document

zu Bakt. Hyg., I. Abt. Orig. A 250,268-276 (1981)

Institute of Parasitology of the Hanover School of Veterinary Medicine

The Sarcocystis muris-Infection as a Model for Research on the

Chemotherapy ofAcute Sarcocystosis ofDomestic Animals*

M. ROMMEL, ANKE SCHWERDTFEGER, and SONJA BLEWASKA

Received Pebruary 10, 1981

Summary

Twelve anticoccidial or antimalarial drugs were tested for their efficacy against variousdevelopmental stages of Sarcocystis muris in NMRI-mice. Schizogonic stages present inthe liver from day 11 to 17 p. i. showed to be most sensitive to drug action. Sulfaquinoxalineplus pyrimethamine, zoalene and Bay g 7183 completely eliminated these stages. A strongthough not 100 per cent efficacy was observed in experiments with primaquine. The otherdrugs tested were less (halofuginone, sulfadoxine plus trimethoprim) or not effective(sulfadimethoxine, amprolium, monensin, aprinocid, sulfaquinoxaline plus diaveridine) inthe used dosages. In trials to improve the Sarcocystis muris-mouse-cat model it was foundthat in NMRI-mice the inoculation dose of 50 sporocysts resulted in the highest infectionrate and intensity of the infection. By the application of less or more sporocysts or byrepeated inoculations poorer infection rates and lower intensities of infection were achieved.Thymus deprived nude mice (NMRI-nu/nu) and the AKR/N-strain were the most susceptible animals in which infection rates of 100 and 95 per cent were achieved by the inoculation of 50 sporocysts. By the application of antilymphocytic serum, cyclophosphamide,irradiation or corticosteroids infection rates of 83, 92, 98 and 100 per cent, respectively,could be achieved in NMRI-mice. For future chemotherapeutical trials an inoculation doseof 50 sporocysts into irradiated NMRI-mice is recommended. It is suggested that the modelis also suitable for the screening of drugs for their efficacyagainst exoerythrocytic schizontsof Plasmodium.

Introduction

Only after the discovery of the mode of transmission of the sarcosporidia itbecame possible to work experimentally with these parasites. In the course of thetremendous research activities following this discovery it was found that the inoculation of large numbers of sporocysts regularly causes severe disease and oftendeaths in domestic animals. Light infections cause reduction of weight gain, andthus for this parasitic infection a considerable economic significance has to besupposed (for lit. see Rommel et a1., 1979).

* Presented at the 3rd German-Japanese Cooperative Symposium on Protozoan Diseases, Kyoto, Japan, Oct. 28.-29. 1980.

zb1. Bakt. Hyg., LAbt. Orig. A 250,268-276 (1981)

Institute of Parasitology of the Hanover School of Veterinary Medicine

The Sarcocystis muris-Infection as a Model for Research on the

Chemotherapy of Acute Sarcocystosis of Domestic Animals *

M. ROMMEL, ANKE SCHWERDTFEGER, and SONJA BLEWASKA

Received F'ebruary 10, 1981

Summary

Twelve anticoccidial or antimalarial drugs were tested for their efficacy against various developmental stages of Sarcocystis muris in NMRI-mice. Schizogonic stages present in the liver from day 11 to 17 p. i. showed to be most sensitive to drug action. Sulfaquinoxaline plus pyrimethamine, zoalene and Bay g 7183 completely eliminated these stages. A strong though not 100 per cent efficacy was observed in experiments with primaquine. The other drugs tested were less (halofuginone, sulfadoxine plus trimethoprim) or not effective (sulfadimethoxine, amprolium, monensin, aprinocid, sulfaquinoxaline plus diaveridine) in the used dosages. In trials to improve the Sarcocystis muris-mouse-cat model it was found that in NMRI-mice the inoculation dose of 50 sporocysts resulted in the highest infection rate and intensity of the infection. By the application of less or more sporocysts or by repeated inoculations poorer infection rates and lower intensities of infection were achieved. Thymus deprived nude mice (NMRI-nu/nu) and the AKR/N-strain were the most susceptible animals in which infection rates of 100 and 95 per cent were achieved by the inoculation of 50 sporocysts. By the application of antilymphocytic serum, cyclophosphamide, irradiation or corticosteroids infection rates of 83, 92, 98 and 100 per cent, respectively, could be achieved in NMRI-mice. For future chemotherapeutical trials an inoculation dose of 50 sporocysts into irradiated NMRI-mice is recommended. It is suggested that the model is also suitable for the screening of drugs for their efficacy against exoerythrocytic schizonts of Plasmodium.

Introduction

Only after the discovery of the mode of transmission of the sarcosporidia it became possible to work experimentally with these parasites. In the course of the tremendous research activities following this discovery it was found that the inoculation of large numbers of sporocysts regularly causes severe disease and often deaths in domestic animals. Light infections cause reduction of weight gain, and thus for this parasitic infection a considerable economic significance has to be supposed (for lit. see Rommel et aI., 1979).

* Presented at the 3rd German-Japanese Cooperative Symposium on Protozoan Diseases, Kyoto, Japan, Oct. 28.-29. 1980.

Sarcocystis muris-Infection 269

There are only a few publications on the chemotherapy of acute sarcocystosis.Fayer and Johnson (1975) and Leek and Fayer (1980) were able to suppress clinicalsigns in experimentally infected calves and lambs by administering 100 mg/kgamprolium for 30 days. Since experiments with domestic animals are very expensive ,it was tried to establi sh a laborator y model for chemotherapeutical trials of theSarcocystis-infection.

Materials and Methods

As paras ite for the model a strain of Sarcocystis muris was chosen which had beenisolated by Ruiz and Frenkel from a house mouse in Costa Rica in 1976. The sporocystsfor the inoculation of mice were recovered from the mucosa of experimentally infectedcats by digestion with tr ypsin (0.25 per cent trypsin DIFCO no 0152-15 in PBS).

