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.

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.

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.

Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems.

Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-gamma production in cells derived from equine lymph nodes. Preincubation of IFN-gamma inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-gamma induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-gamma production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species.

Parasite localization and dissemination in the Babesia-infected host.

Babesia bovis infections in cattle and B. canis infections in dogs are characterized by non-haemolytic anaemia and low parasitaemia during the acute phase of the disease. In this phase of the disease, animals suffer from hypotension followed by disturbances of the coagulation system. This review discusses the hypothesis that may explain the process of parasite localization in the host, and the consequences of such localization. It is suggested that hypotension favours the interaction between infected erythrocytes and the endothelial lining, thus facilitating localization of the infection. In addition, activation of the coagulation system by a parasite-derived molecule (one associated with the surface of infected erythrocytes or a soluble antigen) might consolidate this situation by causing cellular plugs to form. The continued proliferation of parasites in such plugs may then result in the occurrence of capillaries that are particularly heavily parasitised. An explanation is also suggested for the protective effect of vaccines against clinical babesiosis, based on the soluble parasite antigens that are released into the medium in cultures of babesial parasites.

Different Babesia canis isolates, different diseases.

Using surface immunofluorescence isolate-specific antigens were detected on the membrane of erythrocytes infected with Babesia parasites. In addition, the strains reacted differently with Plasmagel in that the European isolate (B.c. canis) could be purified on Plasmagel effectively, whereas infected erythrocytes of the South-African isolate (B.c. rossi) could not. Experimental infection of dogs with Babesia canis isolates from geographically different areas revealed different pathology. The European isolate obtained from France exhibited transient parasitaemia, usually below 1%, associated with low PCV values and congestion of internal organs. Clinical disease was correlated with an effect on the coagulation system, and not with peripheral parasitaemia. Infection of dogs with South-African-derived isolate induced high parasitaemia usually much higher than 1%, which required chemotherapeutic treatment. In these animals clinical disease was correlated with peripheral parasitaemia and not with parameters of the coagulation system. The results show that the etiology of disease caused by these isolates of B.c. canis and B.c. rossi is different. This might have implications for the development of vaccines against these infections.

Vaccination of dogs against Babesia canis infection.

This paper describes the clinico-pathological parameters measured in dogs that were vaccinated against Babesia canis using soluble parasite antigens (SPA) and then challenged. The packed cell volume (PCV) and the plasma creatinine value decreased immediately after challenge. Actual PCV values could be predicted in the first 5-6 days of the infection, assuming that creatinine values were modulated by increase of plasma volume. This association no longer existed after that time, and observations indicated splenic involvement in reduction of numbers of circulating erythrocytes. The anaemia due to B. canis infection appears to be the result of a multifactorial process including plasma volume increase, erythrocyte retention in the spleen and erythrocyte destruction, partly due to parasite proliferation. Vaccination limited the reduction of PCV values, and the development of splenomegaly. Differences in protection between vaccinated and control animals became apparent 6 days after infection, when a memory immune response becomes operative, and the onset of recovery of vaccinated animals correlated with the onset of antibody production against SPA.

Not peripheral parasitaemia but the level of soluble parasite antigen in plasma correlates with vaccine efficacy against Babesia canis.

Groups of five dogs were vaccinated against Babesia canis using soluble parasite (SPA) antigens from in vitro cultures. Although vaccination did not significantly alter peripheral parasitaemia upon challenge, protected animals had lower levels of SPA in the plasma after a challenge infection. The severity of anaemia correlated with the SPA-load during the post-challenge period in that high levels of SPA were associated with low haematocrit values. In addition, it was found that recovery was associated with the production of antibodies against SPA. The results suggest that SPA induce anaemia during B. canis infection, and that vaccination with SPA results in antibody production that can neutralize the effects of SPA after a challenge infection.

Strain variation limits protective activity of vaccines based on soluble Babesia canis antigens.

Groups of five dogs were immunized with vaccines containing soluble parasite antigens (SPA) derived from in vitro culture of Babesis canis parasites, either obtained commercially (Pirodog) or produced at laboratory scale. Both vaccines generated antibodies that reacted with parasitised erythrocytes (PE). Upon challenge infection with homologous parasites, protection was evident from less severe decreases of haematocrit values, and reduced morbidity. Vaccinated animals, however, were not protected against challenge infection with heterologous B. canis parasites. Recovery from challenge infection coincided with the production of antibodies against parasitized erythrocytes. The results suggest that SPA from B. canis carry strain-specific determinants that are crucial for inducing protection in dogs against challenge infection, and explain vaccination failures in the field.

Vaccination of dogs against Babesia canis infection using antigens from culture supernatants with emphasis on clinical babesiosis.

Groups of five dogs were vaccinated with Babesia canis antigens from in vitro culture in combination with saponin as adjuvant. Protection against challenge infection was evident as diminished clinical disease, decrease in parasitaemia, and a less marked fall in haematocrit values. Recovery from infection occurred at the time a memory immune response became effective (from Days 5 to 6 after challenge infection onwards). The effect was dose dependent, the highest antigen dose being most effective. A lysate of normal erythrocytes did not have protective activity, indicating that a parasite component was responsible for protection. Unlike the malaria situation, disease was not associated with elevated levels of tumour necrosis factor in the plasma, nor with hypoglycaemia. Disease appeared to be the result of the activity of a parasite product, which could have triggered reactions which led to sequestration of erythrocytes from the peripheral venous blood. As a result, the packed cell volume decreased, and organs such as lymph nodes and spleen became congested. As soon as immunity had developed there was a rapid increase in the peripheral erythrocyte number, and congestion of the spleen diminished, indicative of restored capillary blood flow. The results further suggest that vaccination with a soluble parasite product blocks the trigger of this pathological process.

Vaccination of dogs against Babesia canis infection using parasite antigens from in vitro culture.

Groups of five dogs were vaccinated with different Babesia canis vaccine formulations. It appeared that partial protection against challenge infection was obtained when using parasite antigens from in vitro culture in combination with saponin. Protection was evident as a decrease in parasitaemia after challenge and was associated with the presence of serum antibodies against Babesia parasites. In addition, parasite antigen derived from in vitro culture supernatant exhibited more protective activity than somatic parasite antigen, in that a less marked fall of haematocrit values was found after challenge infection. The fall of haematocrit value observed in the animals immunized with somatic parasite antigen was not different from that observed in the adjuvant control group.