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.     

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.     

Molecular characterization of a Babesia gibsoni isolate from a Spanish dog

Babesia gibsoni is a morphologically small Babesia species that infects dogs. Molecular techniques have shown that some small Babesia sp. recently described in canids are not related to the original B. gibsoni and they should be assigned to separate taxons. Although the 18s rRNA gene of true B. gibsoni isolates has been studied in the USA, Asia and Australia, no molecular data on the presence and genetic characteristics of B. gibsoni in Europe are available. Blood collected from a Babesia-symptomatic dog from Spain was used for DNA diagnosis by seminested PCR. DNA amplification was positive and the complete 18s rRNA gene of the dog isolate was sequenced, showing 98% homology with B. gibsoni (isolate Asia 1). Evidence from phylogenetic analysis indicated that: The Spanish isolate unambiguously belongs to the B. gibsoni group. The B. gibsoni complex might be diphyletic. In the absence of genetic data from African isolates of B. gibsoni, Asia seems to be the most likely geographical location of origin.     

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.     

吉氏巴贝斯虫cDNA探针的制备及杂交试验

本实验将－６．６ｋｂ的吉氏巴贝斯虫ｃＤＮＡ片段以光照活化法标记光敏生物素，制备成光敏生物素探针。与吉氏巴贝斯虫基因组ＤＮＡ、伊氏锥虫基因组ＤＮＡ、犬白细胞ＤＮＡ的斑点杂交试验表明，该探针可与０．００１ｎｇ以上量的吉氏巴贝斯虫ＤＮＡ杂交，而不与任何浓度的伊氏锥虫ＤＮＡ和犬ＤＮＡ杂交，具有很高的敏感性和特异性。     

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.     

Development of in vitro atovaquone-resistant Babesia gibsoni with a single-nucleotide polymorphism in cytb

An atovaquone (ATV)-resistant Babesia gibsoni was developed by in vitro exposure of uncloned wild type (WT) B. gibsoni to 800 nM ATV for 6 days. Sequence analysis of mitochondrial genes showed a single-nucleotide polymorphism (SNP) at cytb nt363 (G to T) that resulted in the substitution of methionine with isoleucine (M121I), which is one of the SNPs reported in a previous in vivo study. 363T or 363G allele-specific real-time polymerase chain reaction (PCR) revealed that an M121I variant was present in over 99% of the ATV-resistant population. As neither ATV resistance nor gene polymorphisms appeared in the B. gibsoni WT sibling clones, the expression of ATV resistance in this study was suspected to be because of selective multiplication of the B. gibsoni M121I variant. This ATV-resistant B. gibsoni displayed the same sensitivity as the WT B. gibsoni against 5 other drugs, including diminazene aceturate, azithromycin, doxycycline, clindamycin, and proguanil. This is the first report on the in vitro establishment of an ATV-resistant B. gibsoni with gene polymorphisms.     

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.     

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.     

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%.     

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.     

Serosurvey of antiBabesia antibodies in stray dogs and American pit bull terriers and American staffordshire terriers from North Carolina

Stray dogs (n=359) and kennel dogs (n=149) from North Carolina were tested for evidence of antiBabesia antibodies. AntiBabesia antibodies were detected in 21/359 and 22/149 of the stray and kennel dogs, respectively. A total of 57 dogs from both groups were tested for babesiasis by light microscopy and polymerase chain reaction (PCR). Babesia deoxyribonucleic acid (DNA) was detected in 3/28 of the stray dogs and 14/29 of the kennel dogs. When Babesia DNA was detected by PCR, the species-specific PCR results differed from the Babesia species antibody titer results in 6/17 of the PCR-positive dogs. There was no association between antiBabesia antibodies and the presence of ticks. There are currently Babesia gibsoni epizootics affecting American pit bull terrier kennels.     

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.     

Piroplasma infection in dogs in northern Spain

Babesiosis is a world-wide zoonosis caused by tick-borne hematozoan parasites of the genus Babesia. Canine Babesidae have historically been classified as "large Babesia" (Babesia canis) and "small Babesia" (Babesia gibsoni) based on the size of their intraerythrocytic forms. Genetic sequencing technology using the polymerase chain reaction (PCR) has allowed further subdivision. B. gibsoni has three strains: "Asia", "California" and a recently identified small babesial-like parasite, Theileria annae. This newly recognised piroplasm appears to be hyperendemic in northwest Spain. In order to provide some insight into the situation, all the cases diagnosed in our laboratory (NW of Spain) during 2003 were evaluated. Our study (62 samples) shows the existence of a piroplasm morphologically different from B. canis and similar to B. gibsoni, which is genetically related to T. annae. Severe regenerative anemia and thrombocytopenia are almost constant characteristics of infection with T. annae in dogs. In many cases azotemia is found. Abnormally high serum concentrations of urea and creatinin, together with elevated concentrations of inorganic phosphorus, hypoalbuminemia, hypercholesterolemia, proteinuria, high protein/creatinin and presence of hyaline and granular casts in the microscopic examination of urine sediment suggest a glomerular component to the disease. We conclude that observational research and clinical trials should be conducted in order to improve our understanding of this emerging disease in order to provide some insight into the best therapeutic practices.     

吉氏巴贝斯虫单克隆抗体制备的研究

应用淋巴细胞杂交瘤技术制备杂交瘤细胞株 ,经间接萤光抗体法筛选和克隆 ,获得了 5株能稳定分泌吉氏巴贝斯虫特异性抗体的杂交瘤细胞株 ,分别命名为C3B5、M8B7、E9C5、G6 D8、H2 A7。经过鉴定 ,这 5株杂交瘤细胞分泌的单克隆抗体亚类及相对分子质量分别为IgG2b,1 8× 1 0 4 ;IgG2a,1 8× 1 0 4 ;IgG1 ,1 8× 1 0 4 ;IgM ,3 2× 1 0 4 ;IgM ,1 8× 1 0 4 。腹水效价为 1∶1 0 4 ～ 1∶1 0 5。其中E9C5杂交瘤细胞株分泌的单克隆抗体是一种保护性抗体 ,经对实验感染吉氏巴贝斯虫小鼠体内虫体的杀虫试验证明具有较强的杀灭作用。     

A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay

We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection.     

Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.     

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.     

Molecular and phylogenetic characterization of Theileria spp. parasites in autochthonous bovines (Mirandesa breed) in Portugal

A survey was conducted during the months of April-June 2003 in the northeast Portugal (Bragan?a district) in order to characterize the hemoparasite population of an autochthonous Mirandesa breed of Bos taurus. The polymerase chain reaction (PCR) analysis of the bovine blood revealed that 3 out of 116 animals were infected with Theileria and/or Babesia parasites, while reverse line blot hybridisation (RLB) analysis showed that these animals were infected with Theileria buffeli/orientalis. Cloning and sequencing confirmed the RLB results. Database sequence searches combined with phylogenetic analysis of the partial 18S ribosomal RNA gene sequences obtained enabled us to place the parasites in question as members of the T. buffeli/orientalis group, confirming the PCR/RLB diagnosis.     

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.