Br. vet.]. (1975),131,601

EQUINE PIROPLASMOSIS: LEUKOCYTIC RESPONSE TO BABESIA EQUI (LAVERAN, Ig01 ) INFECTION IN CHILE By W.

RUDOLPH,

J.

CORREA,

L.

ZURITA AND

W.

MANLEY

Department of Pathology and Clinical Science, School of Veterinary Medicine, University of Chile, Santiago, Chile SUMMARY

Leukograms from acute field cases in the parasitaemia stage of Babeusa equi infection were analysed. These were compared to leukograms from two 12year-old horses, which were clinically normal and serologically negative to equine piroplasmosis before being inoculated with infected B . equi blood. A wide variation in the leukograms was observed, monocytosis and eosinopenia being the most common variation. Plasma fibrinogen increased early and was sustained throughout the observation period in the experimentally infected horses. INTRODUCTION

Equine piroplasmosis (babesiasis), a world-wide disease, was diagnosed in Chile for the first time in 1970 (Rudolph, 197 I). Since then, many clinical cases, mainly caused by Babesia equi, (Nuttallia equi) have been diagnosed in thoroughbred horses in Santiago Province. It is well known that babesiasis, a hemolytic disease, produces a normocyticnormochromic anaemia of variable severity (Levine, 1961; Sippel et al., 1962; Schalm, 1965), but only limited observations of the leukocytic response have been described. Russian investigators (Ershov, 1965) found a shift to the left in the neutrophils and a mild lymphocytosis as well as monocytosis in 50 per cent of the sick animals. Roberts et al. (1962) observed a leukopenia, neutropenia and lymphopenia just prior to the appearance of parasites in the blood. Taylor et al. (1969) in a review of equine piroplasmosis in the United States, reported a transient leukopenia and lymphopenia which progressed to lymphocytosis in a bout 10 days. The present paper records the leukograms obtained in acute field cases and the leukocyte, packed cell volume (PCV) and fibrinogen changes observed in experimentally infected horses. MATERIALS AND METHODS

Observations were made during parasitemia (B. equi) on 53 acute field cases involving thoroughbred horses ranging in age from 2 to 6 years. Clinical signs

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of the disease were observed in each case and a complete haemogram was obtained within 12 h after blood collection. Experimental infection was carried out in two clinically normal 12-year-old thoroughbred horses negative to the equine piroplasmosis complement fixation test. (B. equi antigen was donated by V.S.D.A.) Both animals received 500 ml of blood infected with B. equi intravenously and 10 ml subcutaneously. Infected blood for horse I was obtained from an acute field case with a parasitized erythrocyte concentration of the order of I per cent. Horse 2 was inoculated with blood from horse I at the height of its parasitaemia ('" 4 per cent). A clinical and haematological examination was performed daily until the horse died or recovered. Total blood cell counts were done using the haemocytometer. Haemoglobin was determined as cyanmethemoglobin (Schalm, 1965) and the microhaematocrit (Adams Autocrit Centrifuge) was used for the PCV determination. Plasma protein and fibrinogen were determined by refractometer methods (Kaneko & Smith, 1967). Blood films prepared on cover slips were stained with Giemsa solution for I h. Five hundred erythrocytes were counted in oil immersion fields using an ocular crossline disc and the percentage of parasitized cells was recorded. Differential leukocyte counts were performed on each blood by two persons following the recommendations of Schalm (1965). Haemograms were interpreted using normal values for thoroughbred horses (Schalm, 1965).

RESULTS

The most common change observed in the leukograms from the acute field cases was a significant increase in relative and absolute values of monocytes and an absence of eosinophils in approximately 60 per cent of the sick horses (Table I and Fig. I). Variations in other leukocyte parameters were much smaller and no relationship was observed between absolute values ofmonocyest and number of parasitized erythrocytes. All changes were somewhat greater in anaemic as compared to non-anaemic horses (Fig. I). A normocytic-normochromic anaemia was observed in the 49 per cent of the total animals analysed. Both experimental horses showed the typical clinical signs of equine piroplasmosis (fever, icterus, depression) and a marked decrease of erythrocytes (Fig. 2). Because horse 2 was down in his stall, could not rise, and was passing blood in his urine, he was killed on the seventh day post-inoculation. The peak of the parasitaemia was observed 5-6 days post-inoculation, and was higher in horse 2 than in horse I. The white blood cell changes were similar in both experimental animals. Total leukocytes, lymphocytes and eosinophils decreased to a variable degree during the first 24-48 h post-inoculation, the leukocytes, increasing later and reaching a peak at 4-5 days. Neutrophils increased abruptly at 24 h, decreasing later below the original level (Fig. 2). Total plasma proteins did not change during the acute stage of the disease,

