156 NEW ZEALAND VETERINARY JOURNAL VOL. 28

Studies in Sarcocystis Species V: A species infecting dogs and goats; observations on the pathology and serology of experimental

sarcocystosis in goats G. H. Collins, * R. H. Sutton+, w. A. G. Charleston

N.Z.vet.l.2B: 156-8

ABSTRACT Eleven goats given dog-derived Sarcocystis sporocysts,

showed iUness from day 18. Doses of 5 x lot' sporocysts caused a progressive temperature rise, with peaks at 6, 11 and 18 days. Necropsy at 18 and 19 days revealed multiple petechiation and schizogony in endothelial cells. In goats given smaller doses, Hb, PCV and TP levels fell from day 17. IFAT titres rose from day 28.

INTRODUCTION The previous paper(l) described the development of a

dog:goat species in goats. Schizogony, which occurred from 18 days after oral dosing with sporocysts, was associated with death, or illness followed by recovery, depending on the num­bers of sporocysts given. This paper reports the clinical, serological and pathological findings in the experimentally induced disease.

MATERIALS AND METHODS (a) Experimental Procedure

Eleven goats, approximately three weeks old, were given various doses of sporocysts (Table I) of a dog:goat species of Sarcocystis(l). Two goats were left undosed as controls. The rectal temperatures of all the animals were recorded each day at approximately the same time.

(b) Haematology Goats 4 to I3 (Table I) were bled at the beginning of the

experiment (day 0) and then twice weekly until day 4.5, or death if earlier, using EDTA vacuum tubes**. Blood was examined by standard procedures, for haemoglobin concen­tration (Hb), packed cell volume (PCV) and hence the mean corpuscular haemoglobin concentration (MCHC), totalleuco­cyte count (WBC), differential leucocyte count (diff. WBC), total protein (TP) and protein fractions. The level ofthe serum enzymes glutamic oxaloacetic transaminase (SOOT) and lactic dehydrogenase (LDH) were estimated.

(c) Serology Goats 4 to I3 were bled using plain vacuum tubes on day 0

then twice weekly up to day 63, or death if earlier, and all the surviving animals at day 129. Serum titres of Sarcocystis antibodies were estimated by an indirect fluorescent antibody test (IF AT) using, as antigen, bradyzoites harvested by pepsin:HCI digestion(lO) from the muscle of goats 6, 7 and 8 killed on day 129. Known positive and negative sera were included in each test as controls. No haematological or sero­logical examinations were made of the blood of goats 1,2 and 3.

(d) Pathology Necropsies were carried out on goats I to 12 (Table I) and

samples of various tissues prepared for histology by standard techniques as described previously(l).

* Department of Veterinary Pathology and Public Health, Massey University, Palmerston North.

+ Present Address: Department of Veterinary Pathology and Public Health, University of Queensland, St. Lucia, Brisbane, Australia.

**Venboject, Terumo Corporation, Japan.

TABLE 1: EXPERIMENTAL INFECTION OF GOATS WITH SPOROCYSTS DERIVED FROM DOGS

Goat Number Dose of sporocysts Date of necropsy

I 5 x ]06 18 k 2 5 x lot' 19 d 3 5 X ]06 19 k 4 6 x 105 34 k 5 6 x 105 24 d 6 15 x ](f 129 k 7 15 x 10" 129 k 8 3 x 10" 129 k 9 3 x 10" 80 k

]0 15 x 103 34 d 11 15 x 103 93 k 12 Not dosed 42 k 13 Not dosed 143 b

k = killed d = died b = biopsied

RESULT~

(a) Temperature The tempatures of goats I, 2 and 3, which were each given

5 x 106sporocysts, rose from day 2 and the mean temperature curve included 3 distinct peaks at days 6, 11 and 18 (Fig. I). Goats 4 and 5, which were each given 6 x lOS sporocysts, showed marked temperature rises between days 18 and 21 followed by a fall to subnormal temperatures; lesser doses resulted in smaller rises between days 18 and 25. The tem­peratures of the control goats remained within the normal range throughout the experiment.

