Veterinary Parasitology, 24 (1987) 157--168 157

Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

T h e P r e v a l e n c e and I d e n t i t y of Sarcocystis in B e e f
Cattle in N e w Zealand
A. B~TTNER 1, W.A.G. CHARLESTON 2, W.E. POMROY and M. ROMMEL ~
Department of Veterinary Pathology and Public Health, Massey University, Palmerston
North (New Zealand)
(Accepted for publication 4 July 1986)

ABSTRACT

BSttner, A., Charleston, W.A.G., Pomroy, W.E. and Rommel, M., 1987. The prevalence
and identity of Sarcocystis in beef cattle in New Zealand. Vet. Parasitol., 24: 157--168.

Muscle tissue from the oesophagus and diaphragm of 500 beef cattle slaughtered
in New Zealand was examined for Sarcocystis infection by microscopic examination of
cysts isolated from muscle samples. All cattle were infected with Sarcocystis; based on
light microscopy of cysts, 98% had thin-walled Sarcocystis cruzi cysts and 79.8% had
thick-walled (Sarcocystis hirsuta/Sarcocystis hominis) cysts. Cysts were also collected
for electron microcopy and transmission experiments. Thick-walled cysts could not be
distinguished as S. hirsuta or S. hominis by light or electron microscopy. Thick-walled
cysts were fed to three cats and one human volunteer; one cat shed sporocysts but not
the human volunteer. Electron microscopy of the cysts revealed many features that have
not been described previously.

INTRODUCTION

Three species of 8arcocystis have been described from the muscles of

cattle (Bos taurus): Sarcocystis cruzi (syn. 3arcocystis bovicanis) of which
canids are definitive hosts; Sarcocystis hirsuta (syn. Sarcocystis bovifelis)
transmitted by cats and Sarcocystis hominis (syn. Sarcocystis bovihominis)
transmitted by man (Levine and Tadros, 1980).
In investigating the prevalence of Sarcocystis in intermediate hosts, the
digestion technique, releasing cystozoites from muscle samples, has proved
to be most reliable. This m e t h o d was first described by Jacobs et al. (1960)
and later modified by various workers (Seneviratna etal., 1970; Erber, 1977;
Box and McGuinness, 1978; Collins et al., 1980). For identification of
species, intact cysts have to be released out of the muscle of infected animals.
Various techniques for this have been described by Ether (1977), Hinaidy
(1980) and Jungmann and Knoch (1980). The cyst wall structure is
considered to be one possible criterion for identifying species. 8arcocystis
I Present address: Ministry of Agriculture and Fisheries, Private Bag, Hastings, N e w
Zealand.
2 Author to whom correspondence should be addressed.
3Present address: Institute of Parasitology, School of Veterinary Medicine, Buenteweg 17,
D-3000 Hannover 71, F.R.G.
0304-4017/87/$03.50 © 1987 Elsevier Science Publishers B.V.
158

species are highly host-specific and species identification may also be

possible by transmitting them to their specific final host ( R o m m e l and
Heydorn, 1974; Gestrich et al., 1975a).
The sarcocysts of S. cruzi can be distinguished readily from those of the
other species in cattle by the structure of the cyst wall as revealed under
light- and electron-microscopy. Under light microscopy (LM) the wall of
S. cruzi cysts appears thin (~ 1.0 pm) with external hair-like protrusions that
are very slender (< 0.5 pm) and difficult to see (Gestrich et al., 1975a;
Mehlhorn et al., 1975b). In contrast, the walls of S. hirsuta and S. hominis
cysts bear a dense palisade-like layer of protrusions on the outer surface so
that they appear distinctly thick-walled (4--6 p m thick) under LM (Gestrich
et al., 1975a, b; Mehlhorn et al., 1975a). Some workers have used the
widths of the cyst wall protrusions as a basis for identifying cysts recov-
ered from naturally infected cattle and for establishing prevalence data
(Boch et al., 1978; Hinaidy et al., 1979).
In this paper we report the prevalence and morphology of microscopic
sarcocysts in muscles of 500 beef cattle mostly from the southern half of
the North Island of New Zealand.

