Gibsoni Infection Research Articles

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.

犬糸状虫症感染予防に来院した犬のバベシア, ヘモバルトネラおよびエールリッビア感染状況調査

Infections with Babesia gibsoni, haemobartonella and Ehrlichia in 536 dogs was examined using a polymerase chain reaction (PCR) specific for each agent in an animal hospital in Sumoto, an endemic area of canine B. gibsoni infection. Ten of 536 dogs (1.9%) tested positive for B. gibsoni, another 16 (3.0%) tested positive for haemobartonella, and none tested positive for Ehrlichia. No mixed infection of B. gibsoni and haemobartonella was detected. Of 16 dogs testing positive for haemobartonella, five were found to be infected with Mycoplasma haemocanis and the other 11 were infected with Candidatus Mycoplasma haematoparvum.

Development of a SYBR Green Real-Time Polymerase Chain Reaction Assay for Quantitative Detection ofBabesia 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.

Development of a practical immunochromatographic test with recombinant P50 for the diagnosis of Babesia gibsoni infection in dogs

An immunochromatographic test (ICT), using recombinant truncated P50 (P50t), for the detection of antibodies to Babesia gibsoni was developed and evaluated. Whereas all sera from specific pathogen-free dogs were clearly negative, all sera from dogs experimentally infected with B. gibsoni were clearly positive in the ICT. In addition, the ICT detected no cross-reactivity with sera from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi, or with Neospora caninum, and Leishmania infantum. Sequential sera from a dog experimentally infected with B. gibsoni were tested with the ICT; it was shown that the specific antibodies are detectable as early as 6 days post-infection (p.i.) and that strong antibody responses remained until the end of the experiment (144 days p.i.). To evaluate the clinical application of the ICT, a total of 54 serum samples collected from domestic dogs that had been identified as having signs of anaemia at veterinary hospitals in Japan, were tested with the ICT, the previously established enzyme-linked immunosorbent assay (ELISA) and with the indirect fluorescent antibody test (IFAT). Twenty-four of the tested samples (44.4%) were positive in both ICT and ELISA, and (51.8%) in IFAT. The concordance between ELISA and ICT was found to be 100%, and 85.7% with IFAT. Taken together, the results above suggest that the ICT using P50t is rapid, simple, accurate, and suitable for use at clinical sites for the diagnosis of B. gibsoni infection in dogs.

Fatal experimental transplacental Babesia gibsoni infections in dogs

A Babesia gibsoni infected bitch was mated with an uninfected dog in order to determine whether this parasite could be vertically transmitted. The bitch delivered a litter of four live and one stillborn pup. The four pups died from congenital babesiosis between 14 and 39 days post-birth. Babesia gibsoni DNA was detected in tissue from all five pups. These results show that vertical transmission occurred by the uterine route and not via the transmammary route. This is the first confirmed report of transplacental Babesia infection in any animal species.

犬の<I>Babesia gibsoni</I>自然感染症例におけるPCR法およびELISA法による診断の比較

Comparative investigation of Babesia (B.) gibsoni infection using the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were performed on 38 dogs suspected of the infection. Sixteen out of 38 dogs (42.1%) were positive by ELISA, while 27 dogs (71.1%) were PCR-positive including 11 ELISAnegative cases additional to the 16 ELISA-positive ones. The protozoa were not detected on blood smears from 4 of 27 PCR-positive cases. PCR method was able to detect the cases even with parasitemia as low as 0.0003% of the red blood cells. These results suggest that the PCR method may be more sensitive for detecting canineB. gibsoniinfection than ELISA and direct microscopic examination. PCR, therefore, is thought to be an effective diagnostic method for the detection ofB. gibsoniinfection in a dog even with low-grade parasitemia.

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.

Efficacy of Combined Atovaquone and Azithromycin for Therapy of Chronic Babesia gibsoni (Asian Genotype) Infections in Dogs

Babesiosis caused byBabesia gibsoni(Asian genotype) is an emerging disease in dogs in the United States. To date, no drugs have been shown to eliminateB gibsoni(Asian genotype) infections from dogs. Twenty‐two dogs that remained persistently infected withB 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 detectableB 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 eliminateB gibsoni(Asian genotype) infections or suppress the parasitemia below the limit of detection in the majority of treated dogs.

SERODIAGNOSIS OF CANINE BABESIA GIBSONI INFECTION BY ENZYME-LINKED IMMUNOSORBENT ASSAY WITH RECOMBINANT P50 EXPRESSED IN ESCHERICHIA COLI

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni-infected, Babesia canis-infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coil is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.

The PCR-based detection of Babesia gibsoni infection in dogs (German shepherds) reared in South Korea.

