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1.
During 1996 a small, ring-shaped, piroplasm was observed in blood smears from 157 dogs in north-west Spain. None of them had previously been in areas endemic for Babesia gibsoni, which was until recently the only small piroplasm known to parasitise dogs. Haematological and serum biochemistry analyses showed that almost all the dogs had an intense regenerative haemolytic anaemia and that in some cases there was evidence of renal failure. A molecular study was made of a sample of the parasite obtained in June 2000. The phylogenetic analysis showed an identity of 100 per cent with the new piroplasm, provisionally denominated as Theileria annae, and 99 per cent with Babesia microti and B. microti-Japan. The results confirm the previous observation of a new form of piroplasm (Theileria annae) which causes disease in dogs in Europe and suggest that it is endemic among the canine population in north-west Spain.  相似文献   

2.
Babesia canis and Babesia gibsoni have, until recently, been considered the only piroplasms that parasitise dogs. However, recent reports indicate that "small" Babesia infections in Spanish dogs are surprisingly frequent and molecular phylogenetic analysis indicates that the infecting agent is closely related to Babesia microti. Because the 18SrDNA sequence was not completely identical to that of B. microti, the new name "Theileria annae" was assigned to the canine agent. No information is available regarding the possible vector of the new piroplasm, T. annae. As part of an effort to identify the tick that may transmit T. annae in northwest Spain we asked veterinary surgeons practising in the region to collect and send to our laboratory ticks from dogs visiting their clinics. Seven hundred and twenty ticks collected from dogs of unknown clinical status during 1998 and 636 ticks collected between November 2001 and March 2002 from 38 dogs infected with T. annae and 131 uninfected dogs were identified. Results from the first study indicated that among the Ixodidae, Ixodes hexagonus clearly predominates over Ixodes ricinus (26.11% versus 6.67%). This observation was consistent with results of the second study, in which I. hexagonus was detected in all infected dogs and 71.8% of non-infected dogs and I. ricinus was not detected in either the infected or non-infected dogs. Results from the 2001-2002 study also indicate that the presence of Dermacentor reticulatus adult females is significantly less frequent among infected than non-infected dogs (OR=0.44; 95% CI: 0.21-0.92). On the other hand, I. hexagonus adult females and males are 6.75 and 4.24 times more likely to be detected among infected than non-infected dogs, respectively, with the association being, in both cases, statistically significant (95% CI: 1.97-23.12 and 1.92-9.36, respectively). I. hexagonus emerges as the main candidate as vector of T. annae because it feeds on dogs more frequently than other ticks and because B. microti is transmitted by Ixodes ticks, both in North America and Europe. In the absence of definitive confirmation of this hypothesis, our observations suggest that I. hexagonus might serve the same role as does Ixodes scapularis (=Ixodes dammini), the vector of B. microti in eastern North America.  相似文献   

3.
Canine piroplasmosis is an emerging disease worldwide, with multiple species of piroplasm now recognised to infect dogs. A nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection and differentiation of each of the piroplasm species currently known to infect dogs on the basis of the 18S ribosomal RNA gene. The assay can potentially amplify and discriminate between Theileria annae, Theileria equi, Babesia conradae, Babesia gibsoni, Babesia sp. (Coco) and each of the Babesia canis subspecies. Non-canine piroplasm species can also potentially be detected using the described assay, however amplification of Neospora caninum was also observed. The PCR was found to have a high detection limit, capable of detecting a 2.7x10(-7)% parasitaemia or the equivalent of 1.2 molecules of target DNA when using DNA extracted from whole EDTA blood and detected a parasitaemia of 2.7x10(-5)% using blood applied to both Flinders Technology Associates (FTA) cards and IsoCodetrade mark Stix. The application of blood samples to filter paper may greatly assist in piroplasm identification in regions of the world where local technologies for molecular characterisation are limited. The assay reported here has the potential to be standardised for routine screening of dogs for piroplasmosis.  相似文献   

