Hemostatic alterations in pigs fed sublethal doses of Sarcocystic miescheriana

Veterin, ary Parasitology, 34 (1989) 1-13 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

1

H e m o s t a t i c A l t e r a t i o n s in P i g s Fed Sublethal Doses of Sarcocystis miescheriana A. DAUGSCHIES, E. ALTFELD and M. ROMMEL

Institute of Parasitology, School of Veterinary Medicine, B~nteweg 17, D-3000 Hannover 71 (F.R.G.) (Accepted for publication 19 January 1989 )

ABSTRACT Daugschies, A., Altfeld, E. and Rommel, M., 1989. Hemostatic alterations in pigs fed sublethal doses of Sarcocystis miescheriana. Vet. Parasitol., 34: 1-13. Effects of non-lethal Sarcocystis miescheriana infections on the blood coagulation system were investigated. Nine pigs were inoculated orally with 2 × 105 sporocysts (Group A ) and nine pigs (Group B) served as non-infected controls. Blood samples were taken from the vena jugularis externa every 2 or 3 days until 19 days post-infection (dpi). The following parameters were investigated: partial thromboplastin time (PTT), prothrombin time (PT), thrombin time (TT), thrombin coagulase time (TCT), fibrinogen (FIB), factor (F) VIII, F XI, F XII, antithrombin III (AT III), oL2macroglobulin (a2 MG), o~2antiplasmin (o~2AP), pre-kallikrein (PK), and the number of circulating thrombocytes. All infected pigs suffered from acute sarcocystiosis between 12 and 19 dpi. Clinical illness was most severe from 14 to 17 dpi. At this time, P T T and FIB increased, and T T and TCT decreased slightly. The activities of the clotting factors increased at 17 and 19 dpi. However, only F VIII activity was significantly higher in the infected pigs than in the controls at 17 and 19 dpi. PK was significantly lower in the infected pigs at 12, 14, and 17 dpi. Thrombocyte counts were reduced with the onset of the acute phase of illness and some pigs had marked thrombocytopenia. These results indicate low-grade disseminated intravascular coagulation (DIC) in the course of mild S. miescheriana infections in pigs.

INTRODUCTION

Certain species of Sarcocystis can cause hemorrhagic anemia in cattle, sheep, goats, and pigs. The cause of anemia is not well understood. The vascular phase of Sarcocystis is thought to alter the hemostatic balance (Barrows et al., 1982; Frelier and Lewis, 1984). Mechanical damage of endothelial cells by intravascular schizonts may cause leakage of blood into the surrounding tissues and thus may contribute to hemorrhagic diathesis seen in acutely affected hosts (Erber and Geisel, 1979; Frelier et a1.,1979; Zielasko et al., 1981; Dubey, 1983). Studies in lethally Sarcocystis-infected animals indicate involvement of DIC, 0304-4017/89/$03.50

© 1989 Elsevier Science Publishers B.V.

2

A. DAUGSCHIEfiET AL.

but some data are contradictory (Prasse and Fayer, 1981; Barrows et al., 1982; Phillips, 1982; Frelier and Lewis, 1984; Mahaffey et al., 1986; Strohlein, 1986). The purpose of the present study was to obtain more detailed information on the effects of non-lethal experimental infections with S. miescheriana on the blood coagulation system in growing pigs. MATERIALS AND METHODS

Eighteen male castrated pigs with a mean body weight of 38 kg were allocated to two groups of nine pigs each. The pigs of Group A were infected orally with 2 X l0 b sporocysts ofS. miescheriana and those of Group B served as noninfected controls. The pigs were kept in individual cages. The two groups were housed in separate barns under controlled climatic conditions. The sporocysts were isolated and stored according to the method of Torp (1979). Two days before inoculation the vena jugularis externa was catheterized in all pigs to avoid hemolysis and stress during blood collection. Blood samples were taken every 2 or 3 days until 19 days post-infection (dpi). The parameters which were examined are listed in Table 1. Blood samples for coagulation tests were diluted with 0.11 M sodium citrate ( 9 + 1 ) as an anticoagulant and centrifuged immediately (10 min, 3000 U min-1). The supernatant blood plasma was kept on ice until assayed. The coagulation tests were done with the Coagulometer KC10 (Heinrich Amelung GmbH, Lemgo, F.R.G. ) which operates on the basis of the ball method. They were completed within 4 h after blood withdrawal. All coagulation tests were TABLE 1

