The prevalence and intensity of Sarcocy~tiSspp infections in sheep

P J O'DONOGHUE and G E FORD

Central Veterinary Laboratories, Department of Agriculture,
Adelaide, South Australia 5000

SUMMARY: A total of 864 sheep sold for slaughter throughout South Australia were examined
for infections with Sarcocysfis spp using macroscopic, microscopic and immunoserological
methods of detection. Macroscopic cysts were found in 6.7% of the sheep ranging in
intensity from 1 to 64 cysts per carcase. Morphometric studies detected 2 types of
macroscopic cysts which differed in their size and cyst wall morphology. Large ovoid cysts
with thick primary cyst walls were identified as S. giganfea (syn. S. ovifelis) whereas small
slender cysts with thin cyst walls were identified as S. medusifofmis. The prevalence of
each species was 4.5% and 3.1 OO/ respectively. In comparison, microscopic cysts were
detected in 93.2% of the sheep and stereological calculations of the intensities of infection
ranged from 20 to 32,700 cysts per unit volume of muscle. Two types of microscopic cysts
were also identified by differences in their cyst wall morphology. Cysts with smooth thin
walls were detected in 88.1% of the sheep whereas cysts with radially-striated thick walls
were found in 74.7%. Although these 2 types appeared to conform to the original descriptions
of S. fenella and S. ovicanis respectively, both were classified as S. tenella(syn. S. ovicanis)
pending further taxonomic studies. Specific antibody was detected in 93.7% of the sheep
by the complement fixation test and in 96.9% by the indirect fluorescent-antibody test
(IFAT). A positive correlation was also found between the prevalence of IFAT antibody and
the prevalence of infections by microscopic cysts.
Aust Vet J 63: 273-278

Introduction in sheep have ranged from 69.8 to 98.4% in complement

Infections of sheep by Sarcocystis spp (Protozoa: Apicom-
plexa) are a cause of concern to the meat industry mainly
because certain species of the parasite form macroscopic cysts
in the striated muscles of sheep. These macroscopic cysts are
classified as carcase lesions in many countries (predominantly
for cosmetic reasons) and infected carcases may be rejected
from export or condemned for human consumption. Abattoir
surveys have recorded infections by macroscopic cysts in 0.8%
and 23% of sheep examined in Australia (Whiting 1972;
Munday 1975) and prevalence reports in other countries have
ranged from 2.3 to 95.7% (Seneviratna et a/ 1975; Meshkov
and Kotomindev 1976). While infections by macroscopic cysts
have been shown to be transmitted by cats (Rommel et al
1972), recent morphological and biochemical studies have
demonstrated that the cysts may be divided into 2 separate
species (Collins e t a / 1979; Atkinson and Collins 1981). How-
ever, no reports on their differential prevalence or abundance
have yet been made.
In addition, sheep may also be infected with other species
of the parasite which only form microscopic cysts in the
musculature. Infections by microscopic cysts have been shown
to be transmitted by dogs (Ford 1974; Munday and Corbould
1974) and recent experimental studies have demonstrated that
they may cause acute and even fatal clinical disease in sheep
(Munday et a/ 1975). abortion in pregnant ewes (Leek and
Fayer 1978) and significant reductions in weight gain and wool
growth in young lambs (Munday 1979, 1984). The prevalence
of infections by these microscopic cysts has been examined in
several countries using a variety of techniques. Prevalence
reports have ranged from 33.2 to 98.5% in trichinoscopic
studies (Retzlaff and Ludwig 1965; Bierschenck 1979), from
18 to 100% in histological studies (Breuer 1966; Kruijf and
Bibo 1976) and from 43.5 to 100% in studies using enzymatic
digestion techniques (Diez-Banos 1978; Fassi-Fehri et a/ 1978).
Two reports also recorded the presence of 2 different types
of microscopic cysts in sheep (Bierschenck 1979; Cerna and
Merhautova 1981). In contrast, no reports on the prevalence
of infections by microscopic cysts have been made in sheep
in Australia.
