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Influence of infective dose, degree of host anaemia and multiparasitism of erythrocytes on Babesia microti and Babesia hylomysci parasitaemias in mice
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Inretnr~lton~t Jt)utnalJ~t I)ar/~l)t~.~t).~r Vat. ~3, No. a~, p. 39'~.4~b1., iv3gL *qrl~tedin Great Britain,
Pergamon I~exs Ltd. ~ 1983 Austrahan Soewty fot" Far#a*tololcy
I N F L U E N C E OF I N F E C T I V E DOSE, D E G R E E OF H O S T A N A E M I A A N D M U L T I P A R A S I T I S M OF E R Y T H R O C Y T E S ON B A B E S I A M I C R O T I A N D B A B E S I A ]~tYLO?/1YSCIPARASITAEMIAS IN M I C E G. D. GRAYand R. S. PHILLIPS Department of Zoology, University of Glasgow, Glasgow, GI 2 8QQ, Scotland (Received 10 August 1981; in revised form 5 January 1983) Abstract--GRAy G. D. ANn PHILLIPSR. S. 1983. Influence of infective dose, degree of host anaemia and multi-parasitism of erythrocytes on Bctbesia mieroti and Babesia hylomysci parasitaemias in mice. International Journal for Par,ts.itology 13: 399-404. Mice were infected with varying doses of the piroplasms Babesia rnieroti or B, hylomysci and the resulting parasitaemias assessed by examination of stained blood smears. High doses resuiled in shorter prepatent periods and increased height of maximum percentage parasitaemias but the duration of the patent B. microti parasitaemia was not affected. High doses of B. hylomysci resulted in death of th: host. The course of percentage parasitaemia did not always accurately reflect the parasite load in the host because during the infection there were changes in the numbers of erythrocytes present, an increase in erythrocyte volume, multi-parasitism of erythroc3qes and indications of large numbers of cell-free parasites at the height of infection. INDEX KEY WORDS: Babesia microti; Babesia hylom)'sci; infective dose; percentage parasitaemias; patterns of infection. INTRODUCTION FACTORSwhich influence the duration and severity of infections with rodent piroplasms include the species of host, the number of passages since primary isolation of the parasite and the size of the infecting inoculum, For example, Nowell (1969) found that the maximum B. microti parasitaemia in rats was 200Z0. The same specie.~ of piroplasm in mice produced maximum par~:.;%~rqias of 50-60o7o (Cox & Young, 1969). In dilf~t::" ~trains of laboratory mice, B, hylornysci soon after isolation from the wild produced m a x i m u m parasitaemias which ranged between 10 and 30°7,, but after further passages in mice maximum par~,sitaemias were as high as 80% (Bafort, Timperman & Molyneux, 1970). Studies o f B. rgdhaini in mice (Overdulve & Antonisse, 1970) an(i of B, boris in cattle (Mahr.ney, Wright & Ketterer, 1973) have shown that the prepatent period of the parasitaemia has an inverse linear relationship with the logzo of the number of infecting organisms. In the experiments reported here we have investigated the role of infecting dose in determining the d u r a t i o n of the prepatent parasilaemia, the rate of increase of the primary patent parasitaemia, the duration of the patent parasitaemia and the packed cell volume durirtg infection with either B. microti or B. hyiomysei. In addition, a comparison has been made between the course of infection with these rodent piroplasms when quantified (1) as the percentage o f the red ceils infected and (2) as an estimate of the actual number
of parasitised red cells in circulation. The course of parasitaemia, measured as a percentage of red blood ceils (RBC) which are infected, has been used frequently to monitor the effect of various treatments on the course of piroplasm infections: for example, to monitor the effect of immune serum (Rogers, 1974; Roberts & Tracey-Patte, 1975; Abdalla, Hussein & Kreler, 1978), and the immunocompetence of mice infected with B. microti (Phillips & Wakelin, 1976; Cox, 1976; Purvis, 1977; Gray & Phillips, 1983). Some of this work has bee.n published (Gray, 1980, Abstract in Parasitology 79, xxvii).
MATERIALS AND METHODS Parasites. The King's strain of Babesia microti, originally isolated (Cox & Young, I969) from the vole, ,Microtus agrestis, was stabilated in our laboratory in liquid nitrogen (-196°C) as GUP (Glasgow University Protozoology) 235. Babesia hylora),sci was originally isolated from the tree rat, H)'lomyseus stella (Bafort et aL, 1970) and was established as GUP 397. Both stocks of parasites have undergone many passages in rodents since primary isolation. To initiate each infection frozen blood was thawed as described previously (Gray & Phillips, 1981) and inoculated intraperitoneally (i.p.) into a single C57B1 mouse which, 4--6 days later, was exsanguinated into heparin (10 i.u./ml of blood) by cardiac puncture under deep ether anaesthesia. The parasitised blood was then diluted as required in Hanks' balanced salt solution (Oxoid) containing l~/o glucose and then inoculated intravenously (i.e.) in a volume of 0.2 ml.
