Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia

Canine babesiosis, caused by intra-erythrocytic Babesia, is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Tunisia. Detection and analysis of Babesia species from naturally infected dogs and ticks recovered from dogs were attempted by reverse line blot hybridization and nucleotide sequence analysis based on 18S rRNA gene. Out of 180 blood samples collected from domestic dogs in 4 villages situated in different bioclimatic zones, 12 were positive for Babesia canis vogeli. In addition, a total of 160 Rhipicephalus sanguineus were analysed; only one male was infected by B. canis vogeli. This is the first report on the detection of DNA belonging to B. canis vogeli in domestic dogs and in R. sanguineus in Tunisia.     

First molecular characterization of Babesia vogeli in two naturally infected dogs of Buenos Aires, Argentina

Large piroplasms (>2.5microm) were detected by direct microscopical investigation in 34 out of 16,767 (0.20%) canine blood smears in the Southern region of Greater Buenos Aires. Genomic DNA was extracted from two parasitemic dogs and the hypervariable 18S RNA gene region of the pathogen was specifically amplified, sequenced, and aligned with corresponding gene sequences available in the GenBank. Phylogenetic trees were constructed and compared. 18S RNA gene sequences reliably segregated in three clearly distinguishable clades representing Babesia canis, Babesia vogeli and Babesia rossi isolates, respectively. The 18S RNA gene sequences of both Babesia isolates from Argentina affiliated to the B. vogeli branch. This finding represents the first molecular evidence of the existence of B. vogeli in Argentina.     

First molecular detection of Babesia vogeli in dogs from Brazil

The present work describes the detection and first molecular characterization of Babesia vogeli in dogs, naturally infected in Brazil and even in South America. Microscopic examination of Giemsa-stained peripheral blood smears collected from dogs originating from four different locations in Brazil revealed the presence of large Babesia merozoites and trophozoites (>2.5 microm). DNA was extracted from infected blood samples and PCR amplifications of the 18S rDNA were carried out. As a reference, DNA from an isolate of B. vogeli originated from Egypt was used. PCR products were purified and sequenced. The DNA sequences demonstrated 100% identity among the Brazilian isolates. Comparisons with the 18S rDNA sequence of the B. vogeli isolate from Egypt and with other B. vogeli sequences from Spain, France, Japan, Australia and South Africa confirmed the affiliation of all Brazilian isolates to the species B. vogeli.     

Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs

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.     

Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus).

Two strains of Rhipicephalus sanguineus acquired Ehrlichia canis by feeding as either larvae or nymphs on acutely infected dogs and, in subsequent instars, transmitted the agent to normal dogs. Three strains of R sanguineus transmitted E canis as adults after their larval and nymphal stages fed on infected dogs. More than 400 adult female ticks were fed on infected dogs as larvae or nymphs or both, but none transmitted E canis transovarially.     

Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland

Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.     

Canine babesiosis in Slovenia: molecular evidence of Babesia canis canis and Babesia canis vogeli

Canine babesiosis, caused by intraerythrocytic Babesia spp., is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Slovenia. Therefore, 238 dogs admitted to the Small animal clinic in Ljubljana from the years 2000 to 2002 were tested for the presence of babesial parasites in the blood. Based on clinical, microscopic and molecular investigations, 14 dogs (5.9%) were determined as being infected with babesiae. Clinical signs relating to acute haemolysis, fever, anorexia, depression and haematological abnormalities such as anaemia and thrombocytopenia were noticed in most of the 14 infected dogs. The morphology of the parasites was indicative of Babesia canis infection. Two subspecies were detected, namely B. canis canis (11 dogs, 4.6%) and B. canis vogeli (3 dogs, 1.3%) using PCR and subsequent sequence analysis of portions of nns rRNA gene. In addition, based on nucleotide sequence analysis, the 11 isolates of B. c. canis could be subdivided into three groups, whereas the three B. c. vogeli isolates were genetically identical. The results of this study demonstrate the presence of canine babesiosis due to B. c. canis and B. c. vogeli in Slovenia.     

First evidence of Anaplasma platys in Rhipicephalus sanguineus (Acari: Ixodida) collected from dogs in Africa

A total of 27 ticks, comprising Rhipicephalus sanguineus (Latreille) (n = 21), Haemaphysalis leachi (Andouin) (n = 4) and Haemaphysalis paraleachi (Camicas, Hoogstraal & El Kammah) (n = 2) were recovered from two clinically healthy female dogs in the Democratic Republic of the Congo. DNA of Anaplasma platys was detected in a female R. sanguineus, using primers derived from the 16S rRNA gene, which amplify members of the family Anaplasmataceae . Anaplasma platys DNA was also detected in the blood of one of the dogs. Phylogenetic analysis based on partial sequences of the 16S rRNA, the gltA and the groEL genes ranged the detected agent within the Anaplasma clade. This is the first reported detection of A. platys in ticks in Africa. This finding raises the question of the possible involvement of R. sanguineus in A. platys infection of dogs.     

