lnternafional Journalfor Parasitology Vol. 20, No. 7. pp. 899-903, 1990 Printed in Great Britain 0

OO2~7519/90 $3.00 + 0.00 Pergamon Press plc 1990 Austndian Socieryfor Pamsifology



and C. DOBSON$

* CSIRO, Division of Tropical Animal Production and t CSIRO, Biometrics Unit, Long Pocket Laboratories, Private Bag No. 3, P.O., Indooroopilly, Queensland 4068, Australia $ Department of Parasitology, University of Queensland, St. Lucia, Queensland 4067, Australia (Received 15 February 1990; accepted 26 May 1990) Abstract-PAmom F., WRIGHT I. G., KERRJ. D. and DOBSON C. 1990. In vitro adherence of erythrocytes infected with Babesia bigemina and Babesia rodhaini to thrombospondin. International Journal for Parasitology 20: 899-903. The adherence of erythrocytes infected with Bubesiu bigemina and Babesiu rodhaini to thrombospondin (TSP) in vitro is demonstrated. Blood with a range of parasitaemias was used and counts of cells which bound to TSP on plastic were significantly different from the controls with both Babesin species. These studies indicated that TSP receptors arc present on the surface of red blood cells infected with the two Babe& species, although these parasites do not alter the membranes of infected erythrocytes obviously and do not cause cerebral symptoms in their hosts. Erythrocytes infected with either B. bigemina or B. rodhaini do not adhere to other erythrocytes in vivo, probably because these parasites induce mild infections in their hosts, but they can adhere to TSP in vitro. INDEX


Bubesiu bigemina; Babesia rodhaini; cytoadherence;



in vitro; thrombospondin

and correlate with phenotypes of the parasites which can induce cell adherence (Magowan, Wollish, Anderson & Leech, 1988; Howard & Gilladoga, 1989). Panton, Leech, Miller & Howard (1987) showed a direct correlation between the expression of 0KM5 antigen on the surface of erythrocytes infected with P. falciparum and the capacity of these cells to adhere in vitro. Erythrocytes infected with malaria have a receptor which promotes adherence in vitro which is a glycoprotein (88 kDa) from the membranes of platelets (Ockenhouse, Tandon, Magowan, Jamieson & Chulay, 1989). Thrombospondin (TSP) is a 450 kDa glycoprotein, found in many cells including monocytes, platelets and endothelial cells, which binds to heparin, fibronectin and collagen (Lawler, 1986) and causes adherence of erythrocytes infected with P. falciparum in vitro (Roberts, Sherwood, Spitalnik, Panton, Howard, Dixit, Frazier, Miller & Ginsburg, 1985). Parrodi, Wright, Bourne & Dobson (1989) demonstrated recently that both virulent and avirulent strains of B. bovis also adhere to TSP in vitro. Here, we studied the capacity of red blood cells, infected with B. bigemina from cattle and B. rodhaini from mice, to adhere to TSP in vitro. These species do not induce the infected cell to form either ‘knobs’ or ‘strands’ and do not cause cerebral symptoms in their hosts (Wright, 1972; Rudzinska & Trager, 1962; Chapman & Ward, 1977).

SEQUESTRATIONof infected red blood cells in brain capillaries is characteristic of acute Plasmodiumfalciparum and Babesia bovis infections (Wright, Goodger & Clark, 1988). Sequestration has also been described for malarial infections in the monkey and mouse (Cox, Semoff & Hommel, 1987; Thumwood, Hunt, Clark & Cowden, 1988). However, the precise mechanism by which infected cells bind to the brain capillaries in both diseases is not fully understood although several factors have been incriminated. Red cells containing mature P. falciparum parasites develop morphological features known as ‘knobs’ (Trager, Rudzinska & Bradbury, 1966) which possess sites that mediate the adhesion of erythrocytes to venous endothelial cells (Luse & Miller, 1971). ‘Strand-like’ alterations have been described on the membrane of red blood cells infected with virulent B. bovis which attach both to other infected cells and to the endothelium (Wright, 1972). A histidine-rich protein (HRP) has been correlated with the presence of knobs on red blood cells infected with P. falciparum and with the capacity of these cells to adhere to endothelial cells in vivo and in vitro (Kilejian, 1979). This protein by itselfis not sufficient to cause adherence in vitro (Rock, Saul, Taylor, Leech, Sherwood & Howard, 1988). Other proteins, of different antigenicity and molecular size, occur on erythrocytes infected with P. 899