TABLE I LEUKOGRAM CHANGES IN ACUTE FIELD CASES OF PIROPLASMOSIS

Classification by Leukocyte counts Leukocytosis 16,655±3976 12 horses (22·6%)

Number of animals

2 1

I

2

Leukopenia 45 00 -4708 2 horses (3.8 %) Leukocytes: normal values 9 156 ± 17 03 39 horses (73.6 %)

13

5

4 4

4

(B. equi),

BASED UPON ABSOLUTE VALUES

Shift to the Left

Neutro philia

Neutro penia

Lympho cytosis

Lympho penia

Mono cytosis

Eosino penia

Yes Yes Yes Yes No No No No Yes No

Yes Yes Yes Yes Yes Yes No No Yes Yes

No No No No No No No No No No

No No Yes No No No Yes No No No

No Yes No No No No No No No No

Yes Yes Yes Yes Yes Yes Yes Yes No No

No Yes Yes Yes Yes No No Yes Yes No

No No

No No

Yes Yes

No No

No No

No No

Yes No

No No Yes Yes Yes No No No No No Yes No No No

No Yes No Yes No No No No No Yes No No Yes No

No No No No No No No No No No No No No No

No No No No No No No No Yes No No No No No

No No No No No No No No No Yes No Yes No Yes

Yes Yes Yes Yes Yes Yes No No No No No No No No

Yes Yes Yes No No No No Yes No Yes No Yes Yes No

ttl

@ ...... Z

ttl 't1 ......

::t'

0

't1 t"'

> Cfl

s:: 0

Cfl ...... Cfl

g> ~

BRITISH VETERINARY JOURNAL,

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5

100

100

50

50

....c'" s

z: C

.... .... c '" ..... c::>

z:

.....

c..>

...."".....

o

o 1L

DL

I INCREASED D DECREASED SL SHIFT TO THE LEFT

SL

IN

DN

L LEUKOCYTES N NEUTROPHILS Ly LYMPHOCYTES

I Ly

DLy

1M

DE

M MONOCYTES E EOSINOPHILS

Fig. I. Frequency of leukogram variations observed in acute field cases of B. equi infection. White bars represent non-anaemic horses and black bars represent anaemic horses.

but fibrinogen increased early and reached its maximum between the third and fifth day post-inoculation (Table II). Monocytosis accompanied and followed the parasitaemia. Eosinopenia developed in horse 2 but eosinophil numbers were variable in horse I (Fig. 2) . Plasma inclusions resembling babesia parasites were observed sporadically in segmented neutrophils (Fig. 3). DISCUSSION

Phagocytosis of parasites by monocytes and neutrophils is a common biologic phenomenon, so an increase in phagocytic cells, mainly monocytes, in a parasitic disease as piroplasmosis may be expected. In our observations on acute field cases, relative and absolute monocytosis and eosinopenia were the most frequent leukocytic variations observed, and is in agreement with Russian reports (Ershov, 1965). At the same time normal values for total leukocytes (73.6 per cent) were more common than was leukocytosis (22·6 per cent) or leukopenia (3.8 per cent) (Table I) . In order to restrict the leukogram study to the acute stage of the naturally

PCV%

To ta l l eukocyte s

per cmm. 'Ol

Immature neulrophils %

MOl ure neulrophils %

Lymphocytes %

Monocyles %

Eosinophils %

Bosophi lS %

Porasitized er ylhrocyles %

Days (post ino culo tion)

.

SO~

PCV%

Total leukocytes per cmm .. 10. 3

40e--r .- .--.-.~

~ 20

Moture neutrophi/s %

-.

8.- . __./.~...

:~f sE.

Immature neutrophils %

4~

-.

o.

2 _._

--.

___

And.~.

. ........

