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Babesia microti and Babesia hylomysci: Spleen and phagocytosis in infected mice
EXPERIMENTAL
PARASITOLOGY
Babesia microti
47, l-12
(1979)
and Babesia hylomysci: Spleen and Phagocytosis in Infected Mice HUSSEINS.
HUSSEIN
Department of Microbiology and Parasitology, Faculty of Veterinary Science, University of Khartoum, P.O. Box 32, Khartoum-North, Sudan (Accepted
for publication
10 August 1978)
HUSSEIN, H. S. 1979. Babe&z microti and Babesia hylomysci: Spleen and phagocytosis in infected mice. Experimental Parasitolugy 47, l-12. The role of the spleen during Babe& microti and B. hylomysci infection was investigated by examining the course of infection in both intact and splenectomized mice. The presence of the spleen was critical during the early stages of infection to control excessive multiplication of either parasite, a role taken over by other lymphoid sites as the infection progressed. Mice splenectomized prior to or within 1 week of B. microti inoculation developed extended infections with some deaths, and others were unable to check their parasitemias. All intact mice, and those splenectomized 1 week after infection with B. microti, recovered completely with subsequent development of sterile immunity. Mice splenectomized prior to or within 1 week of B. hylomysci inoculation succumbed to hyperacute infections: Some of the intact mice, and those splenectomized 12 days after infection, recovered but continued to harbor a low-grade infection with periodical recrudescences. Erythrophagocytosis of infected and uninfected erythrocytes was detected in saline preparations and impression smears of spleen and bone marrow and rarely in blood smears of infected mice. This coincided with anemia, splenomegaly, and relatively high levels of opsonizing antibodies, especially during B. microti infection. The colloidal carbon clearance method was used to investigate the phagocytic activity of the reticuloendothelial system. Carbon clearance rates increased rapidly during both infections, but peak B. hylomysci parasitemia coincided with reticuloendotheIial phagocytic depression and death of the host. Babesia microti stimulated a consistently higher reticuloendothelial phagocytic activity with higher erythrophagocytosis both in the spleen and bone marrow than did B. hylomysci. INDEX DESCRIPTORS: Protozoa, parasitic; Babesia microti; Babe& hylomysci; Mouse; Phagocytosis; Spleen; Bone marrow; Reticuloendothelial system; Colloidal carbon clearance; Opsonin activity; Immunity.
INTRODUCTION
A functional spleen is necessary for the host to control blood-borne protozoa1 infections (Taliaferro 1963) and premunition can easily be broken by splenectomy (Miessner 1931) with subsequent death of the host (Barnett 1965). However, Legg (1935) failed to stimulate recrudescences following splenectomy of cattle with a
latent Babesia argentina infection, and rats and mice infected with Babesia rodhuini may live despite splenectomy (Todorovic et al. 1967; Cox ‘and Young 1969). The role of the spleen in controlling blood protozoa1 infections is probably due to its phagocytic activity (Phillips 1969) or to its ability to form protective ‘antibody (Roberts et al. 1972). This antibody was
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2
HUSSEIN S. HUSSEIN
much less efficient against heterologous parasites, and hence it was concluded that the importance of the spleen was that of a large lymphoid organ monitoring the blood and primed to give a secondary antibody response to antigenic variants as they appeared in the population of parasites. Recently the spleen was reported to have both protective as well as suppressive roles during Plasmodium inui infection in rhesus monkeys ( Wyler et al. 1977). Erythrophagocytosis of normal and infected erythrocytes has been observed in the spleen and bone marrow during B. rodhaini infection and attributed to an autoimmune response (Schroeder et al. 1966). Phagocyti’c hyperactivity of the reticuloendothelial system was also observed during several plasmodial infections (Cox et al. 1963, 1964), although Cantrell et al. (1970) observed that several rats infected with Plasmodium berghei failed to attain such hyperactivity. Macrophages containing infected and uninfected erythrocytes were occasionally seen during routine blood smear examination of mice infected with B. microti and B. hylomysci in the laboratory. ‘Therefore, it was decided to further investigate this phenomenon and to study the role of the spleen and the phagocytic activity of the reticuloendothelial system during these infections. MATERIALS
AXD METHODS
Parasite strains and maintenance. Babesia microti (King’s strain) and B. hylomysci (Antwerp strain) were maintained by periodical syringe passage through mice and as stabilates stored in liquid nitrogen ( -196 C) following the recommendations of Dalgliesh ( 1972). The original strains were obtained respectively from Dr. F. E. G. Cox of London University and Dr. J. M. Bafort when he was at the Liverpool School of Tropical Medicine. Infection and hematological procedures. The mice used were laboratory-bred, male
LACA strain mice, infected with Babesia spp. when 5 to 8 weeks old and weighing 20 to 25 g, each mouse receiving approximately 5 x 10” infected erythrocytes in Alsever’s solution ip. Smears were prepared from tail blood and stained with Leishman’s stain to assess the parasitemia. Recovery from infection is attained when blood smear examination for three ‘consecutive days fails to detect any parasites in case of B. microti or less than 1% parasitemia in B. hylomysci infection. Blood for erythrocyte count and microhematocrit determination was obtained by rupturing the retero-orbital plexus under ether anaesthesia. The packed cell volume (PCV) was determined using a microhematocrit centrifuge and a microhematocrit reader (Hawksley Ltd., London), and the erythrocytes were counted on an improved neubauer hemocytometer (Hawksley Ltd., London). Detection of antibody on erythrocytes. Antibody fixed on erythrocytes was detected using a modification of the method of Ristic (1961). Blood was obtained by ophthalmic puncture and mixed with an equal volume of Alsever’s solution. The erythrocytes were then washed three times in physiological saline. The antibody fixed on the surface of erythrocytes was then detected in a hemolytic system which was set up by adding 0.5 ml of rabbit complement to 0.5 ml of a 2% suspension of erythrocytes in physiological saline in test tubes. The system was then incubated for 4 hr at room temperature (20-24 C). The tubes were then centrifuged at 164g for 5 min. The degree of hemolysis in each tube was then compared with that of a 100% hemolysis using a spectrophotometer (Uvispek H.700, Hilger and Watts, London) at 540-nm wavelength. Control tests were also made using erythrocytes of infected mice plus saline, erythrocytes ‘of infected mice plus heat-inactivated complement, and erythrocytes of normal mice plus complement.
B. microti AND B. hylomysci: Erythrophagocytosis. Evidence of erythrophagocytosis was obtained by examining wet preparations of spleen and bone marrow cells suspended in physiological s.aline. The spleen was teased in cold physiological saline in a petri dish over ice, and the mixture was thoroughly mixed by drawing it up and down in a sterile Pasteur pipet. A drop of the mixture was then placed on a clean slide, covered with a coverslip, and examined under the high dry objective ( x40) of the microscope. A bone marrow plug was forced from the femur by mounting a 25gauge needle in the lower end of the bone after sawing off both ends of the bone. A 2-ml syringe full of physiological saline was then mounted on the needle, and the saline was forced through the bone to push the marrow out ‘of the bone. The marrow plug was then teased, mixed, and similarly examined. Impression smears of the spleen and bone marrow were also prepared, stained with Giemsa’s stain, and examined for phagocytosis . Colloidal carbon clearance. Colloidal carbon (C11/1431, Pelikan Special Biological Ink) was prepared following the procedure of Biozzi et al. (1953). The mice received 1.6 mg of colloidal carbon in gelatin per 10 g body weight in the tail vein. The rate of carbon clearance from the blood was investigated following the technique of Biozzi et al. (1957). Several blood samples were drawn at different time intervals from the retro-orbital plexus following the injection of carbon. The blood was hemolysed in 0.1% Na2C03, and the optical density was read on the spectrophotometer at 540-nm wavelength. The phagocytic index (K) was calculated from the formula:
K = (log G - log G)/(T,
- T,),
where Cl and C, are the concentrations of carbon in the blood at times T1 and T2 after the intravenous injection of colloidal carbon.