German-shepherd dogs from the South Korean provinces of Gangwon and Gyunggi were used in a study exploring both the detection of Babesia gibsoni infection, by use of a PCR-based assay, and the nucleotide sequences of part of the P18 gene of the parasite. Nine (1.8%) of the 501 dogs checked were found PCR-positive. Although the prevalence of the infection appeared slightly higher in the male dogs than in the female (2% v. 1.7%), and in the dogs aged <3 years than in the older animals (2.7% v. 1.1%), these differences were not statistically significant. In addition, the prevalences of infection among the dogs from Gangwon (in the east of the country) and those from Gyunggi (in the west) appeared similar. All nine PCR-positive dogs were confirmed to have B. gibsoni infection by the microscopical examination of thin bloodsmears, which revealed that 0.1%-3% (mean=1.5%) of the dog's erythrocytes harboured the parasite. The nucleotide sequences of the P18 gene of B. gibsoni from each of the PCR-positive dogs were identical to each other and to the corresponding sequence - recorded under accession number AB053292 in the DNA Data Bank of Japan (DDBJ) - previously determined for isolates of B. gibsoni from Japanese dogs. Although the PCR products sequenced in the present study represent only a part of the P18 gene of B. gibsoni, it seems that South Korean and Japanese dogs carry a similar strain of B. gibsoni. These results may help to clarify the phylogenetic position of the B. gibsoni found in South Korea.

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.

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.

High-level expression of truncated surface antigen P50 of Babesia gibsoni in insect cells by baculovirus and evaluation of its immunogenicity and antigenicity.

Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 10(8) cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.

Babesia gibsoni infection among dogs in the southeastern United States.

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. Case series. 33 American Pit Bull Terriers and 87 dogs of various other breeds. 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 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. 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.

Babesia gibsoni infection in a dog from indiana.

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-glutamyltransferase 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.

Morphological changes of Babesia gibsoni grown in canine red blood cell-substituted severe combined immune deficiency mice.

Canine red blood cell-substituted severe combined immune deficiency (Ca-RBC-SCID) mice were prepared for canine Babesia gibsoni infection. The Ca-RBC-SCID mice infected with B. gibsoni developed a high level of parasitemia, and showed clinical symptoms such as anemia and hemoglobinuria, which are similar to those observed in dogs infected with B. gibsoni. The B. gibsoni parasites grown in Ca-RBC-SCID mice showed marked morphological changes, including a significantly larger size of parasites than those in dogs and abundant RBCs containing 4, 8, 16, and 32 parasites. The multiple infection may have resulted from 1 parasite because the posterior end of each parasite in a multiply infected cell was connected. The parasites grown in SCID mice retained their infectivity and virulence to dogs and their morphology was dramatically restored to the original state when they were returned to dogs.

Clinical, Anatomic, and Immunopathologic Characterization of Babesia gibsoni Infection in the Domestic Dog (Canis familiaris)

The pathology associated with acute, chronic, and recrudescent Babesia gibsoni infections was characterized in a group of 6 naturally or experimentally infected, spleen-intact and splenectomized dogs. All experimentally infected dogs became acutely parasitemic, lethargic, anemic, thrombocytopenic, and hemoglobinuric. Anatomic lesions associated, with the disease included diffuse nonsuppurative periportal and centrilobular hepatitis, multifocal necrotizing arteritis, membranoproliferative glomerulonephritis, reactive lymphadenopathy, diffuse erythrophagocytosis, and extramedullary hematopoiesis. The density of CD3+ lymphocytes within the liver sinusoids was markedly increased. Aggregates of large mononuclear cells with immunohistochemical features of activated macrophages were demonstrated in the central veins of the liver. Kupffer cells throughout the hepatic sinusoids appeared hypertrophic and prominent. The density of sinusoidal T lymphocytes, macrophages in central veins, and the degree of Kupffer cell hypertrophy were greatest in the splenectomized dogs. Multifocal deposits of IgM antibody were immunohistochemically demonstrated within the walls of inflamed arteries and renal glomeruli. The results of this study suggest that intense immunostimulation resulting in activation and expansion of T and B lymphocyte populations, macrophage recruitment and activation, vasculitis, glomerulonephritis and anemia contribute to the pathology associated with B. gibsoni infections.

Erythrocyte oxidation in artificial Babesia gibsoni infection

We have already reported that anti-erythrocyte antibody induced by Babesia gibsoni infection (aEAb) showed higher reactivity against aged and/or oxidized erythrocytes than intact ones in vitro (Morita et al., 1995). To clarify the meaning of such a binding character of aEAb in vivo, changes in erythrocyte oxidation were observed in artificially infected dogs. The ratio of methemoglobin concentration against total hemoglobin concentration (metHb%) as the indicator of erythrocyte oxidation was increased by the artificial infection in the intact dogs, suggesting that aEAb played a more important role in erythrocyte destruction in infected dogs. Though parasitemia approximately three times higher was observed in splenectomized dogs than that in intact dogs, metHb% did not increase in the splenectomized dogs. This suggests that the spleen plays a very important role in erythrocyte oxidation in dogs infected with B. gibsoni.

Immunologic characteristics of anti-erythrocyte membrane antibody produced in dogs during Babesia gibsoni infection.

Serum anti-erythrocyte membrane (RBCm) antibodies and anti-RBCm antibodies eluted from erythrocytes of Babesia gibsoni-infected dogs were examined for immunologic characteristics by immunoblotting analysis. Serum anti-RBCm antibodies of the dogs before and after the experimental inoculation with parasitized erythrocytes reacted with cytoskeletal (bands 1, 2, 2.1, 4.1, 4.2 and 5) and transmembrane structural (band 3) proteins of either self or non-self RBCm. Anti-RBCm antibodies eluted from erythrocytes of B. gibsoni-infected dogs reacted with bands 3 and 4.2 of self and non-self RBCm.