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Experimental acute Babesia caballi infections
EXPERIMENTAL PARASITOLOGY 37, 67-77
Experimental
(1975)
Acute Babesia cabal/i
Infections
I. Red Blood Cell Dynamics PATRICIA
C. ALLEN, United (Submitted
WAYNE
M. FRERICHS, AND A. A. HOLBROOK r
States Department for publication
of Agricultwe September
13, 1973)
ALLEN, PATRICIA C., FRERICHS, WAYNE M., AND HOLBROOK, A. A. 1975. Experimental acute Babesia caballi infections. I. Red blood cell dynamics. Experimental Parasitology 37, 67-77. Hematological determinations were made on blood samples from six ponies acutely infected with two dosage levels of Babesia cabaZZi (Group 1: divided into two subgroups of three ponies each). Similar determinations were made on blood samples from three premunized ponies given challenge inoculations (Group 2), and three equidae given uninfected red blood cells (Group 3). A trend towards decreases in RBC counts, hemoglobin concentrations, and hematocrits within one to four days after inoculation (AI) was observed in all groups. However, it was marked only in Group 1. In addition, only in Group 1 was there observed a concerted anemia occurring between Days 7 and 16. Those surviving ponies in Group 1 which developed a higher parasitemia between Days 5 and 6 AI (first parasitemia peak) developed a more severe anemia between Days 7 and 16. Ponies which developed parasitemias higher than 40 X lo3 parasitized cells/mm3 at the first parasitemia peak subsequently died. Free bilirubin in Group 1 animals increased immediately after inoculation, and repeatedly exceeded normal ranges until after Day 20 AI when the RBC counts were rising. Similar changes in free bilirubin did not occur in either Groups 2 or 3. Conjugated bilirubin levels did not exceed normal ranges in any of the experimental animals. Active erythrophagocytosis was evident in histological preparations of lymph node, spleen, liver, and lung from ponies which died. Cytosiderin pigment was present in liver parenchyma, and hematin was scattered throughout lymph nodes and spleen. INDEX DESCRIPTORS: Babesia caballi: Pony; Equidae; Erythrocyte; Hemoglobin; Hemocrit; Free bilirubin; Erythrophagocytosis; Macrophage; Reticuloendothelial system.
Although anemia is recognized as one of the primary clinical signs of Babe& caballi infection (Roberts et al. 1962; Retief 1964; Sippel et al. 1962), little has been reported about infection-associated changes in hematological parameters on a day-to-day basis in experimentally infected equidae. Data of this type have been reported for 1 Animal Parasitology Institute, Agricultural search Service, Beltsville, Maryland 20705.
Babesia infections in cattle (Wright 1972) and dogs ( Maegraith et al. 1957). The purpose of this present study was to define more closely the anemic processes in experimental acute B. caballi infections. MATERIALS
Source of Babesia caballi Babe&a caballi infected RBCs taken from liquid nitrogen storage (Frerichs et al.
Re-
67 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
68
ALLEN,
FRERICHS
AND
TABLE Experimental Experimental groups
HOLBROOK
1
Groups Used to Study Red Blood Cell Dynamics Animal No.
Species
-
Approximate age
Infect)ive dose of piroplasms
of Babesia caballi Infections Rate of transfusion of packed RBCs
Date of experiment
Group 1 Acute infections A
155 154 152
Pony Pony
Acute infect,ions B
153 132 119
Colt Colt ,4dult
I x 109
2 ml,‘kg body wt
2 ,‘5,‘7&3,!‘30,‘72
Pony Pony
Adult Colt Adult
1 x 10”
0.2 ml, ‘kg body w%
2 !5,f72-3/30/72
154 153 119
Pony Pony pony
Colt Adult Adult
1 x 10”
1 ml/kg
161 149 148
Pony Blwro Horse
Adult Adult Adult
None
1 mll’kg body r+?
POllJ'
Pony
Group 2 Challenge infection of premunized ponies
body wt
4/18/X-5/17/72
Group t7 Transfusion with uninfected RBCs
1968) were used to initiate a series of rapid passages in splenectomized ponies and burros until parasitemia was great enough to cause acute infections. The original source of the parasite was infected ticks obtained in 1963 from an enzootic area in Florida. Experimental ponies were infected by intravenous inoculation of a volume of packed RBCs containing the desired number of piroplasms (Table I). Experimental
Animals
All animals were purchased in Maryland, where naturally occurring B. caballi infections have not been found. Serums from animals used for acute infection (Groups 1A and 1B) and from animals which received uninfected RBCs (Group 3) were negative in the CF test (Frerichs et al. 1969). Before and after inoculation, all animals were maintained in a barn held at 20 C, and were fed mixed horse feed (Wayne New Hope complete horse feed, General Offices, Allied Mills, Inc., Chicago, Illinois 60606) twice a day, with water available ad lib. Rectal temperatures were
taken twice daily the experiments. Experimental
at feeding
5,:17/72-6:‘29/72
time during
Groups
The groups of experimental animals are defined in Table I. Group 1 animals had not been exposed previously to B. caballi. Group 2 was comprised of three of the four animals which survived infection in Group 1 and were considered premunized. The animals in Group 3 were chosen specifically to determine whether transfusion of heterologous RBCs alone would produce any changes in blood values similar to those observed in acute infections. Each animal’s blood was cross-matched before transfusion with that of its respective donor. Pony 161 had been sensitized previously to RBCs, and its serum caused slight agglutination of its donor’s RBCs. Serums from Burro 149 and Horse 148 did not agglutinate their respective donor’s RBCs. Collection
of Blood Samples
Blood was collected by jugular venepuncture into evacuated tubes with and
RBC
DYNAMICS
IN
without EDTA (B&D Vacutainers, Becton, Dickinson, Rutherford, New Jersey 07070). Preinoculation samples were collected from all animals to establish baseline values. In Group 1, samples were collected on Days l-30, 33, 37, and 43-50 after inoculation. In Group 2, samples were collected on Days 1-14, 19, and 26. In Group 3, collections were made on Days 1-16, 22, and 29. Hematologic