Various immunoserological tests have also been used to
indirectly demonstrate Sarcocystis spp infections in a variety
of host animals. Reports on the prevalence of specific antibody
Australian Veterinary Journal, Vol. 6 3 , No. 9, September, 1986
fixation tests (Awad 1958; Munday 1975), from 24.7 to 73.3%
in indirect haemagglutination tests (Roscher 1980; Cerva and
Gut 1983) and from 85.0 to 94.8% in indirect fluorescent-
antibody tests (Diez-Banos 1978; Roscher 1980). However, the
diagnostic accuracies of the tests have varied considerably
between studies and no reports have been made on the sen-
sitivities or specificities of the various tests.
The present investigation was therefore performed to de-
termine the prevalence and abundance of the different types
of macroscopic and microscopic cysts occurring in sheep in
South Australia. Two immunoserological tests were also eval-
uated for their accuracy, sensitivity and specificity in the
serodiagnosis of infections.
Materials and Methods
Collection of Samples
Blood and muscle samples were collected at various met-
ropolitan and regional abattoirs from a total of 864 sheep
sold for slaughter from 144 flocks throughout South Australia
between May 1974 and February 1975. The sample sizes were
calculated from the sheep population dynamics to represent
the minimum flock and animal numbers required to estimate
the prevalence of infection to within 1% at a 95% level of
confidence (Cochran 1963). Whole blood was collected at the
moment of slaughter and serum was later harvested by cen-
trifugation and stored frozen until tested. Five different muscle
samples were then collected from each sheep and fixed in 10%
formalin in phosphate-buffered saline (PBS) pH 7.2. These
samples included the lesser diaphragm, the left oblique ab-
dominal muscles, the proximal oesophagus, the apex of the
heart and the posterior muscle groups of the tongue.
Macroscopic Examination
Each carcase was examined by close visual inspection for
infections by macroscopic cysts and all cysts found were col-
lected for morphological studies. The interior and exterior
aspects of each carcase were examined with particular attention
given to the thoracic and abdominal walls and the diaphragm.
The entire length of each oesophagus was inspected and the
posterior muscle groups of each tongue were examined through
a series of cross-sectional incisions.
273
Microscopic Examination
The formalin-fixed muscle samples were processed for his-
tological examination using standard preparative procedures.
However, care was taken that the muscle samples were ran-
domly orientated within the paraffin wax blocks thereby al-
lowing certain stereological calculations to be performed. Sec-
tions were cut at a uniform thickness of 5 Fm and the area
of each section was measured by performing a planimetric
count on an image of the section projected onto a quadratic
test grid (Weibel 1972). The sections were then examined for
microscopic cysts and morphometric observations made at
various levels of magnification. The number of cysts in each
section was also counted (minimum area examined = 1 cm’)
and the intensity of infection was calculated stereologically
using the numerical density formula for determining the num-
ber of spherical particles per unit volume of tissue (Elias et
a1 1971). However, because the microscopic cysts were not
spherical but prolate ellipsoidal in shape, it was necessary to
divide the results by a dimensionless shape coefficient (Weibel
and Gomez 1962) to account for repeated scoring of elongate
cysts. The calculation of this coefficient required that the
mean axial ratio (length: breadth) of the cysts be determined.
Measurements performed on 400 microscopic cysts uniformly
distributed over the entire size range gave an approximate
mean axial ratio of 6: 1 which in turn gave a shape coefficient
of 3.10. Tnese stereological calculations therefore allowed the
intensity results to be expressed as the number of ellipsoid-
shaped cysts per unit volume of muscle. The stereological
procedures and formulae used in this study have subsequently
been documented in detail by Weibel (1979).
Immunoserological Examinations
Serum samples were tested for the presence of specific
antibody against Sarcocystis using the complement fixation
(CF) test and the indirect fluorescent-antibody test (IFAT).