All chemotherapeutical tri als were carried out with female NMRI -mice 1• The animalswere kept in isolated rooms under coccidia-free conditions. Th ey received standard diet(Altromin 1324) and water ad libitum. Following the recomm endati ons of Torp (1979) forchemotherapeutical trials all mice were inoculated with 8000 sporocysts. With this dosean infection rate of 23 to 43 per cent was achieved. With one exception all drugs wereadministered in the maximally tolerated dose through the food. Only halofuginone wasgiven with the drink ing water. In order ro find out which developmental stages are mostseverely affected by the drug s the mice were divided into four group s which were treatedat different times post infection (Table 1). Only substances were tested which were knownto have good efficacies against Eime ria or against exoer ythrocytic schizonts of Plasmodium(lit. see Schwerdtfeger, 1980). The chemotherapeutical experiments were carried out inthree tr ials with each 3 to 5 groups for the testing of drugs and one positive and one negativecontrol group. For each medication period with each drug 40 mice were used.

Table 1. Chemotherapeutic al tri als : Periods of treatment and corresponding developmentalstages of Sarcocystis muris

Group

A

BCD

Period of treatm ent(days post infection )

2-10

11-1718-2728-50

Corresponding developmental stagesof S. muris

Migration of the sporozoites from the gut int othe liver and development to schizontsSchizogonic stages in liver parenchymal cellsMigration of th e merozoites to the musculatureEarly formation of cysts

The NMRI-mice which were used for the experiments for the improvement of the Sarcocystis muris-mouse-cat model were bought from a private company 2. C57 BL/6J Han-,AKR/N Han-, C3H/He Han- and NMRI-nu/nu-mice were received from the CentralInstitute for Experimental Animals in Hanover ' . Rabbitantimouselymphoc yte serum (AntiThy 1.2 F7D5 , monoclonal IgM cytotoxic antibody) originated from OLAC Shaw's FarmBlackthorn /England . The mice were injected subcutaneously with 10 Ill /animal once a

1 Lippische Versuchstierzucht Hagemann, 4901 Exter.2 Versuchstieranstalt WiGa, 8741 Sulzfeld.3 Zentralinstitut fur Versuchstiere, 3000 Hannover 91. We thank Dr. A. Thu nert for

tak ing care of the nude mice.


There are only a few publications on the chemotherapy of acute sarcocystosis. Fayer and Johnson (1975) and Leek and Fayer (1980) were able to suppress clinical signs in experimentally infected calves and lambs by administering 100 mg/kg amprolium for 30 days. Since experiments with domestic animals are very expensive, it was tried to establish a laboratory model for chemotherapeutical trials of the Sarcocystis-infection.

Materials and Methods

As parasite for the model a strain of Sarcocystis muris was chosen which had been isolated by Ruiz and Frenkel from a house mouse in Costa Rica in 1976. The sporocysts for the inoculation of mice were recovered from the mucosa of experimentally infected cats by digestion with trypsin (0.25 per cent trypsin DIFCO no 0152-15 in PBS).

All chemotherapeutical trials were carried out with female NMRI-mice 1• The animals were kept in isolated rooms under coccidia-free conditions. They received standard diet (Altromin 1324) and water ad libitum. Following the recommendations of Torp (1979) for chemotherapeutical trials all mice were inoculated with 8000 sporocysts. With this dose an infection rate of 23 to 43 per cent was achieved. With one exception all drugs were administered in the maximally tolerated dose through the food. Only halofuginone was given with the drinking water. In order to find out which developmental stages are most severely affected by the drugs the mice were divided into four groups which were treated at different times post infection (Table 1). Only substances were tested which were known to have good efficacies against Eimeria or against exoerythrocytic schizonts of Plasmodium (lit. see Schwerdtfeger, 1980). The chemotherapeutical experiments were carried out in three trials with each 3 to 5 groups for the testing of drugs and one positive and one negative control group. For each medication period with each drug 40 mice were used.

Table 1. Chemotherapeutical trials: Periods of treatment and corresponding developmental stages of Sarcocystis muris

Group Period of treatment (days post infection)

Corresponding developmental stages of S. muris

A

B C D

2-10

11-17 18-27 28-50

Migration of the sporozoites from the gut into the liver and development to schizonts Schizogonic stages in liver parenchymal cells Migration of the merozoites to the musculature Early formation of cysts

The NMRI-mice which were used for the experiments for the improvement of the Sarcocystis muris-mouse-cat model were bought from a private company 2. C57 BL/6 J Han-, AKR /N Han-, C3H/He Han- and NMRI-nu/nu-mice were received from the Central Institute for Experimental Animals in Hanover 3. Rabbitantimouselymphocyte serum (AntiThy 1.2 F7D5, monoclonal IgM cytotoxic antibody) originated from OLAC Shaw's Farm Blackthorn /England. The mice were injected subcutaneously with 10,tl /animal once a

1 Lippische Versuchstierzucht Hagemann, 4901 Exter. 2 Versuchstieranstalt WiGa, 8741 Sulzfeld. 3 Zentralinstitut fur Versuchstiere, 3000 Hannover 91. We thank Dr. A. Thunert for

taking care of the nude mice.

270 M. Rommel, A.Schwerdtfeger, and S.Blewaska

week for 4 weeks. X-ray irradiation of mice (total dose 400 r) was done in the Departmentof Medical Physics of the Hanover School of Veterinary Medicine.' 24 hours prior to infection. For chemical immunosuppression dexamethasone (Voren'", BoehringerIngelheim),cyclophosphamide (Endoxan", Asta Werke AG) and azathioprin (Imurek", DeutscheWellcome GmbH) were used. The drugs were administered intraperitoneally from one daybefore to 30 days post infection. Dexamethasone was given in doses of 0.05 mg/animalevery fourth day, cyclophosphamide in doses of 0.35 mg/animal every fourth day andazathioprin in doses of 0.17 mg/mouse every third day (lit. see Bletoaska, 1981).