MATERIALS AND METHODS

Between April 1983 and January 1984, samples of diaphragm muscle

(approximately 30 g) and oesophagus (approximately 10--20 cm of the
caudal end) were collected from 500 unselected cattle slaughtered at a local
meatworks. Most of the cattle were prime beef animals, 18--24 months of age.
The origins of 241 cattle were traced; the source of most of the others could
not be accurately identified as they were submitted by dealers.
Initially, 2-g samples of material collected were subjected to pepsin-HC1
digestion (Jacobs et al., 1960) for 20 min at 40°C. The digest was sieved and
the filtrate Centrifuged at 500 g for 5 min. The supernatant was removed,
the sediment resuspended in a small volume of saline and 2--3 drops of it
examined for Sarcocystis zoites at 400 X magnification. Later, when it was
found that individual cysts could be readily obtained, the pepsin digest
procedure was carried o u t only on samples from which cysts were n o t
recovered.
To obtain individual cysts, tissue samples were examined by a modifica-
tion of the m e t h o d described by Erber (1977). Fifteen-gram samples of
muscle from each oesophagus or diaphragm were finely c h o p p e d and
suspended in 50 ml 0.85% NaCI solution in a flask. A b o u t 50 glass beads (4
mm diameter) were added and the flask shaken vigorously for 10 min in a
mechanical shaker. The contents of the flask were poured through a 2-mm
mesh sieve and the filtrate collected. The flask and sieved material were
washed with a further 20 ml of saline. The filtrate was allowed to stand for
30 min and the sedimented material examined under a dissecting
microscope. Cysts and cyst fragments were transferred to slides in saline, a
 159

coverslip applied and then examined microscopically at 400 X and 1000 ×

magnification. Measurements of the thickness of cyst walls and width of
protrusions were made using a calibrated micrometer eyepiece.
Cysts examined by LM were recorded as thin- or thick-walled and samples
of the latter with protrusion-widths corresponding with those considered by
Boch et al. (1978) as diagnostic of S. hominis (< 0.8/~m; type 1) and S.
hirsuta (> 1.3/~m; t y p e II) were collected for comparative study.
Freshly isolated cysts and cyst fragments were also processed for transmis-
sion- and scanning electron microscopy (TEM and SEM, respectively). Cysts
intended for TEM were placed in a drop of 20% bovine serum albumin which
was then coagulated in 23% glutaraldehyde. After 20 min, 2 X 2 mm blocks
of coagulated material were cut and fixed overnight in Karnovsky's fluid.
After washing three times in phosphate buffer, samples were post-fixed in
1% osmium tetroxide, washed again in phosphate buffer and dehydrated
through alcohols. The blocks were infiltrated with propylene oxide and
embedded in Durcupan-ACM resin (Fluka, AG). Sections were cut at 70--80
nm and stained with uranyl acetate and lead citrate.
Cysts for SEM were fixed overnight in modified Karnovsky's fixative,
washed in phosphate buffer and placed in osmium tetroxide for 1 h. The
cysts, enclosed in folded polycarbonate filter paper, were then dehydrated
through alcohols and critical point dried in an atmosphere of CO2. Cysts
were fixed to aluminum stubs with adhesive tape and sputter-coated with
gold and subsequently examined using a Cwikscan 100 field emission
scanning electron microscope.
A total of 18 thick-walled and 4 thin-walled cysts from different animals
were examined by TEM and a b o u t 50 thick- and thin-walled cysts from 17
animals by SEM.
Four cats and a human volunteer were used in transmission experiments.
One cat had been reared in the laboratory on canned or dried f o o d which
precluded Sarcocystis infection. The remaining cats were obtained at five
weeks of age; these had been conventionally reared but not fed raw meat.
All the animals were fed on processed f o o d and housed in individual cages.
Samples of faeces from the animals and the human volunteer were checked
three times before infection using a combined NaCl-sugar flotation
procedure, but no sporocysts were found.
The cats were given isolated thick-walled cysts and cyst fragments of
t y p e I (width of protrusions < 0.8 /~m) and t y p e II (width of protrusions
> 1.3 #m). One cat received 134 (type I), a second 230 (type I) and a third
190 (type II) cysts or cyst fragments. One cat remained as an uninfected
control. The human volunteer consumed 80 cysts/-fragments (type I) and
two months later 200 g raw beef as well as the thick-walled cysts recovered
from a further 800 g of beef. The second, third and control cats were killed
14 days post-infection (p.i.) and their intestines were subjected to trypsin
digestion (Kepper, 1981); the other cat's and the human volunteer's faeces
were examined for sporocysts up to 21 days p.i.
160