400
G. D. G~,~vand R. S. PHILLIPs
Mice. Male C57BI mice aged 6-10 weeks and supplied by the Centre for Laboratory Animals, Ilniversity of Edinburgh, were u~ed in all experiments. Determination of poekecl cell volume (PCV). The PCV'~ of samples of tail blood from infected and uninfected mice were determined using a micro-haematocrit centrifuge. Measurement o f parasitaemias. Infections ,~ere taonitored by examination of thin smears of tail blood collected daily and stained with Giemsa's stain. Smears ~ere examined at a magnification of ×400 and at least I& uninfected or 10z infected RBC were counted depending on the level of parasitaemia. The percentage parasitaemia (%P~ is the percentage of the red blood cells (RBC) counted ~'hich contain parasites. Four measurements involving parasitaemia ~vere made. These were: (l} the rate of i~ crease of ~'oP from patency in 20~'0 measured by regre_~sion analysis; (2) the pre.2~'* latent period after infection ~as initiated (Warhurst & Fol~ell, 19681; (3) duration of primary patent parasilaeraia above 2% level; 14) maximum parasitaemia. Values for these measurements were obtained for each mouse and the mean and standard deviation were determined for each group, Stalistical comparisons were made between groups using a one-way ~naly~is of variance, Compenaated parasitaemia. In part of this study the actual number of circulating parasitised RBC, i.e. compensated parasitaemia, was calculated from the %P and the total number of circulating RBC estimated from the PCV of infected animals. This compensated parasitaemia of individual mice is equal to the %P multiplied by ~he ralio of the PCV .of that mouse (PCV,) and the mean PCV of all the control uninfected mice (PCV m) ,or, that day, i.e.: compensated parasitaemia -- PCV, × %p, PCV,~ Celt-free parasitaemia. "['he degree of RBC-free parasitaemia was estimated on the following scale: O--no free parasites; + - < 5 in all fields examined; + + - t - 5 per field; + + + ->5 per field. Coulter counter analysis. A Coulter Electronic Particle Counter (Model ZB) with Mean Cell Column (MCV) accessory was used for determining red cell numbers and MCV. Approximately' two drops (100 lall of blood were collected from the tail of each mouse onto a small piece of paraffin film and taken up into a Model R Dual Diluter (Coulter) in preparation for counting. RESULTS
Variation o f infective dose B. microti infection. Each of 8 groups o f 6-8 mige was infected with a dilution of ~,~ood contair~ing 10~ - t 0 ~ B, mieroti-infeeted RBC. No mice given 10~, 10z or I03 parasitised RBC became parasitaemic. For doses above 10 ~ the pre-2070 periods increased by approximately 3 days with each 10-fold decrease in the inoeulum (Tab!e 11. There was no difference in the duration o f the parasitaemias (2-2%) o f the 4 groups given 10~-107 infected RBC (14,9-17.1 days) but these were all significantly shorter ( P < 0.01 ~analysis of variance) than the duration of the infection of the group given 10 a parasites (21.3 days). The rates o f increasing parasitaemia (patency to 20%) were similar in all groups. The relationship between the height o f maximum para~itaemia, the day on which it occurred and the size of the inoculum is
l.l.t', vat. I 3. t983
shown in Fig. la. There was a significant correlation between the day of maximum parasitaemia and its height. The later the maximum occurred, the lower it was. Also, it can be seen that lower infecting doses resulted in lower maximum parasitnemias. B. hyiomysci infection. Five groups of 6--8 mice were inoculated with 104-10 ~ 13. hylomysci infected RBC, The measurements made during infection are shown in Table 1. The duration o f the parasitaemias of the 3 groups which survived were not significantly different (analysis of variance). In contrast with the B. microti infections, the slope of increasing parasitaemias (Fig. l b ) was, on average, much steeper and the angle of the slope decreased with decreasing infective dose. This was reflected in the continually increasing difference between the mean p r e - 2 ~ periods in groups given successively lower infective doses, i.e. 1.39, 2.67, 2.99 and 4.46 days. Further, B. hylomysci killed all of the group given 10~ parasites with an increasing proportion o f the mice surviving as the infecting dose was lowered. The relationship between the height o f m a x i m u m parasitaemia, the day on which it occurred and the size of the inoculum is similar t~, although more striking (Fig. Zb) than that o f tiie tl. microti infections.