3
PHAGOCYTOSIS
Spbnectomy. The mice were sanesthetized with fluothane in an oxygen-fluothane semiclosed system. An incision was made just behind the last rib on the left flank, the spleen was exposed and removed by thermocautery, and the wound was closed by autoclips. The spleen was then measured, weighed, and kept in an ice bath for further use. EXPERIMENTS
AND RESULTS
Role of the Spleen The first experiment followed the course of infection in intact and splenectomized animals. Ten mice were splenectomized 1 week before infection with Babe&a microti and a further 10 before infection with B. hylomysci. Ten intact mice were ‘also inoculated with each parasite. The parasitemia produced by B. microti in splenectomized mice was slightly higher than in intact animals (Table I), However, intact mice survived the infection which became subpatent within 38 days, but five of the splenectomized mice died within 10 days, and the other five continued to show high patent parasitemia for 6 months of examination (mean parasitemia was 25% t 7.8 during the 6 months). Blood subinoculations from the intact mice consistently failed to produce infection in splenectomized recipients. The intact mice were also resistant to ,a challenge infection with approximately lo6 erythrocytes infected with the homologous species of parasite. Blood smears from these mice were examined for 2 weeks following the challenge, and none of them was positive. However, a mild infection (5.3% maximum mean parasitemia k 2.1) which lasted for only 3 days developed following a similar challenge with B. hylomysci. The mice which had been splenectomized prior to B. hylomysci infection developed fulminating infections, and all ‘died within 7 days. A severe infection also developed in the intact mice, but four of the
4
HUSSEIN S. HUSSEIN
10 mice survived and spontaneous recrudescences occurred in them thereafter (Table II). Several severe spontaneous recrudescences occurred in these mice during 6 months of examination. Twelve intact mice which had survived B. hylomysci infection were splenectomized 1 week after recovery, and severe recrudescences (40-60s maximum mean parasitemia during recrudescence) developed in all of them. Periodical spontaneous recrudesTABLE
Parasitemia
(days) 2 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
Intact
mice
2.2 f 5.6 +z 50.1 f 68.2 k 73.6 rt 56.2 f 40.2 f 18.3 + 10.2 f 11.7 + 8.3 zt 9.2 f 8.1 f Fi.2 f 4.3 f 3.2 f 2.1 * 1.1 ?t 0 0 0 0 0 0 0 0 0 0 0 0
0.4 1.3 3.8 8.6 8.2 6.7 3.8 2.5 3.7 4.8 2.1 1.2 3.6 2.8 2.4 1.2 0.8 0.2
Duration of infection (days) 2 3 4 6 8 10 12 14 16 1X 20 22 24 26 28 SO 3‘2 34 36 38 40 42 44 46 50 60
mice
+ 0.8 f 6.5 f 83 + 7.4c zk 6.6d f 7.5 f 2.3 f- 6.5 zk 8.:1 zk 3.6 f 2.1 f 7.4 z!z 5.3 f 7.5 f 3.G It 2.1 f 4.6 It 2.1 * 3.7 + 8.3 f 6.7 h 7.7 jz 2.1 xt 3.8 f 7.G + 4.6 zt 3.2 & 7.8 + 5.6 f 2.3
a Mean of 10 mice f- RI>. PvIean parasitcmia for 6 months in splenectomizcd mice is 25.77 + 9.8c1,. 6 One mouse died. c Three mice died. d One mouse died.
in Intact
Parasitemia Intact
mice
(‘,s)
Splencctomized
mice
-
(x)”
3.2 31.4 60.1 79.7 78.4 63.5 50.1 44.8 42.6 63.7 58.2 55.6 52.1 53.2 52.1 50.1 4X.5 is.2 40.1 43.2 45.1 41.2 39.1 40.2 39.3 36.3 35.3 37.5 33.2 29.5
Infection Mice
__
in Intact
Splenectomized
II
and Splenectomized
I
The Course of Babesia microti Infection and Splenectomizcd Mice Duration of infection
TABLE
Th e c ourse of Rabesia hylom~xi
2.3 f 0.7 6.4 f 1.6 10.4 + 2.5 24.5 + 6.8 35.4 f 7.3 47.6 f 8.S 5S.3 f 8.7n 81.2 f 10.7* 40.3 f 7.9 21.4 zt 3.7 17.5 f 6.7 2.3 f 0.8 2.5 * 0.2 2.2 f 03 0.3 * 0.4 2.6 z!z 1.2 1.3 3.5 * 1.7 5.2 i 10.1 * 3.2 20.2 zt 6.5 30.3 zt 5.7 40.1 * .‘,.A 58.7 zk 7.7 46.5 + 8.9 28.6 z!z 7.5 17.5 zk :3.G 7.3 zt 2.1 1.2 2.3 *
7.2 36.4 65.6 79.5 9::.4
f f zk zt f -d
2.3 6.4 16.4* 12.9b 103
-
--
” Two miw died. h Three mice died. c One mouse died. 11-, a11mice died.
cences were also observed in these mice during 6 months of examination. When a similar number of mice that recovered from B. microti infection were splenectomized during the same period of time, mild recrudescences (5-10s maximum mean parasitemia during recrudescence) developed for only 5 days, and thereafter, the mice remained free of infection during: 6 months of examination. Blood obtained from these mice failed to produce infection in splenectomized recipients, and they were resistant to challenge with the homologous species
B. microti
AND
B.
hylomysci:
of parasite but developed a short-lived (3 days), low-grade infection (5% maximum mean parasitemia) when challenged with B. hybmysci. Another experiment tested the effect of splenectomy on the developing parasitemia. Twenty-four mice were infected with B. microti, and 24 were infected with B. hylomysci. Three from each group were splenectomized at 4-day intervals, and blood smears were prepared from all animals every other day to assess the parasitemia. The three mice infected with B. microti and splenedomized within the first 4 days of infection developed an extended infection similar to that in mice splenectomized prior to the infection (Table I). However, splenectomy after the eighth day of infection had no effe’ct on the course of B. mic&i infection. Splenectomy within 8 days of B. hylomysci infection resulted in the death of infected animals (from fulminating infections) a few days after the operation, but splenectomy at 12 or more days did not have any effect. Erythrophagocytosis Twenty-five mice were infected with B. microti, and 25 were infected with B. hylomysci. Parasitemia, PCV, and erythrocyte counts were determined daily, and the three mice with the highest parasitemia from the B. hylomysci-infected group were killed every other day, while the corresponding three mice from the B. microti group were killed at 3-day intervals. The spleen was removed from every animal, measured, and weighed. The marrow plug was removed from the right femur ‘of every animal, and saline preparations of spleen and bone marrow cells were examined for erythrophagocytosis. A test employing hemolytic systems for detecting antibodies on the surface of erythrocytes (Ristic 1961) was also ‘carried out for each animal,
5
PHAGOCYTOSIS
Erythrophagocytosis was not noticed in any of the saline preparations of spleen and bone marrow cells from normal mice, nor was hemolysis detected in any of the hemolytic systems using washed erythrocytes in the different control regimes (Tables III and IV). Erythrophagocytosis was, however, detected in spleen and bone marrow saline preparations from infected animals, and the highest level of phagocytosis coincided with relatively high levels of antibodies on the erythrocytes and concurrently with enlarged spleens, low PCV, and low erythrocyte counts (Tables III ‘and IV). Impression smears of spleen and bone marrow showed that both infected and noninfected erythrocytes were equally phagocyto,sed. Erythrophagocytosis was not observed after 12 and 30 days of B. hylomysci and B. microti infections, respectively (Tables III and IV). Determination
of the Phugocytic
Index (K)
Thirty mice were infected with B. microti and 30 with B. hylomysci for the assessment of the phagocytic index (K) during each infection. The value of K rose sharply during early B. hylomysci infection, reaching a peak of 0.493 * 0.213 by Day 4 before dropping to subnormal levels (0.007 * 0.002) during peak parasitemia (Fig. 1). During this time most of the mice were very sick and many died. The value of K then rose progressively in surviving animals and was within the normal range by Day 10 ‘of the infection (Fig. 1). Similarly the value of K rose steadily during early B. microti infection, reaching a peak of 0.693 2 0.302 just bef’ore peak parasitemia; a high level was maintained throughout the rest of the infection, and normal levels were reached with recovery (Fig. 2).
HUSSEIN
S. HUSSEIN
.oo ? 2 E :
DISCUSSION
Irvin and Brocklesby (1969) observed continuously high Babes&z microti parasitemias for long periods in both intact and (1977a) splenectomized mice. Hussein showed, however, that intact mice lost the parasite completely as early as 3 weeks after recovery from a primary B. microti infection, and sterile immunity supervened. Continuous high parasitemias (higher than those observed by Irvin and Brocklesby 1969) during B. microti infection were, however, ‘observed in mice splenectomized prior to the infection (Table I ) . These parasitemias were not much higher than those in intact ones, a fact which might be due to the reduction of reticulocytes following splenectomy (Singer 1954) as B. microti is known to favor these cells (Nowell 1969; Hussein 1973). Similar findings were observed during Plasmodium berghei infec-
3 r’ n
0
0.001)1 0
1 2
’ 4
’ 6
Duration
-1c10 q : : z ; .
