Preliminary evaluation of the BrEMA1 gene as a tool for associating babesia rossi genotypes and clinical manifestation of canine Babesiosis.

Babesia rossi, an intraerythrocytic protozoan, causes a severe, often life-threatening disease of domestic dogs. Dogs treated early for B. rossi infection usually recover from the disease, but dogs left untreated or treated at a later stage of infection seldom survive. Dogs infected with B. rossi have varied clinical manifestations that can be categorized as uncomplicated (with a good prognosis) or complicated (with a poor prognosis). One hundred twenty-one blood samples were collected from dogs presented to the Onderstepoort Veterinary Academic Hospital and diagnosed with babesiosis by the use of a thin blood smear. An additional 20 samples were obtained from Babesia-infected dogs from private clinics around the Onderstepoort, Johannesburg, Durban, White River, and Cape Town areas. The samples were screened by PCR targeting the Babesia rossi erythrocyte membrane antigen gene (BrEMA1) and by sequencing of the polymorphic region (i.e., region with a variable number of hexapeptide repeats). Analysis of PCR products revealed 11 different gene profiles, visualized by gel electrophoresis. Twelve distinct BrEMA1 genotypes were identified by sequencing, but the numbers of hexapeptide repeats varied from 6 to 31 (classified as genotype6 to genotype31). The genotypes were retrospectively compared to the clinical case data. The most frequently encountered B. rossi parasites were those attributed to genotype19 (36.2%), genotype28 and genotype29 (20.6% each), and genotype11 (12.7%). These genotypes were also the ones associated with the poorest prognosis. This preliminary finding suggests clinically important differences between the various B. rossi genotypes identified.

Systemic inflammatory responses in dogs experimentally infected with Babesia canis; a haematological study.

A detailed haematological study of dogs that were infected with low, moderate or high numbers of Babesia canis-infected red blood cells was performed in an attempt to elucidate the pathogenesis early after B. canis infection. Results showed that upon infection the C-reactive protein (CRP) level in plasma increased prior to the detection of parasites in the blood indicative of an acute phase reaction. The response was further characterised by fever, fibrinogenaemia, thrombocytopenia and leucopoenia. Thrombocytopenia was associated with increased coagulation time. Infected dogs also developed life threatening hypotension, and dogs that were infected with the highest dose of B. canis-infected red blood cells had to be treated chemotherapeutically. Hypotension was associated with a reduced packed cell volume (PCV). This reduction of PCV correlated with reduced plasma creatinin concentration, suggesting that the plasma volume was increased, affecting both the erythrocyte and creatinin concentration in the plasma. Importantly, the onset of the response but not the dynamics of the response was dependent on the infectious dose i.e. curves obtained with different doses of infected erythrocytes appeared to be shifted in time but had a similar shape. This indicates that infection triggered a preset inflammatory response.

Discovery of a recombinant Babesia canis supernatant antigen that protects dogs against virulent challenge infection.

Soluble parasite antigens (SPA) in supernatants of in vitro cultures of Babesia canis can be used to vaccinate dogs against virulent B. canis infection. The moment that immunity becomes apparent coincides with the appearance of antibodies against SPA in the serum of the vaccinated animals. This so-called vaccination-challenge serum (VC-serum) was used to precipitate antigens from B. canis culture supernatants in agarose gels. This antigen preparation was then used to analyse the reactivity of sera from vaccinated dogs on western blots. RESULTS: showed that the first appearance of antibody reactivity against a protein that migrated at the 39kDa position in SDS-PAGE gels was associated with the moment vaccinated dogs started to recover from a virulent challenge infection. In addition, pulse-chase experiments revealed that a 39-40kDa doublet was released into the supernatant of B. canis cultures starting 15min after the chase. This doublet was specifically precipitated by VC-serum, thus corroborating that the 39-40kDa doublet in SPA preparations was of parasite origin. Partial amino acid sequencing allowed the discovery of the gene that encoded the 39-40kDa doublet (canine Babesia antigen; CBA). The full-length gene was cloned and expressed in E. coli. The recombinant CBA protein (rCBA) was recognized by VC-serum, and antibodies against rCBA precipitated the 39kDa antigen of SPA preparations and of merozoites of B. canis. In addition, anti-rCBA serum reacted with the surface of B. canis merozoites (but not with B. rossi merozoites) in immunofluorescence. Vaccination of dogs with rCBA induced antibodies against rCBA, which recognized B. canis merozoites. Vaccinated dogs were protected against virulent challenge infection by limiting parasite proliferation. As a result, the development of clinical signs was prevented and the animals self-cured. In contrast, six out of seven non-vaccinated control dogs developed relatively high parasitaemia and serious clinical signs associated with poor tissue perfusion. This antigen can be used to replace the SPA antigen in the conventional B. canis vaccines, which eliminates the need for dog blood and serum for vaccine production.

