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

doi:10.1016/0304-4017(94)90114-7

Veterinary Parasitology

Volume 52, Issues 3–4, April 1994, Pages 219-233

https://doi.org/10.1016/0304-4017(94)90114-7 Get rights and content

Abstract

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.

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Cited by (29)

The red fox (Vulpes vulpes), a possible reservoir of Babesia vulpes, B. canis and Hepatozoon canis and its association with the tick Dermacentor reticulatus occurrence
2021, Ticks and Tick-borne Diseases
Citation Excerpt :
Histopathological changes indicate that hypoxia is a more important mechanism of renal damage than hemoglobinuria (Solano-Gallego et al., 2008) and acute tubular necrosis is the dominant pathological process (Zygner et al., 2013). Importantly, hypotension following B. canis infection has been experimentally proven (Schetters et al., 1994). It is suggested that reduced blood flow enhances adherence of infected erythrocytes to the capillary walls and activates the coagulation system leading to formation of blood clots where parasites in entrapped infected erythrocytes develop and have easy access to uninfected red blood cells (Schetters, 2019).
The red fox (Vulpes vulpes) is known to be a reservoir host of various vector-borne protozoan parasites. Yet, the impact of these parasitic infections on the health status of the red fox is unknown. In this study, we compared the prevalence of haemoprotozoa in juvenile and adult foxes from four regions in Poland. The abundance of questing adult Dermacentor reticulatus ticks was shown to differ between regions and include a tick-endemic and non-endemic region, as well as two zones in which D. reticulatus recently expanded: an eastern and a western zone. Spleen and kidney enlargement indices were compared between infected, co-infected and non-infected foxes to evaluate pathogenic output of parasitic infections.
Blood samples were collected from 383 necropsied red foxes and 25 live-trapped red fox cubs. The weights of spleens, kidneys and bodies were recorded for each necropsied individual. Blood and spleen DNA samples were screened by PCR to detect 18S rRNA and cox1 gene fragments of Babesia spp., and 18S rRNA of Hepatozoon spp. Prevalence of infection and mean organ weight indices were compared in relation to fox age and sex, origin (zone) and infection status.
Hepatozoon canis (174/381; 45.7%) and Babesia vulpes (117/381; 30.7%) were the dominant pathogens infecting adult foxes. Babesia canis DNA was detected in 9 (2.4%) individuals. Two fox cubs from Western Poland were infected with B. vulpes (2/14; 14%), while H. canis infection (16/25; 59%) was detected in cubs from all three regions where trapping was carried out. Infection with B. vulpes was associated with significantly higher weights of spleens and kidneys. Spleen weight/ body weight and mean kidney weight/ body weight indices were shown as relevant in evaluating changes occurring during infection with B. vulpes.
Babesia and Hepatozoon spp. infections differed in red foxes from D. reticulatus-endemic, non-endemic and newly inhabited areas. The prevalence of B. canis in foxes from four regions of Poland reflects the prevalence recorded in questing adult D. reticulatus collected in these areas. This finding suggests a contribution of red foxes to the establishment of new foci of B. canis-infected D. reticulatus ticks in areas recently invaded by this tick species. Spleen and kidney enlargement was identified in foxes infected with B. vulpes, particularly in individuals co-infected with B. vulpes and H. canis.
Discovery of a recombinant Babesia canis supernatant antigen that protects dogs against virulent challenge infection
2018, Veterinary Parasitology
Citation Excerpt :
Special attention was given to behaviour, spleen size, size of lymph nodes, colour of the mucous membranes of mouth and eye-lid, and the capillary refill time. Clinical observations were scored as a numeric value (Schetters et al., 1994). From these scores the total clinical score value was calculated for each day.
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.
showed that the first appearance of antibody reactivity against a protein that migrated at the 39 kDa 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 39–40 kDa doublet was released into the supernatant of B. canis cultures starting 15 min after the chase. This doublet was specifically precipitated by VC-serum, thus corroborating that the 39–40 kDa doublet in SPA preparations was of parasite origin. Partial amino acid sequencing allowed the discovery of the gene that encoded the 39–40 kDa 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 39 kDa 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.
Canine babesiosis in Europe: How many diseases?
2012, Trends in Parasitology
Citation Excerpt :