Determinations
Total erythrocytes were counted in hemocytometers according to standard methods (Schalm 1965). Hemoglobin was decyanmethemoglobin termined by the method ( Mattenheimer 1970). Hematocrits were determined by the microcapillary method (International Equipment Company, Boston, Massachusetts 02135). Bilirubin (total and direct ) was determined by a modification of the Jendrassik-Grof procedure ( Mattenheimer 1970). All blood smears were stained with Giemsa and examined for parasites. The number of parasitized cells in 100 oil immersion fields ( 1000x ) was counted. The number of parasitized cells per mm3 was calculated on the basis of the daily RBC count. Postmortem Studies Tissues from animals which died during acute infection (Ponies 155 and 152) were fixed in 10% neutral, buffered formalin, sectioned at 6 pm and stained with hematoxylin and eosin (H&E), azure eosin (A&E ) and Prussian blue. RESULTS
Two (Ponies 155 and 152) of the three ponies which were given 1 x lo” piroplasms died. All three ponies that received 1 x 10s organisms survived. Parasites were found in blood smears from acutely infected ponies on Days l-23 and sporadically thereafter through Day 29. There were two apparent peaks of parasitemia roughly six days apart; the first at 5-6 days, the other
&Zbc?SiU
INFECTIONS
69
at lo-12 days. Pony 152, which died, showed a third peak at Day 17 (Fig. 1). No parasites were observed in any postchallenge blood smears from Group 2 ponies ( premunized animals ), but these animals did react with a transient fever (38.3-38.8 C for two to three days AI). All ponies in Group 1 (acute infections) showed decreases in RBC counts and related values during Days l-4 AI (Fig. 1). The maximum decreases in RBC counts averaged about 2.3% below the respective mean preinoculation counts. Similar decreases were observed in Group 2 ponies and in Horse 148 and Burro 149 from Group 3 (Fig. 2). However, the maximum decreases in RBC counts averaged only 11%) (Group 2 ponies) and 14% (Horse 148 and Burro 149) below the respective mean preinoculation values. The RBC counts for Group 1 ponies tended to return towards normal ranges during Days 4-7 AI. Maximum RBC counts during this time coincided with the first peak in parasitemia (Fig. 1). Pony 155 died during this period. Similar, but less concerted changes in blood values were observed in Group 2 ponies, but no parasites were seen in blood smears. In all acutely infected animals (Group 1)) the RBC counts and related values began a secondary decrease after the first peak in parasitemia. Decreases in RBC counts were moderate and transient for Ponies 153 and 119 (about a 27% average maximum decrease occurring between Days 7 and 9 AI ) . They were more extensive and prolonged for ponies 154 and 132 (about a 59F average maximum decrease occurring between Days 11 and 16 AI). During this time, parasitemia reached a second broader peak (Fig. 1). Pony 152 was unusual in that the secondary decrease in RBC count was delayed for several days. However, just prior to death a 66% decrease in its RBC count was observed. No concerted decreases in RBC counts were observed in either Groups 2 or 3 be-
ALLEN,
70
lnoculoted
9
FRERICHS
p - - -” <_a *----
Days otter inocutotion
HOLBROOK
Inoculated
with I X10’ p~roplasms
Group IA
AND
155 154 152
wth IXIO’
Group 18
Day, after inoculotim
piroplosms
RBC DYNAMICS
IN Babesia
Challenged with IX104 piroplams
INFECTIONS
71
Inoculated with uninfected erythrocytes
Group3 w-_-o
5
IO
15
II9
20
25
30
Days ofter inoculation
FIG. 2. Hemograms of immune (Group 2) and animals inoculated
5
c_ ,--,I49 *__--4
161 148
30
IO 15 20 25 Days ofter inoculation
ponies challenged with 1 X lo4 Babe& caballi with uninfected erythrocytes (Group 3) I
piroplasms
FIG. 1. Hemograms of ponies 1B) Babesia cabal& piroplasms.
inoculated
with
1 X 10” (Group
1A)
and 1 X lo*
(Group
72
ALLEN,
FRERICHS
tween Days 7 and 11 AI. However, transitory decreases were observed in both groups between Days 12 and 15. The maximum decreases averaged about 15% for both groups. After 16 days AI, the RBC counts, hemoglobin concentrations, and hematocrits of the surviving acutely infected ponies (Group 1) returned towards their respective mean preinoculation levels. By Day 29, Ponies 153 and 119 were normal. However, Ponies 154 and 132 were still below normal even 50 days AI. Blood values for animals in Groups 2 and 3 remained normal during this time. Under our experimental conditions, the average free plasma bilirubin level before inoculation was 0.547 mg/lOO ml, with a range (-t 2 SD) of 0.029-1.065 mg/lOO ml. In the acutely infected ponies (Group 1 ), the free bilirubin rose above the normal range; two- to threefold increases were observed through Day 20 AI (Fig. 1) . During the first 20 days AI, the ponies given 1 x 10” piroplasms (Group IA) had free bilirubin levels slightly higher than those given 1 x lo* piroplasms (Group 1B). High values for free bilirubin were observed in Ponies 119 and 132 even beyond 20 days AI. This period of frequent high free bilirubin values (l-20 days AI) coincided with decreases in RBC counts, hemoglobin levels, and hematocrits, and with the presence of parasitized erythrocytes in blood smears (Fig. 1). Free plasma bilirubin did not increase above the normal range in the challenged ponies (Group 2) after inoculation. Two periods of slight increase were observed in the animals receiving unparasitized RBCs (Group 3); one at l-4 days AI and the other at 13-15 day,s AI. These periods coincided with observed dccrcases in RBC c0u11ts, hemoglobin concentrations and hematocrits ( Fig. 1). No hemoglobinemia was obscrvcd in any of the acutely infected animals, and the conjugated bilirubin did not exceed normal ranges.
