Imidocarb: A chemoprophylactic experiment with Babesia canis

Summary

Eight dogs, given imidocarb dipropionate subcutaneously at a dose of 6 mg/kg, were challenged with a sporozoite stabilate of a French strain of Babesia canis, prepared from infected Dermacentor reticulatus ticks, 2, 3, 4 or 5 weeks after treatment. Three control dogs were similarly infected but not preventively treated. One of the controls and one of the dogs treated 5 weeks prior to challenge died of babesiosis. Prepatent and incubation periods were similar in treated and control dogs, and all dogs showed important reductions in the packed cell volume. Relaps'es were commonly seen after recovery from the initial reaction. Although further work is needed before a final conclusion can be drawn to whether imidocarb is suitable as a chemoprophylactic against B. canis infection, it can be used as a curative drug.     

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.     

Ectoparasites of dogs belonging to people in resource-poor communities in North West Province, South Africa

A total of 344 dogs belonging to people in resource-poor communities in North West Province, South Africa, was examined for ectoparasites, and all visible arthropods were collected from the left side of each dog. By doubling these numbers it was estimated that the dogs harboured 14,724 ixodid ticks, belonging to 6 species, 1,028 fleas, belonging to 2 species, and 26 lice. Haemaphysalis leachi accounted for 420 and Rhipicephalus sanguineus for 14,226 of the ticks. Pure infestations of H. leachi were present on 14 dogs and of R. sanguineus on 172 dogs. Small numbers of Amblyomma hebraeum, R. appendiciulatus, R. evertsi evertsi and R. simus were also collected. The predominance of R. sanguitneus accounts for the high prevalence of canine ehrlichiosis (Ehrlichia canis) within the survey region, compared to canine babesiosis (Babesia canis), which is transmitted by H. leachi, and is a much rarer disease.     

Evolution of Acoustic Behaviour in African Bufo (Anura: Bufonidae)

Mating calls are known for 29 species of African Bufo belonging to 11 species groups. Twenty-five African species, representing eight species groups (including four groups or complexes having 2N=22) have calls which Martin (1972) termed Type I. This call type is also found in Schismaderma carens, Nectophrynoides tornieri and N. occidentalis. It is known in only four species of Bufo outside Africa and in Odontophrynus americanus which is thought to be closely related to leptodactylids that gave rise to the genus Bufo. Four African species of Bufo have Type II calls.

Geographic distribution of three call types indicates large radiations of one or two call types in South America, North America and Africa. The European and Asian Bufo faunas appear to be derived primarily from American radiations.

The radiation of bufonids in Africa appears to be equal to that of South America. An explanation of this may be that Bufo or its progenitor evolved prior to the continental separation of South America and Africa.     

A field trial evaluation of the prophylactic efficacy of amitraz-impregnated collars against canine babesiosis (Babesia canis rossi) in South Africa

South African canine babesiosis caused by Babesia canis rossi is a common clinical disease in dogs in South Africa and remains a significant cause of domestic dog mortality. To determine whether tick-repellent, 9% amitraz-impregnated tick collars (Preventic-Virbac) could prevent tick-borne exposure to B. canis rossi, 50 dogs were assigned to two groups. Group 1 (20 dogs), polymerase chain reaction (PCR)--and reverse line blot (RLB)-negative for B. canis rossi, were fitted with amitraz collars and blood samples collected monthly, over a 6-month period, and analysed for B. canis rossi. Group 2 (30 dogs) included 5 dogs selected on a month-by-month basis from a population of dogs from the same geographical area as the group 1 dogs, but with no history of previous tick control, which were blood-sampled together with the treatment group and analysed for B. canis rossi by PCR and RLB, to serve as the control group. Eight of the 30 control dogs (26.6%) were PCR/RLB positive for B. canis rossi, indicating high pathogen exposure during the trial period. All twenty of the treatment group dogs remained negative for B. canis rossi throughout the 6 months of the trial. These results suggest that the use of amitraz-impregnated collars had a significant effect on reducing infection with B. canis rossi.     

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.     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

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.     

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.     

