Soluble parasite antigens from Babesia canis do not directly activate the kallikrein system in dogs infected with Babesia canis

Soluble parasite antigens (SPA) from Babesia canis have been shown to induce protective immunity when used as vaccine. In order to explain the immune mechanisms of vaccination, the precise role of SPA in the pathogenesis of canine babesiosis is under investigation. Earlier studies suggested that the plasma kallikrein system is central in the pathogenesis of babesiosis, malaria and trypanosomosis, and significant plasma kallikrein activation during acute B. bovis and P. knowlesi infections has been described. In the studies presented here dogs were experimentally infected with B. canis to investigate whether the plasma kallikrein system is activated during babesiosis infection. Results showed that prekallikrein levels decreased during episodes of peak parasitaemia. No effect was found on the kallikrein levels. In order to determine whether B. canis SPA could activate plasma kallikrein, dogs were infused with variable amounts of B. canis SPA and plasma samples were taken for (pre-) kallikrein determination. The results indicated that B. canis SPA did not affect plasma (pre-) kallikrein levels. In addition, the effect of B. canis SPA on (pre-) kallikrein levels in normal dog plasma was determined in vitro. Again, no effect on (pre-) kallikrein levels was found. The results suggest that, although the kallikrein pathway may be involved in B. canis-associated pathology, the system is not directly activated by B. canis SPA. Furthermore, infusion of B. canis SPA as well as stroma of normal dog erythrocytes triggered the production of the acute phase reactant, C-reactive protein. This suggests that the inflammatory response that is triggered during B. canis infection could be in part due to the release and exposure of self molecules. The implications of these findings are discussed.     

Differential reactivity of bovine lymphocytes to species of Brucella

The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).     

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.     

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.     

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.     

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.     

Molecular survey of Babesia canis in dogs in Nigeria

An epidemiological study of Babesia canis in dogs in Nigeria was performed. Four hundred blood samples collected from dogs in Nigeria were investigated using nested PCR and sequence analysis. On nested PCR screening, nine samples (2.3%) produced a band corresponding to a 698-bp fragment indicative of B. canis infection. Sequence analysis of the PCR products identified eight samples (2.0%) as B. canis rossi and the ninth (0.3%) as B. canis vogeli. This is the first report of the prevalence of B. canis rossi and B. canis vogeli in dogs in Nigeria.     

Molecular studies on Babesia,Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects

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.     

Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants

Soluble parasite antigens (SPA) from European Babesia canis can be used to protect dogs against a homologous but not heterologous challenge infection. In this study it is shown that when dogs are vaccinated with a mixture of SPA from both, a European B. canis isolate and a South African Babesia rossi isolate, protective immunity against heterologous B. canis infection is induced. Three groups of five beagle dogs each were vaccinated twice with graded doses of SPA derived from in vitro cultures of B. canis and B. rossi, with a 3-week interval. Saponin was used as adjuvant. Three weeks after booster vaccination immunological responsiveness against heterologous B. canis antigen was measured by seroconversion against infected erythrocytes and lymphocyte transformation using SPA. Upon vaccination dogs produced antibodies against infected erythrocytes and lymphoblastogenic responses against SPA in a dose-dependent manner. Dogs were then challenged with heterologous B. canis parasites. Dogs appeared to be protected against challenge infection, which was reflected in less severe decrease of packed cell volume (PCV) and reduced clinical signs. The level of protection to clinical signs (but not excessive PCV drop) was related to the level of SPA in plasma and spleen size, and not related to peripheral parasitaemia. The results suggest that vaccination with this bivalent vaccine primes T-helper cells that recognise common epitopes on SPA from an antigenically distinct B. canis isolate. These cells provide the essential Th signal to mount an effective and timely antibody response against SPA and parasites or parasitised erythrocytes, which prevents the further development of clinical babesiosis.     

Molecular survey of Babesia infection in dogs in Okinawa, Japan

A total of 80 free-roaming dogs on Okinawa Island, Japan, were examined for Babesia infection using the polymerase chain reaction (PCR) and sequence analysis. Of 80 samples, 12 were positive in a Babesia genus-specific PCR. Consequent species-specific PCR for B. canis and B. gibsoni revealed that 5 (6.3%) and 7 (8.8%) dogs were infected with B. canis and B. gibsoni, respectively. Sequence analysis of the PCR products revealed that the 18S rRNA gene sequence of B. canis detected from dogs in Okinawa was very close to B. canis vogeli with sequence similarity of 99.94%.     