All micewere killed 90 days post infection and examined macroscopically and by meansof the trypsin digestion technique for the presence of sarcosporidia. Both, the infectionrate and the intensity of infection were determined.

Results

Chemotherapy

The results of the 3 chemotherapeutical trials are recorded in Table 2 to 4. Themost effective drug was a combination of sulfaquinoxaline and pyrimethamine. Ithad a slight effect on sporozoites, it eliminated the schizogonic stages and the youngcysts completely, and it reduced the intensity of the infection remarkably whenadministered during the period of the migration of the merozoites from the liverto the muscles. The schizogonic stages were also completely eliminated by zoaleneand Bay g 7183 and were almost completely destroyed by the antimalarial primaquine. Lasalocid and halofuginone were less effective. The other drugs had little(sulfadoxine plus trimethoprim, sulfadimethoxine) or no negative influence (amprolium, monensin, aprinocid, sulfaquinoxaline plus diaveridine) on the course ofthe experimental Sarcocystis muris-infection in the used dosages. From Table 5 itcan be seen that the schizogonic stages present from day 11 to 17 p.i. were mostsensitive to chemotherapy.

By some of the drugs the infection rate and/or the intensity of the Sarcocystismuris-infection was increased. Details can be taken from Table 2-4.

Improvement of the model

The effect of the inoculation dose on the infection rate and the intensity of theinfection in NMRI-mice is listed in Table 6. The highest infection rate and thehighest intensity of infection was achieved by administering 50 sporocysts. Less ormore sporocysts or repeated applications resulted in poor infection rates and lowintensities of infection.

The susceptibility of various mouse strains to Sarcocystis muris differed remarkably (Table 7). Thymus-deprived nude mice (NMRI-nu/nu) and the AKR/N-strainwere the most susceptible animals. The inoculation of 50 sporocysts into animalsof these strains resulted in infection rates of 100 and 95 per cent, respectively. Immunosuppression by the application of corticosteroids or by irradiation also induced infection rates of 100 and 98 per cent, respectively, and high intensities ofinfection. The infection rates and the intensities of infection were also increased bythe application of antilymphocytic serum and by cyclophosphamide but not byazathioprin (Table 7).

4 We thank Prof. W.Giese for his support.

270 M. Rommel, A. Schwerdtfeger, and S. Blewaska

week for 4 weeks. X-ray irradiation of mice (total dose 400 r) was done in the Department of Medical Physics of the Hanover School of Veterinary Medicine 4 24 hours prior to infection. For chemical immunosuppression dexamethasone (Voren®, Boehringer Ingelheim), cyclophosphamide (Endoxan®, Asta Werke AG) and azathioprin (Imurek®, Deutsche Wellcome GmbH) were used. The drugs were administered intraperitoneally from one day before to 30 days post infection. Dexamethasone was given in doses of 0.05 mg/animal every fourth day, cyclophosphamide in doses of 0.35 mg/animal every fourth day and azathioprin in doses of 0.17 mg/mouse every third day (lit. see Blewaska, 1981).

All mice were killed 90 days post infection and examined macroscopically and by means of the trypsin digestion technique for the presence of sarcosporidia. Both, the infection rate and the intensity of infection were determined.

Results

Chemotherapy

The results of the 3 chemotherapeutical trials are recorded in Table 2 to 4. The most effective drug was a combination of sulfaquinoxaline and pyrimethamine. It had a slight effect on sporozoites, it eliminated the schizogonic stages and the young cysts completely, and it reduced the intensity of the infection remarkably when administered during the period of the migration of the merozoites from the liver to the muscles. The schizogonic stages were also completely eliminated by zoalene and Bay g 7183 and were almost completely destroyed by the antimalarial primaquine. Lasalocid and halofuginone were less effective. The other drugs had little (sulfadoxine plus trimethoprim, sulfadimethoxine) or no negative influence (amprolium, monensin, aprinocid, sulfaquinoxaline plus diaveridine) on the course of the experimental Sarcocystis muris-infection in the used dosages. From Table 5 it can be seen that the schizogonic stages present from day 11 to 17 p.i. were most sensitive to chemotherapy.

By some of the drugs the infection rate and/or the intensity of the Sarcocystismuris-infection was increased. Details can be taken from Table 2-4.

Improvement of the model

The effect of the inoculation dose on the infection rate and the intensity of the infection in NMRI-mice is listed in Table 6. The highest infection rate and the highest intensity of infection was achieved by administering 50 sporocysts. Less or more sporocysts or repeated applications resulted in poor infection rates and low intensities of infection.

The susceptibility of various mouse strains to Sarcocystis muris differed remarkably (Table 7). Thymus-deprived nude mice (NMRI-nu/nu) and the AKR/N-strain were the most susceptible animals. The inoculation of 50 sporocysts into animals of these strains resulted in infection rates of 100 and 95 per cent, respectively. Immunosuppression by the application ot corticosteroids or by irradiation also induced infection rates of 100 and 98 per cent, respectively, and high intensities of infection. The infection rates and the intensities of infection were also increased by the application of antilymphocytic serum and by cyclophosphamide but not by azathioprin (Table 7).

4 We thank Prof. W. Giese for his support.

Tab

le2

.Eff

icac

yo

far

np

roli

um

,su

lfad

ox

ine

plu

str

imet

ho

prim

,las

aloc

id,

sulf

adim

cth

ox

inc

and.

zoa

lene

ondi

ffer

ent

deve

lop

men

tal

stag

eso

fSa

rcoc

ysti

sm

uris

No

tIn

-A

mp

roli

um

Sulf

ado

xin

cp

lus

Las

alo

cid

Su

lfad

imct

ho

xin

eZ

oal

ene

info

fecr

edT

rim

cth

op

rim

(5p

lus

1)co

n-

can-

AB

CD

A13

CD

AB

CD

AD

CD

AB

CD

trol

trol

gro

up

gro

up

Nu

mb

ero

fmic

e40

79

41

403

939

404

038

4039

394

04

040

3938

40

4039

404

0

Do

se-

323

48

2610

964

(mg

/kg

/bo

dy

wei

ght/

day

)

Nu

mb

ero

fm

ice

++

-j+

++

++

+po

siti

ve1

3417

1315

1517

819

820

827

1827

1117

1311

09

13

Perc

enta

geo

fm

ice

posi

tive

2.5

434

1.5

32.5

38.