The role o f ana,:mia in the pattern o f parasitaemia Babesia microti and B. hylomysci parasitaemias. Two groups of eight mice were infected with 10~ B. microti and two further groups o f eight mice with 104 B. hylomysci infected RBC. A fifth group o f seven mice was not infected. PCV and parasitaemias were determined from one group of mice for each parasite. Two groups infected with each parasite were included in order to demonstrate that regular bleeding did not significantly alter the course o f the parasitaemia. It was found (data not shown) that for both B. microti and 11. hylomysci PCV's fell from about day I0 reaching a minimum which coincided with or was one day later than maximum parasitaemia. For 2-3 days either side of maximum parasitaemia, compensated parasitaemia was significantly less than %/:', A plot o f compensated parasitaem[a against time gave a curve with a peak less welldefined than that o f ~0P. Changes in R B C volume during infection. Groups of 10 mice were infected either with 10~ B. microti-or 10~ 11. hylomysei.infeeted RBC. A further 10 mice not infected were used as controls, Analysis of blood samples collected at intervals was made with a Coulter Counter. Overall the results o f the red blood cell counts paralleled those of the P E W s measured in the previous experiment. There was, however, a difference in both infections which could be accourited for by a change in RBC volume around tile peak o f infection. During the B. microti infection it can be seen (Fig. 2at that the PCV levels fell to approximately 50% o f those of control mice, RBC concentration, however, fell to a minimum o f 26%
67.7 ±
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Pr¢-2~0 period(days) 3~22±0-87 6.11 ± 0 . 5 6 9.3=~0.72 12.0± 1.17 15.4~_0.93 Rate of /ncre~,sing parasitaemia 0.39:~0.09 0 . 4 0 = 0 . 0 6 0 - 3 6 ± 0 - 0 7 0 - 3 4 ± 0 . 0 8 0 - 3 3 ± f f 0 9 (regression coefficicnls)
parasi,aemia
Maximum
%
Number ofB. microriinfec~ed RBC ce}ls in inoculum
NUMBIi~5 01; ~, /l~/cro//- OR B. ~ly/OI17ySf.'i-INFECTEDR ~ C
l0 T
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1-02±0.08
7,02±0,8
32-0 ± 3.9
lO s
10,01 ± 0 . 7
32.5 ± 5,5
iO s
14.47± t.00
20,8 ± 3,7
10 4
5/6
12-2
2/6
I5.3 -+ 1-8
0/6
16.4 ~ 2"6
0/6
t5-8 -+ 1-7
0.71 ±0-10 0-74±0-18 0-44±0-08 0 - 2 8 ± 0 . 1 3
2~96~0,03 4,35~0,11
54.3 ± I0.2 49,8 ± 13.4
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Number o f & hylomy.sciinfected RBC ceils in inocuIum
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FK; I. The relationship between the height of maximum percentage parasitaemia, the day on which it occurred and the size of the infecting dose. Groups of 6-8 mice were infected with 10s (o), 10~ (~), 106 ( I ) , 105(~) or 10 ~ (A) B. mlcrot~- (a) and B. hylomysci- (b) infected RBC on day 0. Each symbol represents the maximum percentage parasitaemia of one mouse, The coefficient of correlalion of B. microti is 0.B2 and for B. hylomy~ci 0.86. Both correlalions are significant (P < 0-01). of normal levels. During the B. hyiomysci (Fig. 2b) the mice became very anaemic with a 90% reduction in RBC concentration. The mice became very ill and bleeding from the tail for PCV determination was not possibl~ during the crisis of infection. There was an increase in mean RBC size up to 35°70 above normal levels. Multiparasitism o f R B C and extracellular parasites. A feature orB. microti and B. hylomysci infections was the presence of more than one parasite per RBC and large numbers of parasites apparently free in the plasma, particularly around the time of maximum parasitaemia. Blood smears from one of the groups infected with B. microti in the previous experiment were examined for multiparasitism of the red blood cells and for extracellular {free) parasites. Red cells with two or more parasites present were noted between days 10 and 30 of the infection, doubly infected cells being most frequent and with an increasing proportion o f red cells containing 3--6 parasites a r o u n d peak parasitaemia. At peak parasitaemia there were almost 25% more parasites in the red cells than parasitised red cells, In blood smears taken between days 14 and 21 there were significant numbers of 'free' parasites to be seen which may indicate free parasites to be present in the plasma of the infected mice. DISCUSSION Two major conclusions can be drawn from these experiments. First, the pattern of parasitaemia is
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Maximum % parasitaemia occurred on day 13, dependent on the number of parasitised RBC in the infecting inoculum, Second, 070P is not always an accurate measure of the parasite load in the host. The experiments with 10-fold dilution of B, rnicroti and B. h.vlomysci confirm and extend the results of Overdulve & Antonisse (1970) that the pre-2% period (or pre-patent period) is inversely proportional to the logan of the number of infecting
:.J,p, VnL, 13, 1983
parasitaemias of rodent piroplasms
