The prevalence of Babesia canis canis in marsh ticks (Dermacentor reticulatus) in the Saarland

An accumulation of autochthonous cases of canine babesiosis caused by Babesia canis has been registered in a small animal clinic in the Saarland since the beginning of 2006, some cases with fatal outcome. This study aims to contribute to the explanation of strong focal occurrence of infections with B. canis in this region.Therefore, patient owners who had presented their dogs in the years 2006 and 2007 because of babesiosis and who had claimed not having left the Saarland with their dogs at least six months before the outbreak of Babesiosis, were asked for their dog walking habits. Accordingly, a selection often tick collection sites of various landscape structures was made.Tick sampling by flagging the vegetation took place every month from March to December 2008. The collected ticks were identified morphologically. In eight of ten collecting sites a total of 397 adult Dermacentor reticulatus ticks were collected from March to December with the highest frequencies during the months of May, October and November. All collected specimens were examined by genus-specific conventional PCR for the presence of Babesia-DNA. In positive samples, the PCR-products were differentiated by sequencing. ten D. reticulatus (2.5%) ticks examined were found positive for DNA of B. canis canis originating from three out of eight collection sites. Consequently, an endemic distribution of D. reticulatus was confirmed and a natural     

West-to-east differences of Babesia canis canis prevalence in Dermacentor reticulatus ticks in Slovakia

Babesia canis canis is the most frequent causative agent of canine babesiosis in Central Europe, frequently causing severe disease. Recently, many new endemic foci of this disease have been reported from European countries. Growing incidence of canine babesiosis was recorded also in Slovakia during the last decade, from first cases in eastern Slovakia ten years ago to recent cases all over the south of the country. We have used nested PCR-RFLP method to study prevalence of B. c. canis in its natural tick vector Dermacentor reticulatus, collected at three geographically isolated lowland areas of southern Slovakia situated in the southeast, southwest, and west of Slovakia, respectively. The highest prevalence of B. c. canis was observed in D. reticulatus from eastern Slovakia (14.7%; n=327), whereas the prevalence in southwest was significantly lower (2.3%; n=1205). Notably, all 874 D. reticulatus ticks collected at Záhorská ní?ina lowland (W Slovakia) were B. c. canis-negative. Recorded differences in Babesia prevalence concurs well with the shift in incidence of clinical cases of canine babesiosis as observed by vet practitioners. Presented results revealed that eastern Slovakia represents an area of high risk of B. c. canis infection, whereas western areas of the country still remain Babesia canis-free.     

The prevention of transmission of Babesia canis canis by Dermacentor reticulatus ticks to dogs using a novel combination of fipronil, amitraz and (S)-methoprene

Four groups of seven dogs were treated topically with a novel combination of fipronil, amitraz and (S)-methoprene in a spot-on formulation (CERTIFECT?, Merial Limited, GA, USA) on 28, 21, 14 and 7 days prior to tick infestation, respectively and acaricidal efficacy and transmission blocking compared with an untreated control group (seven dogs). All dogs were infested with adult Dermacentor reticulatus ticks harbouring Babesia canis canis. Babesia canis canis was transmitted by D. reticulatus to all seven untreated control dogs, confirmed following demonstration of clinical signs, by the detection of B. canis parasites in thin blood smears and B. canis canis PCR-RLB DNA assay on blood and the development of B. canis canis antibody titres by 14-21 days after tick infestation. The majority of treated dogs remained sero-negative for 42 days after infestation. Therefore, the treatment of dogs with CERTIFECT applied up to 28 days prior to infestation with D. reticulatus harbouring B. canis canis, successfully prevented the development of clinical signs of canine babesiosis.     

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay

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.     

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.     

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.     