Animals.Four splenectomized

S. Taurus calves, from 3 to 6 months old were used as donors of infected blood. Uninfected blood was taken from another four splenectomized calves. All the calves were monitored for the presence of blood protozoa before being used. Female, 6-12 weeks old, BALS/C mice were used as donors for infected and uninfected blood, and were kept in appropriate conditions and given food and water ad lib&m. Parasites. The CSIRO Lismore strain of B. bigemina.from a stabilate stored frozen in dimethylsulphoxide (DMSO), was used to infect four of the calves, the blood of which was then used when the parasitaemias ranged between 5 and 25%. A stabilate of B. rudhainiwas injected intraperitoneally (100 ~1) to infect the mice and produce parasitaemias of between 5 and 6%. Blood. Blood was drawn from the jugular vein of calves into sterile flasks and de~brinat~ by gentle agitation with glass beads. Blood was obtained by axillar section from mice under deep ether anaesthesia and was defibrinated in tubes with small glass beads. Human TSP (Lawler, 1986) was used as 50 pg ml-’ bound in replicate 3 mm diameter circles on plastic Petri dishes (Nunclon-~lta, Denmark). Assays. Blood infected with 3. bigemina (parasitaemia 25%) was plated onto four treated dishes, another blood (parasitaemia 14%) onto one dish and another (parasitaemia 5%) onto three dishes. Infected blood from all the animals was tested on untreated plastic together with uninfected blood with and without TSP as controls. ALl assays, for both 3. bigeminaand 3. rodhaini,were done with replicate samples and according to the method of Roberts et al. (1985). Cells which adhered to the plastic were counted across replicates of SO microscope fields ( x 540). Statistical analysis. Numbers of adherent infected and uninfected red blood cells were analysed separately for each experiment using analysis of variance to test for differences. Data are presented as histograms. RESULTS

Adherence of B. bigemina-infected blood At all three parasitaemias, more cells adhered to plastic treated with TSP (P < 0.001) (experiments I8) than to untreated plastic (Figs. 1 and 2). Few cells adhered to the plastic of the control plates and there



- !L.Y&Q FIG. 1.


to TSP





to TSP

FIG. 2. FIGS. I and 2. The effect of TSP on binding of Bubesia bigemina-infected blood to plastic trays. Experiment number 1 was performed with a 15% parasitaemia, experiments 24 with a 5% parasitaemia and experiments 5-8 with a 25% parasitaemia. The counts in all the experiments are from replicate samples and the raw data are represented in histograms.

were no statistically significant differences between control groups. More than 95% of the cells which bound to TSP-treated plastic were parasitized (Fig. 3). Many more cells adhered to the plastic in experiment 1 (Fig. 1) but the count did not differ significantly from those for experiments 2-8. Adherence of B. rodhaini-infected blood Significant differences were found in all four experiments for infected cells adhered to plastic treated with TSP (P < 0.001 for 5%, and P < 0.01 for 6% parasitaemia; Fig. 4). Very few uninfected ceils attached to plastic treated with TSP and very few cells from infected blood attached to the plastic alone. DrSCUssrON

B. bigemina and B. rodhaini both adhere to thrombospondin immobilized on plastic Petri dishes as do erythrocytes containing either P. falciparum (see Roberts et al., 1985) or both virulent and avirulent 3. bovis (see Parrodi et al., 1989). B. rodhaini causes glomerulonephritis, proteinuria, hepatosplenomegaly, severe anaemia and thrombocytopenia in their hosts (Chapman & Ward, 1977). Virulent B. bovis, but neither 3. bigemina nor avirulent B. bovis infections, causes coagulation changes, activation of thrombin and adherence in vivo (Wright & Goodger, 1977; Wright, Goodger, Buffinton, Clark, Parrodi BEWaltisbuhi, 1989). Why then do infections with B. bigemina, 3. rodhaini and most probably Babesia equi and Babesia caballi not cause cytoadherence in vivo? None of these parasites causes the profound circulatory changes like those induced by virulent B. bovis infections nor do they induce apparent changes in the membranes of


of B. bigemina and B. rodhainito thrombospondin


* + 1



L i



FIG. 3. Babesiabigemina-infected erythrocytes bound to a TSP-coated plastic surface (50 pg ml-‘). The sample was stained with Giemsa. mounted on a glass slide and viewed with a 90 x oil immersion objective and 6 x eyepiece. Scale bar = 14 pm.

-I -







lbesia ~-





- In

--J---t Ir_o



to TSP

FIG. 4. The effect of TSP on binding Babesia rodhaini-infected blood to plastic trays. Experiment 1 was performed using blood with a parasitaemia of 6% and experiments 24 with a parasitaemia of 5%. The counis shown are from replicate samples and the raw data are represented in histograms.