Babesia canis canis and Babesia canis vogeli infections in dogs from northern Portugal

Canine babesiosis represents an important veterinary medical problem. This study describes the molecular characterization of babesial parasites detected in eight clinically suspected dogs from northern Portugal, affected by lethargy, muscle tremors, weight loss, pale mucous membranes, hyperthermia or red-coloured urine. Microscopic examination of peripheral blood smears showed large intraerythrocytic piroplasms morphologically compatible with Babesia canis in all eight animals. DNA was extracted from blood on filter paper, and a Babesia spp. infection confirmed by polymerase chain reaction (PCR) amplification of a 408bp fragment of the 18S rRNA gene. Analysis of PCR-derived sequences revealed that seven dogs were infected with B. canis canis and one with B. canis vogeli. This is the first molecular identification report of both the species B. canis and the subspecies B. canis canis and B. canis vogeli in dogs from Portugal.     

Identification of Rickettsia felis in fleas but not ticks on stray cats and dogs and the evidence of Rickettsia rhipicephali only in adult stage of Rhipicephalus sanguineus and Rhipicephalus haemaphysaloides

Rickettsia spp. are zoonotic pathogens and mainly transmitted by various arthropod vectors, such as fleas, ticks, and lice. Previous epidemiological studies indicated that ectoparasites infested on dogs or cats may be infected by Rickettsia spp., and transmit them to human beings accidentally. In this study, the prevalence of Rickettsia infection was evaluated using fleas and ticks from stray dogs and cats in Taiwan. A total of 158 pools made by 451 cat fleas (Ctenocephalides felis) from 37 dogs and 4 cats were used for analysis. Besides, 386 Rhipicephalus ticks collected from the other 62 stray dogs were included in this study. Nymphal and adult ticks were individually analyzed but larvae were separated into 21 pools for molecular detection. Partial sequencing analysis of the gltA gene was applied for Rickettsia identification. The results showed that 44.3% (70/158) of the cat flea pools were harboring Rickettsia DNA. Although 6.9% (13/187) of adult ticks were infected with Rickettsia, neither larval pools nor nymphal ticks were found to contain Rickettsia DNA. According to the results of sequencing analyses, all Rickettsia PCR-positive cat flea pools were infected with R. felis, and all Rickettsia PCR-positive adult ticks were infected with R. rhipicephali. The results of this study demonstrated that C. felis but not Rhipicephlus sanguineus (the brown dog tick) and Rh. haemaphysaloides collected from stray animals in Taiwan could be infected the zoonotic pathogen R. felis. Moreover, R. rhipicephali was only identified in adult stage of Rhipicephalus sanguineus and Rh. haemaphysaloides.     

Duration of antibodies against 24 kd protein of Rhipicephalus sanguineus extract in dogs infested with the adult ticks

A 24 kd protein from Rhipicephalus sanguineus (Rs24p) which was common to larvae, nymphs, male and female whole body and salivary gland extract of males and female was detected specifically in the serum from dogs after repeated infestation with adult R. sanguineus. The duration of antibodies against Rs24p in dogs infested with adults was examined by Western blotting analysis. Anti-Rs24p antibody was detected in two of 4 dogs during the period of 40 days in the first infestation. In the second infestation, all dogs showed positive reaction against Rs24p, but the duration of the antibodies varied greatly among the animals.     

Serial haematology results in transfused and non-transfused dogs naturally infected with Babesia rossi

This prospective longitudinal study investigated the progression of haematological changes in 32 transfused and 54 non-transfused dogs naturally infected with Babesia rossi over the 1st 6 days following diagnosis and treatment. The effect of patient age on the results of complete blood counts was determined. Haematology data were analysed at presentation and at 24 hours, 3 days and 6 days after presentation. Dogs were treated with diminazene aceturate at diagnosis and a blood transfusion was given if deemed clinically required. Mildly to moderately regenerative normocytic normochromic anaemia was observed in all dogs throughout the study period. Transfused dogs more often had an inflammatory leukogram at presentation and at 24 hours, than dogs that were not transfused. In dogs with a left shift, a concurrent normal or decreased segmented neutrophil count was found more commonly than neutrophilia. Severe thrombocytopenia that resolved within a week was common. Blood transfusion alleviated the anaemia, but had no significant effect on white blood cell or platelet responses. Blood cell responses were not significantly influenced by age. In conclusion, the red blood cell and white blood cell responses were less than expected in dogs with babesiosis, given the degree of anaemia and inflammation present. The magnitude of thrombocytopenia and rapid return of the platelet count to normal suggested a possible immune-mediated mechanism for the thrombocytopenia.     