C

c (5)

38

5 10 15 20 DAYS AFTER INFECTION

Figure I.' Mean temperatures of goats I. 2 and 3 given 5 x 106

dog:goat sp. sporocysts. (c-c = mean of controls ± 2 x s.d.: s = ultimate schizogony: (s) = early phase of schizogony?)

1980 NEW ZEALAND VETERINARY JOURNAL 157

(b) Haematology Haemoglobin and PCV values fell from day 17 in goats 4 and

II and the fall was most marked in goats 4 to 7 (Figs. 2 and 3);

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Figure 2: Hb levels of goats 4 and 5 (dose 6 x 105 sporocysts) and goats 6 and 7 (dose 15 x ](yI sporocysts). (c-c = mean of controls ± 2 x s.d.)

4 % 1 c

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10 20 30 40 DAYS AFTER INFECTION

Figure 3: pev values in goats 4 and 5 (dose 6 x 105 sporocysts) and goats 6 and 7 (dose 15 x lif sporocysts). (c-c = mean of controls ± 2 x s.d.)

the MCHC remained within the normal range. Total protein levels fell in all the infected goats examined (4 to II)fromday 3 but levels remained mostly within the normal range. SOOT levels rose slowly during the first five weeks then declined to normal values. No changes from normal values were recorded in WBC. diff. WBC, protein fractions or LDH. Blood values remained within the normal range in the control goats. (c) Serology

No rise in IF AT titre was recorded until day 28. The titres of goats that survived until day 63 (Fig. 4) rose continuously and were apparently still rising when regular observations ceased. At day 129, the titres of goats 6, 7 and 8 were I in 1024, 256 and 1024 respectively. Sera from the control goats were negative to the IF AT at all times including day 129.

(d) Pathology Gross pathological changes were confined to goats 1, 2 and

3 necropsied on days 18 and 19, and goats 4 and 5 on days 34 and 24 respectively. On days 18 and 19, at the time of the ultimate schizogony(l), many small haemorrhages were pre­sent in all tissues. Petechiae were most obvious in subcu­taneous muscle, and on serosal surfaces. The heart and oesophagus were mottled dark red; kidneys were enlarged. No haemorrhages were seen in goat 5 on day 24 but serosal surfaces were flecked with brown pigment. At 34 days the gall bladder of goat 4 was enlarge.d by an accumulation of inspis­sated bile. No macroscopic lesions were found in goats necropsied on days 80, 93 and 129.

Histological examination revealed small haemorrhages in all tissues from the goats necropsied on days 18 and 19. At this time schizonts were present in endothelial cells of small and large blood vessels and were particularly obvious in renal glomeruli(l). The kidneys also showed focal cortical interstitial and perivascular infiltration by lymphocytes and plasma cells. Coagulative necrosis of convoluted tubules was present in goat 2, which died, but was absent from goats 1 and 3 which were killed. At day 24, schizonts were found only in the heart and all tissues contained small focal deposits ofhaemosiderin. Lymphocytic infiltration of kidney was most marked at this time. The muscles of goat 4 killed on day 34 contained many small immature sarcocysts(I). On days 80, 93 and 129 muscles contained mature sarcocysts and were focally infiltrated with mononuclear cells.

DISCUSSION

Goats, experimentally infected with a species of Sarco­cystis derived from dogs showed clinical signs similar to those reported in calves(2) (6) (9) and lambs(8). No symptoms were seen before day 17 and illness was most severe at the time of the ultimate schizogony 18 and 19 days after inoculation. Goats that did not die during or soon after the ultimate schizo­gony made a slow recovery but remained stunted throughout the remainder of the experiment. The effect of sub-clinical sarcocystosis on the growth of goats is being investigated in a further experiment. The schizogony at 18 and 19 days was associated with a rise in temperature and it is likely that two smaller rises noted at approximately 6 and II days in goats 1,2 and 3 (Fig. J) were also caused by phases of schizogony. Fischeri5) also investigated a dog-derived Sarcocystis species in goats and, from the similarity of its features, it is assumed that it is the same species that is present in New Zealand. S. capracanis n. sp., Fischer, 1979 was found to undergo schizo­gony between 10 and 14 days and between 19 and 23 days after infection. Recent investigation has shown that schizogony also occurs in the liver at 5 and 6 days (Collins and Charleston, in prep.). Thus the developmental period following infection comprises three periods of approximately 6 days each termi­nating in schizogony. It is possible that the development of