AND
HOLBROOK
At necropsy, both Ponies 155 and 152 showed edema of the subcutis and marked icterus. The lungs were edematous and petechiated, with pleural effusion, and the hearts showed petechiation and pericardial effusion. The livers were swollen and pale with rounded borders. The spleens were *fibrotic, moderately enlarged, and had some hemorrhages. The kidneys were pale with a few petechiae; the capsules were slightly adherent. Large numbers of macrophages containing phagocytized RBCs were visible in histological preparations of lymph node from both Ponies 155 and 152. Many of the phagacytized RBCs in Pony 155 preparations were still unaltered (Fig. 3), and stained red with A&E and H&E. Many macrophages also contained RBC debris which stained green with A&E but which was not Prussian blue positive. This indicated that RBC degradation at these sites was in its early stages. On the other hand, most of the swollen macrophages in lymph node from Pony 152 contained fragmented RBCs in a more advanced stage of degradation (Fig. 7), which stained yellow with A&E and H&E, and blue with Prussian blue ( hemosiderin, Lillie 1965). Enhanced phagocytic processes were plainly evident in spleens from both animals (Figs. 4 and 8). The spleen from Pony 152 appeared depleted of RBCs (Fig. 8 ), whereas the spleen from Pony 155 was engorged with them (Fig. 4). Macrophages containing debris (yellow, A&E; blue, Prussian blue) were seen in cleared areas within masses of RBCs (Fig. 4). Lymph nodes and spleens from both animals contained crystalline pigment similar to the formaldehyde hcmatin pigment found in malarial infections (Lillie 1965). The livers from both Ponies 155 and 152 exhibited centrilobular necrosis. The sinuses were swollen, and a slight leucocyte infiltration was obsrrvcd. Parcnchymal cells from both animals contained cytosiderin (not Prussian blue positive, Lillie 1965)
RB~ DYNAMICS
IN Babesia
and showed cloudy degeneration (Figs. 5 and 9). Kupffer cells containing degraded RBCs were readily observed within the sinuses, and they appeared more plentiful than in Pony 155. Many more macrophages containing RBC debris (Prussian blue positive, yellow with A&E ) were present within alveolar capillaries of lung from Pony I52 than in lung from Pony 155 (Figs. 6 and IO).
DISXJSSION
A comparison of Figs. 1 and 2 reveals that within Days l-6 AI the patterns of change in hematological parameters were similar in all three experimental groups. All experimental animals except Pony 161 exhibited a noticeable decrease in RBC count during this period. This phenomenon may represent the normal response of the reticuloendothelial system (RES ) to the introduction of excess RBCs (Jandl et al. 1965). The phagocytosis of erythrocytes by macrophages in lymph nodes and spleen is evident in stained sections from Pony I55 which died on Day 7 (Figs. 34). However, the decrease in RBC counts of the acutely infected ponies averaged about twice as great as those in Groups 2 and 3, and only in the acutely infected ponies was there an immediate increase in free plasma bilirubin over the normal range. Thus, although erythrophagocytosis may have taken place in all experimental animals, significant hemolytic reactions developed only in the acute infections. The return of the RBC counts to normal range in Group 1 ponies coincided with the first parasitemia peaks (Fig. 1). That these two events occurred simultaneously indicates that in these animals the RES may have become temporarily saturated with phagocytized cells, thus allowing a buildup in the circulation not only of the total number of RBCs but of the parasitized cells as well. Jandl (1967) showed that such a blocking effect occurs in acquired
INFECTIONS
73
hemolytic anemias and severe transfusion reactions, True anemia developed in the acutely infected ponies (Group 1) after the first parasitemia peak between Days 7 and 16 AI. During this time, parasitemias reached a second broader peak and RBC counts attained minimum values, although these events did not occur simultaneously (‘Fig. 1). Although it would appear that the degree of anemia is correlated with infective dose level, it is difficult to establish such a correlation with the relatively small number of animals in Group 1. However, there does seem to be a strong correlation between the degree of parasitemia evident at the first peak (Days 5-6 AI) and the degree of anemia developing subsequently. Additionally, it would appear that those animals will succumb to infection which attain a parasitemia greater than 40 x lo3 parasitized cells/mm3 at the first peak in parasitemia. Since neither hemogIobinemia nor hemoglobinuria were observed in Group 1 animals during Days 7-16, all significant RBC destruction must have taken place within the RES (Jandl 1967), not intravascularly. The observed anemia could thus be the result of increased phagocytic activity of a stimulated and proliferating RES (George et al. 1966; Topley et al. 1970; Jandl et al. 1965; Kelly et al. 1960), as exemplified by the enlarged spleens of Ponies 155 and 152, and the apparent high activity of macrophages in histopathological preparation from these animals. The development of a second parasitemia peak may again be the result of another temporary blockade of the RES, and represent those parasitized cells which escaped phagocytosis or “pitting” (Conrad 1969; Schnitzer et al. 1972 ) . The progressive involvement in RBC destruction by the RES in organs other than the lymph nodes and spleen were apparent in sections of liver and lung from Ponies 155 and 152. There appeared to be
74
ALLEN,
FRERICHS
AND
HOLBROOK
FIGS. 3-6. Histopathological sections from Pony 155. FIG. 3. Lymph node 300~ (A&E): hlacrophages (hl) contain both undegradcd RBCs (arrow) and RBCs in early stages of decomposition ( * ). FIG. 4. Spleen 300x (.4&E ) engorged with RBCs ( * ) : hiacrophages ( hl ) containing RBC debris; hematin (H). FIG. 5. Liver 300X (A&E): Kupffer cells (K) containing RBC debris; cytosiderin (C) in parenchyma cells. FIG. 6. Lung 300~ (Prussian blue): only a few macrophages containing Prussian blue positive material (P) present in alveolar capillaries.
RBC DYNAMICS
IN
Bahesin
INFECTIONS
FIGS. 7-10. Histopathological sections from Pony 152. FIG. 7. Lymph node 300X (A&E): Macrophages (M) containing RBC debris. FIG. 8. Spleen 405X (A&E) depleted of RBCs: Macrophages (M) containing RBC debris. FIG. 9. Liver 405X (A&E): Kupffer cells (K) containing whole RBCs (“) and debris (arrow); cytosiderin (C). FIG. 10. Lung 300X (Prussian blue): Many macrophages containing Prussian blue positive material (P) present in alveolar capillaries.