538

.54

2.5

2050

2051

.320

.667

.54

5.0

67

.52

8.2

44.7

32.5

27

.50

22.5

32.5

Nu

mb

ero

fv

isi-

++

+++

++

++

V>

ble

cyst

s(X

)pe

r12

05

2.8

41.

032

.452

.756

.12

7.6

28

.54

9.9

56.9

67.5

7.1

26

.538

28.6

41.

22

8.2

47.3

26.5

05

1.1

34.9

'"...,p

osit

ive

mo

use

() 0 ()

Num

ber

of

cys

to-

++

++

<..<:~

zoit

es(X

)p

erg

53.9

28

.272

.216

.620

.955

.412

.111

.620

.926

.933

.33

.938

.34

2.0

10

.030

.616

.52

6.3

9.9

020

.82

6.1

tn·

mus

cula

ture

8(i

nm

illi

on

s)~ ~

. [ ro

+.+

+.+

++

=si

gn

ific

ant

dev

iati

onfr

om

the

infe

cted

Per

iod

so

ftr

eatm

ent:

~ O·an

dn

ot

tre

ated

cont

rol

gro

up

.A

=d

ay2

tol0

p.i

.::>

+=

P<

0.0

5D

=d

ay11

to17

p.i

.

++

=P

<0

.01

C=

day

18

to2

7p

.i.

++

+=

p<

0.0

01D

=da

y28

10

50p

.i.1'

-' '-l

Tab

le 2

. E

ffic

acy

of

amp

roli

un

l, s

ulfa

doxi

ne p

lus

trim

etho

prim

, la

salo

cid

, su

lfad

imct

hox

inc

and

zoal

ene

on d

iffe

rent

dev

elop

men

tal

stag

es o

f S

arco

cyst

is m

uri

s

No

t In

-A

mp

roJi

um

S

ulfa

dox

ine

plus

L

asal

ocid

S

ulfa

dim

eth

ox

ine

Zo

alen

e in

f.

fect

ed

Tri

met

ho

pri

m (5

plu

s 1)

co

n-

can

-A

B

C

D

A

B

C

D

A

B

C

])

A

B

C

D

A

B

C

D

t ro

l tr

ol

gr o

up

gro

up

Nu

mbero

fmic

e 40

7

9

41

40

39

39

40

4

0

38

4

0

39

39

40

4

0 4

0

39

3

8

40

4

0

39

4

0

40

Do

se

323

48

26

10

9 6

4

(mg/

kg

/bo

dy

w

eig

ht/

day)

Nu

mb

er o

f m

ice

+

+

-j

+

+

+++

+

p

osit

ive

34

17

13

15

15

1

7

8 1

9

8 2

0

8 2

7

18

27

11

1

7

13

11

0 9

13

Per

cent

age

of

mic

e p

osit

ive

2.5

43

41

.5

32.5

38

.5

38.5

4

2.5

20

5

0

20

51.3

20

.6

67.5

45

.0

67.5

28

.2

44.7

32

.5

27.5

0

22

.5

32.5

Nu

mb

er o

f vi

s i-

+

+

+++

+

+

+

+

V>

ble

cys

ts (x)

per

12

0 5

2.8

41

.0

32.4

52

.7

56.1

27

.6

28.5

49

.9

56.9

67

.5

7.1

26.5

3

8

28.6

41

.2

28.2

47

.3

26.5

0

51.1

3

4.9

po

..., p

osit

ive

mou

se

()

0 ()

Nu

mb

er o

f cy

sto

-++

+

+

'<

~

zoit

es (x)

per

g

53.9

2

8.2

7

2.2

16

.6

20.9

55

.4

12.1

11

.6

20.9

26

.9

33

.3

3.9

38

.3

42

.0

10.0

3

0.6

16

.5

26.3

9.

9 0

20

.8

26.1

tn

· m

usc

ulat

ure

8 (i

n m

illi

ons)

~

::!.

'" , [ ro

+. +

+. +

++ =

signi

fica

nt d

evia

tio

n f

rom

th

e in

fect

ed

Per

iods

of

trea

tmen

t:

g O·

and

no

t tr

eate

d co

ntr

ol

gro

up

. A

=d

ay

2to

l0p

.i.

:;:l

+ =

P <

0.0

5

B

= d

ay 1

1 to

17

p. i

.

++ =

P <

0.0

1 C

=

day

18

to 2

7 p

.i.

+++

= P

< 0

.001

D

=

day

28

to 5

0 p

. i.

1'-' :--.l

IV '-I

Tab

le3.

Eff

icac

yof

apri

no

cid

,su

lfaq

uin

oxal

ine

plus

diav

erid

ine

and

mo

nen

sin

on

diff

eren

tde

vel

op

men

tal

stag

esof

Sarc

ocys

tis

mur

isN

No

tin

foIn

fecr

edA

rpri

noci

dS

ulfa

quin

oxal

ine

plu

sM

on

ensi

n~

con

tro

lco

ntr

ol

Dia

veri

din

e(4

plus

1)~ 0

gro

up

gro

up

AB

CD

AB

CD

AB

CD

3 3 F-N

um

ber

ofm

ice

4078

4040

3939

4040

4040

4039

4040

:;.-

Vl

Dos

e14

4347

n-

-::r

(mg/

kg/b

ody

~ (1) ....

wei

ght/

day)

0.- ....