158 NEW ZEALAND VETERINARY JOURNAL VOL. 28

species infecting other ruminants also involves three phases of schizogony prior to muscle invasion.

The haematological results are similar to those described for infections by other Sarcocystis species(3) (9) (4) (8) (7) •

Haemoglobin concentration and PCV fell rapidly at the ulti­mate schizogony and the extensive petechiation resulted in anaemia. In sarcocystosis in the goat the severity of the illness appears to be directly related to the numbers of sporocysts and parallels the degree of anaemia.

Antibodies to Sarcocystis were detected by the IF AT from about one month after infection (Fig. 4) and titres rose rapidly thereafter. The ultimate schizogony is followed by the release of large numbers of schizozoites and the resulting parasi­taemia is probably the reason for the subsequent rise in Sarcocystis antibodies. As no regular serological exami­nations were made after day 63 it is not known when the IF AT titres peaked or how long they persisted after 129 days. Further investigations are under way to investigate these aspects and also the relationship between antibody levels and the size of the infective dose of sporocysts.

103 I FAT TI T R E

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ACKNOWLEDGEMENTS

This study was carried out with financial help from the New Zealand Meat Producers Board. Technical assistance was provided by D. Saywell, B. Wiens, J. Wilson and P. Wylie.

REFERENCES I. Collins. G. H. and Charleston. W.A.G. (1979): Studies on Sarcocystis species IV: A

species infecting dogs and goats: development in goats. N'z.veU. 27: 260-2. 2. Fayer. R. and Johnson. A. J. (1973): Development of Sarcocystisfusiformis in calves

infected with sporocysts from dogs. J. Parasit. 59: 1135-7. 3. Fayer. R. and Johnson. A. J. (1975 b): Effect of Amprolium on acute sarcocystosis in

experimentally infected calves. J. Parasit. 61: 932-6. 4. Fayer. R .• Johnson. A. J. and Lunde. M. (1976): Abortion and other signs of disease in

cows experimentally infected with Sarcocystis fusiformjs from dogs. J. In/. Dis. 134: 624-1l.

5. Fischer. F. Die entwicklung von Sarcocystis caprQcanis n. spec. in der ziege. Thesis. D.Med.Vet. Freien Universitat. Berlin 1979. Berlin 1979. 46p.

6. Johnson. A. J., Hildebrandt. P. K. and Fayer. R. (1975): Experimentally induced Sarco<-,ystis infection in calves: pathology. Am.J.llet. Res. 36: 995-9.

7. Leek. R. G. and Fayer. R. (1978): Sheep experimentally infected with Sarcocystis from dogs. Cornell Vet. 68: 1OS-23.

8. Leek. R. G., Fayer. R. and Johnson. A. J. (1977): Sheep experimentally infected with Sarcocystis from dogs.!. Disease in young lambs. J. Parasit. 63:642-50.

9. Mahrt. J. L. and Fayer. R. (f975): Haematologic and serologic changes in calves experimentally infected with Sarcocystisfusi/ormis. J. Parasit. 61: 967-9.

10. Seneviratna. P .. Edward. A. G. and De Giusti. D. L. (1975): Frequency of Sarcocystis spp. in Detroit Metropolitan area. Michigan. Am. J. ver. Res. 36: 337-9.

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Figure 4: IFAT titres of goats 6 and 7 (dose 15 x 104 sporocysts), 8 and 9 (3 x 104 sporocysts) and II (15 x 103 sporocysts).