Babesia canis vogeli,Ehrlichia canis,and Anaplasma platys infection in a dog

A 12‐month‐old male neutered mixed breed dog was presented with a history of diarrhea, lethargy, emaciation, polydypsia, and sniffling. Physical examination findings included pale mucous membranes, increased heart and respiratory rates, and normal rectal temperature (38°C). Hematologic abnormalities included anemia and thrombocytopenia. Biochemical abnormalities included hypoalbuminemia, hyperbilirubinemia, and elevated ALP and ALT activities. A SNAP 4Dx test result was positive for Ehrlichia canis. Babesia canis vogeli organisms were found in the peripheral blood films, while morulae of E canis were not seen. Real‐time polymerase chain reaction testing confirmed the presence of both B c vogeli and E canis organisms, and also was positive for Anaplasma platys infection. The dog recovered following treatment with doxycycline and imidocarb dipropionate, with normal hematology and biochemical profiles.     

Temporal distribution of Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna in Hungary

A survey was carried out over a 4-year period to describe the temporal distribution of three 'anthropophilic' tick species, Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna in Hungary. Altogether 4658 adult ticks belonging to the three species were collected from 1931 red foxes (Vulpes vulpes) killed in an area of about 70,000 km(2) representing all major climatic areas of the country. The seasonal activity of the three species was different. I. ricinus ticks were most active between April and June with an activity peak in May. A less marked increase of activity was also observed in September and October. The highest activity of D. reticulatus ticks was seen between September and November with an activity peak in October, nevertheless, a marked increase of activity could also be observed in April. Small number of I. ricinus and D. reticulatus were collected in all other months. H. concinna ticks were active from May to July with an activity peak in June and completely disappeared between October and March. The temporal distribution of the three tick species might be used for predictions on the seasonality of tick-borne diseases in Hungary.     

Babesia canis canis in dogs from Hungary: detection by PCR and sequencing

Canine babesiosis in Hungary has always been a severe and frequent disease, attributed to infection with Babesia canis transmitted by Dermacentor reticulatus. Identification of the disease agent has been based merely on size and morphology of the intraerythrocytic parasites and no evidence has been found concerning the subspecies (genotype) of B. canis. Therefore, a molecular survey on natural Babesia infection of dogs in Hungary using PCR and sequence analysis was attempted to clarify the subspecies (genotype) and to obtain information on the occurrence of B. canis. A total of 44 blood samples from dogs showing clinical signs of babesiosis were collected. A piroplasm-specific PCR amplifying the partial 18S rRNA gene yielded an approximately 450 bp PCR product in 39 (88.6%) samples. Thirteen positive samples originated from Budapest and 26 from 21 other locations. Five PCR products were chosen randomly for sequencing. The partial 18S rDNA sequences were submitted to GenBank (accession numbers AY611729; AY611730; AY611731; AY611732 and AY611733). The sequences showed 100% homology to one another or differed by one nucleotide. BLAST search against GenBank revealed the highest similarity (99.8 or 100%) with Babesia canis canis. The implication of these data, for the further study and diagnosis of canine babesiosis is discussed.     

Babesia canis canis and Babesia canis vogeli clinicopathological findings and DNA detection by means of PCR-RFLP in blood from Italian dogs suspected of tick-borne disease