organisms. We have extended this observation to show that the height of maximum parasitaemia and in the ease of B. hylomysci the ability of the host to survive infection, i.e, the severity of the disease, is related to the infective dose. This suggests that the ability of the host to c o n t r o l the infection is dependent on the length of time that the host has been infected which in turn is a reflection of the size of the infecting dose. There is a~ interesting contrast between the rates of increasing parasitaemia which do not vary with infecting dose in B. microti-infected mice but which decrease with decreasing infective dose in B. hylomysci-infected mice. A possible explanation for this is that the onset of the host immune response is more rapid in the case of B. microti and thereafter the response to rising parasitaemia is equally effective irrespective of the pre-patent period. The observation that higher infecting doses of B. hyiomysci are fatal certainly indicates that the host response is not effective if the acute phase o1' infection occurs soon after infection. An attempt was made to assess the relationship between ~/0P and the actual numbers of parasitised RBC in the blood during infection of these mice, Initially it was assumed that PCV reflected RBC numbers, it was found that the compensated parasitaemia which w'as calculated from the PCV values did :.at closely follow the same pattern as the ~oP. For example, the sharp peak seen in the 0YaP of B. hylomysci was replaced by a plateau for CP lasting about 7 days. At the height of infection it was subsequently found that RBC volume increased to a maximum 7070 above normal values and thus at this time PCV gave an overestimate of RBC numbers and therefore also of para.sitised RBC numbers. It is not known if the increase in mean red cell volume is the result of macrocytic anaemia or distension of the red cells by the parasite. In summary these observations simply serve to emphasise that % P may sometimes give an exaggerated picture of parasite load. In contrast, it was shown that the actual number of intracellular parasites may be as much as 250/0 grealer than the number of parasitised erythrocytes ann in addition, there may be many cell-free parasites at the height of infection. Both of these observations suggest that even the total number of parasitised erythrocytes is not an accurate measure of total parasite load. In an attempt to avoid these difficulties a direct method o f counting the n u m b e r o f parasitised erythrocytes in a known volume of B. boris; B. bigemina- and Plasmodium berghei-infected blood has been described by Parker (1973) but he noted that the method was not successful for the rodent p~roplasma B. rodhainL The method described by Rodwell & Howard (1981) using bisbenzimide may prove to be a better technique for the estimation of the total load of parasitised erythrocytes. There is a further consideration, not investigated
403
here, which has a bearing on these results. Chapman & Ward (1976) showed that the plasma volume of rats is reduced by 25°70 at the time of maximum B, rodhaini parasitaemia, thereby concentrating the RBC. If this is also true for B. microti and B, hylontysci infections then calculations of parasite numbers will be unknowingly exaggerated by onethird. In contrast, in t3. boris infection an early fall in PCV is ascribed to vasodilation and ~ncreased vascular permeability (Wright & Kerr, 1975). This work has shown differences in the course of two rodent babesia infectious which may reflect differences in the nature of the immune response of the host or of the haemopoietic tissue to respond to the changes brought about by the infection. These are areas for further investigation. Acknowledgements--The authors would like to thank the Medical Research Council for financial support of this work, Prof. F. E. G. Cox for the supply of B. microti and Prof. D. H. Molyneux for the supply of B. hytomys¢i.
REFERENCES ABDALLA H. S., HUSSEIN l'l. S, & KR~tEft J. P. 1978. Babesia rodhaini: passive protection of mice with immune serum. Tropemnedizin and Parasitologie 39: 295-306.
BAvoa'r J, M., TIMP£RMANG, ~z MOtVNEUX D. H. 1970. Babesia hylomysci tLsp., a new Babesia from the Congo transmissible to mice. Aanales de la Soci~t~ beige de rnbdecine tropicale 50:301-3 ~7. CrtAPM^r~ W, E, & W^RD P. A, 1976. Changes in C3 melabolism during protozoan infection (Babesia rodhaim) in rats. Journal oflmmunotogy 116:1284-1288. Cox F. E. G. 1976. Increased virulence of trypanosome infections in mice with malaria or piroplasmosis: immunological considerations, In: Biochemistry o f Parasites and Host-Parasite Relationships (Edited by V^~ t~E~ BosscltE H.), pp, 421--426. Elsevier/North !~olland/AcademicPress, Amsterdam. Co~t F, E. G~ & YouN~ A, S. 1969. Acquired immunity to Babesia microti and Babesia rodhaini in mice. Parasitology 59: 257-268. GRAY(3. D. & PtaILLIPSR. S. 1981. Use of sorbitol in the eryopreservation of babesia. Research in Veterinary Science 30: 388-389. GRAYG. D. & PtUtLIPS R. 5. 1983. Suppression of primary and secondary antibody respor~ses and inhibition of antigen priming during 13abesia microti infections in mice. Parasite lmrnunolog3"5:123-134. Mt'I'|ARDYN. 1973. Effects of stimulating erythropoiesis in mice infected with Babesia rodhaini. Annals of Tropical Medicine and Parasitology 6"/: 301-306. MAUor~EV D. F., WaroHr 1. G. & K~'rrEgEit P. J. 1973. Babesia argentina: 1he infeclivhy of immunogenicity of irradiated blood parasites for splenectomised calves. International Journal for Parasitology 3:209-217. NowEt~. F. 1969. The blood picture resulting from Nuttallia ( = Babesia) rodhaini and Nuttallia [ = Babesia) microti infections in rats and mice. Parasitology 59: 991-I004.
404
G. D. Ga,~v and R. S. Pnt~.tt~s
OVERt~ULV£ J. P. & ANTONISSE H. W. 1970. Measurement of the effect of low temperature on protozoa by titration. II. Titration of Babesia rodhaini, using prepatent period or survival time, before and after storage at -760C. Experimental Parasitology 27: 323 -34 I. P^rtKER R. 1973. A direct c o u n t i n g technique for estimating high parasitaemias in infections o f Babesia argentina, Babesia bigemina and Plasmodium bergheL Annals of Tropical Medicine and Parasitology 67: 387-390. PmLLIPS R. S. & WAKELIIqD. 1976. Trichuris muris: effect of concurrent infections with rodent piroplasms on immune expulsion from mice, Experimental Parasitology 39: 95-100. Purtvls A. C. 1977. Immunodepression in Babesia microti infections. Parasitology 75: 197-205. Roa~Rrs J. A. & TaACEY-PATIE P. D. 1975. Babesia rodhaini: a study of the effects of immune serum. International Journal for Parasitology 5:577-581.
