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.     

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.     

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.     

犬附红细胞体PCR检测方法的建立

目的 步建立犬附红细胞体PCR检测方法。方法 据已发表的附红细胞体基因序列，设计了一对特异性引物，以犬附红细胞体基因组DNA和附红细胞体可疑病犬样品DNA为模板，聚合酶链式反应（PCR）扩增，扩增产物经克隆测序分析。结果 CR扩增产物大小为541bp，序列分析表明与GenBank数据中发表的序列一致，表明这套引物成功扩增出目的基因序列，但正常犬血样品DNA和弓形虫、伊氏锥虫、吉氏巴贝斯虫、犬细小病毒、犬瘟热的DNA样品都不能扩增出目的基因片段。结论 研究建立的PCR方法可以用于犬附红细胞体的检测，为犬附红细胞体病的诊断及分子流行病学的调查提供了新的手段。     

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.     

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.     

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.     

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.     

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 a modified polymerase chain reaction assay for detection of Ehrlichia canis infection.

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days post-exposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.     

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.     

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.     

A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs

We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2 (CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA quantitation over a range of eight orders of magnitude (from 10(2) to 10(9) copies of standard DNA). The reproducibility of the CPV-2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to detect low viral titers of CPV-2 in infected dogs.     

Increased erythrophagocytic activity of macrophages in dogs with Babesia gibsoni infection

To elucidate the mechanism of anemia caused by Babesia gibsoni infection in dogs, the erythrophagocytic activity of macrophages in infected dogs was investigated in vitro. In the present study, macrophages obtained from peripheral blood (PB-macrophages) and bone marrow (BM-macrophages) of splenectomized dogs with chronic B. gibsoni infection were examined. The BM-macrophages in the splenectomized dogs with chronic babesiosis exhibited an increased erythrophagocytic activity compared with those from splenectomized, non-infected dogs. In the infected dogs, erythrophagocytic activities of macrophages against both auto- and iso-erythorcytes from normal dogs were almost the same. Administration of an anti-protozoal drug, diminazene diaceturate, resulted in a decrease of the erythrophagocytic activity of BM-macrophages associated with an increase of the hematocrit value in splenectomized dogs with chronic babesiosis. In splenectomized dogs with acute babesiosis, erythrophagocytic activity of BM-macrophages was also elevated. Such a phenomenon was not, however, observed in splenectomized dogs with onion-induced hemolytic anemia. These results suggest that the erythrophagocytic ability of macrophages in the infected dogs might be accelerated by parasites per se through an unknown mechanism, resulting in severe anemia in spite of low parasitemia.     

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.     

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.     

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.     

Failure of imidocarb dipropionate to clear experimentally induced Ehrlichia canis infection in dogs

The recommended treatment for canine ehrlichiosis is tetracycline or its analog doxycycline, although recent reports have documented ineffective clearing of Erchlichia canis after doxycycline administration. Imidocarb dipropionate is used as an alternative treatment to tetracycline or is used in conjunction with doxycycline. The effectiveness of imidocarb dipropionate in clearing Ehrlichia species from the blood and tissues of dogs with E. canis infection has not been thoroughly evaluated. Fifteen dogs were experimentally infected with E. canis. Ten dogs were treated with imidocarb dipropionate (6.6 mg/kg, IM, 2 injections given 2 weeks apart). Five infected control dogs were not treated. Blood samples from all 15 dogs were E. canis DNA positive by PCR assay by 3 weeks after inoculation (PI), and E. canis antibodies were detected by IFA assay by 1 week PI. Blood platelet counts in all dogs were below the reference interval by 4 weeks PI. E. canis DNA was detected in bone marrow and splenic aspirates by PCR assay 4 weeks PI but not before infection. Bone marrow aspirates were E. canis DNA positive by PCR assay in 14/15 dogs, and splenic aspirates were E. canis DNA positive by PCR assay in 13/15 dogs. Blood samples from all treated and control dogs remained positive for E. canis DNA by PCR assay, and platelet counts remained below preinoculation values 13 weeks PI (6 weeks after 2nd treatment). As administered in this study, imidocarb dipropionate did not clear experimental E. canis infection in dogs.     

Changes in anti-erythrocyte membrane antibody level of dogs experimentally infected with Babesia gibsoni.

To account for the conflict between the excessive destruction of erythrocytes and the number of parasitized erythrocytes in dogs infected with Babesia gibsoni, we examined the correlation between anti-erythrocyte membrane antibody level (AEMAL) and the number of erythrocytes (RBC count) in dogs with experimentally induced babesiosis using hematological examination and an enzyme linked immunosorbent assay (ELISA). In the infected dogs without splenectomy, more prominent reduction in RBC count accompanied with the elevated AEMAL was presented than anticipated from parasitemia until the 21st day. Furthermore, autoagglutinated erythrocytes and spherocytes were demonstrated in blood films. These results suggest that a humoral immunologic mechanism may be involved in a decrease in RBC count in dogs infected with 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.