7G
ALLEN,
FRERICHS
a proliferation of Kupffer cells in the liver of Pony 152 which died on Day 21 AI as compared with Pony 155 which died on Day 7. Many macrophages laden with degraded RBCs were present in lung sections from Pony 152 (Fig. lo), whereas they were rare in lung from Pony 155. The hematological patterns of change exhibited by Pony 152 were delayed for several days as compared with the other animals in Group 1. However, the trends remained similar. The reason for the delays is not known, but there was a concurrent bacterial infection which might have caused this effect. Microcolonies of bacteria were found in sections of lymph node and other tissues. It is interesting to note that the patterns of change observed in Group 2 ponies (challenged, premunized) more closely resemble those of Group 3 than Group 1. No parasitemia was observed, and no prolonged decrease in RBC count occurred after Day 7. We had expected to see some parasitemia and anemia. However, the challenge dose of I x lo4 piroplasms may not have been sufficient to saturate the already proliferated RES of these animals. We emphasize that no severe hemolytic reactions occurred in Group 3 animals. We had expected any adverse effects of transfusion per se (due to incompatability of blood antigens) to be demonstrated in these animals. The hemolytic reactions (presence of free bilirubin in the blood ) in the acutely infected ponies and the early death of Pony 155 are more than likely due only to the presence of B. cabal& in the blood. The animals in Groups 2 and 3 did show transitory decreases in RBC counts and related values between Days 10 and 16 AI (Fig. 2). This decrease coincided with a very slight rise in free bilirubin in Group 3. This phenomenon may also be a natural response of the RES to transfused RBCs (see citations by Berlin 1964)) and may be masked in acutely infected animals by the
AND HOLBROOK
more prominent response to B. cahalli infected cells. We conclude that in experimental B. infections initiated by transfusion caballi of parasitized RBCs, the host’s physiological reaction to the RBCs may influence the patterns of change in erythrocyte counts and related values during Days l-6 AI, perhaps even regulating the time at which the first peak in parasitemia occurs. Hematological changes which occur after this time appear to be due primarily to the reaction of the host to the presence of B. caballi. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of R. Ellis, D. Davis, 0. McGaha, L. Owens, and J. Hoang. We thank D. Thompson for preparing the histologic sections. REFERENCES N. I. 1964. Life span of the red cell. In “The Red Blood Cell,” pp. 423-450. (C. Bishop and D. M. Surgenor, Eds.) Academic Press, New York. CONRAD, M. E. 1969. Pathophysiology of malaria. Hematologic observations in human and animal studies. Annals of Internal Medicine 70, 134-141. FRERICHS, W. M., JOHNSON, A. J., AND HOLBROOK, A. A. 1968. Storage of Babesia cabal& and Babe& equi in liquid nitrogen. Journal of Parasitology 54, 451. FRERICHS, W. M., HOLBROOK, A. A., AND JOFZVSON, A. J. 1969. Equine piroplasmosis: Complement fixation titers of horses infected with Babesia caballi. American Journal of Veterinary Research 30, 697-702. GEORGE, J. N., STOKES, E. F., WICKER, D. J., AND CONRAD, M. E. 1966. Studies of the mechanisms of hemolysis in experimental malaria. Military Medicine 131 (Supplement), 12171224. JANDL, J. H. 1967. The spleen and reticuloendothelial system. In “Pathologic Physiology” (W. A. Sodeman and W. A. Sodeman, Jr., Eds. ), Saunders, Philadelphia. pp. 897-932. JANDL, J. H., FILES, N. M., BARNETT, S. B., AND MACDONALD, R. A. 1965. Proliferative response of the spleen and liver to hemolysis. Journal of Experimental Medicine 122, 299326. BERLIN,
RBC
DYNAMICS
IN
KELLY, L. S. DOBSON, E. L., FINSEY, C. R., AND of the HIRSCH, J. P., 1960. Proliferation the liver. reticuloendothelial system in American Journal of Physiology 198, 11341138. LILLIE, R. D. 1965. “Histopathologic Technic and Practical Histochemistry,” 3rd ed, McGrawHill, New York. MAEGRAITH, B. G., GILLIES, H. M., AND DEVATJKUL, K. 1957. Pathological processes in Babesia infections. Zeitschrift ftiT Tropencanie medizine und Parasitologic 8, 485514. MATTENHEIMER, H. 1970. “Micromethods for the Clinical and Biochemical Laboratory,” pp. 47-51, 88. Ann Arbor Science Publishers, Ann Arbor, Michigan. RETIEF, G. P. 1964. A comparison of equine piroplasmosis in South Africa and the U.S. Journal of the American Veterinary Medical Association 145, 912-916. ROBERTS, E. D., MOREHOUSE, L. G., GAINER, J. H., AND MCDANIEL, H. A. 1962. Equine piroplasmosis. journal of the American Veterinary Medical Association 141, 1323-1329.
&Zh?SiU
INFECTIONS
77
SCHALM, 0. W. 1965. “Veterinary Hematology,” 2nd ed., Lea and Febiger, Philadelphia. SCHNITZER, B., SODE~IAN, T., MEAD, M., AND CONTACOS, P. G. 1972. Pitting function of the spleen in malaria: Ultrastructural observations. Science 177, 175-177. SIPPLE, W. L., COOPERRIDER, D. E., GAINER, J. H., ALLEN, R. W., Mouw, J. E. B., AND TEIGIAND, M. G. 1962. Equine piroplasmosis in the U.S. Medical Journal of the American Veterinary Association 141: 694-698. TOPLEY, E. 1968. Anaemia associated wtih splenomegaly among women villagers in the area where malaria is endemic. East African Medical Journal 45, 190-202. TOPLEY, E., BRUCE-CHAWATT, L. J., AND DORRELL, J, 1970. Haematological study of a rodent malarial model. Journal of Tropical Medicine 73, 1-8. W~GHT, 1. G. 1972. Studies on the pathogenesis of Babesia argentina and Babesia bigemina infections in splenectomized calves. Zeitschrift fiir Parasitenkunde 39, 85-102.