Blood parasites of sheep in the Netherlands. II. Babesia motasi (Sporozoa,Babesiidae)

Summary

A large Babesia species occurs in sheep on the North Sea islands of the Netherlands. The tick Haemaphysalis punctata is a vector. Its pathogenicity appears to be low. It is morphologically similar to a Turkish strain, considered to be B. motasi, which is also transmitted by Haemaphysalis ticks. It differs from the Turkish parasite serologically as well as in cross‐immunity tests and in not being effective to goats.

There may be a group of morphologically similar parasites with serological differences and different infectivity for sheep and goats. As it is impossible to know which one is to be considered as the original B. motasi, we designate the Dutch parasite as B. motasi (Netherlands). Anaplasma mesaeterum was found to occur on the island of Texel as well as on Ameland, where it had been found initially.     

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.     

The suitability of the enzyme-linked immunosorbent assay (ELISA) for the demonstration of babesia antibodies in hyperimmune serums with the use of ectoantigens

From sera of highly parasitised mice and dogs with Babesia rodhaini, B. galagolata and B. canis ectoantigens were isolated by column-chromatography and tested in the ELISA for their serological properties. Hyperimmunsera against the three Babesia species were prepared in rabbits, mice and dogs. Serologic cross-reactions occurred between the investigated Babesia species although distinct titer differences could be observed between B. canis on one hand and B. rodhaini and B. galagolata on the other hand. The ELISA appears to be suitable for serological field surveys of babesiosis.     

Babesia canis rossi infection in a Texas dog

A 5-month-old intact male Boerboel dog, imported from South Africa 1 week previously, was presented to a Texas veterinarian for lethargy, anorexia, and labored breathing. The dog was febrile, anemic, leukopenic, thrombocytopenic, and slightly azotemic. Results of the IDEXX SNAP-4Dx enzyme immunoassay were negative for Dirofilaria immitis antigen and antibodies against Ehrlichia canis, Borrelia burgdorferi, and Anaplasma phagocytophilum. An EDTA blood sample analyzed at Oklahoma State University Center for Veterinary Health Sciences revealed nonregenerative anemia, neutropenia, and large protozoal piroplasms in 0.7% of the RBCs. Piroplasms were 2-5μm long and varied in shape from round to oval to piriform; extracellular merozoites were also observed. Nested PCR was performed on DNA extracted from blood using primers that amplify the 18s rRNA gene from all known Babesia species, and the product was sequenced. Basic Local Alignment Search Tool analysis of the 437 base sequence revealed 99-100% similarity to Babesia canis rossi, 92-93% similarity to Babesia canis canis, and 92% similarity to Babesia canis vogeli. The dog responded well to treatment with imidocarb. PCR analysis of a second blood sample 2 weeks later was negative for Babesia spp. DNA. This case represents the first diagnosis of B. canis rossi infection in the United States.     

Experimental and clinical trials of long acting oxytetracycline in the treatment of canine ehrlichiosis

Summary

Four splenectomized dogs were experimentally infected with Ehrlichia canis and treated at the point of illness with long acting (LA) Oxytetracycline at different dosages. Terramycin LA, when given at a dose of 20mg/kg body weight (deep intramuscularly) twice, at a four‐day interval, was found to have effectively controlled the disease and has replaced the usual 7–14 successive days’ treatment regimen when other groups of tetracycline drugs arc used.

Predej^® 2X* at the rate of 2 mg given intramuscularly concurrently eliminated the local inflammatory reaction caused by the injection of Terramycin LA.

Twenty‐four out of the 26 naturally infected dogs which were treated in a similar way were completely cured; one died before it could receive the full treatment, and another received a second medication five weeks after the first treatment, showing recurrent epistaxis; blood samples taken from this dog were negative for E. canis.     