t,J.P, rot.. 13. 1983
RODWELt B. J. & HOWARD R. J. I981. A fluorescent method for counting BabeMa, Anaplasma, Plasmodiun: and Trypanosoma following DNA staining with 33258 Hoechst (Bisbenzimide). Annals of Tropical Medicine and Parasitology 75:123-130, Ro~E~s R, J. 1974. Serum opsonins and the passive transfer o f protection in BabeMa rodhaini infections of rats. International Journal for Parasitology 4:197-201. WARHURSTD. C. & FOLWELL R. O. 1968. Measurement of the growth rate of the erythr'oeytic stages of Plasmodium berghei and comparisons of the potency of inocula after various treatments. Annals of Tropical Medicine and Parasitology 62: 349-360, Walt3jtr i. G. & KERR J. D. 1975, Effect of trasylol on packed cell volume and plasma kallikrein activation in acute Babesia argentina infection o f splenectomised calves. Zeitschriftfar Parasitenkunde 46: 189-194,
Inretnr~lton~t Jt)utnalJ~t I)ar/~l)t~.~t).~r Vat. ~3, No. a~, p. 39'~.4~b1., iv3gL *qrl~tedin Great Britain,
Pergamon I~exs Ltd. ~ 1983 Austrahan Soewty fot" Far#a*tololcy
I N F L U E N C E OF I N F E C T I V E DOSE, D E G R E E OF H O S T A N A E M I A A N D M U L T I P A R A S I T I S M OF E R Y T H R O C Y T E S ON B A B E S I A M I C R O T I A N D B A B E S I A ]~tYLO?/1YSCIPARASITAEMIAS IN M I C E G. D. GRAYand R. S. PHILLIPS Department of Zoology, University of Glasgow, Glasgow, GI 2 8QQ, Scotland (Received 10 August 1981; in revised form 5 January 1983) Abstract--GRAy G. D. ANn PHILLIPSR. S. 1983. Influence of infective dose, degree of host anaemia and multi-parasitism of erythrocytes on Bctbesia mieroti and Babesia hylomysci parasitaemias in mice. International Journal for Par,ts.itology 13: 399-404. Mice were infected with varying doses of the piroplasms Babesia rnieroti or B, hylomysci and the resulting parasitaemias assessed by examination of stained blood smears. High doses resuiled in shorter prepatent periods and increased height of maximum percentage parasitaemias but the duration of the patent B. microti parasitaemia was not affected. High doses of B. hylomysci resulted in death of th: host. The course of percentage parasitaemia did not always accurately reflect the parasite load in the host because during the infection there were changes in the numbers of erythrocytes present, an increase in erythrocyte volume, multi-parasitism of erythroc3qes and indications of large numbers of cell-free parasites at the height of infection. INDEX KEY WORDS: Babesia microti; Babesia hylom)'sci; infective dose; percentage parasitaemias; patterns of infection. INTRODUCTION FACTORSwhich influence the duration and severity of infections with rodent piroplasms include the species of host, the number of passages since primary isolation of the parasite and the size of the infecting inoculum, For example, Nowell (1969) found that the maximum B. microti parasitaemia in rats was 200Z0. The same specie.~ of piroplasm in mice produced maximum par~:.;%~rqias of 50-60o7o (Cox & Young, 1969). In dilf~t::" ~trains of laboratory mice, B, hylornysci soon after isolation from the wild produced m a x i m u m parasitaemias which ranged between 10 and 30°7,, but after further passages in mice maximum par~,sitaemias were as high as 80% (Bafort, Timperman & Molyneux, 1970). Studies o f B. rgdhaini in mice (Overdulve & Antonisse, 1970) an(i of B, boris in cattle (Mahr.ney, Wright & Ketterer, 1973) have shown that the prepatent period of the parasitaemia has an inverse linear relationship with the logzo of the number of infecting organisms. In the experiments reported here we have investigated the role of infecting dose in determining the d u r a t i o n of the prepatent parasilaemia, the rate of increase of the primary patent parasitaemia, the duration of the patent parasitaemia and the packed cell volume durirtg infection with either B. microti or B. hyiomysei. In addition, a comparison has been made between the course of infection with these rodent piroplasms when quantified (1) as the percentage o f the red ceils infected and (2) as an estimate of the actual number
of parasitised red cells in circulation. The course of parasitaemia, measured as a percentage of red blood ceils (RBC) which are infected, has been used frequently to monitor the effect of various treatments on the course of piroplasm infections: for example, to monitor the effect of immune serum (Rogers, 1974; Roberts & Tracey-Patte, 1975; Abdalla, Hussein & Kreler, 1978), and the immunocompetence of mice infected with B. microti (Phillips & Wakelin, 1976; Cox, 1976; Purvis, 1977; Gray & Phillips, 1983). Some of this work has bee.n published (Gray, 1980, Abstract in Parasitology 79, xxvii).
MATERIALS AND METHODS Parasites. The King's strain of Babesia microti, originally isolated (Cox & Young, I969) from the vole, ,Microtus agrestis, was stabilated in our laboratory in liquid nitrogen (-196°C) as GUP (Glasgow University Protozoology) 235. Babesia hylora),sci was originally isolated from the tree rat, H)'lomyseus stella (Bafort et aL, 1970) and was established as GUP 397. Both stocks of parasites have undergone many passages in rodents since primary isolation. To initiate each infection frozen blood was thawed as described previously (Gray & Phillips, 1981) and inoculated intraperitoneally (i.p.) into a single C57B1 mouse which, 4--6 days later, was exsanguinated into heparin (10 i.u./ml of blood) by cardiac puncture under deep ether anaesthesia. The parasitised blood was then diluted as required in Hanks' balanced salt solution (Oxoid) containing l~/o glucose and then inoculated intravenously (i.e.) in a volume of 0.2 ml.
