C-reactive protein (CRP) concentration as an estimation factor for treatment, were evaluated in four dogs experimentally infected with Babesia gibsoni. The combination therapy successfully eliminated B. gibsoni in peripheral blood in 3 of 4 dogs, however the remaining dog showed obvious uncontrolled relapse after a temporary recovery. In addition, it was shown that CRP levels decreased in an inverse relationship to the recovery of packed cell volume and therefore CRP levels could be used as an optional clinical marker to estimate the response to treatment.     

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.     

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%.     

Efficacy of combined atovaquone and azithromycin for therapy of chronic Babesia gibsoni (Asian genotype) infections in dogs

Babesiosis caused by Babesia gibsoni (Asian genotype) is an emerging disease in dogs in the United States. To date, no drugs have been shown to eliminate B. gibsoni (Asian genotype) infections from dogs. Twenty-two dogs that remained persistently infected with B. gibsoni (Asian genotype) after either imidocarb diproprionate and or diminazine aceturate therapy were identified and randomly and evenly distributed into 2 groups. One group was treated with atovaquone and azithromycin combination therapy, and the other group received a placebo. Eight of 10 dogs in the treatment group had no detectable B. gibsoni (Asian genotype) DNA, as determined by a sensitive and specific polymerase chain reaction (PCR) assay, in any of their posttreatment samples. In contrast, B. gibsoni (Asian genotype) DNA was detectable by PCR in the posttreatment samples from 11 of 11 of the placebo-treated dogs. One dog in the treatment group was excluded from the treatment outcome analysis. This dog had 2 consecutive negative PCR assay results and was euthanized because of ongoing degenerative joint disease prior to completion of the study. No adverse effects of treatment were reported in any dog during the study period. A combination of atovaquone and azithromycin is the 1st described treatment that will either eliminate B. gibsoni (Asian genotype) infections or suppress the parasitemia below the limit of detection in the majority of treated dogs.     

Babesia gibsoni infections in dogs from North Carolina

The recognition of canine babesiosis in North Carolina caused by Babesia gibsoni documents the expansion of the previously reported endemic area of this disease. Clinical signs ranged from severe hemolytic anemia and thrombocytopenia to subclinical infections. No infected dogs had traveled to endemic areas. Antibabesial treatment failed to eradicate the organism from infected dogs.     

Improvement of DNA extraction method for dried blood spots and comparison of four PCR methods for detection of Babesia gibsoni (Asian genotype) infection in canine blood samples

To eradicate canine babesiosis in epidemic areas, mass-screening of the infection situation of Babesia gibsoni including occult infection is necessary. The development of cost-effective method for storage and transport of blood samples is required. A highly efficient DNA extraction procedure from dried blood spots (DBS) onto Whatman 3MM filter paper was developed for the diagnosis of B. gibsoni infection in dog by PCR. In 3 extraction methods, Chelex-based method in combination with saponin washing and phenol-chloroform-isoamyl alcohol extraction (Saponin-PCI method) provided the best results. Sensitivity of the 4 previously described PCR methods for detection of B. gibsoni infection was also compared using serially diluted blood samples of B. gibsoni-infected dogs. The PCR method using Gib599F/Gib1270R primer pair provided the best performance. To evaluate the stability of DNA in DBS, DBS of B. gibsoni-infected dogs stored at room temperature for 2 months. The stability was superior to whole blood samples stored at -20 degrees C for 2 months. This highly efficient DNA extraction method on DBS using Whatman 3MM filter paper has potential to be cost-effective and high performance tool for storage, and molecular diagnosis of clinical blood sample from dog. This procedure in combination with the PCR method using Gib599F/Gib1270R primer pair may greatly assist in diagnosis of B. gibsoni infection in dog populations that are geographically distant.     

Babesia gibsoni infection in a dog from indiana

Abstract: A 10-year-old spayed female mixed-breed dog was presented to the Purdue University Veterinary Teaching Hospital (PUVTH) with complaints of persistent anemia with occasional exacerbations, anorexia, and lethargy. The dog had been presented to the referring veterinarian 2 months prior with multiple bite wounds received during a fight with 3 Pit Bull Terriers. The dog was discharged after the wounds were cleaned and surgically closed. Upon admission to the PUVTH, blood was collected for a complete blood count and biochemical analysis. Microscopic examination of peripheral blood smears revealed intraerythrocytic protozoal parasites consistent with Babesia gibsoni . Molecular analysis confirmed that the organism was B gibsoni and that its 18S ribosomal RNA sequence was identical to that of other B gibsoni isolates from Oklahoma, North Carolina, and Okinawa, Japan. Hematologic changes included moderately severe, regenerative, macrocytic, normochromic anemia, with poikilocytosis, polychromasia, anisocytosis, and a marked increase in nucleated RBCs. Biochemical changes included increased serum alanine aminotransferase, alkaline phosphatase, and gamma glutamyl-transferase activities. The dog was treated with imidocarb, but despite initial clinical improvement, the dog died 2 weeks after the first dose. A necropsy was not performed. The infection in this dog is the first reported case of B gibsoni infection in Indiana. Because of the widespread geographical distribution of the organism, veterinarians and veterinary clinical pathologists throughout the United States should carefully examine Romanowsky-stained blood smears from patients with acute hemolytic anemia for small intraerythrocytic babesial parasites.     

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.     

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.     

Platelet Dysfunction Associated With Immune-Mediated Thrombocytopenia in Dogs

The effect of antiplatelet antibody on in vitro platelet function was investigated in 15 dogs with immune-mediated thrombocytopenia (ITP). Platelet aggregation was assessed after addition of serum from healthy dogs (n = 5) or dogs with ITP (n = 15) to platelet-rich plasma from a healthy donor dog. The aggregation responses to adenosine diphosphate, thrombin, and collagen/epinephrine were measured as the maximum aggregation observed after 2 minutes. In 13 of 15 dogs with ITP, maximal aggregation was significantly inhibited in response to ADP, thrombin, or collagen/epinephrine. The slope of the aggregation curve was decreased after addition of serum from 9 of 15 patients. A polyclonal rabbit anti—dog platelet antiserum induced inhibition of aggregation with all 3 agonists.
Serum from control dogs neither inhibited nor activated platelet aggregation. Aggregation experiments were repeated with all 3 agonists after addition of patient immunoglobulin (lg)G or IgG from a healthy dog to platelet-rich plasma. The IgG fraction from 9 of 10 dogs with ITP suppressed platelet aggregation. The IgG fraction from polyclonal rabbit anti—dog platelet antiserum inhibited platelet aggregation with all agonists. These results suggest that many canine ITP patients have circulating antibodies that, in addition to causing platelet destruction, may cause platelet dysfunction.