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1.
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.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
This study reports on the epidemiology of Babesia gibsoni in American Pit Bull Terriers living in a region of western Victoria in southern Australia. Both American Pit Bull Terriers (n = 100) and other dog breeds (n = 51) were screened for B gibsoni using immunofluorescent antibody testing (IFAT) and/or polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). A questionnaire was also completed by each dog owner, ascertaining the husbandry and habits of the dogs sampled. Fourteen dogs were positive for B gibsoni using IFAT and/or PCR-RFLP and all were American Pit Bull Terriers. Dogs that were male and/or had been bitten by or were biters of other American Pit Bull Terriers were more likely to be B gibsoni positive, thus suggesting that blood-to-blood transmission contributes to the spread of this disease between dogs.  相似文献   

6.
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.  相似文献   

7.
Canine hepatozoonosis is caused by the tick-borne protozoon Hepatozoon spp. The prevalence of the infection in the Aegean coast of Turkey was investigated by examination of blood smear parasitology and polymerase chain reaction (PCR) using blood samples from 349 dogs collected from Central Aydin, Kusadasi, Selcuk, Central Manisa, Bodrum and Marmaris within the Aegean coast of Turkey. The indirect fluorescent antibody test (IFAT) for the detection of Hepatozoon canis antibodies was also used to detect the exposure rate to H. canis. PCR amplifying a 666bp fragment of 18S rRNA gene of Hepatozoon spp. was used in the epidemiological survey. The prevalence of Hepatozoon spp. infection was 10.6% by blood smear parasitology and 25.8% by PCR. IFAT revealed that 36.8% of serum samples were positive for antibodies reactive with Hepatozoon spp. The PCR products of 18S rRNA gene of Hepatozoon spp. isolated from six infected dogs, one isolate originating from each of the six different locations, were sequenced. The results of sequence analysis indicate that they are closely related to Indian and Japanese isolates of H. canis. This is the first epidemiological study on the prevalence of H. canis infection in the dog, in Turkey.  相似文献   

8.
Neospora caninum is a parasite responsible for paresis in dogs. The dog can harbour enkysted parasites in several organs. The detection of N. caninum was performed using 3 different real time PCR systems all amplifying the NC5 DNA region. One system was based on Sybrgreen, one on Plexor technology and the last on Taqman probe. Comparison of the three methods indicated that the detection limit was 1 equivalent genome on pure DNA but that this detection limit increased in the presence of foreign DNA using the Sybrgreen and Plexor systems. Therefore, the Taqman system was chosen to detect N. caninum in liver and spleen of naturally infected dogs. The overall prevalence was 32.2%. Comparison between PCR results and serological results using IFAT showed that among the 28 PCR positive dogs only 9 were seropositive and that 8 seropositive dogs were PCR negative. Therefore serology can underestimate the real carriage in dogs. However, PCR methods must be improved in terms of sensitivity and inhibition problems.  相似文献   

9.
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.  相似文献   

10.
犬附红细胞体PCR检测方法的建立   总被引：2，自引：0，他引：2  
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。  相似文献   

11.
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.  相似文献   

12.
A cDNA encoding the rhoptry-associated protein 1 (RAP-1) homologue was obtained by immunoscreening an expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1740bp. Computer analysis suggested that the sequence contains an open reading frame of 1425bp encoding an expected protein with a molecular weight of 52kDa. Based on the sequence similarity, this putative protein was designated as the B. gibsoni RAP-1 (BgRAP-1). The BgRAP-1 gene was expressed in the Escherichia coli BL21 strain, and the recombinant BgRAP-1 was used as the antigen in the enzyme-linked immunosorbent assay (ELISA). The results can differentiate between the B. gibsoni-infected dog sera and the Babesia canis infected dog sera or the normal dog sera. Furthermore, the antibody response against the recombinant protein was maintained during the chronic stage of infection, indicating that the recombinant BgRAP-1 protein might be a useful diagnostic antigen for the detection of antibodies to B. gibsoni infection in dogs.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
In order to investigate the possible role of dog fleas in the transmission of trypanosomatids, ectoparasites were removed from 59 dogs testing positive for canine zoonotic visceral leishmaniasis according to the indirect fluorescent antibody test (IFAT). Of the fleas collected, 4/207 (1.9%) showed the presence of promastigotes in smears stained by Giemsa, whilst 43/144 (29.9%) exhibited positive polymerase chain reaction (PCR) amplification assays for Leishmania DNA. Fleas (409) from 9 Leishmania chagasi-infected dogs, each hosting more than 20 fleas per animal, were macerated and administered by peritoneal injection or orally to 36 hamsters. After 6 months, the 30 surviving hamsters were sacrificed and liver and spleen fragments were removed for PCA assay and to produce imprint smears, whilst blood samples were subjected to IFAT assay. Sixteen hamsters tested positive for Leishmania infection, 14 on the basis of PCR amplification and four by IFAT assay (two animals testing positive in both assays). Of the infected hamsters, 11/16 (68.7%) had been infected peritoneally and 5/16 (31.2%) orally. The imprint smears for all animals were, however, negative. Since both PCR and IFAT could present cross-reactivity for Leishmania and Leptomonas, the possibility of oral transmission of L. chagasi by fleas cannot be proven unambiguously even though the hamsters developed infection.  相似文献   

16.
Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (>or=1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400-Dog 1 and Sheep n. 342, IFAT 1:50-Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (10(3)oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus.  相似文献   

17.
Molecular characterization of a Babesia gibsoni isolate from a Spanish dog   总被引：1，自引：0，他引：1  
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.  相似文献   

18.
In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.  相似文献   

19.
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.  相似文献   

20.
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%.  相似文献   

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