4.
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.  相似文献   

5.
Following a study on molecular epizootiology of Hepatozoon canis and piroplasmids (Babesia spp. and Theileria spp.) in southern Europe, newly obtained sequences of 18s rRNA gene were used for phylogenetic analysis. Partial sequences were analysed in isolates showing high degree of homology (>99%) with previous GenBank entries: H. canis, B. canis vogeli, B. equi (two isolates, Spain1 and Spain2), T. annulata and Theileria sp. The complete gene sequences were used for B. ovis and B. bovis, that showed lower homology (<95%) with rapport to previously reported species or isolates. A first set of phylogenetic trees constructed with partial 18s rRNA sequences showed that most European isolates clustered unambiguously with previously described species, so that minor sequence dissimilarities found are due probably to strain variations.The second set of phylogenetic trees was made using the complete 18s rRNA sequences of 44 species from GenBank and the newly sequenced B. ovis and B. bovis. The analysis revealed for the first time a division of piroplasmids in five clades: (1) B. microti group, with B. rodhaini, B. felis, B. leo, B. microti and T. annae (proposed name for the group, without taxonomic value: Archaeopiroplasmids), (2) Western USA Theilerid-like group (proposed name: Prototheilerids), (3) Theileria group, containing all Theileria species from Bovinae (proposed name: Theilerids), (4) A first group of Babesia species including B. canis and B. gibsoni from canids together with B. divergens and B. odocoilei (proposed name: Babesids), (5) A second group composed mainly by Babesia species from ungulates: B. caballi, B. bigemina, B. ovis, B. bovis and Babesia sp. from cow (proposed name: Ungulibabesids). The bootstrap support obtained with several analytical procedures for this new dicotomy of Babesiidae was always very high. Taking into account the present phylogenetic analysis and additional paleogeographic, parasitological and zoological evidences, two hypothesis on the origin and evolution of piroplasmids groups are presented.  相似文献   

6.
Canine babesiosis is a tick-borne parasitic disease caused by the intraerythrocytic parasites, Babesia canis and Babesia gibsoni. A lethargic, weak, American Staffordshire Terrier (pit bull) dog, which had regenerative, normocytic, normochromic anemia, was shown by polymerase chain reaction analysis to be infected with B. gibsoni. Transmission electron microscopy of ethylenediamine tetraacetic acid-treated blood disclosed many well-preserved, intraerythrocytic babesia trophozoites. Four morphologic forms of babesia trophozoites are described (small spheres, small rods, irregular forms lacking pseudoinclusions, and large spheres having pseudoinclusions) and are compared with intraerythrocytic forms of B. canis and B. gibsoni described in other light and electron microscopic studies of in vivo and in vitro Babesia infections. This is the first detailed transmission electron microscopic study of canine B. gibsoni-infected red blood cells in North America.  相似文献   

7.
The morphologically small Babesia species isolated from naturally infected dogs in Europe, Japan, and US are described as Babesia gibsoni despite the fact that molecular techniques show that they should be assigned to two or three separate taxons. The morphologically large Babesia isolated from dogs in Europe, Africa, and US were generally classified as B. canis until it was proposed to distinguish three related, albeit genetically distinct subspecies of this genus, namely B. canis canis, B. canis rossi, and B. canis vogeli. The insight into the molecular taxonomy of canine piroplasms is, however, limited because only partial small subunit ribosomal RNA (ssrRNA) sequence data exist for two species from the B. canis group. In this work, we molecularly characterised natural Babesia infections in 11 dogs from Croatia, France, Italy, and Poland. These infections were diagnosed as caused by B. canis canis and B. canis vogeli based on the analysis of the complete sequence of the ssrRNA genes. Phylogenetic analysis confirmed that the large Babesia species of dogs belong the to the Babesia sensu stricto clade, which includes species characterised by transovarial transmission in the tick vectors and by exclusive development inside the mammalian host erythrocytes. The new data facilitate the reliable molecular diagnosis of the subspecies of B. canis.  相似文献   