Babesiosis in dogs and cats--expanding parasitological and clinical spectra

Canine babesiosis caused by different Babesia species is a protozoal tick-borne disease with worldwide distribution and global significance. Historically, Babesia infection in dogs was identified based on the morphologic appearance of the parasite in the erythrocyte. All large forms of Babesia were designated Babesia canis, whereas all small forms of Babesia were considered to be Babesia gibsoni. However, the development of molecular methods has demonstrated that other Babesia species such as Babesia conradae, Babesia microti like piroplasm, Theileria spp. and a yet unnamed large form Babesia spp. infect dogs and cause distinct diseases. Babesia rossi, B. canis and Babesia vogeli previously considered as subspecies are identical morphologically but differ in the severity of clinical manifestations which they induce, their tick vectors, genetic characteristics, and geographic distributions, and are therefore currently considered separate species. The geographic distribution of the causative agent and thus the occurrence of babesiosis are largely dependent on the habitat of relevant tick vector species, with the exception of B. gibsoni where evidence for dog to dog transmission indicates that infection can be transmitted among fighting dog breeds independently of the limitations of vector tick infestation. Knowledge of the prevalence and clinicopathological aspects of Babesia species infecting dogs around the world is of epidemiologic and medical interest. Babesiosis in domestic cats is less common and has mostly been reported from South Africa where infection is mainly due to Babesia felis, a small Babesia that causes anemia and icterus. In addition, Babesia cati was reported from India and sporadic cases of B. canis infection in domestic cats have been reported in Europe, B. canis presentii in Israel and B. vogeli in Thailand. Babesiosis caused by large Babesia spp. is commonly treated with imidocarb dipropionate with good clinical response while small Babesia spp. are more resistant to anti-babesial therapy. Clinical and parasitological cure are often not achieved in the treatment of small Babesia species infections and clinical relapses are frequent. The spectrum of Babesia pathogens that infect dogs and cats is gradually being elucidated with the aid of molecular techniques and meticulous clinical investigation. Accurate detection and species recognition are important for the selection of the correct therapy and prediction of the course of disease.     

Serological evaluation of Ehrlichia canis infections in military dogs in Africa and Reunion Island

Sixty-eight dogs from four African countries and Reunion Island were tested for antibodies against Ehrlichia canis. Twenty-six dogs (50%) in Tunisia, Senegal and Chad were found positive using the indirect fluorescence antibody test. Dogs from both the Central African Republic and Reunion Island were all negative. Thus, this preliminary report confirms the presence of E. canis in Africa. Larger studies will be necessary to evaluate the current epidemiologic situation of canine ehrlichiosis in these countries.     

The indirect fluorescent antibody test for bovine anaplasmosis

Summary

An indirect fluorescent antibody test was used succesfully for the serodiagnosis of experimental Anaplasma infections in cattle. Specific antibodies were detected three to ten days after anaplasma bodies werd found in the blood, and persistedat least 15 weeks post‐infection.

An American and an African stock of A. marginale were used to prepare antigens, and gave comparable results when tested on sera positive to either of these stocks, as well as to an A. centrale‐like stock from Korea.

There were no cross‐reactions with several Theileria, Babesia, Trypanosoma and Eperythrozoon species.     

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.     

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.     

Serological survey for antibodies reactive with Ehrlichia canis and E. chaffeensis in dogs from the Bloemfontein area, South Africa

Sera from 161 dogs in the Bloemfontein area in South Africa were tested for the presence of antibodies reactive with Ehrlichia canis and E. chaffeensis by indirect fluorescent antibody testing. Overall, 68 (42%) of the dogs had significant antibody titres (> or = 1/64) against E. canis and 61 (38%) had significant titres (> or = 1/64) against E. chaffeensis. Seven (11%) dogs had higher titres to E. chaffeensis than E. canis (1/2048 and 1/1024 (2 dogs); 1/1024 and 1/512 (2 dogs); 1/2048 and 1/512; 1/512 and 1/256 and 1/512 and < 1/64, respectively). The remaining seropositive dogs had equal (n = 26; 42%) or 2-(n = 17; 25%), 3-(n = 13; 2%) or 4-fold (n = 5; 7%) higher titres against E. canis. Dogs from economically depressed, high-density suburbs (60/112; 48%) had significantly higher prevalences of antibodies against E. canis than those from more affluent, low-density suburbs (8/49; 14%) (chi 2 = 19.38, p < 0.001). Higher titres to E. chaffeensis than E. canis were found in dogs from affluent, low-density suburbs (3/49) and in dogs from economically depressed, high-density suburbs (4/112).