The C F technique was based on a microtest method (Anon
1965) and was essentially similar to that described by Munday
and Corbould (1974). The antigen consisted of a soluble extract
obtained by centrifugation from frozen and thawed cystozoites
harvested from large macroscopic cysts. The antigen prepara-
tions were standardised by titrations against several reference
serum samples and then diluted to their highest working di-
lutions. Complement was obtained from guinea-pig serum and
diluted to 5 times the 50% haemolytic dose. The test serum
samples were inactivated at 58°C for 30 min immediately prior
to testing. Equal volumes of antigen, complement and titrated
test serum were then incubated together on microtitre plates
at 37°C for 30 min. Similar volumes of a 3% suspension of
sheep erythrocytes optimally sensitised in haemolysin were then
added to each well and the reagents further incubated at 37°C
for 30 min. The test results were then read and the end-point
titre of each test serum taken as the last titration at which
complete lysis has not yet occurred. End-point titres greater
than or equal to 1/8 were regarded as indicative of infection.
The IFAT technique was modified from that used to detect
specific antibody against Toxoplasma in various animals (Su-
zuki et a1 1965; Munday and Corbould 1971). The antigen
consisted of intact cystozoites harvested from large macro-
TABLE 1
Levels of detection of Sarcocystls infections in slaughter sheep
Method of No. sheep Prevalence
detection examined of infection
Macroscopic 864 6.7%
examination
Microscopic 864 93.2%
examination
Complement 064 93.7%
fixation test
indirect fluorescent- 864 96.9%
anti body test
214
scopic cysts from sheep. The cystozoites were washed several
times in phosphate-buffered saline (PBS) pH 7.2, air-dried
onto microscope slides and fixed in 10% methanol for I min.
The antigen slides were covered with serial dilutions of each
test serum and incubated at 37°C for 30 min. The slides were
then thoroughly washed in PBS and covered with rabbit anti-
sheep immunoglobulins conjugated to fluorescein isothio-
cyanate. They were incubated for a further 30 min at 37°C
and then washed thoroughly with PBS, flushed with distilled
water and mounted in carbonate-buffered glycerol pH 9.0.
The test slides were examined under a light microscope using
transmitted ultra-violet illumination and the end-point titre
was determined for each test serum as the highest dilution at
which uniform circumferential fluorescence of the cystozoites
was last detected. An end-point titre of 1/16 or higher was
regarded as being indicative of infection.
Numeric Analyses
The ranges and geometric means of all numeric data are
given where appropriate. Qualitative assessments of parasite
morphology were also supplemented where possible with quan-
titative morphometric observations. Measurements were per-
formed on all macroscopic cysts found in the sheep as well
as on the first 10 microscopic cysts encountered in each his-
tological section. Cyst sizes were recorded as their minimum
cross-sectional diameters.
The immunoserological test results were compared to the
results of the macroscopic and microscopic examinations to
determine the relative efficacy of the tests in diagnosing in-
fections. The accuracy of each test was measured as the
proportion of animals which gave true positive and negative
test results whereas sensitivity was expressed as the proportion
of infected animals which gave positive results and specificity
was expressed as the proportion of uninfected animals which
gave negative tests results (Trajstman 1979).
Correlations were also sought between the macroscopic,
microscopic and immunoserological methods of detection by
multiple regression analyses using the computerized Statistical
Subroutine Package No. 6400 from the Control Data Library.
Results
The results of the various methods used to detect Sarcocystis
infections in the slaughter sheep are summarised in Table 1.
Detection of Macroscopic Cysts
Infection with macroscopic cysts was detected in 58 of the
864 sheep and the intensities of infection ranged from 1 to
64 cysts per carcase (Table 1). A total of 661 cysts were
detected ranging in size from 180 to 2750 pm in diameter.
These infections were classified as light, moderate or heavy
depending upon the number of cysts detected per carcase.
Light infections ( < 5 cysts/carcase) were detected in 27 sheep
whereas moderate infections (5 to 15 cysts) and heavy infec-
tions ( > 15 cysts) were found in 5 and 26 sheep respectively.
Similar classifications were applied during meat inspection at
the abattoirs to decide the fates of infected carcases. The
moderately-infected carcases were rejected from the meat ex-
intensity Diameter
of infection of cysts
1-64 cystslcarcase 160-2750 pm
(X = 12) (X = 957)
20-32700 cystslcm3 15-115 pm
(X = 800) (X = 35)
Q 11512 titre
(X = 1/25)
< 111024 titre
(% = 1/50)
Australian Velerinary Journal, Vol. 63, No. 9, September, 1986
port market whereas the heavily-infected carcases were con-
demned as being unfit for human consumption.