-141 / \
H
\
-1. 0
I
I
I
1
123456789~ Duration
of
I
I
I
Infection
I
I
(Days)
FIG. 1. Variations in the phagocytic index (K) during Babe& hylomysci infection in mice. Phagocytic
index,
l -
centage), l ----
l l
,
(K);
parasitemia
70 (per-
\\ \\
Of I)I.,..,. rlo I : O.Ol-:’ sL / 0’ ! E ” : E 0” : i :; 1 1 1 6 10 12
.O
1 15
0.1 20
of Infectlonbyd
FIG. 2. Variations in the phagocytic index (K) during Babe&z microti infection in mice. Phagocytic index, 0 0 (K); parasitemin $3 (percentage), 0 ---0.
tion in mice (Singer 1954). B. hylomysci which favors mature erythrocytes (Hussein 1976), developed a hyperacute infection in splenectomized mice which died within few days (Table II). Splenectomy had little effect when performed after either infection had become well-established. This might indicate that the spleen is essential only during the early stages of infection, possibly to check excessive multiplication of either parasite through phagocytosis and the elaboration of protective antibodies as observed by Todorovic et al. (1967) and Roberts et al. ( 1972) during similar infections, Recently Gravely et al. (1976) observed dramatic decreases in B cells in spleens of rats infected with P. berghei early in the infection, but their numbers returned to normal as the infection progressed. These cells are possibly mobilized and transformed into
2.9 39.0 73.0 76.9 58.3 19.1 6.3 1.1
0 zt zt * zk f f h f
1.2 6.3 7.5 5.4 11.2 9.6 3.2 0.9
Parasitemia (% f SD)
1.32 2.93 2.78 1.73 1.32 1.34 1.78 2.1 2.1
51 46 36 21 21 25 28 38 45
0 zk ++ +++ +-t+ ++ ++ ++ 0
xt f f f f f f f f
7.00 6.63 4.75 2.93 2.09 2.87 3.37 4.48 6.50
0 0 f + ++ + + + 0
Bone marrow
Erythrophagocytosisb Spleen
PCV (%I
RBC count (X lOB/ mm3) * SD
76.2 82.8 343.3 308.3 504.4 730.0 663.3 783.3 216.6
i f f f f f zk f f
12.3 24.3 80.2 90.3 125.6 225.4 234.2 215.2 78.4
Weight (mg f SD)
Spleen
15 15 26 26 33 28 32 39 20
x x x X X X x X x
Size (mm)
5 5 9 8 9 12 11 1S 8 7.8 56.2 100 100 100 100 64.8
0 f 4.5 f 11.1 f 0 f 0 f 0 f 0 f 8.1 0
RBC of infected mice + complement 0 0 0 0 0 0 0 0 0
RBC of infected mice + saline
erythro-
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
containing
RBC of normal mice + complement
SD)
RBC of infected mice + heatinactivated complement
lysisc (% f
Fixed on
Immune
between Anemia, Parasitemia, Erythrophagocytosis, and Levels of Antibody Erythrocyte Surface during Babesiu microti Infection in Micea
III
a All figures are arithmetic means f SD. b Erythrophagocytosis : 0, no phagocytes containing erythrocytes; f, l-5% phagocytes containing erythrocytes; +, lO-2O% phagocytes cytes ; + +, 30-40’% phagocytes containing erythrocytes, and + + +, 50% and more phagocytes containing erythrocytes. c Immune lysis : The level of antibody fixed on the surface of erythrocytes is proportional to the degree of lysis.
0 3 6 9 12 15 18 21 31
Duration of infection (day.9
The Relationship
TABLE
2 0 cn z
1
3 2 C*
z
g Pj
$’ 2c. 9
3
.Y
2 g z
0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 T'TI 0 O'ZI S'L F.9
0 T X'ZI =F OOT =F 1'9.i r G-91 =F Z'S 0 ~113UI
-alduro:, + a:,rur
L x 9 x 8 x 9x 9 x 9 X Px
sz PZ PZ IZ AL 61 zt
1'01
L'S X'P!) 2'OL C'h'P TLC %.';I
=l= O'OLZ =F O'WZ =F 0'0% =F 0'01: 1 =F 0.901 =F y'pg > . =F 0'09
0 Zt + 0 0 0
B. microti AND B. hylomysci: antibody-producing cells early in the infection upon stimulation by parasite antigens. Moreover, as the infection progresses, the spleen is known to be more concerned with erythropoiesis than with immunological functions (Dokow et al. 1974; Poels 1977). The spleen was also ‘observed to exert a protective role during early stages of quartan malaria (Plasmodium inui infection) in rhesus monkeys, but had a suppressive role later in the infection, and unlike splenectomized animals, intact ones were unable to achieve self-cure (Wyler et al. 1977). During chronic stages of rodent babesiosis ( B. microti infection), however, the opposite occurred; intact mice were able to achieve self-cure, but splenectomized ones were not (Table I). Splenectomy performed within 1 week of recovery from B. microti infection in the present study resulted in recrudescences which were not as severe as those observed by Miessner (1931) and Barnett (1965) following splenectomy of cattle recovered from B. bigemina infection. However, splenectomy of mice 3 or 4 weeks following recovery from B. microti infection does not always result in recrudescences as sterile immunity may supervene ( Hussein 1977a). The spleen seems to be essential for the initiation of sterile immunity which fails to develop in mice splenectomized prior to and within 1 week of B. microti infection during the present study. Moreover, splenectomy after the infection has become well established does not seem to affect the development of sterile immunity, possibly because the spleen function might have been taken over by other organs (Taliaferro 1956) or because the spleen cells might have migrated to other tissues follmowing primary immunization (Cannon and Wissler 1967). Thus the presence of the spleen appears to be critical in the early response to B. microti infection, but other sites may supersede it in the maintenance of immunity over longer periods. The development of sterile immunity to B. microti
PHAGOCYTOSIS
9
infection is in accordance with the findings of Cox and Young (1969) that immunity to babesiosis in mice, unlike that against cattle babesiosis, is complete in the rodent host. However, the immunity to B. hylomysci infection in mice seemed to be an exception and similar to that of bovine babesiosis ( Hussein 1977a). The possible roles of the spleen in the immunity to the related malaria parasites have been discussed by Brown ( 1969) and Brown and Phillips ( 1974), who suggested that the key role of the spleen in rodent malaria is to develop immunity to new antigenie variants appearing in the parasite population. This does not seem to be the case with either B. microti infection where solid sterile immunity eliminates the parasite completely from the host (Cox and Young 1969; Hussein 1977a), or with B. hylomysci infection where severe spontaneous recrudescences are common (Table II and Hussein 1977a) and the relapsing parasitemia is likely to be accompanied by antigenic variations which the spleen was not able to control. Erythrophagocytosis was common during both infections but was more pronounced during B. microti infection and coincided with severe anemia, splenomegaly, and relatively high levels of antibodies on the surface of erythrocytes (Tables III and IV). Similar observations were made by Schroeder et al. (1966) during B. rodhaini infection in the rat and these workers ascribed the condition to an autoimmune response. The demonstration of antibodies on the surface of the erythrocytes and their relationship to erythrophagocytosis might indicate that the anemia during mouse babesiosis might be, at least in part, due to an autoimmune response as both infected and noninfected erythrocytes were observed to be equally phagocytosed during the present study. Noninfected erythrocytes might have been injured during the course of infection or coated with antigen-antibody
10
HUSSEIN
complexes and hence phagocytosed. Similar observations were made by Ristic (1961) during bovine anaplasmosis. Moreover, eirculating macrophages and neutrophils as well as Kupffer cells of the liver have been observed to be active in erythrophagocytosis and consequently involved in the anemia associated with babesiosis (Holbrook et al. 1968; Simpson 1970, 1974). Erythrophagocytosis of infected and uninfected erythrocytes was once postulated to explain the excess anemia observed during malaria (Zuckerman 1966; Cox et al. 1966). However, Chow and Kreier (1972) and Hamburger and Kreier (1976) were unable to demonstrate significant erythrophagocytosis during P. berghei infection using an in vitro system, even in the presence of immune serum. Phagocytosis of free Babesia spp, parasites and its comparison to erythrophagocytosis are yet to be thoroughly investigated. However, Roberts and Tracey-Patte (1974) observed that the parasite and its red cell are removed simultaneously and that it is not necessary for the parasite to be extracellular in order to be phagocytosed. This was later confirmed by Abdalla et al. (1978) who indicated that protective antibodies can penetrate the infected erythrocyte to interact with B. rodhaini .and bring about erythrophagocytosis. In support of the findings of Cox et al. (1963, 1964), the phagocytic activity of the reticuloendotbelial system increased during both infections, but peak B. hylomysci parasitemia was accompanied by reticuloendothelial phagocytic depression ( Fig. 1)) with subsequent death of the host. This might indicate that the death of the host during B. hylomysci infection may be, at Ieast partly, due to overwhelming of the reticuloendothelial system by excessive multiplication of the parasite. Cantrell et al. (1970) explained the loss of phagocytic hyperactivity during rodent malaria as being due to the fact that the parasites act both as a stimulus and a load
S. HUSSEIN
to the phagocytic system, and owing to their potential for growth, their role as a load may exceed their roIe as a stimulus and hence bring about a nonhyperactive state. They also suspected that a nonhyperactive state in infected rats might be due to the exhaustion of some substance essential for phagocytosis. Such a substance was isolated, purified, and characterized as a-2globuhn by Blumenstock et al. (1976). These authors also suggested that a deficiency of this factor occurring during tumor growth, trauma, or shock would precipitate a state of reticuIoendothelia1 dysfunction. Therefore, the reticuloendothelial depression observed during B. hylomysci infection in mice could be attributed to a possible deficiency in the a-2-globulin which might have occurred during the state of shock observed in infected mice by Hussein ( 1977b). A state of shock never occurred during B. microti infection in mice (Hussein 1973). This might explain the absence of reticuloendothelial phagocytic depression during this infection, as there might be no deficiency in the a-2-globulin factor due to the absence of a state of shock. Further work is needed to investigate the variations in the level of the a-2-globulin factor during either infection. The loss of phagocytic hyperactivity observed by Cantrell et al. (1970) did not adversely affect the ability of rats to recover from P. berghei infection, and the authors suggested that recovery was due to acquired immunity that is effective without phagocytic hyperactivity. This does not seem to be the case with B. hylomysci infection in the mouse, a host known to be less able to mount an effective immunological response to bIood protozoa1 infections than the rat (Gravely et al. 1976; Hamburger and Kreier 1976). Later Cantrell and Elko (1976) observed that the ability to maintain phagocytic hyperactivity toward colloidal carbon during P. berghei infection varied within individuals of the same strain as well as among
B. microti AND B. hylomysci: different rat strains. This does not seem to be the case with B. hylomysci infection in LACA strain mice, where a11infected animals showed reticuloendothelial phagocytic depression. However, variations within the different strains of mice remains to be investigated. The multiplication rate of B. microti is slower than that of B. hylomysci (Hussein 1973)) and hence the reticuloendothelial system is quite capable of dealing with this parasite. This is exemplified by the fact that ph,agocytic activity never fell in mice infected with B. microti while it did in mice with B. hylomysci infection (Figs. 1 and 2). Moreover, B. microti seems to be a more efficient stimulant to the reticuloendothelial system than B. hylomysci. This is indicated by the consistently superior phagocytic index (Figs. 1 and 2) and by the higher rate of erythrophagocytosis both in the spleen and bone marrow (Tables III and IV). Such increased phagocytosis may be initially protective, so that the development and mobilization of other immunological defences of the host may be permitted. This might well be the reason that, unlike intact mice with B. hylomysci and B. rodhaini infections, intact mice usually recover from Babesia microti infection. ACKNOWLEDGMENTS The author wishes to thank Professor M. J. Clarkson of Liverpool University for advice and Professor J. P. Kreier, of the Ohio State University for valuable discussions and constructive criticism of the manuscript.