Blood clotting parameters examined during acute sarcocystiosis in pigs Parameter

Abbreviation

Determined dpi

No. of pigs per group examined

Partial thromboplastin time Prothrombin time Thrombin time Fibrinogen Factor VIII Factor XI Factor XII Thrombin coagulase time Antithrombin III a2 macroglobulin o~2antiplasmin Pre-kallikrein Thrombocyte counts

PTT PT TT FIB F VIII F XI F XII TCT AT III ~z MG o~2 AP PK

0, 3, 5, 7, 10, 12, 14, 17, 19 0, 3, 5, 7, 10, 12, 14, 17, 19 0, 3, 5, 7, 10, 12, 14, 17, 19 0, 3, 5, 7, 10, 12, 14, 17, 19 0, 3, 5, 7, 10, 12, 14, 17, 19 0, 3, 5, 7, 10, 12, 14, 17, 19 0, 3, 5, 7, 10, 12, 14, 17, 19 0, 7, 12, 14, 17, 19 0, 7, 12, 14, 17, 19 0, 7, 12, 14, 17, 19 0, 7, 12, 14, 17, 19 0, 7, 12, 14, 17, 19 3, 5, 7, 10, 12, 14, 17, 19

9 9 6 9 9 9 9 6 9 9 9 9 6

SARCOC YSTIS MIESCHERIANA AND HEMOSTASIS IN PIGS

3

done at least in duplicate. Precision and accuracy of these assays were controlled with commercial human control plasma (Behringwerke AG, Marburg, F.R.G.; Boehringer Mannheim, Mannheim, F.R.G.). Partial thromboplastin time (PTT) was determined after incubation of blood plasma with a mixture of kaolin and cephalin which act as activators of the intrinsic pathway of coagulation for 3 min at 37 ° C in plastic tubes. Thereafter, a solution of calcium chloride (CaC12, 0.025 mol 1-1 ) was added to the activated plasma sample and P T T was measured in seconds required until coagulation was detectable. Prothrombin time (PT) was measured by a one-step assay. The plasma samples were incubated at 37°C for 2 min and then Ca-thromboplastin which selectively activates the extrinsic part of the coagulation cascade was added and the time measured simultaneously until coagulation occurred. A standard curve was calculated by regression analysis of the values obtained with pooled plasma from three non-infected pigs that had been diluted 1 + 1, 1 + 2, 1 + 3 and 1 + 7 with a 0.9% sodium chloride solution. The time required for clotting after mixing the four dilutions with Ca-thromboplastin were defined as 50 ( 1 + 1 ) , 33 ( 1 + 2 ) , 25 ( 1 + 3 ) and 12.5% ( 1 + 7 ) of normal activity and the PT values of the individual pigs were expressed respectively. Thrombin time (TT) measurements were carried out using a one-step assay. Plasma was incubated at 37°C for 2 min and then mixed with thrombin reagent, and the time until coagulation occurred was measured. Thrombin coagulase time (TCT) was determined similarly with the exception that thrombin coagulase was used instead of thrombin reagent. Both tests give information on the conversion of fibrinogen to fibrin. Fibrinogen (FIB) was determined in plasma that had been incubated for 1 min at 37 ° C. Excess thrombin was mixed with the plasma and the time was measured until coagulation was detectable. The plasma concentration of FIB was then read from a table delivered by the manufacturer of the reagent. The accuracy of the test system was controlled with human standard plasma (Boehringer Mannheim, Mannheim, F.R.G. ). Determination of the blood clotting factors F VIII, F XI, and F XII was done by a modification of the P T T assay. Plasma was diluted 1 + 9 with imidazole buffer (pH 7.4) and diluted plasma was incubated at 37 °C for 3 min with a mixture of kaolin and cephaline and with human plasma deficient for the factor to be assayed. Then CaC12 was added and the clotting time was measured. Calibration of the assay systems was done by determining the clotting times of pooled plasma from three healthy pigs which had been diluted previously with a 0.9% solution of sodium chloride 1+9, 1+19, 1+39, 1+79 and 1+199. The clotting times which were measured in these dilutions were defined as 100% (1+9),50% (1+19),25% (1+39), 12.5% ( l + 7 9 ) , a n d 5 % (1+199) of normal activity of the respective clotting factor and calibration curves were calculated by regression analysis of the logarithms of per cent normal activity