;;,.,0

Monocytes %

~~t~·--·--·-·~ sot .--.

~~~~.--.-.--.--.--

20 ~ l Ot

O~-.--..--·-

Eosinophils %

,

.~

30.:

Lymphocytes %

Horse 2

•

• ._.-

4~

2. 4t 2.

O ~- . - - . - - . - - . - - . - - .

Bosophils %

Parasitized erythrocytes

%

ot-. . . . . . .. --.-·-.-·-.

2100 ~

00

.-.-.

.uthonized

trw

._.-, 2

3

4

I

,

S

6

Day. I post inoculOlioo)

Fig. 2. Leukogram changes in relation to parasitized erythrocytes in blood of experimental horses infected with B. equi. Leukocytes from horse I showed some degree of aggregation.

O'l

o

O'l

-OJ

::0 >-3

TABLE II TOTAL PLASMA PROTEINS AND FIBRINOGEN IN HORSES EXPE R IMENTALLY INFECTED WITH

D ays after inoculation

Plasma proteins (gm %) Fibrinogen (mg %) PP:F-ratio Plasma proteins (gm %) Fibrinogen (mg %) PP :F-ratio

PP = Plasma proteins_ F = Fibrinogen _

0

2

3

4

5

6

7

en

B a besia equi

8

9

32

H ORSE I

7-5 300 24 -0

7-5 200 36 -5

7-7 400 18-2

7-5 500 14-0

6-8 200 33- 0

7-1 400 16- 7

7-1 200 34-5

7-4 600 I 1-3

HOR SE

7-6 600 I I -5

7-2 600

6-7 400 15"7

6-2 500 I 1-4

I I -I

7-1 600 10-8

7-4 500 13 -8

7-6 300 24 -3

~

>-3 ~ ::0

Z ;...

::0 ~

2 7-4 600 I I -3

7- 0 400 16-5

::t: 0 M

U1

EQUINE PIROPLASMOSIS

Fig. 3. Segmented neutrophil showing a cytoplasmic inclusion, which resembles a babesia parasite.

occurring disease, sick horses with parasitaemia were selected. Nevertheless, since half of them already showed anaemia, it is obvious that some were in a more advanced stage of the disease. This variation in the stage of the disease probably resulted in some of the variation in the leukogram (Fig. I, Table I) . The incubation p eriod observed in the experimentally infected animals was shorter than the incu ba tion period reported for naturally infected animals (Levine, 1961; Retief, 1964) . This is probably related to the relatively large inoculums received by the experimental horses. The leukocytic change observed in both animals was similar despite the differing number of parasites injected. However, horse 2, which received more parasites, showed a higher parasitaemia (maximum 14 per cent) and it was necessary to kill the animal in extremis. Nevertheless, the percentage parasitaemia was substantially less than that usually reported in clinical infections due to B. equi (Ershov, 1965). The early and marked increase of mature neutrophils 24 h post-inoculation cannot be due to a specific reaction to the inoculated babesias. Since lymphocytes and eosinophils were below the normal range, a stress reaction against the total blood inoculated, including toxins and other substances circulating in the diseased animal, must be considered. On the other hand, the increasing percentage of band cells and the appearance of metamyelocyte could be a response to the depletion of neutrophil storage. This reaction lasted for 4 days in horse 2 but only 2 days in horse 1. The early leukopenia and lymphopenia observed is partially in agreement with the observations of Roberts et at. (1962), who inoculated 1 litre of B. caballi infected blood subcutaneously into a 12year-old horse and observed leukopenia, neutropenia and lymphopenia just prior to the appearance of parasites in blood. Monocytes increased after artificial infection, following the increasing

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parasitaemia to a maximum 4 to 5 days after challenge. However, when the number of monocytes was compared with the percentage of parasitized erythrocytes in the acute field cases, no relationship was observed; this is not in agreement with Ershov (1965) who described an inverse relationship between number of monocytes and parasitized erythrocytes. Total values for plasma proteins were not affected by the babesiosis infection but fibrinogen values increased and plasma protein: fibrinogen ratios decreased significantly beginning 48 h after challenge and continuing during the observation period. Since increases of plasma fibrinogen may be indicative of a disease in general and more specifically of an inflammatory process (Schalm, Smith & Kaneko, 1970), its elevation in a disease like piroplasmosis was not unexpected. Observations on fibrinogen levels in equine piroplasmosis have not been reported