400
G. D. G~,~vand R. S. PHILLIPs
Mice. Male C57BI mice aged 6-10 weeks and supplied by the Centre for Laboratory Animals, Ilniversity of Edinburgh, were u~ed in all experiments. Determination of poekecl cell volume (PCV). The PCV'~ of samples of tail blood from infected and uninfected mice were determined using a micro-haematocrit centrifuge. Measurement o f parasitaemias. Infections ,~ere taonitored by examination of thin smears of tail blood collected daily and stained with Giemsa's stain. Smears ~ere examined at a magnification of ×400 and at least I& uninfected or 10z infected RBC were counted depending on the level of parasitaemia. The percentage parasitaemia (%P~ is the percentage of the red blood cells (RBC) counted ~'hich contain parasites. Four measurements involving parasitaemia ~vere made. These were: (l} the rate of i~ crease of ~'oP from patency in 20~'0 measured by regre_~sion analysis; (2) the pre.2~'* latent period after infection ~as initiated (Warhurst & Fol~ell, 19681; (3) duration of primary patent parasilaeraia above 2% level; 14) maximum parasitaemia. Values for these measurements were obtained for each mouse and the mean and standard deviation were determined for each group, Stalistical comparisons were made between groups using a one-way ~naly~is of variance, Compenaated parasitaemia. In part of this study the actual number of circulating parasitised RBC, i.e. compensated parasitaemia, was calculated from the %P and the total number of circulating RBC estimated from the PCV of infected animals. This compensated parasitaemia of individual mice is equal to the %P multiplied by ~he ralio of the PCV .of that mouse (PCV,) and the mean PCV of all the control uninfected mice (PCV m) ,or, that day, i.e.: compensated parasitaemia -- PCV, × %p, PCV,~ Celt-free parasitaemia. "['he degree of RBC-free parasitaemia was estimated on the following scale: O--no free parasites; + - < 5 in all fields examined; + + - t - 5 per field; + + + ->5 per field. Coulter counter analysis. A Coulter Electronic Particle Counter (Model ZB) with Mean Cell Column (MCV) accessory was used for determining red cell numbers and MCV. Approximately' two drops (100 lall of blood were collected from the tail of each mouse onto a small piece of paraffin film and taken up into a Model R Dual Diluter (Coulter) in preparation for counting. RESULTS
Variation o f infective dose B. microti infection. Each of 8 groups o f 6-8 mige was infected with a dilution of ~,~ood contair~ing 10~ - t 0 ~ B, mieroti-infeeted RBC. No mice given 10~, 10z or I03 parasitised RBC became parasitaemic. For doses above 10 ~ the pre-2070 periods increased by approximately 3 days with each 10-fold decrease in the inoeulum (Tab!e 11. There was no difference in the duration o f the parasitaemias (2-2%) o f the 4 groups given 10~-107 infected RBC (14,9-17.1 days) but these were all significantly shorter ( P < 0.01 ~analysis of variance) than the duration of the infection of the group given 10 a parasites (21.3 days). The rates o f increasing parasitaemia (patency to 20%) were similar in all groups. The relationship between the height o f maximum para~itaemia, the day on which it occurred and the size of the inoculum is
l.l.t', vat. I 3. t983
shown in Fig. la. There was a significant correlation between the day of maximum parasitaemia and its height. The later the maximum occurred, the lower it was. Also, it can be seen that lower infecting doses resulted in lower maximum parasitnemias. B. hyiomysci infection. Five groups of 6--8 mice were inoculated with 104-10 ~ 13. hylomysci infected RBC, The measurements made during infection are shown in Table 1. The duration o f the parasitaemias of the 3 groups which survived were not significantly different (analysis of variance). In contrast with the B. microti infections, the slope of increasing parasitaemias (Fig. l b ) was, on average, much steeper and the angle of the slope decreased with decreasing infective dose. This was reflected in the continually increasing difference between the mean p r e - 2 ~ periods in groups given successively lower infective doses, i.e. 1.39, 2.67, 2.99 and 4.46 days. Further, B. hylomysci killed all of the group given 10~ parasites with an increasing proportion o f the mice surviving as the infecting dose was lowered. The relationship between the height o f m a x i m u m parasitaemia, the day on which it occurred and the size of the inoculum is similar t~, although more striking (Fig. Zb) than that o f tiie tl. microti infections.