REFERENCES H. S., HUSSEIN, H. S., AND -IER, J. P. 1978. Babesia rodhaini: Passive protection of mice with immune serum. Tropenmedizin und Parasitologic 29. BARNETT, S. F. 1965. The chemotherapy of Babesia bigemina infections in cattle. Research in Veterinary Science 6, 397-415. BIOZZI, G., BENACERRAF, B., AND HALPERN, B. N. 1953. Quantitative study of the granulopectic activity of the reticuloendothelial system. II. A study of the kinetics of the granulopectic activABDALLA,
PHAGOCYTOSIS
11
ity of R.E.S. in relation to the dose of carbon injected. Relationship between the weight of organs and their activity. British Journal of Experimental Pathology 34,441457. Bxozzr, G., HALPERN, B. N., BENACERRAF, B., AND STIFFLE, C. 1957. Phagocytic activity of the reticuloendothelial system in experimental infections. In “Physiology of the Reticuloendothelial System.” (Edited under the direction of B. N. Halpern by B. Benacerraf and J. D. Delafresnaye), pp. 204-225. Springfield, Illinois. BLUMENSTOCK, F., SABA, T. M., WEBBER, P., AND CHO, E. 1976. Purification and biochemical characterization of a macrophage stimulating alphae-globulin opsonic protein. Journal of the Reticuloendothelial Society 19, 157-172. BROWN, I. N. 1969. Immunological aspects of malaria infection. Aduances in Immunology 11, 267-349. BROWN, I. N., AND PHILLIPS, R. S. 1974. Immunity to Plasmodium berghei in rats: Passive serum transfer and the role of the spleen. Infection aml Immunity 10, 1213-1218. CANNON, D. C., AND WISSLER, R. W. 1967. Spleen cell migration in immune response of the rat. Archives of Pathology 84, 109-118. CANTRELL, W., ELKO, E. E., AND HOPFF, B. M. 1970. Plasmodium berghei: Phagocytic hyperactivity of infected rats. Experimental Parasitology 28,291-297. CANTRELL, W., AND ELKO, E. E. 1976. Plasmodium berghei: Phagocytic activity in two strains of rats. Experimental Parasitology 40, 281-285. CHOW, J. S., AND KREIER, J. P. 1972. Pkwnodium berghei: Adherence and phagocytosis by rat macrophages. Experimental Parasitology 31, 1318. Cox, F. E. G., BILBEY, D. L. J., AND NICOL, T. 1964. Reticuloendothelial activity in mice infected with Plusmodium uinckei. Journal of PTOtozoobgy 11,229-230. Cox, F. E. G., NICOL, T., AND BILBEY, D. L. J. 1963. Reticuloendothelial activity in Haemamoeba (Plasmodium) gallinucea infections. ]ournal of Protozoology 10, 107-109. Cox, F. E. G., AND YOUNG, A. S. 1969. Acquired immunity to Babesia microti and Babesia rodhaini in mice. ParasitoZogy 59, 257-268. Cox, H. W., SCHROEDER, W. F., AND RISTIC, M. 1966. Hemagglutination and erythrophagocytosis associated with Plasmodium berghei infection of rats. Journal of ProtozooZogy 13, 327-332. DALGLIESH, R. J. 1972. Theoretical and practical aspects of freezing parasitic protozoa. Australian Veterinary Journal 48, 233-239. D~KOW, S., GOLENSER, J., GREENBLATT, C. L., AND SPIRA, D. T. 1974. Changes in rat splenocyte
12
HUSSEIN
population during infection with Plasmodium berghei. Journal of Protozoology 21, 463. GRAVELY, S. M., HAMBURGER, J., AND KREIER, J. P. 1976. T and B cell population changes in young and adult rats infected with Plasmodium berghei. Infection and Immunity 14, 178-183. HAMBURGER, J., AND KREIER, J. P. 1976. Plasmodium berghei: Use of free blood stage parasites to demonstrate protective humoral activity in the serum of recovered rats. Experimental Parasitology 40, 158-169. HOLBROOK, A. A., JOHNSON, A. J., AND MADDEN, P. A. 1968. Equine piroplasmosis: Intra-erythrocytic development of Babe& cab& (Nuttall) and Babesia equi (Laveran). American Journal of Veterinary Research 29, 297-303. HUSSEIN, H. S. 1973. “Studies on Rodent Babesiosis Caused by Babesia hylomysci Bafort, Timperman and Molyneux, 1970 and Babesia microti Ph.D. Thesis, University of (Franca, 1910).” Liverpool. HUSSEIN, H. S. 1976. Babesia hylomysci in mice: Preference for erythrocytes of a particular age group and pathogenesis of the anemia. Zeits&rift fur Purasitenkunde 50, 103-108. HUSSEIN, H. S. 1977a. The nature of immunity against Babesia hylomysci and B. microti infection in mice. Annals of Tropical Medicine and Parasitology 71, 249-253. HUSSEIN, H. S. 1977b. The pathology of Babesia hylomysci infection in mice. I. Clinical signs and liver lesions. Journal of Comparatioe Pathology 87, 161-167. IRVIN, A. D., AND BROCKLESBY, D. W. 1969. Continuous high parasitemia in mice infected with Babesia (Nuttallia) microti. Journal of Parasitology 55, 1190. LEGG, J, 1935. The occurrence of bovine babesiellosis in Northern Australia. C.S.I.R.O. Pamphlet No. 56, l-48. MLESSNER, H. 1931. Piroplasmosen und Splenektomie. Archiv fiir Wissenschaftliche und practische Tierheilkunde 63, 68-90. NOWELL, F. 1969. The blood picture resulting from Nuttallia (=Babesia) rodhaini and Nuttallia (=Babesia) microti infections in rats and mice. Parasitology 61, 425433. PHILLIPS, R. S. 1969. The role of the spleen in relation to natural and acquired immunity to infections of Babes&r rodhaini in the rat. Parasitology 59, 637-648.