RESULTS

Prevalence of infection

Intact sarcocysts or cyst fragments were recovered from 498 of the 500
cattle; pepsin digestion of the samples of the remaining two animals showed
that they too were infected, as free Sarcocystis zoites were recovered.
Thin- and thick-walled cysts were easily distinguished by L.M. The pre-
valence and distribution of these two categories of cysts in the two sites
sampled are shown in Table I. Thin-walled cysts were found significantly
(P < 0.001) more often in the oesophagus than in the diaphragm (97.4%
and 51.8%, respectively). The prevalence of thick-walled cysts in the
diaphragm was significantly (P < 0.05) higher than in the oesophagus. The
occurrence of single and mixed infections in the material sampled is shown
in Table II. The origins of 241 cattle are indicated in Fig. 1; there was no
suggestion of regional differences in cyst prevalence.

Thin-walled cysts

Thin-walled cysts were readily identifiable by LM having a cyst wall <~ 1

/~m in thickness. By LM and TEM the thin-walled cysts were morphologically

• TE KUITI

TAIHAPE

Fig. 1. Origins of 241 cattle from the North Island of New Zealand.
 161

TABLE I
Prevalence of thin- and thick-walled Sarcocystis cysts in the oesophagus and diaphragm
of 500 cattle

Oesophagus Diaphragm Total

No. % No. % No. %

Thin-walled cysts 487 97.4 259 51.8 490 98.0

Thick-walled cysts 321 64.2 353 70.6 399 79.8

TABLE H

Occurrence of single and mixed infections with thin- and thick-walled Sarcocystis cysts
in the oesophagus and diaphragm of 500 cattle

Oesophagus Diaphragm Total

No. % No. % No. %

Thin-walled cysts 172 34.4 93 18.6 99 19.8

Thick-walled cysts 6 1.2 187 37.4 8 1.6
Mixed infections 315 63.0 166 33.2 391 78.2
Cyst isolation negative 7 1.4 54 10.8 2 0.4

indistinguishable from those of S. cruzi described by Gestrich et al. (1975a),

Mehlhorn et al. (1975b) and Dubey (1982a).
SEM of intact cysts showed these hair-like protrusions to be densely
packed on the cyst-surface (Fig. 2).

Thick-walled cysts

Thick-walled cysts belong either to S. hirsuta or S. hominis. They are

characterised by having a palisade-like layer of protrusions which densely
cover the entire cyst surface. Thus the cyst wall appears thick-walled and,
depending on the width of the individual protrusions, more or less striated
under the light microscope.
Measurements of the thickness of the cyst wall including the protrusions,
and the width of the protrusions, were made on 305 cysts derived from 31
samples of diaphragm and oesophagus. The distribution of cyst wall
thickness and protrusion widths are shown in Figs. 3 and 4, respectively. Cyst
wall thicknesses ranged from 2.0 to 7.0 ~m (5.01 + 0.68 pro) and were
approximately normally distributed. Protrusion widths ranged from 0.6
to 2.2 ~m (0.99 + 0.27 pro) and also formed a continuous, though skewed,
distribution.
During the examination of released cysts, it was observed that the
162

Fig. 2. Thin-walled cyst (S. cruzi), protrusions densely packed on the cyst surface (SEM).

Nc~
NO.
120 7
z
100
100
8O
80
60
60
4O
4O
20
20

0 O,
0 " ~ 5 / 2 " 0 2"5 3:0 35 4"0 ~S SO 5~ 6"0 6"5 ZO Ism C).2 ().4 0.6 0.8 1.0 1.2 1.4 1.6, 1.8 2.0 2.2 prn

Fig. 3. Distribution of cyst wall thickness of thick-walled cysts.