Experimental
(1975)
Acute Babesia cabal/i
Infections
I. Red Blood Cell Dynamics PATRICIA
C. ALLEN, United (Submitted
WAYNE
M. FRERICHS, AND A. A. HOLBROOK r
States Department for publication
of Agricultwe September
13, 1973)
ALLEN, PATRICIA C., FRERICHS, WAYNE M., AND HOLBROOK, A. A. 1975. Experimental acute Babesia caballi infections. I. Red blood cell dynamics. Experimental Parasitology 37, 67-77. Hematological determinations were made on blood samples from six ponies acutely infected with two dosage levels of Babesia cabaZZi (Group 1: divided into two subgroups of three ponies each). Similar determinations were made on blood samples from three premunized ponies given challenge inoculations (Group 2), and three equidae given uninfected red blood cells (Group 3). A trend towards decreases in RBC counts, hemoglobin concentrations, and hematocrits within one to four days after inoculation (AI) was observed in all groups. However, it was marked only in Group 1. In addition, only in Group 1 was there observed a concerted anemia occurring between Days 7 and 16. Those surviving ponies in Group 1 which developed a higher parasitemia between Days 5 and 6 AI (first parasitemia peak) developed a more severe anemia between Days 7 and 16. Ponies which developed parasitemias higher than 40 X lo3 parasitized cells/mm3 at the first parasitemia peak subsequently died. Free bilirubin in Group 1 animals increased immediately after inoculation, and repeatedly exceeded normal ranges until after Day 20 AI when the RBC counts were rising. Similar changes in free bilirubin did not occur in either Groups 2 or 3. Conjugated bilirubin levels did not exceed normal ranges in any of the experimental animals. Active erythrophagocytosis was evident in histological preparations of lymph node, spleen, liver, and lung from ponies which died. Cytosiderin pigment was present in liver parenchyma, and hematin was scattered throughout lymph nodes and spleen. INDEX DESCRIPTORS: Babesia caballi: Pony; Equidae; Erythrocyte; Hemoglobin; Hemocrit; Free bilirubin; Erythrophagocytosis; Macrophage; Reticuloendothelial system.
Although anemia is recognized as one of the primary clinical signs of Babe& caballi infection (Roberts et al. 1962; Retief 1964; Sippel et al. 1962), little has been reported about infection-associated changes in hematological parameters on a day-to-day basis in experimentally infected equidae. Data of this type have been reported for 1 Animal Parasitology Institute, Agricultural search Service, Beltsville, Maryland 20705.
Babesia infections in cattle (Wright 1972) and dogs ( Maegraith et al. 1957). The purpose of this present study was to define more closely the anemic processes in experimental acute B. caballi infections. MATERIALS
Source of Babesia caballi Babe&a caballi infected RBCs taken from liquid nitrogen storage (Frerichs et al.
Re-
67 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
68
ALLEN,
FRERICHS
AND
TABLE Experimental Experimental groups
HOLBROOK
1
Groups Used to Study Red Blood Cell Dynamics Animal No.
Species
-
Approximate age
Infect)ive dose of piroplasms
of Babesia caballi Infections Rate of transfusion of packed RBCs
Date of experiment
Group 1 Acute infections A
155 154 152
Pony Pony
Acute infect,ions B
153 132 119
Colt Colt ,4dult
I x 109
2 ml,‘kg body wt
2 ,‘5,‘7&3,!‘30,‘72
Pony Pony
Adult Colt Adult
1 x 10”
0.2 ml, ‘kg body w%
2 !5,f72-3/30/72
154 153 119
Pony Pony pony
Colt Adult Adult
1 x 10”
1 ml/kg
161 149 148
Pony Blwro Horse
Adult Adult Adult
None
1 mll’kg body r+?
POllJ'
Pony
Group 2 Challenge infection of premunized ponies
body wt
4/18/X-5/17/72
Group t7 Transfusion with uninfected RBCs
1968) were used to initiate a series of rapid passages in splenectomized ponies and burros until parasitemia was great enough to cause acute infections. The original source of the parasite was infected ticks obtained in 1963 from an enzootic area in Florida. Experimental ponies were infected by intravenous inoculation of a volume of packed RBCs containing the desired number of piroplasms (Table I). Experimental
Animals
All animals were purchased in Maryland, where naturally occurring B. caballi infections have not been found. Serums from animals used for acute infection (Groups 1A and 1B) and from animals which received uninfected RBCs (Group 3) were negative in the CF test (Frerichs et al. 1969). Before and after inoculation, all animals were maintained in a barn held at 20 C, and were fed mixed horse feed (Wayne New Hope complete horse feed, General Offices, Allied Mills, Inc., Chicago, Illinois 60606) twice a day, with water available ad lib. Rectal temperatures were
taken twice daily the experiments. Experimental
at feeding
5,:17/72-6:‘29/72
time during
Groups
The groups of experimental animals are defined in Table I. Group 1 animals had not been exposed previously to B. caballi. Group 2 was comprised of three of the four animals which survived infection in Group 1 and were considered premunized. The animals in Group 3 were chosen specifically to determine whether transfusion of heterologous RBCs alone would produce any changes in blood values similar to those observed in acute infections. Each animal’s blood was cross-matched before transfusion with that of its respective donor. Pony 161 had been sensitized previously to RBCs, and its serum caused slight agglutination of its donor’s RBCs. Serums from Burro 149 and Horse 148 did not agglutinate their respective donor’s RBCs. Collection
of Blood Samples
Blood was collected by jugular venepuncture into evacuated tubes with and
RBC
DYNAMICS
IN
without EDTA (B&D Vacutainers, Becton, Dickinson, Rutherford, New Jersey 07070). Preinoculation samples were collected from all animals to establish baseline values. In Group 1, samples were collected on Days l-30, 33, 37, and 43-50 after inoculation. In Group 2, samples were collected on Days 1-14, 19, and 26. In Group 3, collections were made on Days 1-16, 22, and 29. Hematologic