Evolution of clinical, haematological and biochemical findings in young dogs naturally infected by vector-borne pathogens

Longitudinal studies evaluating the evolution of clinical, haematological, biochemical findings in young dogs exposed for the first time to multiple vector-borne pathogens have not been reported. With the objective of assessing the evolution of clinical, haematological and biochemical findings, these parameters were serially monitored in naturally infected dogs throughout a 1-year follow-up period. Young dogs, infected by vector-borne pathogens based on cytology or polymerase chain reaction, were examined clinically and blood samples were obtained at seven different follow-up time points. Dogs were randomized to group A (17 dogs treated with a spot-on formulation of imidacloprid 10% and permethrin 50%) or to group B (17 dogs untreated). In addition, 10 4-month-old beagles were enrolled in each group and used as sentinel dogs. At baseline, Anaplasma platys was the most frequently detected pathogen, followed by Babesia vogeli, Bartonella spp., Ehrlichia canis and Hepatozoon canis. Co-infections with A. platys and B. vogeli, followed by E. canis and B. vogeli, A. platys and H. canis and A. platys and Bartonella spp. were also diagnosed. In dogs from group B, abnormal clinical signs were recorded at different time points throughout the study. No abnormal clinical signs were recorded in group A dogs. Thrombocytopenia was the most frequent haematological alteration recorded in A. platys-infected dogs, B. vogeli-infected dogs and in dogs co-infected with A. platys and B. vogeli or A. platys and Bartonella spp. Lymphocytosis was frequently detected among dogs infected with B. vogeli or co-infected with A. platys and B. vogeli. Beagles were often infected with a single pathogen rather than with multiple canine vector-borne pathogens. There was a significant association (p<0.01) between tick infestation and A. platys or B. vogeli, as single infections, and A. platys and B. vogeli or A. platys and Bartonella spp. co-infections. This study emphasizes the clinical difficulties associated with assigning a specific clinical sign or haematological abnormality to a particular canine vector-borne disease.     

Brucella suis biotype 1 infection in a dog

Brucella suis biotype 1 was isolated from the semen of a dog with hindlimb weakness and a large, firm, left epididymis. A semen sample was oligospermic, with many neutrophils, the numbers of which decreased in serial sampling. A card agglutination test for B abortus and a rapid slide agglutination test for B canis were positive. The modified 2-mercaptoethanol slide agglutination test for B canis and the agar gel immunodiffusion test, using B canis cell wall antigen, were negative. At necropsy, chronic granulomatous inflammation was found in, and B suis biotype 1 was isolated from, the left epididymis and prostate gland.     

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.     

Genetic comparison of Brucella canis isolates by the MLVA assay in South Korea

The multiple-locus VNTR analysis (MLVA) assay is a method frequently employed as a molecular epidemiological tool for Brucella genetic fingerprinting. The purpose of this study was to assess the genotyping of 77 B. canis isolates from 14 different dog breeding farms in Korea by the MLVA assay and to compare the epidemiological relationships between the Korean isolates and foreign ones. Simpson's diversity index for 17 loci showed a range from 0 to 0.846 in 77 B. canis isolates. B. canis isolates in Korea were observed to have high genetic diversity at the most variable loci and were divided into 30 distinct genotypes by phylogenetic analysis. Some B. canis isolates were closely related to previously typed isolates in other countries. The MLVA assay can be helpful to analyze the epidemiological correlation of B. canis isolates in domestic pet animals and to track the geographic origin by comparing the genetic patterns with foreign isolates. Therefore, the MLVA assay will be useful as a tool for control and preventive measures of canine brucellosis.     

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.     

Eradication of brucellosis in the beagle]

An infectious disease caused by the Brucella canis germ was determined in Czechoslovakia in 1973 in laboratory breeding stock of the beagle dog. The clinical symptoms were slinking between the 50th and 57th day of pregnancy and six-week serosanguinolent discharge or greenish gray mucoid discharge after the abortion and extensive hemorrhages and edemata under the skin of the aborted fetuses. The disease was proved serologically and also by isolating the B. canis germ. Histological symptoms found in bitches--were only non-specific inflammatory changes in nodes and some organs, in dogs--inflammation of the prostate, epididymides and testicles. Measures taken to eradicate this disease involved isolation and culling of the serologically positive dogs, creating a new, repeatedly negative breeding stock and strict hygienic and work procedures. The incidence of positive serological findings decreased from 43.0% in 1974 to 8.0% in the first half of 1976. As a positive titre of antigens we consider titres starting from 1:20 though the presence of the B canis germ was found in titres even higher than 1 :160, but we must expect the possibility of the initial stages of the disease.     

Immunologic properties of soluble materials from Brucella species.