4

A. DAUGSCHIES ET AL.

and clotting time. The per cent activities of F VIII, F XI and F XII in the blood plasma of the individual pigs were calculated from these calibration curves. Antithrombin III (AT III), a2 macroglobulin (a2 MG) and a2 antiplasmin (a2 AP) were determined spectrophotometrically (405 nm, 25°C) with chromogenic substrates in citrated plasma which had been stored at - 20 ° C for 3-12 weeks. The substrates for these assays were: tosyl-glycyl-prolyl-arginin-p-nitranilid-acetate (AT III), tosyl-glycyl-prolyl-lysin-p-nitranilid-acetate ( a2 AP) and carbobenzoxy-valyl-glycyl-argininin-p-nitranilid-acetate (a2 MG ). In principle, samples for determination of AT III, a2 AP or a2 MG which physiologically act as protease inhibitors were incubated in excess with the respective protease (thrombin, plasmin, or trypsin). This results in the formation of inhibitor-protease complexes with no (AT III-thrombin, a2 A P plasmin) or little (a2 MG-trypsin) proteolytic activity, p-Nitroanilin can be split off from the chromogenic substrates by the remaining proteolytic activity in the sample. This proteolytic activity can be measured with a photometer as an increase of extinction which is directly (a2 MG) or inversely (a2 AP, AT III) proportional to the concentration of the inhibitor in the blood plasma. Precision and accuracy of the methods were checked with Precichrom I/II (Boehringer Mannheim, Mannheim, F.R.G. ), a control plasma of human origin. Pre-kallikrein (PK), the precursor of kallikrein, was activated in the sample by incubation with a cold solution of dextran sulfate (0.025 g 1-') at 0 °C for 12 min. Then the chromogenic substrate benzoyl-prolyl-phenylalanyl-argininp-nitranilid-acetate was added and the total catalytic activity of activated kallikrein in the sample was measured photometrically (405 nm, 37°C) as an increase of extinction. Spontaneous activity of kallikrein in the blood plasma was assayed without activation of PK. With the exception of thromboplastin (Sigma Chemie GmbH, Deisenhofen, F.R.G.) all reagents and substrates used in this study are manufactured by Boehringer Mannheim (Mannheim, F.R.G. ). Circulating thrombocytes were counted microscopically in whole blood ( 1.3 mg EDTA ml-1 blood) immediately after collection of the sample using standard methods. The data of both infected and non-infected pigs were compared by one-factorial variance analysis. The time-dependent course of the respective values in Group A and B over the period of the experiment was evaluated statistically by Student's t-test for paired observations. Since the values of P T T , TCT, F XII, F VIII, FIB and the thrombocyte counts followed a log-norm distribution, as shown by the Lilliefors test, they had to be transformed logarithmically prior to parametric statistical analysis. RESULTS

Inoculated pigs developed mild clinical illness from 12 to 19 dpi. The disease was most severe between 14 and 17 dpi characterized by reduced appetite, ex-

SARCOCYSTIS MIESCHERIANA AND HEMOSTASIS IN PIGS

5

haustion and fever. Thereafter, all pigs recovered, but food consumption remained low in some pigs until the termination of the experiment on 19 dpi. No clinical signs were observed in the pigs of the control group.

Coagulation tests In the infected pigs (Group A) P T T (Fig. 1) was reduced from 7 to 10 dpi ( P < 0 . 0 5 ) , but increased thereafter (P<0.001). At 14 dpi P T T was significantly (P < 0.01 ) longer in the infected pigs when compared to the P T T of the non-infected controls. From 14 to 17 dpi P T T dropped (P<0.01) in the infected pigs to reach values comparable to those of the non-infected controls. There were no significant alterations of P T T in the non-infected controls (Group B ) during the experiment. There was no significant effect of the infection on P T in the course of this experiment. In seconds 3,1.

3.o2,92,82,7" 2,6 0

3

5

7

10

12

14

17

19 dpi

Fig. 1. P T T of pigs inoculated with 2 X l0 s sporocysts of Sarcocystis m iescheriana (A) and of noninfected pigs (B). seconds 26

25 24 23 22 21 20 19 18

0

3

5

7

10

12

14

17

19 dpi

Fig. 2. T T of pigs inoculated with 2 X 10 5 sporocysts of Sarcocystis miescheriana (A) and of noninfected pigs (B).