Fig. 4. Distribution of widths of cyst wall protrusions of thick-walled cysts.

apparent width o f individual protrusions could be increased by increasing the

pressure o n the coverslip. The explanation for this emerged from SEM
examination of these cysts as described below.
TEM examination of the cyst wall showed that all cysts examined had
basically the same structure. The protrusions were b o u n d e d exteriorly by
a relatively s m o o t h electron-dense layer, the primary cyst wall comprising
a unit membrane underlain by osmiophilic substance (Fig. 5a). In some cysts
 163

the primary cyst wall was very irregular (Fig. 5b), either because of small
vesicle-like invaginations of the unit membrane (Fig. 5a, b) or the irregular
course of the primary cyst wall. These vesicle-like invaginations were always
present at the base of the protrusions and the adjacent cyst wall. Areas
where the unit membrane formed these invaginations were not underlaid
with osmiophilic substance.
Within the protrusions, fibrillar and tubule-like structures were present
(Fig. 5a--d). Some protrusions were entirely filled with fibrils and in others
they were mainly confined to the base of the protrusions (Fig. 5c). In some
sections, adjacent fibrils were joined together by crossbars forming pairs
which appeared like striated bands in longitudinal sections. Such pairs ran

Fig. 5a--d. T E M sections o f thick-walled cysts, a, b, c: protrusions (P), c u t longitudinally,

with fibrillar and tubule-like elements (F); PC ffi primary cyst wall, IN = vesicle-like
invaginations, GS = ground substance, d: protrusions c u t transversely; same cyst as c.
164

approximately parallel from the tip to the base of the protrusions (Fig. 5a, c)
and in cross section appeared to form a network of structures (Fig. 5d).
The width of the protrusions as seen under TEM was very variable 10.5--1.4
~m). In most cysts, protrusions were closely packed on the primary cyst wall
(0.03--0.15~pm apart, Fig. 5a), b u t in others they were more widely spaced
(> I pm, Fig. 5b).
Cysts initially classified as S. hirsuta and S. hominis on the basis of
protrusion widths measured under light microscopy, revealed no features
under TEM that were suggestive of distinctive morphologies.
SEM of cysts revealed a number of interesting features. The cyst wall
protrusions were flattened and tongue-like (Figs. 6 and 7b). The tips of the
protrusions, as seen from the surface view of the cyst, varied in appearance
(Fig. 7a--d); in some cases they were thin and overlapped one another like
tiles on a roof, while in others the tips showed varying degrees of enlarge-
m e n t and surface irregularity.
Narrow bridges between individual protrusions were occasionally seen in
SEM as shown in Fig. 7d. Again no differences were observed between
putative S. hirsuta and S. hominis cysts.
The transmission experiments shed little light on the identity o f the thick-
walled cysts. One cat alone passed a few sporocysts in its faeces on Days

Fig. 6. Thick-walled cyst. Break in cyst wall exposing tongue-like protrusions; SEM.
 165

Fig. 7a---d. SEM of thick-walled cysts, a: view of the tips of the protrusions; b: cyst with
thin flattened protrusions; c: showing tips of protrusions overlapping one another; d:
showing narrow bridges (arrows) between individual protrusions.

11--17 p.i.; no sporocysts were recovered from the intestinal digests of the
kittens or from the faeces of the human volunteer.

DISCUSSION

Although somewhat time-consuming, the procedure for recovering cysts

and cyst fragments from muscle samples proved very successful, yielding cyst
material suitable for further examination from > 99% of animals.
No attempt was made to passage the thin-walled cysts, but morphological-
ly they were indistinguishable from those of S. cruzi described by others
(Gestrich et al., 1975a; Mehlhorn et al., 1975b; Dubey, 1982a) and there is
166

no reason to d o u b t their identity. Considering the small a m o u n t of muscle