Nu

mb

erof

mic

ert

i'+

++

++

++

++

++

++

(JQ (1)

posi

tive

018

1119

2517

1112

1317

1928

1710

~.... ..,P

erce

ntag

eof

;:l 0.-

mic

epo

siti

ve0

2327

.547

.564

.143

.627

.530

32.5

42.5

47.5

71.

842

.525

~ 0::1

Nu

mbe

rof

visi

ble

++

++

(b

cyst

s(x

)p

erpo

si-

029

.67

1.4

22.1

42.2

45.7

21.8

49.6

33.7

82.5

53.4

16.5

54.6

49~ .., '"

tive

mou

se?;

""" ..,

Nu

mb

ero

fcy

sto-

++

zoit

es(x

)p

etg

011

.933

.810

.635

.932

.28.

529

.116

.355

.727

.37.

522

.230

.5m

uscu

latu

re(i

nm

illio

ns)

+,

++

,+

++

=si

gn

ific

an

tde

viat

ion

from

the

infe

cted

Peri

ods

oftr

eatm

ent:

and

no

ttr

eate

dco

ntr

olg

rou

p.

A=

day

2to

10p

.i.

+=

P<

0.05

B=

day

11to

17p

.i.

++

=P

<0

.01

C=

day

18to

27p.

I.+

++

=P

<0.

001

D=

day

28

to50

p.i

.

IV

'-I

Tab

le 3

. Eff

icac

y o

f ap

rino

cid,

su

lfaq

uin

ox

alin

e pl

us d

iave

ridi

ne a

nd

mo

nen

sin

on

dif

fere

nt

deve

lop

men

tal

stag

es o

f Sa

rcoc

ysti

s m

uris

N

No

tin

f.

Infe

cted

A

rpri

noci

d S

ulfa

quin

oxal

ine

plu

s M

on

ensi

n

~

con

tro

l co

ntr

ol

Dia

veri

dine

(4

plus

1)

~

0 g

rou

p

gro

up

A

B

C

D

A

B

C

D

A

B

C

D

3 3 F-

Nu

mb

er o

f m

ice

40

78

40

40

39

39

40

40

40

40

40

39

40

40

>

V

l

Dos

e 14

43

47

n ::

r (m

g/kg

/bod

y ~

(1) ....

wei

gh

t/da

y)

p..

....

Nu

mb

er o

f m

ice

rti'

++

+

++

+

+

+

+

++

+

+

(JQ

(1

)

posi

tive

0

18

11

19

25

17

11

12

13

17

19

28

17

10

~ .... I>'

Per

cent

age

of

;:l p..

mic

e po

siti

ve

0 23

27

.5

47.5

64

.1

43.6

27

.5

30

32.5

42

.5

47.5

71

.8

42.5

25

~

0::1

Nu

mb

er o

f vi

sibl

e +

++

+

(b

cyst

s (x

) p

er p

osi-

0 29

.6

71.4

22

.1

42.2

45

.7

21.8

49

.6

33.7

82

.5

53.4

16

.5

54.6

49

~

I>' '"

tive

mou

se

?;""

I>

'

Nu

mb

er o

f cy

sto-

++

zo

ites

(x)

per

g

0 11

.9

33.8

10

.6

35.9

32

.2

8.5

29.1

16

.3

55.7

27

.3

7.5

22.2

30

.5

mus

cula

ture

(i

n m

illi

ons)

+,

+ +,

+ +

+ =

sig

nif

ican

t de

viat

ion

fro

m t

he i

nfec

ted

Per

iods

of

trea

tmen

t:

and

no

t tr

eate

d c

on

tro

l gr

oup

. A

= d

ay

2 to

10

p.i

. +

= P

< 0

.05

B =

day

11

to 1

7 p

.i.

+ +

= P

< 0

.01

C =

day

18

to 2

7 p

.i.

+ +

+ =

P <

0.0

01

D =

day

28

to 5

0 p

.i.

:;; NT

able

4.

Eff

icac

yo

fB

ayg

7183

,h

alof

ugin

one

,pr

imaq

uin

ean

dsu

lfaq

uin

ox

alin

ep

lus

py

rim

eth

amin

eon

dif

fere

nt

dev

elo

pmen

tal

stag

eso

fSa

rcoc

ysti

sm

uris

~ '" '" ? ::rN

oti

nf.

Infe

cted

Bay

g71

83H

alo

fug

ino

neP

rim

aqui

ne

Su

lfaq

uin

ox

alin

epl

us'< '!"

con

tro

lco

ntr

ol

Py

rim

etha

min

e(3

.3pl

.1)

r-g

rou

pg

rou

pA

nC

DA

BC

DA

BC

])A

DC

D:» ~ c a. '!" :»

Nu

mb

ero

fm

ice

4080

4040

403

940

39

4040

40

383

940

3840

4040

N '""D

ose

0

(mg

/kg/

bod

y-

-6

0.28

331

170

wei

gh

t/da

y)

Nu

mb

ero

fm

ice

+++

++

++

++

++

++

++

++

++

+po

siti

ve0

2816

013

1532

2311

1822

212

106

027

0

Per

cent

age

of

mic

epo

sitiv

e0

3540

032

.53

8.5

805

927

.545

555.

330

.825

15.8

067

.50

Nu

mb

ero

fvi

sibl

e+

++

+++

++

++

cyst

s(x)

per

pos

i-0

24.

59.

50

29.7

9.5

11.1

9.1

39.

110

.714

.21

29.8

34.

18

.20

1.4

0V

>Il

'ti

vem

ouse

'"('l 0N

um

ber

of

cyst

o-

++

++

++

++

++

+('

l'<

zoit

cs(x

)pe

rg

08

3.3

010

.92

.72.

41.

78

.23

.14

.40

.87.

816

.13

.10

1.2

0~ V;

.m

uscu

latu

re3

(in

mil

lion

s)l::

:::l

.":' ;- ;:;-

-l-,+

+,+

++=

sig

nif

ican

td

evia

tion

fro

mth

ein

fect

edP

erio

ds

of

trea

tme

nt:

:4 O·an

dn

ottr

eate

dco

ntr

ol

gro

up.