S. HUSSEIN
POELS, L. G. 1977. Plasmodium berghei: Polyribosome profiles in the spleens of infected mice. Experimental Parasitology 41, 83-88. Rrs,rrc, M. 1961. Studies in anaplasmosis. III. An autoantibody and symptomatic macrocytic anemia. American Journal of Veterinary Research 22, 871-876. ROBERTS, J. A., KERR, J. D., AND TRACEY-PATTE, P. 1972. Functions of the spleen in controlling infections of Babesia rodhaini in mice. International Journal for Parasitology 2, 217-226. ROBERTS, J. A., AND TRACEY-PATIF, P. 1974. Babesia rodhaini: Effects of the immune system in mice. Experimental Parasitology 35, 119-124. SCHROEDER, W. F., Cox, H. W., AND RISTIC, M. 1966. Anemia, parasitemia, erythrophagocytosis and hemagglutinins in Babesia rodhaini infection. Annals of Tropical Medicine and Parasitology 60, 31-38. SIMPSON, C. F. 1970. Electron microscopic comparison of Babesia spp. and hepatic changes in ponies and mice. American Journal of Veterinary Research 31, 1763-1768. SIMPSON, C. F. 1974. Phagocytosis of Babesia canis by neutrophils in the peripheral circulation. American Journal of Veterinary Research 35, 701-704. SINGER, I. 1954. The effect of splenectomy or phenylhydrazine on infections with Plasmodium berghei in the white mouse. Journal of Infectious Diseases 94, 159-163. TALIAFEMO, W. H. 1956. Functions of the spleen in immunity. American Journal of Tropical Medicine and Hygiene 5, 391410. TALIAFERRO, W. H. 1963. Cellular and humoral factors in immunity to protozoa. In “Immunity to Protozoa” (P. C. C. Garnham, A. E. Pierce, and I. Roitt, eds.), pp. 22-38. Blackwell, Oxford. TODOROVIC, R., FERHIS, D., AND RISTIC, M. 1967. Roles of the spleen in acute plasmodial and babesial infections in rats. Experimental Parasitology 21, 354-372. WYLER, D. J., MILLER, L. H., AND SCHMIDT, L. H. 1977. Spleen function in quartan malaria (due to Plasmodium inui): Evidence for both protective and suppressive roles in host defense. Journal of Infectious Diseases 135, 8693. ZUCKERMAN, A. 1966. Recent studies in factors involved in malarial anemia. Military Medicine 131 (Special Issue), 1201-1216.
PARASITOLOGY
Babesia microti
47, l-12
(1979)
and Babesia hylomysci: Spleen and Phagocytosis in Infected Mice HUSSEINS.
HUSSEIN
Department of Microbiology and Parasitology, Faculty of Veterinary Science, University of Khartoum, P.O. Box 32, Khartoum-North, Sudan (Accepted
for publication
10 August 1978)
HUSSEIN, H. S. 1979. Babe&z microti and Babesia hylomysci: Spleen and phagocytosis in infected mice. Experimental Parasitolugy 47, l-12. The role of the spleen during Babe& microti and B. hylomysci infection was investigated by examining the course of infection in both intact and splenectomized mice. The presence of the spleen was critical during the early stages of infection to control excessive multiplication of either parasite, a role taken over by other lymphoid sites as the infection progressed. Mice splenectomized prior to or within 1 week of B. microti inoculation developed extended infections with some deaths, and others were unable to check their parasitemias. All intact mice, and those splenectomized 1 week after infection with B. microti, recovered completely with subsequent development of sterile immunity. Mice splenectomized prior to or within 1 week of B. hylomysci inoculation succumbed to hyperacute infections: Some of the intact mice, and those splenectomized 12 days after infection, recovered but continued to harbor a low-grade infection with periodical recrudescences. Erythrophagocytosis of infected and uninfected erythrocytes was detected in saline preparations and impression smears of spleen and bone marrow and rarely in blood smears of infected mice. This coincided with anemia, splenomegaly, and relatively high levels of opsonizing antibodies, especially during B. microti infection. The colloidal carbon clearance method was used to investigate the phagocytic activity of the reticuloendothelial system. Carbon clearance rates increased rapidly during both infections, but peak B. hylomysci parasitemia coincided with reticuloendotheIial phagocytic depression and death of the host. Babesia microti stimulated a consistently higher reticuloendothelial phagocytic activity with higher erythrophagocytosis both in the spleen and bone marrow than did B. hylomysci. INDEX DESCRIPTORS: Protozoa, parasitic; Babesia microti; Babe& hylomysci; Mouse; Phagocytosis; Spleen; Bone marrow; Reticuloendothelial system; Colloidal carbon clearance; Opsonin activity; Immunity.
INTRODUCTION
A functional spleen is necessary for the host to control blood-borne protozoa1 infections (Taliaferro 1963) and premunition can easily be broken by splenectomy (Miessner 1931) with subsequent death of the host (Barnett 1965). However, Legg (1935) failed to stimulate recrudescences following splenectomy of cattle with a
latent Babesia argentina infection, and rats and mice infected with Babesia rodhuini may live despite splenectomy (Todorovic et al. 1967; Cox ‘and Young 1969). The role of the spleen in controlling blood protozoa1 infections is probably due to its phagocytic activity (Phillips 1969) or to its ability to form protective ‘antibody (Roberts et al. 1972). This antibody was
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2
HUSSEIN S. HUSSEIN
much less efficient against heterologous parasites, and hence it was concluded that the importance of the spleen was that of a large lymphoid organ monitoring the blood and primed to give a secondary antibody response to antigenic variants as they appeared in the population of parasites. Recently the spleen was reported to have both protective as well as suppressive roles during Plasmodium inui infection in rhesus monkeys ( Wyler et al. 1977). Erythrophagocytosis of normal and infected erythrocytes has been observed in the spleen and bone marrow during B. rodhaini infection and attributed to an autoimmune response (Schroeder et al. 1966). Phagocyti’c hyperactivity of the reticuloendothelial system was also observed during several plasmodial infections (Cox et al. 1963, 1964), although Cantrell et al. (1970) observed that several rats infected with Plasmodium berghei failed to attain such hyperactivity. Macrophages containing infected and uninfected erythrocytes were occasionally seen during routine blood smear examination of mice infected with B. microti and B. hylomysci in the laboratory. ‘Therefore, it was decided to further investigate this phenomenon and to study the role of the spleen and the phagocytic activity of the reticuloendothelial system during these infections. MATERIALS
AXD METHODS
Parasite strains and maintenance. Babesia microti (King’s strain) and B. hylomysci (Antwerp strain) were maintained by periodical syringe passage through mice and as stabilates stored in liquid nitrogen ( -196 C) following the recommendations of Dalgliesh ( 1972). The original strains were obtained respectively from Dr. F. E. G. Cox of London University and Dr. J. M. Bafort when he was at the Liverpool School of Tropical Medicine. Infection and hematological procedures. The mice used were laboratory-bred, male
LACA strain mice, infected with Babesia spp. when 5 to 8 weeks old and weighing 20 to 25 g, each mouse receiving approximately 5 x 10” infected erythrocytes in Alsever’s solution ip. Smears were prepared from tail blood and stained with Leishman’s stain to assess the parasitemia. Recovery from infection is attained when blood smear examination for three ‘consecutive days fails to detect any parasites in case of B. microti or less than 1% parasitemia in B. hylomysci infection. Blood for erythrocyte count and microhematocrit determination was obtained by rupturing the retero-orbital plexus under ether anaesthesia. The packed cell volume (PCV) was determined using a microhematocrit centrifuge and a microhematocrit reader (Hawksley Ltd., London), and the erythrocytes were counted on an improved neubauer hemocytometer (Hawksley Ltd., London). Detection of antibody on erythrocytes. Antibody fixed on erythrocytes was detected using a modification of the method of Ristic (1961). Blood was obtained by ophthalmic puncture and mixed with an equal volume of Alsever’s solution. The erythrocytes were then washed three times in physiological saline. The antibody fixed on the surface of erythrocytes was then detected in a hemolytic system which was set up by adding 0.5 ml of rabbit complement to 0.5 ml of a 2% suspension of erythrocytes in physiological saline in test tubes. The system was then incubated for 4 hr at room temperature (20-24 C). The tubes were then centrifuged at 164g for 5 min. The degree of hemolysis in each tube was then compared with that of a 100% hemolysis using a spectrophotometer (Uvispek H.700, Hilger and Watts, London) at 540-nm wavelength. Control tests were also made using erythrocytes of infected mice plus saline, erythrocytes ‘of infected mice plus heat-inactivated complement, and erythrocytes of normal mice plus complement.
B. microti AND B. hylomysci: Erythrophagocytosis. Evidence of erythrophagocytosis was obtained by examining wet preparations of spleen and bone marrow cells suspended in physiological s.aline. The spleen was teased in cold physiological saline in a petri dish over ice, and the mixture was thoroughly mixed by drawing it up and down in a sterile Pasteur pipet. A drop of the mixture was then placed on a clean slide, covered with a coverslip, and examined under the high dry objective ( x40) of the microscope. A bone marrow plug was forced from the femur by mounting a 25gauge needle in the lower end of the bone after sawing off both ends of the bone. A 2-ml syringe full of physiological saline was then mounted on the needle, and the saline was forced through the bone to push the marrow out ‘of the bone. The marrow plug was then teased, mixed, and similarly examined. Impression smears of the spleen and bone marrow were also prepared, stained with Giemsa’s stain, and examined for phagocytosis . Colloidal carbon clearance. Colloidal carbon (C11/1431, Pelikan Special Biological Ink) was prepared following the procedure of Biozzi et al. (1953). The mice received 1.6 mg of colloidal carbon in gelatin per 10 g body weight in the tail vein. The rate of carbon clearance from the blood was investigated following the technique of Biozzi et al. (1957). Several blood samples were drawn at different time intervals from the retro-orbital plexus following the injection of carbon. The blood was hemolysed in 0.1% Na2C03, and the optical density was read on the spectrophotometer at 540-nm wavelength. The phagocytic index (K) was calculated from the formula:
K = (log G - log G)/(T,
- T,),
where Cl and C, are the concentrations of carbon in the blood at times T1 and T2 after the intravenous injection of colloidal carbon.