Determinations
Total erythrocytes were counted in hemocytometers according to standard methods (Schalm 1965). Hemoglobin was decyanmethemoglobin termined by the method ( Mattenheimer 1970). Hematocrits were determined by the microcapillary method (International Equipment Company, Boston, Massachusetts 02135). Bilirubin (total and direct ) was determined by a modification of the Jendrassik-Grof procedure ( Mattenheimer 1970). All blood smears were stained with Giemsa and examined for parasites. The number of parasitized cells in 100 oil immersion fields ( 1000x ) was counted. The number of parasitized cells per mm3 was calculated on the basis of the daily RBC count. Postmortem Studies Tissues from animals which died during acute infection (Ponies 155 and 152) were fixed in 10% neutral, buffered formalin, sectioned at 6 pm and stained with hematoxylin and eosin (H&E), azure eosin (A&E ) and Prussian blue. RESULTS
Two (Ponies 155 and 152) of the three ponies which were given 1 x lo” piroplasms died. All three ponies that received 1 x 10s organisms survived. Parasites were found in blood smears from acutely infected ponies on Days l-23 and sporadically thereafter through Day 29. There were two apparent peaks of parasitemia roughly six days apart; the first at 5-6 days, the other
&Zbc?SiU
INFECTIONS
69
at lo-12 days. Pony 152, which died, showed a third peak at Day 17 (Fig. 1). No parasites were observed in any postchallenge blood smears from Group 2 ponies ( premunized animals ), but these animals did react with a transient fever (38.3-38.8 C for two to three days AI). All ponies in Group 1 (acute infections) showed decreases in RBC counts and related values during Days l-4 AI (Fig. 1). The maximum decreases in RBC counts averaged about 2.3% below the respective mean preinoculation counts. Similar decreases were observed in Group 2 ponies and in Horse 148 and Burro 149 from Group 3 (Fig. 2). However, the maximum decreases in RBC counts averaged only 11%) (Group 2 ponies) and 14% (Horse 148 and Burro 149) below the respective mean preinoculation values. The RBC counts for Group 1 ponies tended to return towards normal ranges during Days 4-7 AI. Maximum RBC counts during this time coincided with the first peak in parasitemia (Fig. 1). Pony 155 died during this period. Similar, but less concerted changes in blood values were observed in Group 2 ponies, but no parasites were seen in blood smears. In all acutely infected animals (Group 1)) the RBC counts and related values began a secondary decrease after the first peak in parasitemia. Decreases in RBC counts were moderate and transient for Ponies 153 and 119 (about a 27% average maximum decrease occurring between Days 7 and 9 AI ) . They were more extensive and prolonged for ponies 154 and 132 (about a 59F average maximum decrease occurring between Days 11 and 16 AI). During this time, parasitemia reached a second broader peak (Fig. 1). Pony 152 was unusual in that the secondary decrease in RBC count was delayed for several days. However, just prior to death a 66% decrease in its RBC count was observed. No concerted decreases in RBC counts were observed in either Groups 2 or 3 be-
ALLEN,
70
lnoculoted
9
FRERICHS
p - - -” <_a *----
Days otter inocutotion
HOLBROOK
Inoculated
with I X10’ p~roplasms
Group IA
AND
155 154 152
wth IXIO’
Group 18
Day, after inoculotim
piroplosms
RBC DYNAMICS
IN Babesia
Challenged with IX104 piroplams
INFECTIONS
71
Inoculated with uninfected erythrocytes
Group3 w-_-o
5
IO
15
II9
20
25
30
Days ofter inoculation
FIG. 2. Hemograms of immune (Group 2) and animals inoculated
5
c_ ,--,I49 *__--4
161 148
30
IO 15 20 25 Days ofter inoculation
ponies challenged with 1 X lo4 Babe& caballi with uninfected erythrocytes (Group 3) I
piroplasms
FIG. 1. Hemograms of ponies 1B) Babesia cabal& piroplasms.
inoculated
with
1 X 10” (Group
1A)
and 1 X lo*
(Group
72
ALLEN,
FRERICHS
tween Days 7 and 11 AI. However, transitory decreases were observed in both groups between Days 12 and 15. The maximum decreases averaged about 15% for both groups. After 16 days AI, the RBC counts, hemoglobin concentrations, and hematocrits of the surviving acutely infected ponies (Group 1) returned towards their respective mean preinoculation levels. By Day 29, Ponies 153 and 119 were normal. However, Ponies 154 and 132 were still below normal even 50 days AI. Blood values for animals in Groups 2 and 3 remained normal during this time. Under our experimental conditions, the average free plasma bilirubin level before inoculation was 0.547 mg/lOO ml, with a range (-t 2 SD) of 0.029-1.065 mg/lOO ml. In the acutely infected ponies (Group 1 ), the free bilirubin rose above the normal range; two- to threefold increases were observed through Day 20 AI (Fig. 1) . During the first 20 days AI, the ponies given 1 x 10” piroplasms (Group IA) had free bilirubin levels slightly higher than those given 1 x lo* piroplasms (Group 1B). High values for free bilirubin were observed in Ponies 119 and 132 even beyond 20 days AI. This period of frequent high free bilirubin values (l-20 days AI) coincided with decreases in RBC counts, hemoglobin levels, and hematocrits, and with the presence of parasitized erythrocytes in blood smears (Fig. 1). Free plasma bilirubin did not increase above the normal range in the challenged ponies (Group 2) after inoculation. Two periods of slight increase were observed in the animals receiving unparasitized RBCs (Group 3); one at l-4 days AI and the other at 13-15 day,s AI. These periods coincided with observed dccrcases in RBC c0u11ts, hemoglobin concentrations and hematocrits ( Fig. 1). No hemoglobinemia was obscrvcd in any of the acutely infected animals, and the conjugated bilirubin did not exceed normal ranges.