The purpose in this study was to examine the immunogenic properties of various preparations of aqueous ether extracts of Brucella suis and Brucella canis. The B suis strain 3b and B canis strain RM-6-66 were grown on tryptose agar, and aqueous ether extracts were prepared from the cells. The ether was removed, and the extracts were clarified by centrifugation for 10 hours at 144,000 X g and fractionated by gel chromatography. The B suis endotoxin-containing precipitate, obtained from aqueous ether extracted material by ethanol precipitation, and fraction 1, prepared from ultracentrifugal supernate by column chromatography, protected mice against homologous infection. The B canis aqueous ether-extracted material also protected mice against B suis infections.     

A PCR-based epidemiological survey of Hepatozoon canis in dogs in Nigeria

The prevalence of Hepatozoon canis infections in dogs in Nigeria was surveyed using molecular methods. DNA was extracted from blood samples obtained from 400 dogs. A primer set that amplified the Babesia canis 18S rRNA gene, which has high similarity to the H. canis 18S rRNA gene, was used for the PCR. As a result, samples from 81 dogs (20.3%) produced 757 bp bands, which differed from the 698 bp band that corresponded to B. canis infection. The sequence of the PCR products of 10 samples were determined, all of which corresponded with the H. canis sequence.     

Concurrent babesiosis and ehrlichiosis in the dog: blood smear examination supplemented by the indirect fluorescent antibody test, using Cowdria ruminantium as antigen

Giemsa-stained, peripheral blood smears of 67 dogs, showing clinical signs typical of babesiosis or reminiscent of concurrent babesiosis and ehrlichiosis, were examined for the presence of Babesia canis and Ehrlichia canis. Since Cowdria ruminantium cross-reacts with Ehrlichia, the sera of these dogs were also subjected to the indirect fluorescent antibody (IFA) test in which C. ruminantium was used as antigen. Fifty-five per cent of these dogs had mixed infections of B. canis and E. canis, as judged by blood smear examination and serology. The serum of 32% of these dogs with mixed infections reacted positively in the IFA test. Six out of 9 dogs, the blood smears of which were negative for both B. canis and E. canis, were serologically positive for E. canis. Furthermore, sero-conversion from a negative in the initial serum sample to titres of up to 1:160 in a subsequent sample was recorded in 9 out of 13 dogs with suspected mixed infection on blood smear.     

Detection of Anaplasma platys and Babesia canis vogeli and their impact on platelet numbers in free-roaming dogs associated with remote Aboriginal communities in Australia

OBJECTIVE: To detect Anaplasma platys and Babesia canis vogeli infection, using polymerase chain reaction (PCR)-based assays, in free-roaming dogs associated with eight Aboriginal communities in remote areas of Australia and to determine the impact of infection through the assessment of platelet numbers. PROCEDURES: Blood samples from 215 dogs were screened by PCR for A platys and B canis vogeli using established genus-specific DNA primers for the 16S and 18S rRNA genes respectively. Both A platys DNA and B canis vogeli DNA were confirmed from the screening PCR either by sequencing or by the use of species-specific primers. Peripheral blood films from 92 of the 215 dogs were used to estimate platelet numbers through an indirect method. RESULTS: Of 215 dogs, 69 (32%) were positive for A platys, 22 (10%) for B canis vogeli and 24 (11%) for both. The two organisms were detected singularly and as coinfection in all communities. For the 92 dogs in which peripheral blood films were examined, the mean estimated platelet counts for the non-infected dogs was 318 x 10(9)/L, those infected with A platys alone was 256 x 10(9)/L, those with B canis vogeli alone was 276 x 10(9)/L and those infected with both parasites was 169 x 10(9)/L. In young dogs, infection produced significantly decreased mean platelet counts when compared to uninfected dogs. Thrombocytopenia (< 200 x 10(9)/L) was detected in 18 (51%) dogs infected with A platys alone, 3 (33%) dogs infected with B canis vogeli alone, 13 (72%) dogs coinfected, and 8 (27%) uninfected dogs. CONCLUSIONS: A platys and B canis vogeli infection, either singularly or together, was widespread in free roaming dogs associated with remote Aboriginal communities in the Northern Territory and north-western New South Wales. Moreover, both A platys and B canis vogeli infections were associated with a reduction in mean platelet numbers in dog populations, particularly in young dogs. The fact that 51% of dogs infected with A platys alone and 72% dogs coinfected were thrombocytopenic compared to 27% of uninfected dogs suggests that the organism alone or in combination with B canis vogeli has the potential to cause thrombocytopenia and perhaps contribute to a clinical bleeding disorder in infected dogs.