Morphometric observations performed on the macroscopic
cysts revealed several distinctive features. The cysts were not
uniformly distributed in size and 2 different populations were
evident when the cyst diameters were plotted against their
frequencies (Figure 1). The 2 size populations (small and large
cysts) had respective modes of 350 and 1250 pm in diameter.
Large cysts were found predominantly in the oesophagus and
to a lesser extent in the tongue and skeletal musculature
whereas small cysts were found in all striated muscle groups
examined except cardiac muscle. Upon histological examina-
tion, the small and large cysts were found to differ in the Figure 2. Large macroscopic cyst identified a s S. gigantea
appearance of their cyst walls (Table 2). The large ovoid cysts exhibiting thick primary (p) cyst wall with irregular protrusions
were surrounded by thick primary and secondary cyst walls and thick secondary (s)cyst wall. Haematoxylin and eosin x
whereas the small slender cysts were bounded by thin primary 150.
cyst walls and only rarely by structures resembling thin sec- Figure 3. Small macroscopic cyst identified as S. medusiformis
ondary cyst walls (Figures 2, 3). All macroscopic cysts were exhibiting thin primary (p) cyst wall. Haematoxylin and eosin
septate and their cystozoites were uniform in size (mean di- x 150.
mensions of 12.3 pm in length by 4.1 pm in width). Infections
by large macroscopic cysts were detected in 3 1 sheep, infections was calculated using the formula for prolate ellipsoids (Weibel
by small macroscopic cysts were found in 19 sheep and mixed 1979) to equal only 0.01% of 1 cm’. The range of intensity
infections by both types were observed in 8 sheep. values was also not biased towards heavy or light infections
but was normally distributed around the mean. Differences
in the intensities of infection were observed, however, between
TABLE 2 the various muscles examined (Table 3). Heavier infections
Characteristics of the different types of macroscopic cysts were found in cardiac muscle than in tongue, oesophagus,
detected
diaphragm or skeletal muscle. Infections were also more prev-
Tvoe 1 alent in cardiac and tongue musculature than in diaphragm,
oesophagus o r skeletal muscle.
Morphological characters
cyst size large macroscopic small macroscopic Morphometric studies performed on microscopic cysts also
cyst shape ovoid-ellipsoid prolate ellipsoid revealed several distinctive features. A negative skew distri-
cyst diameter 800-2750 pm 180-800 pm bution was found for the sizes of the cysts when their diameters
(Z = 1250) (Z = 350) were plotted against their frequencies (Figure 4). The majority
primary cyst wall present present of the cysts were less than 50 pm in diameter and 2 modes
appearance thick thin in their distribution were apparent (28 and 36 pm in diameter).
diameter 1-2 pm 0.5-1.0 pm Although the prevalence and intensities of infection varied
(n = 1.8) (Z = 0.6)
secondary cyst wall present rarely encountered
appearance thick thin (when present)
diameter 1-2 pm 0.2-1 pm
~
IF = 1.41 IP = 0.5)
Prevalence 4.5% 3.1 o/‘
of infection
Taxonomic S. gigantea S. medusiformis
classification (syn. S. ovifelis)

Detection of Microscopic Cysts

Infection with microscopic cysts was detected in 805 of the
864 sheep examined (Table 1). A total of 26,565 microscopic
cysts were detected ranging in size from 15 to 115 pm in I
diameter. The average intensity of infections was 800 cysts 0 40 80
Cyst diameter ( y m )
per cm’ of muscle. Although this may seem extraordinarily
high, the volume occupied by 800 microscopic cysts with Figure 4. Frequency distribution of microscopic cysts detected
average dimensions of 210 x 35 pm (mean axial ratio of 6:l) in sheep.

150

150

g
u.
100

in sheep
L! 1000
Cyst diameter ( yrn)
2000 3000

Figure 1. Frequency distribution of macroscopic cysts detected

Australian Veterinary Journal, Vol. 63, No. 9, September, 1986

Figure 5. Microscopic cyst classified as S. tenella exhibiting
thick primary cyst wall with prominent radial striations. Hae-
matoxylin and eosin x 500.