3
PHAGOCYTOSIS
Spbnectomy. The mice were sanesthetized with fluothane in an oxygen-fluothane semiclosed system. An incision was made just behind the last rib on the left flank, the spleen was exposed and removed by thermocautery, and the wound was closed by autoclips. The spleen was then measured, weighed, and kept in an ice bath for further use. EXPERIMENTS
AND RESULTS
Role of the Spleen The first experiment followed the course of infection in intact and splenectomized animals. Ten mice were splenectomized 1 week before infection with Babe&a microti and a further 10 before infection with B. hylomysci. Ten intact mice were ‘also inoculated with each parasite. The parasitemia produced by B. microti in splenectomized mice was slightly higher than in intact animals (Table I), However, intact mice survived the infection which became subpatent within 38 days, but five of the splenectomized mice died within 10 days, and the other five continued to show high patent parasitemia for 6 months of examination (mean parasitemia was 25% t 7.8 during the 6 months). Blood subinoculations from the intact mice consistently failed to produce infection in splenectomized recipients. The intact mice were also resistant to ,a challenge infection with approximately lo6 erythrocytes infected with the homologous species of parasite. Blood smears from these mice were examined for 2 weeks following the challenge, and none of them was positive. However, a mild infection (5.3% maximum mean parasitemia k 2.1) which lasted for only 3 days developed following a similar challenge with B. hylomysci. The mice which had been splenectomized prior to B. hylomysci infection developed fulminating infections, and all ‘died within 7 days. A severe infection also developed in the intact mice, but four of the
4
HUSSEIN S. HUSSEIN
10 mice survived and spontaneous recrudescences occurred in them thereafter (Table II). Several severe spontaneous recrudescences occurred in these mice during 6 months of examination. Twelve intact mice which had survived B. hylomysci infection were splenectomized 1 week after recovery, and severe recrudescences (40-60s maximum mean parasitemia during recrudescence) developed in all of them. Periodical spontaneous recrudesTABLE
Parasitemia
(days) 2 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
Intact
mice
2.2 f 5.6 +z 50.1 f 68.2 k 73.6 rt 56.2 f 40.2 f 18.3 + 10.2 f 11.7 + 8.3 zt 9.2 f 8.1 f Fi.2 f 4.3 f 3.2 f 2.1 * 1.1 ?t 0 0 0 0 0 0 0 0 0 0 0 0
0.4 1.3 3.8 8.6 8.2 6.7 3.8 2.5 3.7 4.8 2.1 1.2 3.6 2.8 2.4 1.2 0.8 0.2
Duration of infection (days) 2 3 4 6 8 10 12 14 16 1X 20 22 24 26 28 SO 3‘2 34 36 38 40 42 44 46 50 60
mice
+ 0.8 f 6.5 f 83 + 7.4c zk 6.6d f 7.5 f 2.3 f- 6.5 zk 8.:1 zk 3.6 f 2.1 f 7.4 z!z 5.3 f 7.5 f 3.G It 2.1 f 4.6 It 2.1 * 3.7 + 8.3 f 6.7 h 7.7 jz 2.1 xt 3.8 f 7.G + 4.6 zt 3.2 & 7.8 + 5.6 f 2.3
a Mean of 10 mice f- RI>. PvIean parasitcmia for 6 months in splenectomizcd mice is 25.77 + 9.8c1,. 6 One mouse died. c Three mice died. d One mouse died.
in Intact
Parasitemia Intact
mice
(‘,s)
Splencctomized
mice
-
(x)”
3.2 31.4 60.1 79.7 78.4 63.5 50.1 44.8 42.6 63.7 58.2 55.6 52.1 53.2 52.1 50.1 4X.5 is.2 40.1 43.2 45.1 41.2 39.1 40.2 39.3 36.3 35.3 37.5 33.2 29.5
Infection Mice
__
in Intact
Splenectomized
II
and Splenectomized
I
The Course of Babesia microti Infection and Splenectomizcd Mice Duration of infection
TABLE
Th e c ourse of Rabesia hylom~xi
2.3 f 0.7 6.4 f 1.6 10.4 + 2.5 24.5 + 6.8 35.4 f 7.3 47.6 f 8.S 5S.3 f 8.7n 81.2 f 10.7* 40.3 f 7.9 21.4 zt 3.7 17.5 f 6.7 2.3 f 0.8 2.5 * 0.2 2.2 f 03 0.3 * 0.4 2.6 z!z 1.2 1.3 3.5 * 1.7 5.2 i 10.1 * 3.2 20.2 zt 6.5 30.3 zt 5.7 40.1 * .‘,.A 58.7 zk 7.7 46.5 + 8.9 28.6 z!z 7.5 17.5 zk :3.G 7.3 zt 2.1 1.2 2.3 *
7.2 36.4 65.6 79.5 9::.4
f f zk zt f -d
2.3 6.4 16.4* 12.9b 103
-
--
” Two miw died. h Three mice died. c One mouse died. 11-, a11mice died.
cences were also observed in these mice during 6 months of examination. When a similar number of mice that recovered from B. microti infection were splenectomized during the same period of time, mild recrudescences (5-10s maximum mean parasitemia during recrudescence) developed for only 5 days, and thereafter, the mice remained free of infection during: 6 months of examination. Blood obtained from these mice failed to produce infection in splenectomized recipients, and they were resistant to challenge with the homologous species
B. microti
AND
B.
hylomysci:
of parasite but developed a short-lived (3 days), low-grade infection (5% maximum mean parasitemia) when challenged with B. hybmysci. Another experiment tested the effect of splenectomy on the developing parasitemia. Twenty-four mice were infected with B. microti, and 24 were infected with B. hylomysci. Three from each group were splenectomized at 4-day intervals, and blood smears were prepared from all animals every other day to assess the parasitemia. The three mice infected with B. microti and splenedomized within the first 4 days of infection developed an extended infection similar to that in mice splenectomized prior to the infection (Table I). However, splenectomy after the eighth day of infection had no effe’ct on the course of B. mic&i infection. Splenectomy within 8 days of B. hylomysci infection resulted in the death of infected animals (from fulminating infections) a few days after the operation, but splenectomy at 12 or more days did not have any effect. Erythrophagocytosis Twenty-five mice were infected with B. microti, and 25 were infected with B. hylomysci. Parasitemia, PCV, and erythrocyte counts were determined daily, and the three mice with the highest parasitemia from the B. hylomysci-infected group were killed every other day, while the corresponding three mice from the B. microti group were killed at 3-day intervals. The spleen was removed from every animal, measured, and weighed. The marrow plug was removed from the right femur ‘of every animal, and saline preparations of spleen and bone marrow cells were examined for erythrophagocytosis. A test employing hemolytic systems for detecting antibodies on the surface of erythrocytes (Ristic 1961) was also ‘carried out for each animal,
5
PHAGOCYTOSIS
Erythrophagocytosis was not noticed in any of the saline preparations of spleen and bone marrow cells from normal mice, nor was hemolysis detected in any of the hemolytic systems using washed erythrocytes in the different control regimes (Tables III and IV). Erythrophagocytosis was, however, detected in spleen and bone marrow saline preparations from infected animals, and the highest level of phagocytosis coincided with relatively high levels of antibodies on the erythrocytes and concurrently with enlarged spleens, low PCV, and low erythrocyte counts (Tables III ‘and IV). Impression smears of spleen and bone marrow showed that both infected and noninfected erythrocytes were equally phagocyto,sed. Erythrophagocytosis was not observed after 12 and 30 days of B. hylomysci and B. microti infections, respectively (Tables III and IV). Determination
of the Phugocytic
Index (K)
Thirty mice were infected with B. microti and 30 with B. hylomysci for the assessment of the phagocytic index (K) during each infection. The value of K rose sharply during early B. hylomysci infection, reaching a peak of 0.493 * 0.213 by Day 4 before dropping to subnormal levels (0.007 * 0.002) during peak parasitemia (Fig. 1). During this time most of the mice were very sick and many died. The value of K then rose progressively in surviving animals and was within the normal range by Day 10 ‘of the infection (Fig. 1). Similarly the value of K rose steadily during early B. microti infection, reaching a peak of 0.693 2 0.302 just bef’ore peak parasitemia; a high level was maintained throughout the rest of the infection, and normal levels were reached with recovery (Fig. 2).
HUSSEIN
S. HUSSEIN
.oo ? 2 E :
DISCUSSION
Irvin and Brocklesby (1969) observed continuously high Babes&z microti parasitemias for long periods in both intact and (1977a) splenectomized mice. Hussein showed, however, that intact mice lost the parasite completely as early as 3 weeks after recovery from a primary B. microti infection, and sterile immunity supervened. Continuous high parasitemias (higher than those observed by Irvin and Brocklesby 1969) during B. microti infection were, however, ‘observed in mice splenectomized prior to the infection (Table I ) . These parasitemias were not much higher than those in intact ones, a fact which might be due to the reduction of reticulocytes following splenectomy (Singer 1954) as B. microti is known to favor these cells (Nowell 1969; Hussein 1973). Similar findings were observed during Plasmodium berghei infec-
3 r’ n
0
0.001)1 0
1 2
’ 4
’ 6
Duration
-1c10 q : : z ; .