8.
Clinical symptoms produced by Mycoplasma spp. and piroplasmids in cats are sometimes similar. Diagnosis of these pathogens is difficult by microscopic procedures and molecular methods have been used as an alternative. We present in this work, the development of new molecular procedures for diagnosis of the aforementioned organisms, together with a molecular characterization of isolates found in southern European cats.A single PCR-RFLP procedure was designed for diagnosis of Mycoplasma spp. and a seminested PCR-RFLP was designed for diagnosis of piroplasmids. The 16S or 18S rRNA genes of isolates found in clinical samples were partially sequenced in all positive cases.Mycoplasma spp. was detected in 9 (30%) out of 30 symptomatic cats from Spain. Sequencing indicated that 66.6% of these isolates can be ascribed to Mycoplasma haemofelis and only 33.3% to Mycoplasma haemominutum. Partial 16S rRNA sequences obtained in Spanish isolates were very similar to those previously published from the UK and the USA.The presence of piroplasmids (Babesia and Theileria spp.) was studied in 16 cats from Spain (n=13) and Portugal (n=3). Animals analyzed were 10 cats with immunosuppressive viral infection (either FeLV or FIV), 5 asymptomatic cats and 1 cat with Babesia-compatible symptoms. Asymptomatic cats were all PCR-negative. Partial sequencing of 18S rRNA gene demonstrated that the Babesia-symptomatic cat was infected with Babesia canis canis whereas 3 (30%) out of the 10 cats with immunosuppressive viral infection were coinfected with piroplasmids (1 with B. canis canis, 1 with Theileria annae, and 1 with B. canis canis and T. annae both).  相似文献   

9.
Babesia gibsoni genotype Asia is a small, tick-transmitted intraerythrocytic protozoan that parasitizes dogs. Reports suggest that it is increasingly diagnosed in the United States. The clinical outcome of infection with this piroplasm is often variable, leading us to hypothesize that the different clinical outcomes resulting from B. gibsoni genotype Asia infection are due to genetically distinguishable strains that differ in virulence. As a first step to assess the genetic variability of B. gibsoni isolates originating from the southeastern United States, we sequenced the rRNA first internal transcribed spacer region of recent isolates from Georgia and Alabama, and compared these sequences with isolates originating from Japan and Australia. All isolates examined proved to be genetically identical at the first internal transcribed spacer region, although this region differed distinctly from other Babesia species and closely related apicomplexan species. Although negating our hypothesis, this information gives us insight into the recent evolutionary history and spread of B. gibsoni genotype Asia in dogs in the U.S. Our research suggests that the gradual rise in prevalence of canine babesiosis due to B. gibsoni genotype Asia in the United States may be a result of clonal expansion of a single strain within a susceptible host population.  相似文献   

10.
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   

11.
The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied. In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77 per cent of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied. In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30 per cent of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found. In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira 1 from Brazil.  相似文献   

12.
Small piroplasms as a cause of canine babesiosis in southern California were first documented in 1990. Initially these piroplasms were considered to be Babesia gibsoni, the only small Babesia parasite known to infect dogs at that time. In the following decade, the use of molecular analysis made it clear that small canine Babesia in fact are comprised of at least three distinct species, and the isolates from dogs in southern California were not B. gibsoni. Molecular, antigenic, and morphological characteristics of the southern California species of canine piroplasm supported naming it as a distinct species, Babesia conradae. The renaming of this species prompted this literature review of small canine piroplasms in California in order to clarify clinical, diagnostic, epidemiological, and molecular characteristics of B. conradae in comparison to other small canine piroplasms. Clinical symptoms of B. conradae are similar to those of B. gibsoni; however, B. conradae infections may be more pathogenic, resulting in higher parasitaemia and more pronounced anaemia when compared with B. gibsoni-infected dogs. The immunofluorescent antibody test is the most commonly used test to diagnose B. conradae. It is important to specify which small Babesia species to test for since there is little serological cross reactivity between the small canine Babesia antigens or cross-detection in the newer molecular tests. Molecular characterization of B. conradae, based principally on the 18S small subunit rRNA gene, and recently the second internal transcribed spacer region, demonstrate that B. conradae is most closely related to piroplasms recovered from humans and animals in the western United States.  相似文献   