6

A. DAUGSCHIESET AL.

In seconds

~,2

k0 3,8

~_~-~ h - - - - _ ~

A B

3,6 ~4

7

0

12

14

17

19 dpi

Fig. 3. TCT of pigs inoculated with 2 X 105sporocysts of Sarcocystis miescheriana (A) and of noninfected pigs (B). In % activity 5,S-

l.,O-

0

3

5

7

10

12

14

17

19 clpi

Fig. 4. F XII activity of pigs inoculated with 2 X 105 sporocysts of Sarcocystis rniescheriana (A) and of non-infected pigs (B).

T T (Fig. 2 ) decreased significantly in the infected pigs from 12 to 14 dpi and in the non-infected controls from 14 to 17 dpi ( P < 0.05). Statistically significant differences between the two groups of pigs were not found at any time. However, there was a tendency to reduced T T values at 14 dpi and to increased T T values at 19 dpi in the infected pigs when compared to the values of the control pigs. Mean T C T values (Fig. 3) were lower in the infected pigs than in the controls almost throughout the experiment. T C T did not vary significantly in the controls, but increased in the infected pigs ( P < 0.05) from 7 to 12 dpi. At 14 dpi T C T was shorter ( P < 0.05 ) in the infected pigs than in the controls. The mean values of F XII activity (Fig. 4) did not differ significantly between the two groups at any time. The F XII values of the controls did not change significantly throughout the experiment. Those of the infected pigs decreased ( P < 0.01 ) from 12 to 14 dpi, and thereafter increased from 14 to 17 dpi and from 17 to 19 dpi ( P < 0 . 0 5 ) .

SARCOCYSTIS MIESCHERIANA AND HEMOSTASIS IN PIGS

7

% activity 200

120 100 80 60

0 3 5 7 I0 12 14 17 19 dpi Fig. 5. F X I activity of pigs inoculated with 2 × l0 s sporocysts of Sarcocystis miescheriana (A) a n d of non-infected pigs (B).

In % activity 5,0-

4,5-

L,0-

3,5"

0

3

5

7

10

12

14

17

19 dpi

Fig. 6. F VIII activity of pigs inoculated with 2 × l0 s sporocysts of Sarcocystis miescheriana (A) a n d of non-infected pigs (B).

Neither in the infected pigs nor in the controls did F XI values (Fig. 5) vary significantly, and there were no significant differences between the two groups of pigs at any time. However, three out of nine infected pigs had remarkably high F XI values at 17 and 19 dpi. The F VIII activity (Fig. 6) increased in the infected pigs significantly from 7 to 10 dpi and from 14 to 17 dpi (P<0.05) while there were no significant alterations in the controls. At 17 and 19 dpi the mean F VIII activities were higher in the infected pigs than in the controls ( P < 0.05 ). FIB values (Fig. 7) of the infected pigs increased from 12 to 14 dpi (P < 0.05) and declined thereafter (14-17 dpi, P < 0.01 ). At 3 and 14 dpi the FIB values of the infected pigs were higher than those of the non-infected controls (P < 0.05). At 19 dpi the FIB values of the infected pigs were reduced, but this could not be verified statistically. Photometric tests

AT III values (Fig. 8) declined in the infected pigs from 14 to 17 dpi and increased at the same time in the non-infected controls (P < 0.05 ). The mean

8

A. DAUGSCHIES ET AL.

In{rr i/d0 6,1

~

5,9 5,8 i

5,7. 5,6 5,5 5,~

"

5,3 5,2 5,1 5,0

0 3 5 7 10 12 14 17 19 dpi Fig. 7. F I B v a l u e s of pigs inoculated with 2 × 10 5 s p o r o c y s t s o f Sarcocystis rniescheriana (A) a n d of n o n - i n f e c t e d pigs ( B ) .

IUlml 17

6

15

B 1211109-

0

7

12

14

17

19 dpi

Fig. 8. A T III v a l u e s of pigs inoculated w i t h 2 × 10 5 s p o r o c y s t s of Sarcocystis miescheriana (A) and of n o n - i n f e c t e d pigs ( B ) .