previously. The variable leukograms from the acute field cases of piroplasmosis resembled partially the variations observed at different post-inoculation times in the experimental horses. The differences from those reported in the available English literature on this subject could be related to the mild character of piroplasmosis in Chile. Despite the fact that more than 90 per cent of the piroplasmosis observed in Chile since 1970 has been caused by B. equi, which is recognized as the more pathogenic piroplasma of horses (Levine, 196 I), no mortality has been observed in the field and the clinical signs in most of the horses were mild. The absence of the classical vector (tick-borne) in Chile (Tagle, 1971) q)Uld account for a milder response to an otherwise more pathogenic babesia. ACKNOWLEDGEMENT

The authors are grateful to the Veterinarian Staff of Club Hipico and Hipodromo of Santiago and to the Institucciones Hipicas de Chile for its financial assistance. REFERENCES ERSHOV, V. S. (1965). Parasitology and Parasitic Diseases of Livestock. Moscow: State Publishing House for Agricultural Literature. KANEKO, J. J. & SMITH, R. ( 1967). Calif. Vet. :U, 2 I. LEVINE, N. D. ( 1961). Protozoan Parasites of Domestic Animals and Man. Minnesota: Burgers Publishing Co. RETIEF, G. P. (1964). J. Am. vet. med. Ass. 145,912. ROBERTS, E. D., MOREHOUSE, L. G., GAINER, J . H . & McDANIEL, H. A. ( 1962). J. Am. vet. med. Ass. 141, 1232. RUDOLPH, W. (1971 ). Bol. Chile. Parasit. 26,66. SCHLAM, 0. W. ( 1965). Veterinary Hematology, 2nd edn., Philadelphia: Lea & Febiger. SCHALM, 0. W., SMITH, R . & KANEKO, J. J. (1970) . Calif. Vet. 24,9· SIPPEL, W. L., COOPERRIDER, D. E., GAINER, J. H., ALLEN, R. W., Mouw, J. E. & TIEGLAND, M. B. (1962). J. Am. vet. med. Ass. 141,694. TAGLE, I. (1971). Bol. Chile. Parasit. 28,46. TAYLOR, W. M., BRYANT, J. E., ANDERSON, J. B. & WILLERS, H . K . H. ( 1969). J. Am. vet. riled. Ass. 155,915. (Accepted for publication 13 August 1974)

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Piroplasmose equine: Reponse leueoeytaire II. l'infeetion provoque par Babesia equi (Laveran, IgoI) au Chili (Rudolph et al.) ResUlDe. On analysa les numerations leucocytaires des cas aigus survenus dans les champs du stade parasitaire de l'infection par Babesia equi. On les compara a des numerations leu cocytaires de 12 chevaux ages de 12 ans, qui etaient cliniquement normaux et dont la serologie etait negative it la piroplasmose equine avant d'etre inocuIes avec du sang infecte par B. equi. On observa une large variation des numerations leucocytaires, la monocytose et l'eosinopenie etant la variation la plus courante. Le fibrinogene plasmatique s'elevait precocement et se maintenait pendant la periode d'observation chez les chevaux infectes experimentalement. PiroplasIDose bei Pferden. Leukozytenreaktion auf Babesia-equi-(Laveran, IgoI )-Infektion in Chile (Rudolph et al.) ZusaDlDlenfassung. Das weisse Blutbild freilebender Pferde, die sich im parasitiiren Stadium einer Babesia-equi-Infektion befanden, wurde analysiert und mit dem 12 Jahre alter, klinisch normaler Pferde verglichen, deren Serum negativ auf Piroplasmosis equi reagierte, bevor sie mit Blut inokuliert worden waren, dass mit B. equi infiziert war. Die weissen Blutbilder variierten stark. Die am hiiufigsten bemerkte Variation betraf monozytose und Eosinopenie. Plasmafibrinogen war frtihzeitig vermehrt. Dies blieb so wiihrend der ganzen Beobachtungszeit der experimentell infizierten Pferde. Piroplasmosis equina: La respuesta leueocitiea a la Weedon por Babesia equi (Laveran, IgoI) en Chile (Rudolph et al.) ResUIDen. Se analizaron leucogramas de casos agudos naturales en la etapa parasitemia de la infecci6n por Babesia equi. Estos se compararon con leucogramas obtenidos de dos caballos de 12 alios de edad, que eran clinicamente normales y serologicamente negativos en cuanto a la piroplasmosis equina hasta que se les inocul6 con sangre infectada con B. equi. Se observo una amplia variacion en los leucogramas, siendo monocitosis y eosinopenia la variac ion mas comlin. Los fibrinogenes plasmaticos aumentaron temprano y se mantuvieron a este nivel durante el periodo de observacion en el caso de los caballos infectados experimentalmente.