Figure 6. Microscopic cyst classified as S. tenella exhibiting
smooth thin primary cyst wall. Haematoxylin and eosin x 500.
275
 TABLE 3 TABLE 5
Levels of detection of microscopic cysts in various striated Efficacy of CF test in detecting infections by Sarcocystis spp
muscles in sheep
cyst
Muscle
examined
No. sheep Prevalence Intensity
examined of infection (cystslcm3) di:rz;er Seroloav Infected
Infection status
(macroscopic and microscopic examination)
Not infected Total
Heart 864 83.2o/‘ 1410 35.3
Tongue 864 81.8% 983 35.8 CF + 760 50 810
Oesophagus 864 59.0% 620 35.2 CF - A5 9 54
Diaphragm 864 64.1Yo 557 33.2 805 59 A64
Skeletal muscle 864 54.5% 433 34.5
accuracy = 7691864 = 0.89
sensitivity = 7601805 = 0.94
specificity = 9/59 = 0.15
TABLE 4
Characteristics of the different types of microscopic cysts
detected
TABLE 6
Type 1 Type 2 Efficacy of IFAT in detecting infections by Sarcocystls spp in
sheep
Morphological characters
cyst size microscopic microscopic Infection status
cyst shape prolate ellipsoid prolate ellipsoid (macroscooic and microscoDic examination)
cyst diameter 15-115pm 15-115 pm
(% = 35.7) (n = 34.9) Serology Infected Not infected Total
primary present present IFAT + 792 45 837
cyst wall IFAT - 13 14 37
appearance thick, thin,
radially-striated smooth 805 59 864
diameter 1.0-2.5pm 0.5-1.0prn
(2 = 2.1) (% = 0.6) accuracy = 8061864 = 0.96
secondary absent absent sensitivity = 7921805 = 0.98
cvst wall specificity = 14159 = 0.24
Prevalence 74.7% 88.1Y o
of infection
the various techniques occurred between the microscopic ex-
Taxonomic both types classified a s the species amination and the IFAT (correlation coefficient = 0.595).
classification S. tenella (svn. S. ovicanid The number of sheep positive to the IFAT correlated well
with the number of sheep found to be infected with micro-
scopic cysts.
between the different muscle groups examined, the cysts de-
tected in the various muscles did not differ significantly in Discussion
size (Table 3). Upon histological examination, however, 2 types The morphometric studies resulted in the detection of 4
of microscopic cysts were identified by differences in the different types of cysts formed by Surcocystis spp in the sheep
morphology of their cyst walls (Table 4). Approximately 15% examined. Unfortunately, the nomenclature applicable to these
of the microscopic cysts were surrounded by thick primary different cyst types has been the subject of debate for some
cyst walls with prominent radial striations whereas the re- time. Prior to the last decade, it was generally thought that
mainder of the cysts were bounded by thin smooth primary only one species occurred in sheep and most reports referred
cyst walls (Figures 5 , 6). Both cyst types were uniformly to the parasite as S. tenellu (Railliet 1886a). However, when
distributed over the entire size range and they were found in recent cross-transmission studies indicated that more than one
all muscle groups examined. All cysts appeared to be septate species may occur in sheep (Munday and Rickard 1974), the
and their cystozoites were uniform in size (mean dimensions nomenclature applicable to each species became confused.
of 11.9 pm in length by 3.4 pm in width). Infections by cysts Several authors proposed new species names based on the
with thick radially-striated walls were found in 44 sheep, results of transmission studies (Heydorn et ul 1975) whereas
infections by cysts with thin smooth walls were detected in others resurrected old names (Ashford 1977). Following a
160 sheep and mixed infections by both types were found in comprehensive examination of the literature, it is our opinion
601 sheep. that the original descriptions of species occurring in sheep d o
provide sufficient information for their correct historical clas-
Detection of Specific Antibody sification.
