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 - - -”

Experimental acute Babesia caballi infections. I. Red blood cell dynamics.

EXPERIMENTAL PARASITOLOGY 37, 67-77 Experimental (1975) Acute Babesia cabal/i Infections I. Red Blood Cell Dynamics PATRICIA C. ALLEN, United (S...
4MB Sizes 0 Downloads 0 Views