Vaccination against large Babesia species from dogs.

The original observation of Sibinovic that soluble parasite antigens (SPA) of B. canis could be used to protect dogs against challenge infection formed the starting point for the development of an effective vaccine. With the advent of in vitro cultivation techniques for haemoprotozoan parasites an important tool became available for the commercial production of the vaccine antigens. A first generation vaccine was developed for dogs, but it appeared that the level of protection induced was not complete. In contrast to what was found with the SPA from serum/plasma of infected animals, protection induced with SPA from a single Babesia canis strain protected against a homologous challenge infection only. Further research led to the discovery that a combination of SPA of B. canis and SPA of B. rossi induced a broad spectrum of immunity. This improved vaccine, Nobivac Piro, not only induces protection against heterologous B. canis infection, but also against heterologous B. rossi infection.

Chemotherapy against babesiosis.

Babesiosis is caused by a haemotropic protozoal parasite of the genus Babesia, member of the phylum Apicomplexa and transmitted by the bite of an infected tick. There are many Babesia species affecting livestock, dogs, horses and rodents which are of economic significance. Infections can occur without producing symptoms, but babesiosis may also be severe and sometimes fatal caused by the intraerythrocytic parasite development. The disease can cause fever, fatigue and haemolytic anemia lasting from several days to several months. There are a number of effective babesiacides, but imidocarb dipropionate (which consistently clears the parasitaemia; often the only available drug on the market) and diminazene aceturate are the most widely used. Some Babesia spp. can infect humans, particularly Babesia microti and Babesia divergens, and human babesiosis is a significant emerging tick-borne zoonotic disease. Clinical manifestations differ markedly between European and North American diseases. In clinical cases, a combination of clindamycin and quinine is administered as the standard treatment, but also administration of atovaquone-azithromycin is successful. Supportive therapy such as intravenous fluids and blood transfusions are employed when necessary. More specific fast-acting new treatments for babesiosis have now to be developed. This should be facilitated by the knowledge of the Babesia spp. genome and increased interest for this malaria-like parasite.

Onset and duration of immunity against Babesia canis infection in dogs vaccinated with antigens from culture supernatants.

It has previously been shown that dogs can be vaccinated against heterologous Babesia canis infection using a vaccine containing soluble parasite antigens (SPA) from in vitro cultures of B. canis and B. rossi that are adjuvanted with saponin. In the present study the onset and duration of immunity of vaccinated dogs were studied. Results showed that 3-26 weeks after initial vaccination, dogs effectively limit the level of SPA in plasma upon challenge infection, which was reflected in limited duration and extent of clinical manifestations. There was no statistically significant effect of vaccination on the parasite load in the circulation, which was determined from blood smears. It was further shown that the level of immunity of primary vaccinated dogs (priming and booster vaccination with a 6-week interval) and that of repeatedly vaccinated dogs (a single additional vaccination 6 months after primary vaccination) is comparable. From this study it is concluded that vaccination with this preparation induces protective immunity against clinical babesiosis from 3 weeks after booster vaccination onwards, and remains effective for a period of at least another 6 months. A single booster vaccination is sufficient to maintain immunity for at least another 6 months.

[Human babesiosis]. / Babésioses humaines.

Babesia is one of the most ubiquitous and widespread blood parasite in the world based on numbers and distribution of species in animals. The clinical presentation may vary according to the incriminated species. In some states of the USA this kind of infection is endemic; the number of cases reported in Europe is inferior but more life-threatening. A better understanding of parasite specificities such as cycle and pathogenicity allowed to suggest treatment guidelines adapted to the different clinical and microbiological situations.

Classification of Babesia canis strains in Europe based on polymorphism of the Bc28.1-gene from the Babesia canis Bc28 multigene family.