Antibody against Surcocystis spp was detected in 93.7% of Microscopic cysts from sheep were first named Surcocystis
the sheep by the C F test and in 96.9% by the IFAT (Table (Miescheriu) tenella by Railliet (1886a) who described cysts
1). Antibody titres ranged as high as 1/512 in the CF test and with thin non-striated walls found in squash preparations of
1/1024 in the IFAT. The accuracies of the immunoserological sheep musculature by Moule (1886). Transmission studies per-
tests in diagnosing infections were assessed by comparing their formed many years later demonstrated that microscopic cysts
results with those of the macroscopic and microscopic ex- from sheep were infective to dogs (Ford 1974; Munday and
aminations. The relative accuracies of the C F test and the Corbould 1974) whereas repeated attempts to infect cats were
IFAT were 0.89 and 0.96 respectively. This means that al- unsuccessful (Munday and Rickard 1974). On the basis of this
though the C F test accurately diagnosed the presence or ab- host specificity, it was proposed that microscopic cysts from
sence of infections in a total of 769 sheep, false positive and sheep be renamed S. ovicunis (Heydorn et a/ 1975). However,
negative reactions were detected in 95 sheep (Table 5 ) . In these authors described S. ovicunis as forming microscopic
comparison, the IFAT accurately diagnosed the status of in- cysts bounded by thick radially-striated cyst walls which is
fection in 806 sheep and false reactions were only detected in markedly different from the original description of S. tenelfu
58 sheep (Table 6). The sensitivities of both the C F test and given by Railliet (1886a). Light and electron microscopic stud-
the IFAT were high (0.94 and 0.98 respectively) but their ies have subsequently shown that cysts with thick radially-
specificities were low (0. IS and 0.24 respectively). striated cyst calls contained numerous “palisade-like’’ protru-
Relationships were also sought between the results of the sions of their primary cyst walls whereas cysts with thin non-
various methods of examination by multiple regression anal- striated cyst walls contained numerous “hair-like” primary
yses. The only significant positive correlation found between cyst wall protrusions (Mehlhorn et ul 1975; Boch et ul 1979;
216 Australian Veterinary Journal, Vol. 63, No. 9, September, 1986
Erber 1982; O’Donoghue et al 1986). These differences in cyst
wall morphology may be regarded as secondary phenotypic
characters only because they relate to differences between
structures surrounding colonies of organisms rather than to
differences between the organisms themselves. I f such phen-
otypic differences are regarded as sufficient criteria for the
separate taxonomic classification of the cysts, the valid names
for the thin-walled and thick-walled cysts would be S. fenella
and S. ovicanis respectively. However, since no other phen-
otypic (or genotypic) characters have yet been found to support
their separate classification, both cyst types could be regarded
as different morphotypes (or different strains) of the same
species. In our opinion, more than one phenotypic character
should be used to classify different species therefore both
types of microscopic cysts found in our study were classified
as the single species S. tenella (synonym S. ovicanis) pending
further taxonomic studies.
Macroscopic cysts from sheep, on the other hand, were
originally named Sarcocysris (Balbiania) gigantea by Railliet
(1886b) who described large ovoid cysts from the oesophagi
of sheep. Years later, ultrastructural studies revealed these
cysts to be bounded by thick primary cyst walls containing
numerous “cauliflower-like’’ protrusions and also by thick
secondary cyst walls (Mehlhorn and Scholtyseck 1973). Ex-
perimental transmission studies demonstrated that these cysts
were also infective to cats only (Rommel et al 1972) and a
proposal was made to rename them as S. ovifelis (Heydorn
el al 1975). However, the original species name must take
priority in accordance with the International Code of Zoo-
logical Nomenclature. The large macroscopic cysts bounded
by thick primary and secondary cyst walls detected in our
study were therefore classified as the species S. giganlea (syn.
S. ovifelis) on the basis of their morphological characteristics.
More recently, small slender macroscopic cysts from the
skeletal muscles of sheep were found to differ from the large
ovoid cysts in the ultrastructure of their cyst walls. The small
cysts were bounded by thin primary cyst walls containing
numerous “snake-like” protrusions and it was proposed that
they be named S. medusiformis (Collins et al 1979). The
separate taxonomic classification of these cysts was later con-
firmed by isoenzyme electrophoretic studies which demon-
strated distinct biochemical differences between the large and
small macroscopic cysts from sheep (Atkinson and Collins
1981; O’Donoghue et al 1986). All of the small macroscopic
cysts detected in our study were bounded by thin primary cyst
walls but structures resembling thin secondary cyst walls were
occasionally found around several of these cysts. Previous
reports have not described secondary cyst walls around S.
medusiformis cysts (Collins et al 1979; Moore 1980) whereas
thin secondary cyst walls (and thick primary cyst walls) have
been found around small developing cysts of S. gigantea as
early as 10 months after infection (Munday and Obendorf
1984). Nevertheless, because the primary cyst walls of the cysts
found in our study were uniformly thin and relatively smooth
in appearance, the cysts were all classified as belonging to the
species S. medusiformis pending more comprehensive mor-
phological or ultrastructural studies.