A=

day

2to

10p

.i.

:::l

+=

p<

0.0

5II

=d

ay11

to17

p.i

.++

=p

<0

.01

C=

day

18to

27p

.i.++

+=

P<

0.0

01

D=

day

28to

50p

.i.

IV '-I

w

:;; N

Tab

le 4

. E

ffic

acy

of

Bay

g 7

18

3,

hal

ofu

gin

on

e, p

rim

aqu

ine

and

sulf

aqu

ino

xal

ine

plu

s p

yri

me

tham

ine

on

dif

fere

nt

dev

elo

pm

enta

l st

ages

of

Sarc

ocys

tis

mur

is

~ '" .. ~

:t

'<

'!" :- ;r.

~

C

:::!.

'!"

;r.

Nu

mb

er o

f m

ice

N ~

Dos

e (m

g/k

g/b

od

y

wei

gh

t/d

ay)

Nu

mbe

r o

f m

ice

posi

tive

Per

cen

tag

e o

f m

ice

pos

itiv

e

Nu

mb

er

of

visi

ble

cy

sts (

x)

per

po

si

tiv

e m

ous

e

Nu

mb

er o

f cy

sto

zo

ites

(x)

per

g

mu

scul

atur

e (i

n m

illi

on

s)

No

t in

fo

Infe

cted

co

ntr

ol

con

tro

l g

rou

p

gro

up

A

40

80

40

o 2

8

16

o 3

5

40

+

o 2

4.5

9

.5

+

o 8

3.3

Bay

g 7

183

B

C

40

40

6

+++

0 13

0 32

.5

0 29

.7

0 10

.9

+. +

+,

+++

= si

gnif

ican

t d

evia

tio

n f

rom

th

e in

fect

ed

and

no

t tre

ated

con

tro

l g

rou

p.

-I-=

P

< 0

.05

+

+ =

P

< 0

.01

++

+ =

P

< 0

.00

1

D

39

15

38

.5

+ 9.5

+

2.7

Hal

ofu

gin

on

e

A

B

C

40

3

9 4

0

0.2

83

++

+

32

2

3

11

80

5

9

27

.5

+

++

11

.1

9.1

39.1

++

++

+

2.4

1.

7 8

.2

Per

iod

s o

f tr

eatm

ent:

A

=

da

y 2

to 1

0 p

.i.

B

=

day

11

to 1

7 p

.i.

C =

da

y 18

to

27 p

.i.

D =

d

ay 2

8 to

50

p.i

.

Pri

maq

uin

e

D

A

B

C

40

4

0 3

8

39

31

+

++

+

18

22

2

12

45

55

5.3

3

0.8

+

10.7

1

4.2

2

9.8

3.1

4.4

0

.8

7.8

D

40

10

25

34

.1

16.1

Su

lfaq

uin

ox

alin

e p

lus

Py

rim

eth

amin

e (3

.3 p

I. 1)

A

BC

D

38

4

0 40

4

0

170

+

+++

++

+ +

++

6

0 2

7

0

15.8

0

67

.5

0

+

+++

8

.2

0 1.

4 0

+

++

+

3.1

0

1.2

0

V>

I"

... (") 0 (")

'< ~

<n'

3 ~ ~.

~ ;- (;'

~ o·

::>

N " ""'

Tab

le5

.Eff

icac

yof

12dr

ugs

on

diff

eren

tde

velo

pmen

tal

stag

esof

Sarc

ocys

tis

mur

isin

NM

RI-

mic

e.In

ocu

lati

ondo

se=

8000

spor

ocys

ts~

2-1

1(s

poro

zoit

es)

Sub

stan

ce

sulf

aqui

noxa

line

plus

pyri

met

ham

ine

(3.3

plus

1)

zoal

ene

Bay

g71

83

prim

aqui

nedi

phos

phat

e

lasa

loci

d

halo

fugi

none

sulf

adox

ine

plu

str

imet

hopr

im(5

plus

1)

sulf

adim

etho

xine

ampr

oliu

m

mon

ensi

n

apri

noci

d

sulf

aqui

noxa

line

plus

diav

erid

ine

(4p

lus

1)

Do

se(m

g/k

g/b

ody

wei

ght/

day

)

170.

5

63.9

5.6

31.4

25.8 0.2

83

47.

9

109

.2

32

2.9

47.3

14.4

43.3

+/X

+1

+/

+/

+/

+1

Tre

atm

ent

days

po

stin

ocul

atio

n(p

aras

itic

stag

esdo

min

atin

g)1

1-1

71

8-2

72

8-5

0(s

chiz

onts

)(m

eroz

oite

s)(y

oung

cyst

s)

++

+/X

XX

++

+/

++

+/x

xx

++

+/x

xx

Ix+

++

/xx

x+

/+

++

/xx

x+

++

/x+

/+

+/

+/

+/x

Ix+

/

~ ;;0 o S S p.. ?'" V)

r> ::r ~ ... ~ ~ '".... ~ ::> p..

~ tl:l

ib ::E ~ en ;>;'"~

+=

redu

ctio

nof

inte

nsit

yo

fin

fect

ion

x=

redu

ctio

nof

infe

ctio

nra

te+

/xlo

wly

sign

ific

ant

++

/xx

sign

ific

ant

++

+/x

xx

high

lysi

gnif

ican

t

Tab

le 5

. E

ffic

acy

of

12

dru

gs

on

dif

fere

nt d

evel

opm

enta

l stage~ o

f Sa

rcoc

ysti

s m

uris

in N

MR

I-m

ice.