AT III values of the infected pigs were lower than those of the controls at 17 dpi (P<0.05). Sarcocystiosis had no significant effect on the ~2 MG values. c~2 AP varied in both groups of pigs but these changes could not be correlated with the sarcosporidial infection. There were no significant differences between infected and non-infected pigs throughout the experiment. The PK values (Fig. 9) of the non-infected pigs of Group B remained fairly constant during this study while those of the infected pigs decreased from 7 to 12 dpi (P<0.01). From 12 dpi onwards the mean PK values of the infected

SARCOCYSTISMIESCHERIANA AND HEMOSTASIS IN PIGS T

u In'd. 1,2

i

1

1,1 1,0 O,g

0,8 03

0,6 0,5 0,/.

0

7

12

14

19dpi

17

Fig. 9. P K values of pigs inoculated with 2 × 10 5 sporocysts of Sarcocystis miescheriana (A) a n d of non-infected pigs (B). In counts =103/,u1 6,5

6,0 s,s

5,0¸

J

4,5

/-,0

o

3

s

7

io

12

i~

i?

19 ®i

Fig. 10. T h r o m b o c y t e counts of pigs inoculated with 2 × 10 s sporocysts of Sarcocystis miescheriana (A) and of non-infected pigs (B).

pigs were lower than those of the non-infected controls; the differences being statistically significant at 12, 14, and 17 dpi (P < 0.05). No spontaneous activity of kallikrein could be demonstrated in the plasma of infected and noninfected pigs. Thrombocyte counts The mean number of circulating thrombocytes (Fig. 10) did not change significantly in both groups of pigs in the course of the experiment. However, the

10

A. DAUGSCHIES ET AL.

infected pigs had a decreasing tendency of thrombocyte counts from 10 to 14 dpi. At 14 dpi, the mean values of the infected pigs were significantly lower ( P < 0.05) than those of the controls and they remained low thereafter. Individual infected pigs showed transient thrombocytopenia with values below 5 X 104 thrombocytes #l- 1 whole blood. DISCUSSION

The S. miescheriana infection had an influence on several parameters of the blood coagulation system with the onset of the acute phase. P T T is a screening assay for the intrinsic pathway of the coagulation cascade. This pathway is activated when plasma is exposed to collagenous fibers or endothelial cell fragments (Gersmeyer and Yasargil, 1978). At the beginning, small amounts of F XII are activated to F XIIa. F XIIa converts prekallikrein into kallikrein and the latter in turn activates more F XII. Subsequently, F XI, F IX and F VIII are activated. The following steps of the coagulation cascade are common to both the intrinsic and extrinsic pathway. The infected pigs had increased P T T values at 14 dpi which indicates that the intrinsic pathway of the coagulation cascade was hampered. Similar results were obtained previously in Sarcocystis-infected cattle (Frelier and Lewis, 1984) and pigs (Barrows et al., 1982) while Prasse and Fayer (1981) could not find an influence of S. cruzi infection on P T T in cattle. Kallikrein, which activates F XII by a positive feedback mechanism, is of significance for the initiation of the intrinsic pathway of coagulation ("contact activation" ) and of fibrinolysis (Delvos and Miiller-Berghaus, 1985 ). The reduced PK values found in infected pigs indicate activation of the kallikreinkinin system which has major hemodynamic functions and plays an important role in the pathogenesis ofDIC (Mason and Colman, 1971 ). Activated kallikrein is inhibited rapidly in vivo and continuous conversion of PK to kallikrein may have resulted in a depletion of PK. Reduced contact activation of the intrinsic pathway of coagulation due to PK depletion may have caused the increase of P T T at 14 dpi although the activities of the other clotting factors were unaffected (F XI, F VIII) or were even elevated (FIB, TT) at this time. Wright and Goodger (1977) interpreted their findings in Babesia-infected cattle similarly. The activities of the clotting factors F XII, F XI and F VIII were elevated at 17 and 19 dpi. This reflects a hypercoagulable condition on these days resulting from overcompensation of accelerated consumption of hemostatic factors, a paradoxical situation often found in low-grade DIC (Hamilton et al., 1978). The extrinsic pathway of the coagulation cascade was not altered by the Sarcocystis infection as indicated by normal PT values. This is in contrast to the results of previous studies (Prasse and Fayer, 1981; Barrows et al., 1982; Frelier and Lewis, 1984). Only one of these studies (Barrows et al., 1982 ) dealt