Macroscopic cysts were detected in 6.7% of the 864 sheep
examined and the prevalence of infections by S. gigantea and
S. medusiformis was 4.5% and 3.1% respectively. Both types
of infections were responsible for the condemnation of a total
of 26 carcases from human consumption and the rejection of
a further 5 carcases from the export trade. Nine carcases were
condemned due to heavy infections by S. gigantea, 14 due to
infections by S. medusiformis and 3 due to infections by both
species. Four carcases were rejected due to moderate infections
by S. gigantea and one due to infection by S. medusiformis.
Although the condemned carcases were processed for pet food
and the rejected carcases were trimmed free of lesions and
sold on local markets, significant economic losses were in-
curred due to loss of trade, loss of quality product and
increased handling costs. Local marketing authorities in South
Australia have estimated that carcases condemned or rejected
due to infections by Sarcocystis spp only realise approximately
Australian Velerinary Journal, Vol. 63, No. 9, September, 1986
half the value of uninfected carcases. The average market
price at the time of our study was $A 15 per carcase (Aitchison
1975) therefore the financial loss resulting from the 26 con-
demnations and 5 rejections was calculated to be $A 232. This
value represents an approximate 1.8% loss from a potential
earnings of $A 12,960 for the 864 carcases examined in our
study. During the 1974175 fiscal year in South Australia, it
was estimated that 2.5 million sheep were sold for slaughter
from a total population of 16.5 million sheep (Aitchison 1975).
I f similar condemnation and rejection rates applied to all
sheep slaughtered during this period and if similar losses were
incurred, the total loss in earnings would have exceeded $A
670,000 for South Australia alone. Clearly, infections by mac-
roscopic cysts are a cause for concern to the Australian meat
industry and future research should be oriented towards de-
termining the epidemiology of infections and examining mech-
anisms for their possible prevention.
In comparison, the prevalence of infections by microscopic
cysts was found to be very high and infections by S. tenella
were found in 93.2% of the 864 sheep examined. Similar levels
of infection have been reported in sheep in other countries
using a variety of microscopic examination techniques, in-
cluding trichinoscopy, histology and enzymatic digestion of
muscle samples (cf Meshkov 1973; Fassi-Fehri el al 1978;
Bierschenck 1979). Recent comparative studies have shown
that enzymatic digestion techniques appear to be the most
sensitive in detecting infections due to the larger volumes of
musculature which can be examined for the presence of cys-
tozoites (Bierschenck 1979; Gut 1982). However, these diges-
tion techniques do not allow morphological observations to
be performed on intact cysts therefore recourse was made in
our study to the examination of multiple histological sections.
Nonetheless, microscopic cysts were detected in abundance
and 2 types of cysts were found to differ in their cyst wall
morphology. Cysts with thick radially-striated cyst walls were
detected in 74.7% of the sheep whereas cysts with thin non-
striated cyst walls were detected in 88.1%. Two previous
studies have also reported the detection of thick-walled and
thin-walled cysts in 63.9% and 84.8% of 336 sheep examined
in West Germany (Bierschenck 1979) and in 90.5% and 9.5%
of 21 sheep examined in Czechoslovakia (Cerna and Merhau-
tova 1981). lnfections by S. fenella (including both morpho-
types) therefore appear to be world-wide in distribution in
sheep-producing areas which differ markedly in geography,
climate and sheep management practices.