-141 / \
H
\
-1. 0
I
I
I
1
123456789~ Duration
of
I
I
I
Infection
I
I
(Days)
FIG. 1. Variations in the phagocytic index (K) during Babe& hylomysci infection in mice. Phagocytic
index,
l -
centage), l ----
l l
,
(K);
parasitemia
70 (per-
\\ \\
Of I)I.,..,. rlo I : O.Ol-:’ sL / 0’ ! E ” : E 0” : i :; 1 1 1 6 10 12
.O
1 15
0.1 20
of Infectlonbyd
FIG. 2. Variations in the phagocytic index (K) during Babe&z microti infection in mice. Phagocytic index, 0 0 (K); parasitemin $3 (percentage), 0 ---0.
tion in mice (Singer 1954). B. hylomysci which favors mature erythrocytes (Hussein 1976), developed a hyperacute infection in splenectomized mice which died within few days (Table II). Splenectomy had little effect when performed after either infection had become well-established. This might indicate that the spleen is essential only during the early stages of infection, possibly to check excessive multiplication of either parasite through phagocytosis and the elaboration of protective antibodies as observed by Todorovic et al. (1967) and Roberts et al. ( 1972) during similar infections, Recently Gravely et al. (1976) observed dramatic decreases in B cells in spleens of rats infected with P. berghei early in the infection, but their numbers returned to normal as the infection progressed. These cells are possibly mobilized and transformed into
2.9 39.0 73.0 76.9 58.3 19.1 6.3 1.1
0 zt zt * zk f f h f
1.2 6.3 7.5 5.4 11.2 9.6 3.2 0.9
Parasitemia (% f SD)
1.32 2.93 2.78 1.73 1.32 1.34 1.78 2.1 2.1
51 46 36 21 21 25 28 38 45
0 zk ++ +++ +-t+ ++ ++ ++ 0
xt f f f f f f f f
7.00 6.63 4.75 2.93 2.09 2.87 3.37 4.48 6.50
0 0 f + ++ + + + 0
Bone marrow
Erythrophagocytosisb Spleen
PCV (%I
RBC count (X lOB/ mm3) * SD
76.2 82.8 343.3 308.3 504.4 730.0 663.3 783.3 216.6
i f f f f f zk f f
12.3 24.3 80.2 90.3 125.6 225.4 234.2 215.2 78.4
Weight (mg f SD)
Spleen
15 15 26 26 33 28 32 39 20
x x x X X X x X x
Size (mm)
5 5 9 8 9 12 11 1S 8 7.8 56.2 100 100 100 100 64.8
0 f 4.5 f 11.1 f 0 f 0 f 0 f 0 f 8.1 0
RBC of infected mice + complement 0 0 0 0 0 0 0 0 0
RBC of infected mice + saline
erythro-
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
containing
RBC of normal mice + complement
SD)
RBC of infected mice + heatinactivated complement
lysisc (% f
Fixed on
Immune
between Anemia, Parasitemia, Erythrophagocytosis, and Levels of Antibody Erythrocyte Surface during Babesiu microti Infection in Micea
III
a All figures are arithmetic means f SD. b Erythrophagocytosis : 0, no phagocytes containing erythrocytes; f, l-5% phagocytes containing erythrocytes; +, lO-2O% phagocytes cytes ; + +, 30-40’% phagocytes containing erythrocytes, and + + +, 50% and more phagocytes containing erythrocytes. c Immune lysis : The level of antibody fixed on the surface of erythrocytes is proportional to the degree of lysis.
0 3 6 9 12 15 18 21 31
Duration of infection (day.9
The Relationship
TABLE
2 0 cn z
1
3 2 C*
z
g Pj
$’ 2c. 9
3
.Y
2 g z
0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 T'TI 0 O'ZI S'L F.9
0 T X'ZI =F OOT =F 1'9.i r G-91 =F Z'S 0 ~113UI
-alduro:, + a:,rur
L x 9 x 8 x 9x 9 x 9 X Px
sz PZ PZ IZ AL 61 zt
1'01
L'S X'P!) 2'OL C'h'P TLC %.';I
=l= O'OLZ =F O'WZ =F 0'0% =F 0'01: 1 =F 0.901 =F y'pg > . =F 0'09
0 Zt + 0 0 0
B. microti AND B. hylomysci: antibody-producing cells early in the infection upon stimulation by parasite antigens. Moreover, as the infection progresses, the spleen is known to be more concerned with erythropoiesis than with immunological functions (Dokow et al. 1974; Poels 1977). The spleen was also ‘observed to exert a protective role during early stages of quartan malaria (Plasmodium inui infection) in rhesus monkeys, but had a suppressive role later in the infection, and unlike splenectomized animals, intact ones were unable to achieve self-cure (Wyler et al. 1977). During chronic stages of rodent babesiosis ( B. microti infection), however, the opposite occurred; intact mice were able to achieve self-cure, but splenectomized ones were not (Table I). Splenectomy performed within 1 week of recovery from B. microti infection in the present study resulted in recrudescences which were not as severe as those observed by Miessner (1931) and Barnett (1965) following splenectomy of cattle recovered from B. bigemina infection. However, splenectomy of mice 3 or 4 weeks following recovery from B. microti infection does not always result in recrudescences as sterile immunity may supervene ( Hussein 1977a). The spleen seems to be essential for the initiation of sterile immunity which fails to develop in mice splenectomized prior to and within 1 week of B. microti infection during the present study. Moreover, splenectomy after the infection has become well established does not seem to affect the development of sterile immunity, possibly because the spleen function might have been taken over by other organs (Taliaferro 1956) or because the spleen cells might have migrated to other tissues follmowing primary immunization (Cannon and Wissler 1967). Thus the presence of the spleen appears to be critical in the early response to B. microti infection, but other sites may supersede it in the maintenance of immunity over longer periods. The development of sterile immunity to B. microti
PHAGOCYTOSIS
9
infection is in accordance with the findings of Cox and Young (1969) that immunity to babesiosis in mice, unlike that against cattle babesiosis, is complete in the rodent host. However, the immunity to B. hylomysci infection in mice seemed to be an exception and similar to that of bovine babesiosis ( Hussein 1977a). The possible roles of the spleen in the immunity to the related malaria parasites have been discussed by Brown ( 1969) and Brown and Phillips ( 1974), who suggested that the key role of the spleen in rodent malaria is to develop immunity to new antigenie variants appearing in the parasite population. This does not seem to be the case with either B. microti infection where solid sterile immunity eliminates the parasite completely from the host (Cox and Young 1969; Hussein 1977a), or with B. hylomysci infection where severe spontaneous recrudescences are common (Table II and Hussein 1977a) and the relapsing parasitemia is likely to be accompanied by antigenic variations which the spleen was not able to control. Erythrophagocytosis was common during both infections but was more pronounced during B. microti infection and coincided with severe anemia, splenomegaly, and relatively high levels of antibodies on the surface of erythrocytes (Tables III and IV). Similar observations were made by Schroeder et al. (1966) during B. rodhaini infection in the rat and these workers ascribed the condition to an autoimmune response. The demonstration of antibodies on the surface of the erythrocytes and their relationship to erythrophagocytosis might indicate that the anemia during mouse babesiosis might be, at least in part, due to an autoimmune response as both infected and noninfected erythrocytes were observed to be equally phagocytosed during the present study. Noninfected erythrocytes might have been injured during the course of infection or coated with antigen-antibody
10
HUSSEIN
complexes and hence phagocytosed. Similar observations were made by Ristic (1961) during bovine anaplasmosis. Moreover, eirculating macrophages and neutrophils as well as Kupffer cells of the liver have been observed to be active in erythrophagocytosis and consequently involved in the anemia associated with babesiosis (Holbrook et al. 1968; Simpson 1970, 1974). Erythrophagocytosis of infected and uninfected erythrocytes was once postulated to explain the excess anemia observed during malaria (Zuckerman 1966; Cox et al. 1966). However, Chow and Kreier (1972) and Hamburger and Kreier (1976) were unable to demonstrate significant erythrophagocytosis during P. berghei infection using an in vitro system, even in the presence of immune serum. Phagocytosis of free Babesia spp, parasites and its comparison to erythrophagocytosis are yet to be thoroughly investigated. However, Roberts and Tracey-Patte (1974) observed that the parasite and its red cell are removed simultaneously and that it is not necessary for the parasite to be extracellular in order to be phagocytosed. This was later confirmed by Abdalla et al. (1978) who indicated that protective antibodies can penetrate the infected erythrocyte to interact with B. rodhaini .and bring about erythrophagocytosis. In support of the findings of Cox et al. (1963, 1964), the phagocytic activity of the reticuloendotbelial system increased during both infections, but peak B. hylomysci parasitemia was accompanied by reticuloendothelial phagocytic depression ( Fig. 1)) with subsequent death of the host. This might indicate that the death of the host during B. hylomysci infection may be, at Ieast partly, due to overwhelming of the reticuloendothelial system by excessive multiplication of the parasite. Cantrell et al. (1970) explained the loss of phagocytic hyperactivity during rodent malaria as being due to the fact that the parasites act both as a stimulus and a load