examined, an observed prevalence of 98% S. cruzi infection indicates that
virtually all cattle in the sample had been exposed to this species. This is
comparable with the prevalences recorded in Germany (66%; Boch et al.,
1978) and Austria (70%; Hinaidy et al., 1979).
Thick-walled cysts were recovered from 79.8% of animals, b u t these could
not be identified as S. hirsuta and/or S. hominis on morphological grounds.
The continuous distribution of the measurements of the thickness of cyst
walls and the widths of individual protrusions provided no basis for the
separation of the cysts recovered into two types. The continuous distribu-
tion of protrusion widths (from 0.6 to 2.2 ~m) is in contrast to the observa-
tions by Boch et al. (1978) that they form two entirely distinct populations
(~ 0.8 g m and t> 1.3 ~m in width), on the basis of which they identified
cysts as S. hominis and S. hirsuta, respectively, by light microscopy of fresh-
ly isolated cysts.
Scanning EM of these cysts shows the cyst surface to be densely covered
with protrusions which are flattened and tongue-like. It is difficult, there-
fore, to be sure one is measuring only one row of protrusions in fresh
preparations. The shape of the protrusions also accounts for the observation
that their width could be altered by varying the pressure of the coverslip
on the cyst. Accurate measurement of protrusion widths is, therefore,
extremely difficult. Nevertheless, there was no indication of two populations
in our measurements.
It is difficult to be certain, furthermore, if the criteria used by Boch et al.
(1978) are valid in all circumstances. There are very few published descrip-
tions of sarcocysts of these species: the development of the cyst wall has
been studied for S. hominis up to 98 days p.i. (Mehlhorn et al., 1975a)
and for S. hirsuta up to day 160 p.i. (Gestrich et al., 1975b; Boch et al.,
1978). Protrusion widths have been recorded only from these studies, once
for S. hominis, twice for S. hirsuta. These descriptions are a meagre basis for
distinguishing cysts of indeterminate age, particularly as it cannot be
assumed that cyst morphology does not change significantly with age.
Studies to be published on the structure and identity of macroscopic sarco-
cysts from cattle indicate that very considerable changes occur (BSttner,
1984).
Differences in the fine-structure of the cyst walls of S. hirsuta and S.
hominis have been described by Gestrich et al. (1975b) and Mehlhorn et al.
(1975a), though again of relatively y o u n g cysts. The differences relate
particularly to fibriUar structures within the protrusions. Many of the cysts
examined by TEM in this study resembled the descriptions of S. hirsuta,
b u t none showing the features of S. hominis were seen. While all cysts
examined showed essentially similar structures, there were considerable
variations in detail. Whether these differences resulted from cysts of differing
ages or species being examined, or from delays in fixation that were un-
avoidable in some cases, could n o t be determined.
 167

A t t e m p t s to transmit the thick-walled cysts to potential definitive hosts

were almost totally unsuccessful and no definite conclusions can be drawn
as to the identity of these cysts from the transmission experiments. A
major problem is the collection of sufficiently large numbers of cysts to
constitute an infective dose large enough for the production of sporo-
cysts in detectable numbers, particularly in faeces. Most of the cysts iso-
lated were 1--2 m m in length and contained what appeared to be fully
developed zoites; t h e y were generally larger than those described by Dubey
(1982b) as infective.
However, in work with S. gigantea, McKenna (1984) f o u n d that < 0.1%
of zoites fed to cats could be accounted for in terms of sporocysts produced
in faeces, so our difficulties could perhaps have been c o m p o u n d e d by a
similarly poor success rate though we have no direct evidence for this.
At this stage the identity of the thick-walled cysts examined in this study
is unclear. We have f o u n d no basis for categorising these cysts into groups
that could be interpreted in terms of species. It is possible, of course, t h a t
they were all of one species, most probably S. hirsuta, and that the variations
in cyst structure observed were primarily due to age. If S. hominis was
present, it could n o t be distinguished.

ACKNOWLEDGEMENTS

Our thanks are due to to the proprietors of Crown Meats Ltd., Feilding
and their staff, as well as Ministry of Agriculture and Fisheries staff
employed there, for their cooperation during this work. Thanks also to
D. Hopcroft for his skilled assistance with the EM work; to S. Thomas and
S. Calder for technical assistance and T. Law for photographic work. The
study was carried o u t during tenure (A. B~ttner) of a Rotary F o u n d a t i o n
Scholarship.

REFERENCES

Boch, J., Laupheimer, K.E. and Erber, M., 1978. Drei Sarkosporidienarten bei Schlach-
trindern in Siiddeutschland. Berl. Miinch. Tier~ztl. Wschr., 91: 426--431.
BSttner, A., 1984. Vorkommen sowie licht und elektronenmikroskopische Differen-
zierung yon Sarkosporidienarten hie Schlachtrindern auf der Nordinsel Neuseelands.
Vet. med. Diss., Hannover, 71 pp.
Box, E.D. and McGuinness, T.B., 1978. Sarcocystis in beef from retail outlets demon-
strated by digestion technique. J. Parasitol., 64: 161--162.
Collins, G.H., Charleston, W.A.G. and Wiens, B.G., 1980. Studies on Sarcocystis species.
6. A comparison of three methods for detection of Sarcocystis species in muscle.
N. Z. Vet. J., 28: 173.
Dubey, J.P., 1982a. Development of the ox--coyote cycle of Sarcocystis cruzi. J.
Protozool., 29: 591--601.
Dubey, J.P., 1982b. Development of the ox--coyote cycle of S. hirsuta. Proc. Helminthol.
Soc. Wash., 49: 295--304.
168