13.
Canine babesiosis.   总被引：6，自引：0，他引：6  
Canine babesiosis is a tickborne, protozoal, hemoparasitic disease that can cause varying degrees of hemolytic anemia, splenomegaly, thrombocytopenia, and fever. Babesia organisms frequently are classified as large or small. Large Babesia infections are attributed to one of three subspecies of Babesia canis. All small Babesia infections previously were attributed to B gibsoni, but molecular analysis and DNA sequencing have revealed that there are at least three small piroplasms infecting dogs. Correctly identifying the infectious agent is important for treatment planning and prognosis.  相似文献   

14.
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   

15.
Molecular epizootiology of piroplasmids (Babesia spp., Theileria spp.) and Hepatozoon canis was studied in mammals from southern Europe (mainly from Spain, but also from Portugal and France). Partial amplification and sequencing of the 18s rRNA gene was used for molecular diagnosis. In some particular cases (B. ovis and B. bovis) the complete 18s rRNA gene was sequenced. Blood samples were taken from domestic animals showing clinical symptoms: 10 dogs, 10 horses, 10 cows, 9 sheep and 1 goat. In addition, DNA samples were isolated from blood of 12 healthy dogs and from spleen of 10 wild red foxes (Vulpes vulpes). The results of the survey were the following: Piroplasmid infections: Approximately from 50 to 70% of wild or domestic mammals (symptomatic) were infected.Piroplasmids detected in ruminants were:COW: B. bovis, T. annulata and Theileria sp. (type C). Sheep and goat: B. ovis. Piroplasmids present in canids were: Babesia canis vogeli, Babesia canis canis, Theileria annae and B. equi. The only piroplasmid found in asymptomatic dogs was B. equi. Piroplasmids found in horse were: B. equi and B. canis canis.H. canis infections in canids: H. canis was absent of domestic dog samples, whereas all foxes studied were infected by this protozoa.Genetic analysis showed that most of piroplasmid and Hepatozoon isolates from southern Europe matched unambigously with previously described species, as demonstrated by the high level sequence identity between them, usually between 99 and 100%. Minor differences, usually detected in hypervariable regions of 18s rRNA gene are probably due to strain variations or rare genetic polymorphisms. A possible exception was B. bovis, which shows a relatively lower degree of homology (94%) with regard to other B. bovis isolates from several countries. The same is true for B. ovis, that showed a 94% identity with regard to Babesia sp. from South African cow and a 92% with rapport to B. bovis from Portugal.  相似文献   

16.
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   

17.
Here we report a case of canine babesiosis with unusual morphology of the causative agent. A male, seven-week-old Labrador retriever puppy, exhibiting severe anaemia and haemoglobinuria, was presented at the Clinic of Internal Medicine in February 2011. The puppy was euthanised. The most relevant pathological changes were icterus, severe splenomegaly, generalised lymphadenopathy and haemoglobin nephrosis. Samples were collected from various organs for histology within one hour post mortem. Impression smears were also prepared from the spleen after overnight storage at 4 °C. Tissue sections and smears showed the presence of multiple, coccoid intraerythrocytic bodies that measured 1-2 μm and resembled small babesiae. No large piroplasms were seen. DNA was extracted from the spleen, and a conventional PCR was performed for the amplification of a 450-bp region of the 18S rRNA gene of piroplasms. The causative agent was identified as Babesia canis canis, with 99% sequence identity to other European isolates. Sequence identity to B. gibsoni was only 91%. This is the first account to verify that the morphology of the large canine piroplasm, B. canis, can be uniformly small babesia-like post mortem or following the storage of tissue samples.  相似文献   

18.
Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.  相似文献   

19.
The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.  相似文献   

20.
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   

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