S. HUSSEIN
to the phagocytic system, and owing to their potential for growth, their role as a load may exceed their roIe as a stimulus and hence bring about a nonhyperactive state. They also suspected that a nonhyperactive state in infected rats might be due to the exhaustion of some substance essential for phagocytosis. Such a substance was isolated, purified, and characterized as a-2globuhn by Blumenstock et al. (1976). These authors also suggested that a deficiency of this factor occurring during tumor growth, trauma, or shock would precipitate a state of reticuIoendothelia1 dysfunction. Therefore, the reticuloendothelial depression observed during B. hylomysci infection in mice could be attributed to a possible deficiency in the a-2-globulin which might have occurred during the state of shock observed in infected mice by Hussein ( 1977b). A state of shock never occurred during B. microti infection in mice (Hussein 1973). This might explain the absence of reticuloendothelial phagocytic depression during this infection, as there might be no deficiency in the a-2-globulin factor due to the absence of a state of shock. Further work is needed to investigate the variations in the level of the a-2-globulin factor during either infection. The loss of phagocytic hyperactivity observed by Cantrell et al. (1970) did not adversely affect the ability of rats to recover from P. berghei infection, and the authors suggested that recovery was due to acquired immunity that is effective without phagocytic hyperactivity. This does not seem to be the case with B. hylomysci infection in the mouse, a host known to be less able to mount an effective immunological response to bIood protozoa1 infections than the rat (Gravely et al. 1976; Hamburger and Kreier 1976). Later Cantrell and Elko (1976) observed that the ability to maintain phagocytic hyperactivity toward colloidal carbon during P. berghei infection varied within individuals of the same strain as well as among
B. microti AND B. hylomysci: different rat strains. This does not seem to be the case with B. hylomysci infection in LACA strain mice, where a11infected animals showed reticuloendothelial phagocytic depression. However, variations within the different strains of mice remains to be investigated. The multiplication rate of B. microti is slower than that of B. hylomysci (Hussein 1973)) and hence the reticuloendothelial system is quite capable of dealing with this parasite. This is exemplified by the fact that ph,agocytic activity never fell in mice infected with B. microti while it did in mice with B. hylomysci infection (Figs. 1 and 2). Moreover, B. microti seems to be a more efficient stimulant to the reticuloendothelial system than B. hylomysci. This is indicated by the consistently superior phagocytic index (Figs. 1 and 2) and by the higher rate of erythrophagocytosis both in the spleen and bone marrow (Tables III and IV). Such increased phagocytosis may be initially protective, so that the development and mobilization of other immunological defences of the host may be permitted. This might well be the reason that, unlike intact mice with B. hylomysci and B. rodhaini infections, intact mice usually recover from Babesia microti infection. ACKNOWLEDGMENTS The author wishes to thank Professor M. J. Clarkson of Liverpool University for advice and Professor J. P. Kreier, of the Ohio State University for valuable discussions and constructive criticism of the manuscript.
REFERENCES H. S., HUSSEIN, H. S., AND -IER, J. P. 1978. Babesia rodhaini: Passive protection of mice with immune serum. Tropenmedizin und Parasitologic 29. BARNETT, S. F. 1965. The chemotherapy of Babesia bigemina infections in cattle. Research in Veterinary Science 6, 397-415. BIOZZI, G., BENACERRAF, B., AND HALPERN, B. N. 1953. Quantitative study of the granulopectic activity of the reticuloendothelial system. II. A study of the kinetics of the granulopectic activABDALLA,
PHAGOCYTOSIS
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ity of R.E.S. in relation to the dose of carbon injected. Relationship between the weight of organs and their activity. British Journal of Experimental Pathology 34,441457. Bxozzr, G., HALPERN, B. N., BENACERRAF, B., AND STIFFLE, C. 1957. Phagocytic activity of the reticuloendothelial system in experimental infections. In “Physiology of the Reticuloendothelial System.” (Edited under the direction of B. N. Halpern by B. Benacerraf and J. D. Delafresnaye), pp. 204-225. Springfield, Illinois. BLUMENSTOCK, F., SABA, T. M., WEBBER, P., AND CHO, E. 1976. Purification and biochemical characterization of a macrophage stimulating alphae-globulin opsonic protein. Journal of the Reticuloendothelial Society 19, 157-172. BROWN, I. N. 1969. Immunological aspects of malaria infection. Aduances in Immunology 11, 267-349. BROWN, I. N., AND PHILLIPS, R. S. 1974. Immunity to Plasmodium berghei in rats: Passive serum transfer and the role of the spleen. Infection aml Immunity 10, 1213-1218. CANNON, D. C., AND WISSLER, R. W. 1967. Spleen cell migration in immune response of the rat. Archives of Pathology 84, 109-118. CANTRELL, W., ELKO, E. E., AND HOPFF, B. M. 1970. Plasmodium berghei: Phagocytic hyperactivity of infected rats. Experimental Parasitology 28,291-297. CANTRELL, W., AND ELKO, E. E. 1976. Plasmodium berghei: Phagocytic activity in two strains of rats. Experimental Parasitology 40, 281-285. CHOW, J. S., AND KREIER, J. P. 1972. Pkwnodium berghei: Adherence and phagocytosis by rat macrophages. Experimental Parasitology 31, 1318. Cox, F. E. G., BILBEY, D. L. J., AND NICOL, T. 1964. Reticuloendothelial activity in mice infected with Plusmodium uinckei. Journal of PTOtozoobgy 11,229-230. Cox, F. E. G., NICOL, T., AND BILBEY, D. L. J. 1963. Reticuloendothelial activity in Haemamoeba (Plasmodium) gallinucea infections. ]ournal of Protozoology 10, 107-109. Cox, F. E. G., AND YOUNG, A. S. 1969. Acquired immunity to Babesia microti and Babesia rodhaini in mice. ParasitoZogy 59, 257-268. Cox, H. W., SCHROEDER, W. F., AND RISTIC, M. 1966. Hemagglutination and erythrophagocytosis associated with Plasmodium berghei infection of rats. Journal of ProtozooZogy 13, 327-332. DALGLIESH, R. J. 1972. Theoretical and practical aspects of freezing parasitic protozoa. Australian Veterinary Journal 48, 233-239. D~KOW, S., GOLENSER, J., GREENBLATT, C. L., AND SPIRA, D. T. 1974. Changes in rat splenocyte
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HUSSEIN
population during infection with Plasmodium berghei. Journal of Protozoology 21, 463. GRAVELY, S. M., HAMBURGER, J., AND KREIER, J. P. 1976. T and B cell population changes in young and adult rats infected with Plasmodium berghei. Infection and Immunity 14, 178-183. HAMBURGER, J., AND KREIER, J. P. 1976. Plasmodium berghei: Use of free blood stage parasites to demonstrate protective humoral activity in the serum of recovered rats. Experimental Parasitology 40, 158-169. HOLBROOK, A. A., JOHNSON, A. J., AND MADDEN, P. A. 1968. Equine piroplasmosis: Intra-erythrocytic development of Babe& cab& (Nuttall) and Babesia equi (Laveran). American Journal of Veterinary Research 29, 297-303. HUSSEIN, H. S. 1973. “Studies on Rodent Babesiosis Caused by Babesia hylomysci Bafort, Timperman and Molyneux, 1970 and Babesia microti Ph.D. Thesis, University of (Franca, 1910).” Liverpool. HUSSEIN, H. S. 1976. Babesia hylomysci in mice: Preference for erythrocytes of a particular age group and pathogenesis of the anemia. Zeits&rift fur Purasitenkunde 50, 103-108. HUSSEIN, H. S. 1977a. The nature of immunity against Babesia hylomysci and B. microti infection in mice. Annals of Tropical Medicine and Parasitology 71, 249-253. HUSSEIN, H. S. 1977b. The pathology of Babesia hylomysci infection in mice. I. Clinical signs and liver lesions. Journal of Comparatioe Pathology 87, 161-167. IRVIN, A. D., AND BROCKLESBY, D. W. 1969. Continuous high parasitemia in mice infected with Babesia (Nuttallia) microti. Journal of Parasitology 55, 1190. LEGG, J, 1935. The occurrence of bovine babesiellosis in Northern Australia. C.S.I.R.O. Pamphlet No. 56, l-48. MLESSNER, H. 1931. Piroplasmosen und Splenektomie. Archiv fiir Wissenschaftliche und practische Tierheilkunde 63, 68-90. NOWELL, F. 1969. The blood picture resulting from Nuttallia (=Babesia) rodhaini and Nuttallia (=Babesia) microti infections in rats and mice. Parasitology 61, 425433. PHILLIPS, R. S. 1969. The role of the spleen in relation to natural and acquired immunity to infections of Babes&r rodhaini in the rat. Parasitology 59, 637-648.
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