The aims of this study were to determine the presence of Babesia spp. in blood samples from Italian dogs with clinical signs compatible with tick-borne diseases by means of PCR-restriction fragment length polymorphism (RFLP) and describe the clinicopathological findings of dogs with Babesia infection. We evaluated the majority of canine babesiosis cases by means of clinical history, physical examination, hematological, biochemical, serum electrophoresis, urinalysis and hemostatic tests. Forty-five out of 164 canine blood samples studied were positive to Babesia PCR-RFLP with the following results: Babesia canis canis (n=34) and Babesia canis vogeli (n=11). The majority of B. c. canis infections were detected in Northern Italy (29.1%; 30/103). B. c. vogeli cases were detected mainly in Central and Southern Italy (16.3%; 10/61). Only one B. c. vogeli was detected in Northern Italy (0.9%; 1/103). Three positive samples to B. c. canis and four positive samples to B. c. vogeli were selected for sequencing of a fragment of the 18S rRNA gene (410bp) for further molecular characterization. The sequence obtained from all seven dogs was 99/100% homologous to sequences from B. c. canis and B. c. vogeli, respectively, present in GenBank. Sixty-two percent of dogs infected with B. c. canis had recently travelled on a hunting trip to East European countries. The main acute clinical signs were dehydration, apathy, anorexia and fever. The majority of dogs infected with B. c. canis presented at initial clinical examination mild to severe thrombocytopenia, hyperfibrinogenemia, mild to moderate normocytic-normochromic non-regenerative anemia, hemolysis and neutropenia. The urinalysis showed hemoglobinuria in 13/19 dogs suggesting intravascular hemolysis. Dogs with B. c. canis infection had high levels of C-reactive protein. Hypoalbuminemia was present in 17/26 dogs. The 11 cases of B. c. vogeli infection did not present a homogenous clinicopathological pattern. B. c. vogeli infections were observed in young dogs causing hemolytic anemia and in adult/old does that frequently presented predisposing factors such as splenectomy or immunocompromised conditions. In conclusion, this study demonstrates the presence of B. c. canis and B. c. vogeli in Italian sick dogs and differences in clinicopathological pattern in these two species of B. canis.     

Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada

To identify the tick-borne pathogens in dogs from Grenada, we conducted a serologic survey for Ehrlichia canis in 2004 (104 dogs) and a comprehensive serologic and molecular survey for a variety of tick-borne pathogens in 2006 (73 dogs). In 2004 and 2006, 44 and 32 dogs (42.3% and 43.8%) were seropositive for E. canis, respectively. In 2006, several tick-borne pathogens were identified by serology and PCR. DNA of E. canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, and Bartonella sp. were identified in 18 (24.7%), 14 (19.2%), 5 (7%), 5 (7%), and 1 (1.4%) dogs, respectively. Six (8.2%) dogs were seropositive for Bartonella vinsonii subsp. berkhoffii. All dogs were seronegative and PCR-negative for Rickettsia spp. Coinfection with two or three pathogens was observed in eight dogs. Partial 16S rRNA E. canis and A. platys sequences were identical to sequences in GenBank. Partial 18S rRNA gene sequences from the Grenadian H. canis were identical to each other and had one possible mismatch (ambiguous base) from H. canis detected from Spain and Brazil. Grenadian B. c. vogeli sequences were identical to B. c. vogeli from Brazil and Japan. All of the detected pathogens are transmitted, or suspected to be transmitted, by Rhipicephalus sanguineus. Results of this study indicate that dogs from Grenada are infected with multiple tick-borne pathogens; therefore, tick-borne diseases should be included as differentials for dogs exhibiting thrombocytopenia, leukopenia, fever, or lethargy. One pathogen, E. canis, is also of potential public health significance.     

Three groups of Babesia canis distinguished and a proposal for nomenclature

Summary

Two stocks of large Babesiae from dogs originating in France, transmitted by Dermacentor reticulatus, two from North Africa, having Rhipicephalus sanguineus as vector, and one from South Africa, transmitted by Haemaphysalis leachi, were compared in cross‐immunity tests in dogs and in the indirect fluorescent antibody test (IFAT).

The French and North African stocks did not immunise against the South African one, while the North African stocks did not protect against a French one. The South African stock partially protected against a French one.

The three groups could be clearly distinguished in the IFAT These differences have practical implications for existing and future vaccines against canine babesiosis and for the serological diagnosis of atypical and chronic cases.

It is proposed to use a trinomial system of nomenclature for these groups: Babesia canis canis (Piana and Galli‐Valerio, 1895), Babesia canis vogeli Reichenow, 1937, and Babesia canis rossi (Nuttall, 1910), having Dermacentor, Rhipicephalus and Haemaphysalis ticks as their vectors respectively.     