SARCOC YSTIS MIESCHERIANA AND HEMOSTASIS IN PIGS

11

with sarcosporidial infections in pigs and these pigs were infected lethally while the pigs in our study were inoculated with a sublethal dose of sporocysts. T C T and T T were shortened and FIB values were elevated in the infected pigs at 14 dpi indicating an activation of the final steps of the coagulation cascade with no remarkable fibrinolysis. The elevated FIB values probably reflect the acute phase response to endothelial damage caused by the developing and rupturing schizonts ("acute phase protein"). Other authors (Barrows et al., 1982; Phillips, 1982; Frelier and Lewis, 1984) reported hypofibrinogenaema in Sarcocystis-infected hosts during acute illness. Most of their animals died of acute sarcocystiosis which might explain why their results differ from ours. DIC is often accompanied by elevated levels of FIB (Hamilton et al., 1978). High FIB values were also found in malaria (Abilgaard et al., 1975; Stuart, 1978; Phillips et al., 1986), trypanosomosis (Olubayo and Mugera, 1985; Forsberg et al., 1987), babesiosis (Allen et al., 1974; Goodger and Wright, 1977; Moore and Williams, 1979) and pest in pigs (Heene et al., 1971 ). It can be speculated that the hypofibrinogenaemia reported in Sarcocystis-infected hosts previously was due to a rapid conversion of FIB to fibrin during the terminal stage of DIC in the dying animals. The values of the inhibitors of coagulation or of fibrinolysis were only slightly or not altered at all by the sarcosporidial infection. The reduced AT III values at 17 dpi can be interpreted as a consumption of this inhibitor resulting from increased production and activation of clotting factors. The reduction of the numbers of circulating thrombocytes in the infected pigs is consistent with the results of other authors (Prasse and Fayer, 1981; Barrows et al., 1982; Mahaffey et al., 1986). This has to be interpreted as the result of an enhanced adhesion and aggregation of thrombocytes to cover the endothelial damage caused by the rupturing schizonts. Aggregating thrombocytes secrete a variety of substances including FIB ("release reaction"). Thrombocytic FIB may have contributed to the elevated FIB values in the infected pigs. McKay (1973) reported that reduced thrombocyte counts and elevated levels of fibrinogen and other clotting factors are typical for chronic DIC following a mild pro-coagulatory stimulus. Aggregating thrombocytes also secrete ADP and TxB2 which enhance the aggregability of thrombocytes (Delvos and Miiller-Berghaus, 1985 ). The latter is one of the most potent vasoconstrictors. Platelet factor 3, which is also released, is known to be essential for the activation of several clotting factors. The effects of non-lethal infections with S. miescheriana on the blood coagulation system in pigs reported in this paper indicate a hypercoagulable condition and chronic low-grade DIC. In the course of lethal infections, DIC may be acute and more pronounced leading to death. ACKNOWLEDGEMENTS

This study was supported by a research grant from the German Research Council (D.F.G.). Special thanks are given to Martina Walter for excellent

12

A. DAUGSCHIE~ ET AL.

technical assistance given during this study and to Michael Wolfhagen for producing the graphs.