In

ocu

lati

on

do

se =

80

00 s

po

rocy

sts

Sub

stan

ce

sulf

aqui

noxa

line

plu

s p

yri

met

ham

ine

(3.3

plu

s 1)

zoal

ene

Bay

g 7

183

pri

maq

uin

e d

iph

osp

hat

e

lasa

loci

d

halo

fugi

none

su.J

fado

xine

plu

s tr

imet

ho

pri

m (

5 p

lus

1)

sulf

adim

etho

xine

amp

roli

um

mon

ensi

n

apri

noci

d

sulf

aqui

noxa

line

plu

s di

aver

idin

e (4

plu

s 1)

Do

se

(mg

/kg

/bo

dy

w

eigh

t/da

y)

170.

5

63.9

5.6

31

.4

25.8

0.28

3

47.9

109.

2

322.

9

47.

3

14.4

43.3

+ =

re

du

ctio

n o

f in

ten

sity

of

infe

ctio

n

x =

re

du

ctio

n o

f in

fect

ion

rat

e

Tre

atm

ent

days

po

st i

no

cula

tio

n (

par

asit

ic s

tage

s d

om

inat

ing

) 2-

11

11

-17

1

8-2

7

28

-50

(s

poro

zoit

es)

(sch

izon

ts)

(mer

ozoi

tes)

(y

oung

cys

ts)

+ /x

+

++

/xx

x

++

+/

++

+/x

xx

+/

++

+/x

xx

/x

+/

++

+/x

xx

+

/ +

++

/xx

x

+ +

+/x

+

/ +

/ +

+/

+/

+/

+/x

/x

+/

+/

+ / x

lo

wly

sig

nifi

cant

+

+ / x

x

sign

ific

ant

+ +

+ /

x x

x hig

hly

sig

nifi

cant

~

~

:;z:l o § ~ ?>

en

g..

~ .. e- f ... ., ::

l P. :n o:l

(b

~ ., '" :>':"

.,


Table 6. Sarcocystis muris: Influence of the inoculation dose on the infection rate and theintensity of the infection in NMRI-mice

Inoculation dose(sporocysts)

Number of Infection rateanimals per trial (per cent)

Intensityof infection

51050

100500

10001000050000

1000005 every 2nd day for 30 days

50 every 2nd day for 30 days50 every 2nd week for 4 weeks2 x 5 3 days apart2 x 50 3 days apart

5050505050505050404950494950

323074702426283028162

164122

++++++++++++++++++++++

+ = light infection (1 to 300 cysts per mouse)++ = medium infection (more then 300 cysts per mouse)

+ + + = very strong infection with impairment of locomotion

Table 7. Sarcocystis muris: Influence of mouse-strain and immuno-suppressive treatmenton infection rate and intensity of infection. Inoculation dose = 50 sporocysts

Strain of mouse and treatment Number of Infection rate Intensityanimals per trial (per cent) of infection

NMRI-WiGa 20 70 ++C57BLj6J Han 20 75 ++AKRjNHan 20 95 +++C3HjHeHan 20 25 +NMRI-nujnu 19 100 +++NMRI-WiGa + ALS 18 83 ++NMRI-WiGa + dexamethasone 51 100 +++NMRI-WiGa + cyclophosphamide 49 92 +++NMRI-WiGa + azathioprin 48 54 +++NMRI-WiGa + irradiated 58 98 +++

+ = light infection (1 to 300 cysts per mouse)++ = medium infection (more then 300 cysts per mouse)


Discussion

It could be shown that several anticoccidial or antimalarial drugs have a goodefficacy against Sarcocystis muris when administered from day 11 to day 17 postinfection. During this period the schizogonic stages are present in the liver. These


Table 6. Sarcocystis muris: Influence of the inoculation dose on the infection rate and the intensity of the infection in NMRI-mice

Inoculation dose Number of Infection rate Intensity (sporocysts) animals per trial (per cent) of infection

5 50 32 ++ 10 50 30 ++ 50 50 74 ++

100 50 70 ++ 500 50 24 +

1000 50 26 + 10000 50 28 + 50000 50 30 +

100000 40 28 + 5 every 2nd day for 30 days 49 16 ++

50 every 2nd day for 30 days 50 2 + 50 every 2nd week for 4 weeks 49 16 ++ 2X5 3 days apart 49 41 + 2 x 50 3 days apart 50 22 +++

+ = light infection (1 to 300 cysts per mouse) ++ = medium infection (more then 300 cysts per mouse)

+++ = very strong infection with impairment of locomotion

Table 7. Sarcocystis muris: Influence of mouse-strain and immuno-suppressive treatment on infection rate and intensity of infection. Inoculation dose = 50 sporocysts

Strain of mouse and treatment Number of Infection rate animals per trial (per cent)

NMRI-WiGa 20 70 C57BL/6J Han 20 75 AKR/NHan 20 95 C3H/HeHan 20 25 NMRI-nu/nu 19 100 NMRI-WiGa + ALS 18 83 NMRI-WiGa + dexamethasone 51 100 NMRI-WiGa + cyclophosphamide 49 92 NMRI-WiGa + azathioprin 48 54 NMRI-WiGa + irradiated 58 98

+ = light infection (1 to 300 cysts per mouse) + + = medium infection (more then 300 cysts per mouse)


Discussion

Intensity of infection

++ ++ +++ + +++ ++ +++ +++ +++ +++

It could be shown that several anti coccidial or antimalarial drugs have a good efficacy against Sarcocystis muris when administered from day 11 to day 17 post infection. During this period the schizogonic stages are present in the liver. These

276 M. Rommel, A.Schwerdtfeger, and S.Blewaska

are the pathogenic stages in Sarcocystis infections of domestic animals. As wellanticoccidial as antimalarial drugs showed to be effective, the most reliable substances being sulfaquinoxaline plus pyrimethamine, zoalene and Bay g 7183. Stagespresent from day 2 to 10 and from day 18 to 27 post infection could not be completely eliminated by any of the drugs tested. The good efficacy of sulfaquinoxalineplus pyrimethamine against young cystic stages present from day 28 to 50 of theinfection is surprising. These results need, however, confirmation. There is no explanation for the enhancement of the infection rate and/or the intensity of the infection by some of the drugs.