Molecular detection of tick-borne haemopathogens in shelter dogs and Rhipicephalus sanguineus (sensu lato) ticks from Peninsular Malaysia

Ticks are important vectors in transmitting various pathogens and they could jeopardize the health and welfare of humans and animals worldwide. The present study aimed to investigate the presence of important tick-borne haemopathogens (TBH) in dogs and ticks via polymerase chain reaction (PCR) assays. A total of 220 blood samples and 140 ticks were collected from 10 animal shelters in Peninsular Malaysia. Of 220 blood samples, 77 (35 %) were positive to TBH, of which 20 % were E. canis, 12 % were A. platys, 7 % were B. gibsoni and 7 % were B. vogeli. All ticks were identified as Rhipicephalus sanguineus with five samples (3.57 %) positive with TBH. Co-infections of TBH (0.45–9.55 %) in dogs were also observed in this study.     

Evidence of an acute phase response in dogs naturally infected with Babesia canis

The erythrocyte sedimentation rate (ESR), white blood cell count (WBC), haematocrit (HCT) and platelet number (PLT) were quantified and compared with the acute phase proteins (APPs) in dogs naturally infected with Babesia canis and healthy dogs. Both groups were treated with imidocarb dipropionate on the day of admission and both groups were monitored for all parameters on the admission day and on the first, second, third, fourth and seventh days in order to determine the presence of an acute phase reaction, to assess the diagnostic value of these markers in uncomplicated canine babesiosis and to evaluate the use of APPs in treatment monitoring. It was demonstrated that an acute phase response occurs in dogs naturally infected with Babesia canis, with significant increases in the concentration of major acute phase proteins. The serum concentration of C-reactive protein (CRP), serum amyloid A (SAA) and the erythrocyte sedimentation rate (ESR) decreased daily after treatment and approached reference range values by the eighth day. PLT and haematocrit (HCT) increased daily after treatment and approached reference range values by the fourth day. WBC and haptoglobin increased after treatment and then decreased from the third and fourth days, respectively, to the eighth day. The diagnostic sensitivity of CRP, SAA and PLT was significantly higher compared to haptoglobin, ESR, HCT and the WBC count. CRP and SAA were of clinical use in monitoring the response to antibabesial treatment.     

Confirmation of occurrence of Babesia canis vogeli in domestic dogs in South Africa

The prevalence of Babesia infections in domestic dogs in South Africa was studied using reverse line blot hybridization and 18S sequence analysis. Babesia canis vogeli was confirmed for the first time in domestic dogs in South Africa. Out of a total of 297 blood samples collected from domestic dogs in Bloemfontein, East London, Johannesburg, Durban and from the Onderstepoort Veterinary Academic Hospital, 31 were positive for Babesia canis rossi, whereas B. c. vogeli was detected in 13 dogs. None of the dogs carried both parasites. The detection of B. c. vogeli has implications with regard to prevalence and varied clinical manifestation of canine babesiosis in South Africa.     

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.     

Molecular and serological detection of Ehrlichia canis and Babesia vogeli in dogs in Colombia

Ehrlichiosis and babesiosis are tick-borne diseases, caused mainly by Ehrlichia canis and Babesia canis, respectively, with a worldwide occurrence in dogs, whose main vector is the brown-dog tick, Rhipicephalus sanguineus. The present work aimed to detect the presence of E. canis and Babesia sp. in 91 dog blood samples in Colombia, by molecular and serological techniques. We also performed sequence alignment to indicate the identity of the parasite species infecting these animals. The present work shows the first molecular detection of E. canis and B. vogeli in dogs from Colombia. Immunoglobulin-G (IgG) antibodies to E. canis and Babesia vogeli were found in 75 (82.4%) and 47 (51.6%) sampled dogs, respectively. Thirty-seven (40.6%) and 5 (5.5%) dogs were positive in PCR for E. canis and Babesia sp., respectively. After sequencing, amplicons showed 99% of identity with isolates of E. canis and B. vogeli. The phylogenetic trees based on 16S rRNA-Anaplasmataceae sequences and 18S rRNA-piroplasmid sequences supported the identity of the found E. canis and B. vogeli DNAs, respectively. The present work shows the first molecular detection of E. canis and B. vogeli in dogs in Colombia.     

Development of Babesia gibsoni in the midgut of larval tick, Rhipicephalus sanguineus.

Studies were made on the development of Babesia gibsoni in the midgut of the larval tick, Rhipicephalus sanguineus. Six hr after repletion, merozoites of B. gibsoni, freed from erythrocytes, were observed in the midgut contents of the tick. After that, within 24 hr, those merozoites were transformed into the ring-forms which were relatively large, 2-3 microns in diameter. Later, the ring forms developed into the spherical forms which were subelliptical in shape and 4-6 microns in diameter. Within 2-4 days, the elongated forms, 5-8 microns in length, were found. At this time, some of the binucleated fusion form has assumed a form intermediate between the spherical and elongated-forms. About 5-6 days after repletion, large round or elliptic zygotes, 8-10 microns in diameter, were observed in the tick gut.