Erber, M., 1977. M6glichkeiten des Nachweises und der Differenzieurung yon zwei
Sarkozystenarten des Schweines. Berl. Mtinch. TierSxztl. Wschr., 90: 480--482.
Gestrich, R., Heydorn, A.-O. and Baysu, N., 1975a. Beitr~'ge zum Lebenszyklus der
Sarkosporidien. VI. Untersuchungen zur Artendifferenzierung bei Sarcocystis
fusiformis und Sarcocystis tenella. Berl. Mtinch. TieHirztl. Wschr., 8 8 : 1 9 1 - - 1 9 7 and
201--204.
Gestrich, R., Mehlhorn, H. and Heydorn, A.-O., 1975b. Licht und elektronenmikros-
kopische Untersuchungenan Cysten yon Sarcocystis fusiformis in der Musku]aturyon
K~]bern nach experimenteller Infektion mit Oocysten und Sporocysten der grossen
Form yon Isospora bigemina der Katze. Zbl. Bakt. Hyg., I. Abt. Orig. A, 233: 261--
276.
Hinaidy, K.H., 1980. Vereinfachte Homogenatmethode zum Nachwies yon Sarko-
sporidien bei Schlachtrindern. Wien. TierSxztl. Mschr., 67: 54--55.
Hinaidy, K.H., Burgu, A., Supperer, R. and Kallab, K., 1979. Sarkosporidienbefall des
Rindes in Osterreich. Wein,Tier~irtztl.Mschr., 66: 181--184.
Jacobs, L., Remington, J.S. and Melton, M.L., 1960. A survey of meat samples from
swine, cattle and sheep for the presence of encysted Toxoplasma. J. Parasitol., 46:
23--28.
Jungmann, R. and Knoch, W., 1980. MSglichkeiten des Sarkosporidiennachweises unter
besonderer Berticksichtigung der Artendifferenzierung. Mhfte. Vet. Med., 35: 947--
949.
Kepper, A., 1981. Untersuchungen tiber den Verlauf der Antik6rpertiter bei ein- und
mehrmals mit Sarcocystis muris inokulierten NMRI-Maiisen mit Hilfe des indirekten
Immunfluoreszenzantik6rpertestes (IFAT). Vet. reed. Diss., Hannover, 60 pp.
Levine, N.D. and Tadros, W., 1980. Named species and hosts of Sarcocystis (Protozoa:
Apicomplexa:Sarcocystidae). Syst. Parasitol., 2: 41--49.
McKenna, P.B., 1984. Sarcocystis gigantea: Studies on sporocyst production, excystation
and viability. Ph.D. Thesis, Massey University, Palmerston North, New Zealand.
Mehlhorn, H., Heydorn, A.-O. and Gestrich, R., 1975a. Licht-und elektronen-
mikroskopische Untersuchungen an Cysten von Sarcocystis fusiformis in der
Muskulatur yon K~/lbern nach experimenteller Infektion mit Oocysten und
Sporocysten von Isospora hominis Railliet et Lucet, 1891. 1. Zur Entstehung der
Cyste und der Cystenwand. Zbl. Bakt. Hyg., I. Abt. Orig. A, 231: 301--322.
Mehlhorn, H., Heydorn, A.-O. and Gestrich, R., 1975b. Licht-und elektronenmikros-
kopische Untersuchungen an Cysten von Sarcocystis fusiformis in der Muskulatur
yon K~lbern nach experimenteUer Infektion 1-nit Oocysten und Sporocysten der
grossen Form von Isosopora bigemina des Hundes. 1. Zur Entstehung der Cyste und
der Cystenwand. Zbl. Bakt. Hyg., I. Abt. Orig. A, 232: 392--409.
Rommel, M. and Heydorn, A.-O., 1974. Neue Erkentnisse zur Epidemiologie der Sar-
kosporidiose bei Mensch und Tier. Fortschr. Veterin~med., 20: 104--106.
Seneviratna, P., Edwards, A.G. and DeGiusti, D.L., 1970. Frequency of Sarcocystis spp.
in Detroit, Metropolitan area, Michigan. Am. J. Vet. Res., 36: 337--339.