REFERENCES Abilgaard, C., Harrison, J., DeNardo, S., Spangler, W. and Gribble, D., 1975. Simian Plasmodium knowlesi malaria: studies of coagulation and pathology. Am. J. Trop. Med. Hyg., 24: 764-768. Allen, P.C., Frerichs, W.M. and Holbrook, A.A., 1974. Experimental acute Babesia caballi infections. Exp. Parasitol., 37: 373-379. Barrows, P.L., Prestwood, A.K. and Green, C.E., 1982. Experimental Sarcocystis suicanis infections: disease in growing pigs. Am. J. Vet. Res., 43: 1409-1412. Delvos, U. and Miiller-Berghaus, G., 1985. Die Regulation der Blutgerinnung. Naturwissenschaften, 22: 461-469. Dubey, J.P., 1983. Clinical sarcocystosis in calves fed Sarcocystis hirsuta sporocysts from cats. Vet. Pathol., 20: 90-98. Erber, M. and Geisel, O., 1979. Untersuchungen zur Klinik und Pathologie der Sarcocystis suicanis-Infektion beim Schwein. Berl. Muench. Tieraerztl. Wochenschr., 92: 197-202. Forsberg, C.M., Valli, V.E.O., Gentry, P.W. and Donworth, R.M., 1987. The pathogenesis of Trypanosoma congolense infection in calves. Vet. Pathol., 16: 229-242. Frelier, P.F. and Lewis, R.M., 1982. Hematologic and coagulation abnormalities in acute bovine sarcocystosis. Am. J. Vet. Res., 45: 40-48. Frelier, P.F., Mayhew, I.G. and Pollock, R., 1979. Bovine sarcocystosis: pathologic features of naturally occurring infections with Sarcocystis cruzi. Am. J. Vet. Res., 40:651-657. Gersmeyer, E.F. and Yasargil, E.C., 1978. Schock und hypotone KreislaufstSrungen. Georg Thieme Verlag, Stuttgart. Goodger, B.V. and Wright, I.G., 1977. Acute Babesia bigemina infection: changes in fibrinogen catabolism. Z. Parasitenkd., 53: 53-61. Hamilton, P.J., Stakler, A.L. and Douglas, A.S., 1978. Disseminated intravascular coagulation: a review. J. Clin. Pathol., 31: 609-619. Heene, D., Hoffmann-Fezer, G., Hoffmann, R., Weiss, E., Miiller-Berghaus, G. and Lasch, H.G., 1971. GerinnungsstSrungen bei akuter Schweinepest. Beitr. Pathol., 144: 259-271. Mahaffey, E.A., George, J.W., Duncan, J.R., Prasse, K.W. and Fayer, R., 1986. Hematologic values in calves infected with Sarcocystis cruzi. Vet. Parasitol., 19: 275-280. Mason, J.W. and Colman, R.W., 1971. The role of hageman factor in disseminated intravascular coagulation induced by septicemia, neoplasia, or liver disease. Thromb. Diath. Haemorrh., 26: 325 331. McKay, D.J., 1973. Intravascular coagulation - - acute and chronic - - disseminated and local. In: G. Schmer and P.E. Strandjord (Editors), Coagulation: Current research and Clinical Applications. Academic Press, New York, pp. 45-68. Moore, D.J. and Williams, M.C., 1979. Disseminated intravascular coagulation: a complication of Babesia canis infection in the dog. J. S. Afr. Vet. Assoc., 50: 265-275. Olubayo, R.O. and Mugera, G.M,, 1985. Pathogenesis of hemorrhages in Trypanosoma vivax infection in cattle. I. Disseminated intravascular coagulation. Bull. Anim. Health Prod. Afr., 33: 211-217. Phillips, H.R., 1982. The pathophysiology of Sarcocystis tenella infections in specific-pathogenfree (Sporozoa) sheep. Ph.D. Thesis, University of Adelaide. Phillips, R.E. and Warrell, D.A., 1986. The pathophysiology of severe falciparum malaria. Parasitol. Today, 2:271 282.

SARCOC~YSTISMIESCHERIANA AND HEMOSTASIS IN PIGS

13

Prasse, K.W. and Fayer, R., 1981. Hematology of experimental acute Sarcocystis bovicanis infection in calves. II. Serum biochemistry and hemostatis studies. Vet. Pathol., 18: 358-367. Strohlein, D.A., 1986. Contributions to the biology of experimental Sarcocystis suicanis infection in domestic swine. Ph.D. Thesis, University of Georgia. Stuart, J., 1978. Intravascular coagulation in falciparum malaria. Br. Med. J., 2: 774. Torp, C., 1979. Untersuchungen fiber den Einflu~ der Sarcocystis muris-Infektion auf die Tr/ichtigkeit und das Aufzuchtergebnis von NMRI-M~iusen. Vet. Med Diss., Hannover, F.R.G. Wright, I.G. and Goodger, B.V., 1977. Acute Babesia bigemina infection: changes in coagulation and kallikrein parameters. Z. Parasitenkd., 53: 63-73. Zielasko, B., Petrich, J., Trautwein, G. and Rommel, M., 1981. Untersuchungen tiber pathologisch-anatomische Ver~inderungen und die Entwicklung der Immunit~t bei der Sarcocystis suicanis-Infektion. Berl. Muench. Tieraerztl. Wochenschr., 94: 223-228.