The results of our experiments encourage the testing of additional substancesagainst S. muris and to retest the best drugs of the present trial to find the minimaleffective dose. The effective drugs should then be tested in cases of acute sarcocystosis of domestic animals.

Our trials to improve the model Sarcocystis muris-mouse-cat have shown thatwith an inoculation dose of only 50 sporocysts maximal infection rates can beachieved. Furthermore it was found that the infection rate very much depends fromthe strain of mouse used. 100 per cent and 95 per cent infection rates were achievedin nude mice and in the AKR/N Han-strain, respectively, the other strains testedbeing less susceptible. The two highly susceptible races are, however, too expensivefor routine screening work.

By irradiation or by the application of corticosteroids the susceptibility of cheapNMRI-mice can be increased considerably to infection rates of 98 and 100 per cent,respectively. Sinceirradiation has to be done only once, in contrast to the continousapplication of the corticosteroids, irradiation seems to be the method of choice forthis type of screening.

Since it was found that drugs with an efficacy against exoerythrocytic stages ofPlasmodium were also active against the schizonts of Sarcocystis muris in liverparenchymal cells, the described model can also be used for the search for newantimalarials.

References

1. Bletoaska, S.: Versuche zur Steigerung der Befallsintensitat und -exrensitat der Sarcocystis muris-Infektion in Mausen. Med. vet. Diss., Hannover (1981)

2. Fayer, R. and A.J.Johnson: Effect of AmproIium on acute sarcocystosis in experimentally infected calves. J.Parasit. 61 (1975) 932-936

3. Leek, R. G. and R.Fayer: AmproIium for prophylaxis of ovine Sarcocystis. J. Parasit. 66(1980) 100-106

4. Rommel, M., A. O. Heydorn und M. Erber: Die Sarkosporidiose der Haustiere und desMenschen. Berl. MUnch. tierarztl. Wschr. 92 (1979) 457-464

5. Ruiz, A. and J.K. Frenkel: Recognition of cyclic transmission of Sarcocystis muris bycats. J. infect. Dis. 133 (1976) 409-418

6. Schwerdtfeger, A.: Untersuchungen zur Chemotherapie der Sarcocystis-muris-Infektion.Med. vet. Diss., Hannover (1980)

7. Torp, C.: Untersuchungen iiber den Einfluf der Sarcocystis muris-Infektion auf dieTrachtigkeit und das Aufzuchtsergebnis von NMRI-Mausen. Med. vet. Diss., Hannover(1979)

Professor Dr. Michel Rommel, Institut fiir Parasitologie, Tierarztliche Hochschule,Biinrewcg 17, D-3000 Hannover 71

276 M. Rommel, A. Schwerdtfeger, and S. Blewaska

are the pathogenic stages in Sarcocystis infections of domestic animals. As well anticoccidial as antimalarial drugs showed to be effective, the most reliable substances being sulfaquinoxaline plus pyrimethamine, zoalene and Bay g 7183. Stages present from day 2 to 10 and from day 18 to 27 post infection could not be completely eliminated by any of the drugs tested. The good efficacy of sulfaquinoxaline plus pyrimethamine against young cystic stages present from day 28 to 50 of the infection is surprising. These results need, however, confirmation. There is no explanation for the enhancement of the infection rate and/or the intensity of the infection by some of the drugs.

The results of our experiments encourage the testing of additional substances against S. muris and to retest the best drugs of the present trial to find the minimal effective dose. The effective drugs should then be tested in cases of acute sarcocystosis of domestic animals.

Our trials to improve the model Sarcocystis muris-mouse-cat have shown that with an inoculation dose of only 50 sporocysts maximal infection rates can be achieved. Furthermore it was found that the infection rate very much depends from the strain of mouse used. 100 per cent and 95 per cent infection rates were achieved in nude mice and in the AKR/N Han-strain, respectively, the other strains tested being less susceptible. The two highly susceptible races are, however, too expensive for routine screening work.

By irradiation or by the application of corticosteroids the susceptibility of cheap NMRI-mice can be increased considerably to infection rates of 98 and 100 per cent, respectively. Since irradiation has to be done only once, in contrast to the continous application of the corticosteroids, irradiation seems to be the method of choice for this type of screening.

Since it was found that drugs with an efficacy against exoerythrocytic stages of Plasmodium were also active against the schizonts of Sarcocystis muris in liver parenchymal cells, the described model can also be used for the search for new antimalarials.

References

1. Blewaska, S.: Versuche zur Steigerung der Befallsintensitat und -extensitat der Sarcocystis muris-Infektion in Mausen. Med. vet. Diss., Hannover (1981)

2. Fayer, R. and A.J.Johnson: Effect of Amprolium on acute sarcocystosis in experimentally infected calves. J. Parasit. 61 (1975) 932-936

3. Leek, R. G. and R.Fayer: Amprolium for prophylaxis of ovine Sarcocystis. J. Parasit. 66 (1980) 100-106

4. Rommel, M., A. O. Heydorn und M. Erber: Die Sarkosporidiose der Haustiere und des Menschen. Berl. MUnch. tierarztl. Wschr. 92 (1979) 457-464

5. Ruiz, A. and J. K. Frenkel: Recognition of cyclic transmission of Sarcocystis muris by cats. J. infect. Dis. 133 (1976) 409-418

6. Schwerdtfeger, A.: Untersuchungen zur Chemotherapie der Sarcocystis-muris-Infektion. Med. vet. Diss., Hannover (1980)

7. Torp, C.: Untersuchungen Uber den EinfluB der Sarcocystis muris-Infektion auf die Trachtigkeit und das Aufzuchtsergebnis von NMRI-Mausen. Med. vet. Diss., Hannover (1979)

Professor Dr. Michel Rommel, Institut fUr Parasitologie, Tierarztliche Hochschule, BUnteweg 17, D-3000 Hannover 71

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