The role o f ana,:mia in the pattern o f parasitaemia Babesia microti and B. hylomysci parasitaemias. Two groups of eight mice were infected with 10~ B. microti and two further groups o f eight mice with 104 B. hylomysci infected RBC. A fifth group o f seven mice was not infected. PCV and parasitaemias were determined from one group of mice for each parasite. Two groups infected with each parasite were included in order to demonstrate that regular bleeding did not significantly alter the course o f the parasitaemia. It was found (data not shown) that for both B. microti and 11. hylomysci PCV's fell from about day I0 reaching a minimum which coincided with or was one day later than maximum parasitaemia. For 2-3 days either side of maximum parasitaemia, compensated parasitaemia was significantly less than %/:', A plot o f compensated parasitaem[a against time gave a curve with a peak less welldefined than that o f ~0P. Changes in R B C volume during infection. Groups of 10 mice were infected either with 10~ B. microti-or 10~ 11. hylomysei.infeeted RBC. A further 10 mice not infected were used as controls, Analysis of blood samples collected at intervals was made with a Coulter Counter. Overall the results o f the red blood cell counts paralleled those of the P E W s measured in the previous experiment. There was, however, a difference in both infections which could be accourited for by a change in RBC volume around tile peak o f infection. During the B. microti infection it can be seen (Fig. 2at that the PCV levels fell to approximately 50% o f those of control mice, RBC concentration, however, fell to a minimum o f 26%
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parasi,aemia
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7,02±0,8
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FK; I. The relationship between the height of maximum percentage parasitaemia, the day on which it occurred and the size of the infecting dose. Groups of 6-8 mice were infected with 10s (o), 10~ (~), 106 ( I ) , 105(~) or 10 ~ (A) B. mlcrot~- (a) and B. hylomysci- (b) infected RBC on day 0. Each symbol represents the maximum percentage parasitaemia of one mouse, The coefficient of correlalion of B. microti is 0.B2 and for B. hylomy~ci 0.86. Both correlalions are significant (P < 0-01). of normal levels. During the B. hyiomysci (Fig. 2b) the mice became very anaemic with a 90% reduction in RBC concentration. The mice became very ill and bleeding from the tail for PCV determination was not possibl~ during the crisis of infection. There was an increase in mean RBC size up to 35°70 above normal levels. Multiparasitism o f R B C and extracellular parasites. A feature orB. microti and B. hylomysci infections was the presence of more than one parasite per RBC and large numbers of parasites apparently free in the plasma, particularly around the time of maximum parasitaemia. Blood smears from one of the groups infected with B. microti in the previous experiment were examined for multiparasitism of the red blood cells and for extracellular {free) parasites. Red cells with two or more parasites present were noted between days 10 and 30 of the infection, doubly infected cells being most frequent and with an increasing proportion o f red cells containing 3--6 parasites a r o u n d peak parasitaemia. At peak parasitaemia there were almost 25% more parasites in the red cells than parasitised red cells, In blood smears taken between days 14 and 21 there were significant numbers of 'free' parasites to be seen which may indicate free parasites to be present in the plasma of the infected mice. DISCUSSION Two major conclusions can be drawn from these experiments. First, the pattern of parasitaemia is
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Maximum % parasitaemia occurred on day 13, dependent on the number of parasitised RBC in the infecting inoculum, Second, 070P is not always an accurate measure of the parasite load in the host. The experiments with 10-fold dilution of B, rnicroti and B. h.vlomysci confirm and extend the results of Overdulve & Antonisse (1970) that the pre-2% period (or pre-patent period) is inversely proportional to the logan of the number of infecting
:.J,p, VnL, 13, 1983
parasitaemias of rodent piroplasms
organisms. We have extended this observation to show that the height of maximum parasitaemia and in the ease of B. hylomysci the ability of the host to survive infection, i.e, the severity of the disease, is related to the infective dose. This suggests that the ability of the host to c o n t r o l the infection is dependent on the length of time that the host has been infected which in turn is a reflection of the size of the infecting dose. There is a~ interesting contrast between the rates of increasing parasitaemia which do not vary with infecting dose in B. microti-infected mice but which decrease with decreasing infective dose in B. hylomysci-infected mice. A possible explanation for this is that the onset of the host immune response is more rapid in the case of B. microti and thereafter the response to rising parasitaemia is equally effective irrespective of the pre-patent period. The observation that higher infecting doses of B. hyiomysci are fatal certainly indicates that the host response is not effective if the acute phase o1' infection occurs soon after infection. An attempt was made to assess the relationship between ~/0P and the actual numbers of parasitised RBC in the blood during infection of these mice, Initially it was assumed that PCV reflected RBC numbers, it was found that the compensated parasitaemia which w'as calculated from the PCV values did :.at closely follow the same pattern as the ~oP. For example, the sharp peak seen in the 0YaP of B. hylomysci was replaced by a plateau for CP lasting about 7 days. At the height of infection it was subsequently found that RBC volume increased to a maximum 7070 above normal values and thus at this time PCV gave an overestimate of RBC numbers and therefore also of para.sitised RBC numbers. It is not known if the increase in mean red cell volume is the result of macrocytic anaemia or distension of the red cells by the parasite. In summary these observations simply serve to emphasise that % P may sometimes give an exaggerated picture of parasite load. In contrast, it was shown that the actual number of intracellular parasites may be as much as 250/0 grealer than the number of parasitised erythrocytes ann in addition, there may be many cell-free parasites at the height of infection. Both of these observations suggest that even the total number of parasitised erythrocytes is not an accurate measure of total parasite load. In an attempt to avoid these difficulties a direct method o f counting the n u m b e r o f parasitised erythrocytes in a known volume of B. boris; B. bigemina- and Plasmodium berghei-infected blood has been described by Parker (1973) but he noted that the method was not successful for the rodent p~roplasma B. rodhainL The method described by Rodwell & Howard (1981) using bisbenzimide may prove to be a better technique for the estimation of the total load of parasitised erythrocytes. There is a further consideration, not investigated
403
here, which has a bearing on these results. Chapman & Ward (1976) showed that the plasma volume of rats is reduced by 25°70 at the time of maximum B, rodhaini parasitaemia, thereby concentrating the RBC. If this is also true for B. microti and B, hylontysci infections then calculations of parasite numbers will be unknowingly exaggerated by onethird. In contrast, in t3. boris infection an early fall in PCV is ascribed to vasodilation and ~ncreased vascular permeability (Wright & Kerr, 1975). This work has shown differences in the course of two rodent babesia infectious which may reflect differences in the nature of the immune response of the host or of the haemopoietic tissue to respond to the changes brought about by the infection. These are areas for further investigation. Acknowledgements--The authors would like to thank the Medical Research Council for financial support of this work, Prof. F. E. G. Cox for the supply of B. microti and Prof. D. H. Molyneux for the supply of B. hytomys¢i.