However, prerenal azotemia with no structural kidney damage can also lead to elevated serum creatinine concentrations, and creatinine values are therefore insufficiently specific to establish renal failure. In addition, in experimental B. canis infection it was shown that creatinine levels decrease in the early phase of the disease [27,75]. Liver failure is commonly diagnosed from elevated concentrations of liver enzymes, but this in our opinion is also not specific.
Babesiosis, recognized since ancient times as an important disease of livestock and more recently as an emerging disease in dogs worldwide, is caused by intraerythrocytic protozoa of the genus Babesia and is transmitted by ticks. The pathophysiology of canine babesiosis has been extensively studied but many questions remain unanswered, especially regarding the diversity of disease manifestations in different European countries. Continued investigation of the similarities and differences in host–parasite interplay in canine babesiosis in different European countries should lead to a better understanding of the disease process, potentially leading to better prediction of disease outcome and the development of new treatment modalities. From the European point of view it is important to conduct these studies on Babesia canis.
Soluble parasite antigens from Babesia canis do not directly activate the kallikrein system in dogs infected with Babesia canis
2011, Veterinary Parasitology
Citation Excerpt :
Every 48 h, subcultures were performed at 1% (v/v) PE in a fresh culture medium supplemented with 10% nds for each culture type. Parasitaemia was determined daily by evaluation of blood smears that were stained with May-Grünwald/Giemsa solutions and expressed as the log number of B. canis-PE per 105 erythrocytes (Schetters et al., 1994). SPA of the B. canis strain A were produced in large scale in vitro cultures in cell factories 10 (CF10, Nunc).
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.
Systemic inflammatory responses in dogs experimentally infected with Babesia canis; a haematological study
2009, Veterinary Parasitology
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.
Immunity against Babesia rossi infection in dogs vaccinated with antigens from culture supernatants
2007, Veterinary Parasitology
Soluble parasite antigens (SPA) from different Babesia species have been shown earlier to induce protective immunity when used as vaccine. However, initial attempts to produce such vaccine against Babesia rossi infection using SPA from B. rossi culture supernatants were not or only partially successful. Here we show that when dogs were vaccinated with a vaccine comprising SPA from B. rossi combined with SPA from Babesia canis protective immunity against experimental challenge infection was induced. Immunity was reflected in reduced clinical signs that resolved spontaneously, and reduction of parasitaemia and SPA in the blood. Not a single infected erythrocyte could be found in blood smears of dogs that had been repeatedly boosted (three vaccinations in total). In contrast, three out of four control dogs required chemotherapeutic treatment to prevent death. The fourth control dog showed a transient parasitaemia that resolved spontaneously. Vaccination did not prevent the development of a transient anaemia. It is concluded that a vaccine containing a mixture of SPA obtained from in vitro culture supernatants of B. rossi and B. canis induces protection in dogs against heterologous challenge infection with B. canis (as shown before) or B. rossi.

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Veterinary Parasitology

Abstract

References (18)

Babesia argentina: immunization of cattle with a killed antigen against infection with a heterologous strain

Vet. Parasitol.

Babesia canis: The life cycle and laboratory maintenance in its arthropod and mammalian hosts

Int. J. Parasitol.

Malarial parasites induce TNF production by macrophages

Immunology

Soluble malaria antigens are toxic and induce the production of tumour necrosis factor in vivo

Immunology

Malaria exoantigens induce T-independent antibody that blocks their ability to induce TNF

Immunology

Immunity and immunopathology in babesiosis

Possible roles of tumor necrosis factor in the pathology of malaria

Am. J. Pathol.

Malaria mimicry with tumor necrosis factor

Am. J. Pathol.

Spurious Babesia antigens

Nature

Cited by (29)

The red fox (Vulpes vulpes), a possible reservoir of Babesia vulpes, B. canis and Hepatozoon canis and its association with the tick Dermacentor reticulatus occurrence

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

Canine babesiosis in Europe: How many diseases?

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

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

Immunity against Babesia rossi infection in dogs vaccinated with antigens from culture supernatants