AND
HOLBROOK
At necropsy, both Ponies 155 and 152 showed edema of the subcutis and marked icterus. The lungs were edematous and petechiated, with pleural effusion, and the hearts showed petechiation and pericardial effusion. The livers were swollen and pale with rounded borders. The spleens were *fibrotic, moderately enlarged, and had some hemorrhages. The kidneys were pale with a few petechiae; the capsules were slightly adherent. Large numbers of macrophages containing phagocytized RBCs were visible in histological preparations of lymph node from both Ponies 155 and 152. Many of the phagacytized RBCs in Pony 155 preparations were still unaltered (Fig. 3), and stained red with A&E and H&E. Many macrophages also contained RBC debris which stained green with A&E but which was not Prussian blue positive. This indicated that RBC degradation at these sites was in its early stages. On the other hand, most of the swollen macrophages in lymph node from Pony 152 contained fragmented RBCs in a more advanced stage of degradation (Fig. 7), which stained yellow with A&E and H&E, and blue with Prussian blue ( hemosiderin, Lillie 1965). Enhanced phagocytic processes were plainly evident in spleens from both animals (Figs. 4 and 8). The spleen from Pony 152 appeared depleted of RBCs (Fig. 8 ), whereas the spleen from Pony 155 was engorged with them (Fig. 4). Macrophages containing debris (yellow, A&E; blue, Prussian blue) were seen in cleared areas within masses of RBCs (Fig. 4). Lymph nodes and spleens from both animals contained crystalline pigment similar to the formaldehyde hcmatin pigment found in malarial infections (Lillie 1965). The livers from both Ponies 155 and 152 exhibited centrilobular necrosis. The sinuses were swollen, and a slight leucocyte infiltration was obsrrvcd. Parcnchymal cells from both animals contained cytosiderin (not Prussian blue positive, Lillie 1965)
RB~ DYNAMICS
IN Babesia
and showed cloudy degeneration (Figs. 5 and 9). Kupffer cells containing degraded RBCs were readily observed within the sinuses, and they appeared more plentiful than in Pony 155. Many more macrophages containing RBC debris (Prussian blue positive, yellow with A&E ) were present within alveolar capillaries of lung from Pony I52 than in lung from Pony 155 (Figs. 6 and IO).
DISXJSSION
A comparison of Figs. 1 and 2 reveals that within Days l-6 AI the patterns of change in hematological parameters were similar in all three experimental groups. All experimental animals except Pony 161 exhibited a noticeable decrease in RBC count during this period. This phenomenon may represent the normal response of the reticuloendothelial system (RES ) to the introduction of excess RBCs (Jandl et al. 1965). The phagocytosis of erythrocytes by macrophages in lymph nodes and spleen is evident in stained sections from Pony I55 which died on Day 7 (Figs. 34). However, the decrease in RBC counts of the acutely infected ponies averaged about twice as great as those in Groups 2 and 3, and only in the acutely infected ponies was there an immediate increase in free plasma bilirubin over the normal range. Thus, although erythrophagocytosis may have taken place in all experimental animals, significant hemolytic reactions developed only in the acute infections. The return of the RBC counts to normal range in Group 1 ponies coincided with the first parasitemia peaks (Fig. 1). That these two events occurred simultaneously indicates that in these animals the RES may have become temporarily saturated with phagocytized cells, thus allowing a buildup in the circulation not only of the total number of RBCs but of the parasitized cells as well. Jandl (1967) showed that such a blocking effect occurs in acquired
INFECTIONS
73
hemolytic anemias and severe transfusion reactions, True anemia developed in the acutely infected ponies (Group 1) after the first parasitemia peak between Days 7 and 16 AI. During this time, parasitemias reached a second broader peak and RBC counts attained minimum values, although these events did not occur simultaneously (‘Fig. 1). Although it would appear that the degree of anemia is correlated with infective dose level, it is difficult to establish such a correlation with the relatively small number of animals in Group 1. However, there does seem to be a strong correlation between the degree of parasitemia evident at the first peak (Days 5-6 AI) and the degree of anemia developing subsequently. Additionally, it would appear that those animals will succumb to infection which attain a parasitemia greater than 40 x lo3 parasitized cells/mm3 at the first peak in parasitemia. Since neither hemogIobinemia nor hemoglobinuria were observed in Group 1 animals during Days 7-16, all significant RBC destruction must have taken place within the RES (Jandl 1967), not intravascularly. The observed anemia could thus be the result of increased phagocytic activity of a stimulated and proliferating RES (George et al. 1966; Topley et al. 1970; Jandl et al. 1965; Kelly et al. 1960), as exemplified by the enlarged spleens of Ponies 155 and 152, and the apparent high activity of macrophages in histopathological preparation from these animals. The development of a second parasitemia peak may again be the result of another temporary blockade of the RES, and represent those parasitized cells which escaped phagocytosis or “pitting” (Conrad 1969; Schnitzer et al. 1972 ) . The progressive involvement in RBC destruction by the RES in organs other than the lymph nodes and spleen were apparent in sections of liver and lung from Ponies 155 and 152. There appeared to be
74
ALLEN,
FRERICHS
AND
HOLBROOK
FIGS. 3-6. Histopathological sections from Pony 155. FIG. 3. Lymph node 300~ (A&E): hlacrophages (hl) contain both undegradcd RBCs (arrow) and RBCs in early stages of decomposition ( * ). FIG. 4. Spleen 300x (.4&E ) engorged with RBCs ( * ) : hiacrophages ( hl ) containing RBC debris; hematin (H). FIG. 5. Liver 300X (A&E): Kupffer cells (K) containing RBC debris; cytosiderin (C) in parenchyma cells. FIG. 6. Lung 300~ (Prussian blue): only a few macrophages containing Prussian blue positive material (P) present in alveolar capillaries.
RBC DYNAMICS
IN
Bahesin
INFECTIONS
FIGS. 7-10. Histopathological sections from Pony 152. FIG. 7. Lymph node 300X (A&E): Macrophages (M) containing RBC debris. FIG. 8. Spleen 405X (A&E) depleted of RBCs: Macrophages (M) containing RBC debris. FIG. 9. Liver 405X (A&E): Kupffer cells (K) containing whole RBCs (“) and debris (arrow); cytosiderin (C). FIG. 10. Lung 300X (Prussian blue): Many macrophages containing Prussian blue positive material (P) present in alveolar capillaries.