Prevalence of Babesia canis, Borrelia afzelii, and Anaplasma phagocytophilum infection in hard ticks removed from dogs in Warsaw (central Poland)

The purposes of this study were to specify the occurrence and prevalence of Babesia canis, Borrelia burgdorferi sensu lato, and Anaplasma phagocytophilum in ticks removed from dogs in Warsaw, and to determine the Borrelia species occurring in Ixodes ricinus ticks. Among 590 collected ticks, 209 were identified as I. ricinus, and 381 as Dermacentor reticulatus. DNA of B. canis was detected in 11% of D. reticulatus ticks. We found that 6.2% of I. ricinus ticks harbored B. burgdorferi s.l. specific DNA and 2.9% harbored A. phagocytophilum DNA. In these samples sequencing of the detected Borrelia amplicon confirmed infection with Borrelia afzelii genospecies. New sequences were submitted to the GenBank database (accession no. EU152128, EU152127, EU152126). This work is the first detection of B. afzelii and A. phagocytophilum in ticks from Warsaw, and the first survey for the prevalence of B. canis, B. afzelii, and A. phagocytophilum in ticks in central Poland.     

Comparative efficacy of the combination fipronil-(S)-methoprene and the combination permethrin-imidacloprid against Dermacentor reticulatus, the European dog tick, applied topically to dogs

This study compared the efficacy of two of the most widely veterinary-dispensed topical products for control of ticks on dogs: fipronil-(S)-methoprene and imidacloprid-permethrin. Eighteen healthy beagle dogs of both sexes were divided into three groups of six dogs. Group 1 served as the untreated control. Dogs in group 2 were treated with fipronil 10% + (S)-methoprene 9% w/v applied once on day 0, and those in group 3 were treated with imidacloprid 8.8% and permethrin 44% w/v, applied once on day 0. All dogs were infested with approximately 50 unfed Dermacentor reticulatus ticks on days 1, 7, 14, 21, 28, 35, and 42, and ticks remaining were counted and removed 48 hours after each infestation. The 48-hour efficacy of the fipronil-(S)-methoprene combination remained at 100% at all assessment points through and including day 37, declining to 95.30% on day 44. The 48-hour efficacy of the imidacloprid-permethrin combination peaked at 86.46% on day 9, decreased to 73.37% by the third week, and fell to 63.53% by the end of the study (day 44). Significantly (P < .05) fewer ticks were recovered from dogs treated with fipronil-(S)-methoprene or imidacloprid-permethrin than from untreated control dogs at each infestation. Notably, the dogs treated with fipronil-(S)-methoprene had significantly (P < .05) lower tick counts at every assessment than the dogs treated with imidacloprid-permethrin.     

A survey for infection with Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi, and Babesia canis in feral and client-owned dogs in the Turks and Caicos Islands, British West Indies

The frequency of infection with Dirofilaria immitis and Babesia canis and seropositivity to Ehrlichia canis and Borrelia burgdorferi in feral and client-owned dogs was determined. Feral dogs were 14.8 and 11.2 times more likely to be seropositive to D. immitis and E. canis, respectively, than were client-owned dogs. None of the dogs tested positive for B. burgdorferi or B. canis.     

Resistance and immunity of dogs against Babesia canis in an endemic area

Canine babesiosis is increasing in incidence and prevalence in some areas in France and is now a major problem for dogs. Sex and age do not have any influence on the animals' susceptibility to the disease. Some breeds are more resistant (Beagle, Fox terrier, Porcelain, Teckel, mongrel dogs) and others are more susceptible (Spaniel, Cocker, Griffon, Yorkshire terrier, Doberman, Pekinese); however, none of them is completely resistant. Dogs which live in endemic areas can synthesize antibodies against Babesia canis, sometimes at high levels, without any sign of disease.     

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.