parasitized red blood cells (Goodger & Wright, 1977; Rudzinska & Trager, 1962; Chapman & Ward, 1977). Avirulent B. bovis and B. bigemina infections do not induce either thrombocytopenia or circulatory changes including thrombin activation (Wright et al., 1989; Goodger & Wright, 1977). It is likely that the absence of thrombocyte-parasite interactions probably limits the amount of TSP liberated into the circulation. Our present data certainly indicate that both B. bigemina and B. rodhaini can adhere in the presence of TSP, which supports the view that both species induce some changes in the membrane of the red cell which includes the insertion of a parasite TSP

receptor. Splenectomized calves infected with B. bovis are less likely to develop cerebral symptoms than nonsplenectomized animals. This difference is believed to relate to the level of lipid peroxidation. Severe lipid peroxidation, presumably as a result of stimuli to macrophages induced by parasites, occurs during acute B. bovis but not in the less acute B. bigemina infections (Wright et al., 1988). Virulent P. falciparum and B. bovis alter the membranes of infected erythrocytes (Trager et al., 1966; Wright, 1972). In malaria, ‘knobs’ on the infected red cell surface have been correlated with the expression and presence of receptors for cytoadherence. However, it is also known that the presence or absence of ‘knobs’ on erythrocytes infected with several species of Plasmodium does not necessarily determine their capacity to adhere to cells (Smith & Theakson, 1970; David, Hommel, Miller, Udeinya & Oligino, 1983; Udeinya, Graves, Carter, Aikawa & Miller, 1983; Cox et al., 1987; Udomsangpetch, Aikawa, Berzins, Wahlgren & Perlmann, 1989; Biggs, Culvenor, Ng, Kemp & Brown, 1989). Virulent strains of B. bovis cause a generalized circulatory disturbance, activate the coagulation system, and cause the release of vasoactive peptides. They also promote the consumption of complement, hypotension, vasodilation and terminal haemolysis (Wright, 1979). Similar pathology is found in P. falciparum infections (Wright et aI., 1988). The spleen plays an important role in the cytoadherence of P. falciparum (see David et al., 1983) while in virulent B. bovis, cerebral sequestration is not usually seen in splenectomized animals. Fibronectin, the third com-



ponent of complement, conglutinin, parasite antigens, IgG and cryofibrinogen are all associated with cytoadherence induced by B. bovis in cattle (Wright et al., 1988). It is these pathological changes which act together in cattle infected with virulent B. bovis to promote sequestration and clogging of brain capillaries by infected blood cells. Virulent B. bovis causes marked procoagulant activity where phosphatidyl serine binds irreversibly to fibrin monomer which helps to explain the presence of fibrin in infected erythrocytes; the reaction could also bind infected cells to endothelial fibrin (Wright et al., 1988). Changes in the lipid composition of plasma and parasitized erythrocytes of cattle infected with B. bovis and lipid peroxidation may also promote thrombin generation and exacerbate the reaction in platelet-poor plasma (Commins, Goodger, Waltisbuhl & Wright, 1988). Thrombocytopenia is commonly seen in both B. bovis and P. falciparum infections (Wright et al., 1988), and may correlate with the release of TSP by platelets. Thrombocytopenia complicated by intravascular coagulation has also been observed in Babesia canis infections in dogs (Guelfi, Dubois & Boneu, 1984). Thrombocytopenia and a rise in plasma fibrinogen also occur in both mild and severe forms of B. equi and B. caballi infections (Waal, van Heerden & Potgieter, 1987). We conclude that TSP receptors are present on the surfaces of red blood cells infected with a number of different species of Babesia (see Parrodi et al., 1989). Although both B. bigemina and B. rodhaini infections produce high parasitaemias, they do not produce cerebral symptoms. This is likely to be due to their failure to produce significant lipid peroxidation which would trigger a cascade of events with resultant liberation of TSP. Acknowledgements-Human thrombospondin (TSP) (Lawler, 1979) was a eenerous eift from Dr L. H. Miller (NIH. Beth&da, Maryland, U.S.A). The authors would also hke to thank Consejo National de Ciencia y Tecnologia CONACYT (MEXICO), the Universidad Autonoma de Morelos (MEXICO) and the CSIRO-University of Queensland Collaborative Research Program for their support. REFERENCES BIGGS B. A., CULVENORJ. G., NC J. S., KEMP D. J. & BROWN G. V. 1989. Plasmodium falciparum: cytoadherence of a knobless clone. Experimental Parasitology 69: 189-197. CHAPMAN W. E. & WARD P. A. 1977. Babesia rodhaini. Requirement of complement for penetration of human erythrocvtes. Science 196167-70. CO&INS G. A., G~~DCER B. V., WALTISBUHLD. J. &WRIGHT I. G. 1988. Babesia bovis: studies of parameters influencing microvascular stasis of infected erithrocytes. Research I’; Veterinary Science 44: 226-228. Cox J., SEMOFFS. & HOMMELM. 1987. Plasmodium chabaudi: a rodent malaria model for in vivo and in vitro cytoadherence of malaria parasites in the absence of knobs. Parasite Immunology 9: 543-561. DAVID P. H., HOMMEL M., MILLER L. H., UDEINYA I. V. & OLIGINO L. D. 1983. Parasite sequestration in Plasmodium ^ falciparum malana: spleen and antlbody modulation of

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In vitro adherence of erythrocytes infected with Babesia bigemina and Babesia rodhaini to thrombospondin.

The adherence of erythrocytes infected with Babesia bigemina and Babesia rodhaini to thrombospondin (TSP) in vitro is demonstrated. Blood with a range...
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