The vast majority of clinical babesiosis cases in dogs in Europe is caused by Babesia canis. Although dogs can be vaccinated, the level of protection is highly variable, which might be due to genetic diversity of B. canis strains. One of the major merozoite surface antigens of B. canis is a protein with a Mr of 28 kDa that belongs to the Bc28 multigene family, that comprises at least two genes, Bc28.1 and a homologous Bc28.2 gene. The two genes are relatively conserved but they are very distinct in their 3' ends, enabling the design of specific primers. Sequencing of the Bc28.1 genes from 4 genetically distinct B. canis laboratory strains (A8, B, 34.01 and G) revealed 20 mutations at conserved positions of which three allowed the classification of B. canis strains into three main groups (A, B and 34.01/G) by RFLP. This assay was subsequently used to analyze blood samples of 394 dogs suspected of clinical babesiosis from nine countries in Europe. All blood samples were first analyzed with a previously described assay that allowed detection of the different Babesia species that infect dogs. Sixty one percent of the samples contained detectable levels of Babesia DNA. Of these, 98.3% were positive for B. canis, the remaining cases were positive for B. vogeli. Analysis of the Bc28.1 gene, performed on 178 of the B. canis samples, revealed an overall dominance of genotype B (62.4%), followed by genotypes A (37.1%) and 34 (11.8%). Interestingly, a great variation in the geographical distribution and prevalence of the three B. canis genotypes was observed; in the North-East genotype A predominated (72.1% A against 27.9% B), in contrast to the South-West where genotype B predominated (10.3% A against 89.7% B). In the central part of Europe intermediate levels were found (26.0-42.9% A against 74.0-57.1% B, from West to East). Genotype 34 was only identified in France (26.9% among 78 samples) and mostly as co-infection with genotypes A or B (61.9%). A comparative analysis of the classification of 35 B. canis strains in genotypes A and B using a previously described 18SrDNA-derived PCR-RFLP test revealed a partial but no direct correlation with the classification based on polymorphism of the Bc28.1-gene described here.

Antithrombin III antigen in human platelets.

Immunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 10(9) platelets with a mean value of 70.3 +/- 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

Association between sequence polymorphism in an epitope of Babesia divergens Bd37 exoantigen and protection induced by passive transfer.

In Europe, Babesia divergens is the major agent responsible for babesiosis in cattle and can occasionally infect splenectomised humans. Recently, we reported the characterisation of a 37 kDa exoantigen (Bd37) anchored in the merozoite membrane of B. divergens by a glycosylphosphatidyl-inositol. After phospholipase hydrolyse of the glycosylphosphatidyl-inositol anchor, the Bd37 antigen could be isolated in the plasma of the infected host and from the in vitro culture supernatants. Immunisation of mice with a gel-filtration protective fraction of B. divergens exoantigens, produced a monoclonal antibody (MAb), called F4.2F8-INT, directed against Bd37. In the present study, we report data on passive protection using MAb F4.2F8-INT. This MAb was able to completely protect against virulent challenges with B. divergens isolates Rouen 1987 (Rouen87) and Weybridge 8843 (W8843) but had no protective effect against another French isolate from Massif Central (6303E). Physical characterisation of the epitope recognised by F4.2F8-INT allowed us to explain the differences observed between these isolates by western blotting and passive protection. These results suggest that the antigen carrying this epitope could be used as a target in the development of a recombinant vaccine against B. divergens babesiosis.

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.

Identification and characterization of corpuscular, soluble and secreted antigens of a Venezuelan isolate of Anaplasma marginale.