REFERENCES ABDALLA H. S., HUSSEIN l'l. S, & KR~tEft J. P. 1978. Babesia rodhaini: passive protection of mice with immune serum. Tropemnedizin and Parasitologie 39: 295-306.
BAvoa'r J, M., TIMP£RMANG, ~z MOtVNEUX D. H. 1970. Babesia hylomysci tLsp., a new Babesia from the Congo transmissible to mice. Aanales de la Soci~t~ beige de rnbdecine tropicale 50:301-3 ~7. CrtAPM^r~ W, E, & W^RD P. A, 1976. Changes in C3 melabolism during protozoan infection (Babesia rodhaim) in rats. Journal oflmmunotogy 116:1284-1288. Cox F. E. G. 1976. Increased virulence of trypanosome infections in mice with malaria or piroplasmosis: immunological considerations, In: Biochemistry o f Parasites and Host-Parasite Relationships (Edited by V^~ t~E~ BosscltE H.), pp, 421--426. Elsevier/North !~olland/AcademicPress, Amsterdam. Co~t F, E. G~ & YouN~ A, S. 1969. Acquired immunity to Babesia microti and Babesia rodhaini in mice. Parasitology 59: 257-268. GRAY(3. D. & PtaILLIPSR. S. 1981. Use of sorbitol in the eryopreservation of babesia. Research in Veterinary Science 30: 388-389. GRAYG. D. & PtUtLIPS R. 5. 1983. Suppression of primary and secondary antibody respor~ses and inhibition of antigen priming during 13abesia microti infections in mice. Parasite lmrnunolog3"5:123-134. Mt'I'|ARDYN. 1973. Effects of stimulating erythropoiesis in mice infected with Babesia rodhaini. Annals of Tropical Medicine and Parasitology 6"/: 301-306. MAUor~EV D. F., WaroHr 1. G. & K~'rrEgEit P. J. 1973. Babesia argentina: 1he infeclivhy of immunogenicity of irradiated blood parasites for splenectomised calves. International Journal for Parasitology 3:209-217. NowEt~. F. 1969. The blood picture resulting from Nuttallia ( = Babesia) rodhaini and Nuttallia [ = Babesia) microti infections in rats and mice. Parasitology 59: 991-I004.
404
G. D. Ga,~v and R. S. Pnt~.tt~s
OVERt~ULV£ J. P. & ANTONISSE H. W. 1970. Measurement of the effect of low temperature on protozoa by titration. II. Titration of Babesia rodhaini, using prepatent period or survival time, before and after storage at -760C. Experimental Parasitology 27: 323 -34 I. P^rtKER R. 1973. A direct c o u n t i n g technique for estimating high parasitaemias in infections o f Babesia argentina, Babesia bigemina and Plasmodium bergheL Annals of Tropical Medicine and Parasitology 67: 387-390. PmLLIPS R. S. & WAKELIIqD. 1976. Trichuris muris: effect of concurrent infections with rodent piroplasms on immune expulsion from mice, Experimental Parasitology 39: 95-100. Purtvls A. C. 1977. Immunodepression in Babesia microti infections. Parasitology 75: 197-205. Roa~Rrs J. A. & TaACEY-PATIE P. D. 1975. Babesia rodhaini: a study of the effects of immune serum. International Journal for Parasitology 5:577-581.
t,J.P, rot.. 13. 1983
RODWELt B. J. & HOWARD R. J. I981. A fluorescent method for counting BabeMa, Anaplasma, Plasmodiun: and Trypanosoma following DNA staining with 33258 Hoechst (Bisbenzimide). Annals of Tropical Medicine and Parasitology 75:123-130, Ro~E~s R, J. 1974. Serum opsonins and the passive transfer o f protection in BabeMa rodhaini infections of rats. International Journal for Parasitology 4:197-201. WARHURSTD. C. & FOLWELL R. O. 1968. Measurement of the growth rate of the erythr'oeytic stages of Plasmodium berghei and comparisons of the potency of inocula after various treatments. Annals of Tropical Medicine and Parasitology 62: 349-360, Walt3jtr i. G. & KERR J. D. 1975, Effect of trasylol on packed cell volume and plasma kallikrein activation in acute Babesia argentina infection o f splenectomised calves. Zeitschriftfar Parasitenkunde 46: 189-194,