7G
ALLEN,
FRERICHS
a proliferation of Kupffer cells in the liver of Pony 152 which died on Day 21 AI as compared with Pony 155 which died on Day 7. Many macrophages laden with degraded RBCs were present in lung sections from Pony 152 (Fig. lo), whereas they were rare in lung from Pony 155. The hematological patterns of change exhibited by Pony 152 were delayed for several days as compared with the other animals in Group 1. However, the trends remained similar. The reason for the delays is not known, but there was a concurrent bacterial infection which might have caused this effect. Microcolonies of bacteria were found in sections of lymph node and other tissues. It is interesting to note that the patterns of change observed in Group 2 ponies (challenged, premunized) more closely resemble those of Group 3 than Group 1. No parasitemia was observed, and no prolonged decrease in RBC count occurred after Day 7. We had expected to see some parasitemia and anemia. However, the challenge dose of I x lo4 piroplasms may not have been sufficient to saturate the already proliferated RES of these animals. We emphasize that no severe hemolytic reactions occurred in Group 3 animals. We had expected any adverse effects of transfusion per se (due to incompatability of blood antigens) to be demonstrated in these animals. The hemolytic reactions (presence of free bilirubin in the blood ) in the acutely infected ponies and the early death of Pony 155 are more than likely due only to the presence of B. cabal& in the blood. The animals in Groups 2 and 3 did show transitory decreases in RBC counts and related values between Days 10 and 16 AI (Fig. 2). This decrease coincided with a very slight rise in free bilirubin in Group 3. This phenomenon may also be a natural response of the RES to transfused RBCs (see citations by Berlin 1964)) and may be masked in acutely infected animals by the
AND HOLBROOK
more prominent response to B. cahalli infected cells. We conclude that in experimental B. infections initiated by transfusion caballi of parasitized RBCs, the host’s physiological reaction to the RBCs may influence the patterns of change in erythrocyte counts and related values during Days l-6 AI, perhaps even regulating the time at which the first peak in parasitemia occurs. Hematological changes which occur after this time appear to be due primarily to the reaction of the host to the presence of B. caballi. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of R. Ellis, D. Davis, 0. McGaha, L. Owens, and J. Hoang. We thank D. Thompson for preparing the histologic sections. REFERENCES N. I. 1964. Life span of the red cell. In “The Red Blood Cell,” pp. 423-450. (C. Bishop and D. M. Surgenor, Eds.) Academic Press, New York. CONRAD, M. E. 1969. Pathophysiology of malaria. Hematologic observations in human and animal studies. Annals of Internal Medicine 70, 134-141. FRERICHS, W. M., JOHNSON, A. J., AND HOLBROOK, A. A. 1968. Storage of Babesia cabal& and Babe& equi in liquid nitrogen. Journal of Parasitology 54, 451. FRERICHS, W. M., HOLBROOK, A. A., AND JOFZVSON, A. J. 1969. Equine piroplasmosis: Complement fixation titers of horses infected with Babesia caballi. American Journal of Veterinary Research 30, 697-702. GEORGE, J. N., STOKES, E. F., WICKER, D. J., AND CONRAD, M. E. 1966. Studies of the mechanisms of hemolysis in experimental malaria. Military Medicine 131 (Supplement), 12171224. JANDL, J. H. 1967. The spleen and reticuloendothelial system. In “Pathologic Physiology” (W. A. Sodeman and W. A. Sodeman, Jr., Eds. ), Saunders, Philadelphia. pp. 897-932. JANDL, J. H., FILES, N. M., BARNETT, S. B., AND MACDONALD, R. A. 1965. Proliferative response of the spleen and liver to hemolysis. Journal of Experimental Medicine 122, 299326. BERLIN,
RBC
DYNAMICS
IN
KELLY, L. S. DOBSON, E. L., FINSEY, C. R., AND of the HIRSCH, J. P., 1960. Proliferation the liver. reticuloendothelial system in American Journal of Physiology 198, 11341138. LILLIE, R. D. 1965. “Histopathologic Technic and Practical Histochemistry,” 3rd ed, McGrawHill, New York. MAEGRAITH, B. G., GILLIES, H. M., AND DEVATJKUL, K. 1957. Pathological processes in Babesia infections. Zeitschrift ftiT Tropencanie medizine und Parasitologic 8, 485514. MATTENHEIMER, H. 1970. “Micromethods for the Clinical and Biochemical Laboratory,” pp. 47-51, 88. Ann Arbor Science Publishers, Ann Arbor, Michigan. RETIEF, G. P. 1964. A comparison of equine piroplasmosis in South Africa and the U.S. Journal of the American Veterinary Medical Association 145, 912-916. ROBERTS, E. D., MOREHOUSE, L. G., GAINER, J. H., AND MCDANIEL, H. A. 1962. Equine piroplasmosis. journal of the American Veterinary Medical Association 141, 1323-1329.
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SCHALM, 0. W. 1965. “Veterinary Hematology,” 2nd ed., Lea and Febiger, Philadelphia. SCHNITZER, B., SODE~IAN, T., MEAD, M., AND CONTACOS, P. G. 1972. Pitting function of the spleen in malaria: Ultrastructural observations. Science 177, 175-177. SIPPLE, W. L., COOPERRIDER, D. E., GAINER, J. H., ALLEN, R. W., Mouw, J. E. B., AND TEIGIAND, M. G. 1962. Equine piroplasmosis in the U.S. Medical Journal of the American Veterinary Association 141: 694-698. TOPLEY, E. 1968. Anaemia associated wtih splenomegaly among women villagers in the area where malaria is endemic. East African Medical Journal 45, 190-202. TOPLEY, E., BRUCE-CHAWATT, L. J., AND DORRELL, J, 1970. Haematological study of a rodent malarial model. Journal of Tropical Medicine 73, 1-8. W~GHT, 1. G. 1972. Studies on the pathogenesis of Babesia argentina and Babesia bigemina infections in splenectomized calves. Zeitschrift fiir Parasitenkunde 39, 85-102.