Anaplasma marginale is the etiological agent of anaplasmosis, a tick-transmitted disease with an important economic impact that affects cattle throughout the world. Although, North American isolates of A. marginale and their antigens have been extensively studied, relatively little information is available on the antigenic composition of South American isolates. The characterization of diverse geographical isolates of A. marginale will result in a thorough antigenic profile and may lead to the identification of additional diagnostic and immunoprophylactic tools. Short-term cultures of a Venezuelan isolate (Ta) of A. marginale were maintained for up to 13 days in vitro. During that period, the A. marginale remained viable and were propagated in the bovine erythrocyte culture system. During the initial days of culture, cell division and reinvasion were evidenced by a significant rise in parasitemia up to a 50%. A. marginale antigens were identified by metabolic labeling with (35S) methionine, followed by fractionation and immunoprecipitation with homologous and heterologous bovine sera. This yielded a complete antigenic set for the Ta isolate of A. marginale, including soluble, secreted and corpuscular polypeptide antigens. Fifteen immunodominant polypeptides were recognized by the bovine sera in the soluble and corpuscular fractions with relative molecular weights of 200, 150, 100-110, 86, 60, 50, 47, 40, 37, 33, 31, 25, 23, 19 and 16kDa. Seven polypeptides were present in the exoantigen fraction. The 31 and 19kDa antigens were recognized by the ANAR76A1 and ANAF16C1 monoclonal antibodies, respectively which are specific for MSP-4 and MSP-5 from North American isolates of A. marginale. Metabolic labeling with (14C) glucosamine prior to immunoprecipitation with bovine sera allowed the identification of glycoprotein antigens of 200, 100-150, 60, 55, 50, 45-43, 37, 33, 31, 22, 19 and 16kDa in the soluble fraction.

In vitro excystation of Giardia from humans: a scanning electron microscopy study.

The in vitro excystation of Giardia lamblia on cysts isolated from human feces was studied. After purification by sucrose gradient, cysts were incubated in a pepsin-acid solution, then placed in a modified HSP3 medium where excystation occurred within a few minutes. The excystation procedure was studied by continuous observations by light microscopy and sequential observations by scanning electron microscopy (SEM). The in vitro excystation was stopped at timed intervals during incubation by addition of a large amount of 1% glutaraldehyde. The excystation process began by the cyst wall opening at one pole. Flagella protruded rapidly, the parasite emerged progressively from the cyst envelope, posterior end first, the empty cyst collapsed and shrank. Although flagella emerging from the organism were distinguishable, the cell body had not yet shown all the morphological features of the G. lamblia trophozoite. A radical rearrangement of the organism occurred gradually: initially oval in shape, the parasite became round, then elongated, flattened, and underwent cytokinesis. The daughter trophozoites acquired their typical morphological features: the shape, the adhesive disc with the C-shaped structure distinctly visible on the ventral surface, and the definite placement of the flagella. These observations obtained on G. lamblia by SEM were comparable to those obtained with G. muris.

Large scale survey of bovine babesiosis due to Babesia divergens in France.

Data on the epidemiology of bovine babesiosis in France were collected from two sources: a questionnaire sent to large animal veterinary surgeons and blood samples collected from cattle treated for clinical babesiosis. Babesia divergens was common and widespread. B major was found in only one sample. The disease was particularly widespread in north west, south west and central France. The mean clinical incidence was 0.4 per cent. The bimodal seasonality of the clinical cases was correlated with that of the tick vector (Ixodes ricinus). Of the affected animals 72.6 per cent were more than three years old.

Babesia divergens: an ELISA with soluble parasite antigen for monitoring the epidemiology of bovine babesiosis.

An enzyme linked immunosorbent assay (ELISA) for bovine babesiosis caused by Babesia divergens was developed to analyse the evolution of the serological status of cattle living in an enzootic area. The antigen used was a soluble extract of B. divergens obtained from in vitro culture. Specificity, evaluated with negative sera, was 96.6%. The ELISA was compared to indirect immunofluorescence analysis (IFA) on naturally or experimentally infected animals. It appeared that IFA was positive for at least seven or eight weeks; on the contrary, B. divergens-specific antibodies were only transitorily detected by ELISA. Furthermore, the ELISA was more sensitive than the IFA for the detection of post-infection antibody increases, particularly when infection-pressure was low. These results suggest that IFA and ELISA could be complementary tools in epidemiological surveys; indeed, this ELISA could be the most efficient tool to detect new infections in cohort monitoring.

Secreted antigens of the amastigote and promastigote forms of Leishmania infantum inducing a humoral response in humans and dogs.

To study the antigens secreted by promastigote and amastigote forms of Leishmania infantum which are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitro cultured parasites, metabolically labelled with [35S]methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitro culture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum. Nevertheless, the humoral response directed against the excreted-secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secreted antigens of the amastigote forms are immunoprecipitated with either human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion-secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitro life cycle.

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.