The economic impact that these infections have on sheep
production is difficult to assess. Microscopic cysts are not
classified as carcase lesions therefore they do not contribute
to carcase rejection or condemnation rates. However, recent
experimental studies have shown that S. tenella can cause
severe and even fatal clinical disease in sheep during the period
of parasite merogonous proliferation prior to muscle cyst
formation (Munday et al 1975). Experimental infections have
also been shown to cause abortion in ewes (Leek and Fayer
1978) and to affect meat and wool production in lambs (Mun-
day 1979, 1984). I t must be noted, however, that these results
were obtained following experimental infections and that few
cases of naturally-occurring clinical disease directly attribut-
able to the parasite have been reported in sheep (Hartley and
Blakemore 1974; Morgan et al 1984; Stubbings and Jeffrey
1985). The severity of natural field infections may therefore
be limited by certain hostlparasite interactions. Recent studies
have shown that primary infections of sheep by S. tenella can
produce a partial protective immunity against subsequent le-
thal challenge (Phillips 1982; Leek and Fayer 1983). Sheep
repeatedly exposed to natural infections may therefore develop
a persistent protective immunity against acute clinical disease.
The majority of infections observed in the field would there-
fore be subclinical in nature but it is not yet known whether
such infections significantly affect sheep production.
Comprehensive surveys on infections by Sarcocystis spp are
limited to a large extent by the time and effort required to
diagnose infections. Conventional methods involve the labo-
rious and time-consuming examination of muscle samples taken
217
from the host at post-mortem. Various immunoserological
tests have therefore been examined as alternative means for
the rapid diagnosis of infections in live animals. The CF test
and IFAT used in our study detected specific antibody against
Sarcocystis spp in 93.7% and 96.9% of the 864 sheep re-
spectively. Both tests have previously been used to detect
specific antibody in 69.8%, 69.9%, 99.7% and 98.4% of 245,
223, 339 and 304 sheep examined by the C F test (Awad 1958;
Piekarski et a/ 1961; Munday and Corbould 1974; Munday
1975) and in 85.0%, 85.8% and 94.8% of 674, 106 and 366
sheep examined by the IFAT (Arru et al 1978; Diez-Banos
1978; Roscher 1980). Unfortunately, these tests cannot be used
to diagnose infections by individual species of the parasite as
most antigen preparations used have been shown to be genus-
specific and not species-specific (Reiter et a1 1981; Weiland el
a/ 1982; O’Donoghue and Weyreter 1984). Despite the ap-
parent success of both the C F test and the IFAT in detecting
specific antibody, both tests have been shown to vary in their
diagnostic accuracy. Two major components which affect the
accuracy of a test are its sensitivity and specificity. A lack of
sensitivity gives rises to false negative results whereas a lack
of specificity leads to false positive results (cf. Trajstman
1979). The sensitivities of both the C F test and the IFAT were
found to be high but their specificities were low resulting in
a total of 50 false positive reactions in the C F test and 45 in
the IFAT. The demonstration of specific antibody in sheep
in which no cysts were detected suggests that the level of
histological detection used in our study was not high enough
to detect all infections. This was found to be the case for 10
sheep which gave false positive reactions to both immuno-
serological tests. Tissues collected from these sheep were re-
sectioned and histological examination revealed the presence
of low numbers of microscopic cysts in all 10 sheep. Fur-
thermore, a significant positive correlation was also found
between the prevalence of infections as determined by the
IFAT and the histological examination. These results suggest
that the IFAT may be used to provide general sero-epide-
miological information about Sarcocystis spp infections in
sheep without having to make recourse to the post-mortem
examination of muscle samples.
Acknowledgments
This study was supported by a research grant from the Australian
Meat Research Committee and was performed in the Veterinary Pa-
thology Division of the Institute of Medical and Veterinary Science
before its transfer to the South Australian Department of Agriculture.
The authors wish to thank the various abattoir staff and Department
of Primary Industry personnel who aided in the collection of samples
and M e w s M G O’Callaghan and K M Randall for their assistance
in the preparation of the histological sections.
References
Anon (1965) - Standard Diagnostic Complement Fixation and Ad-
aptation to Microtest, Pub Hlth Monograph no 74, US Dept Hlth,
Educ and Welfare
Aitchison, D L J (1975) - South Australian Year Book, Australian
Bureau of Statistics, South Australian Office, Adelaide
Arru, E, Cosseddu, A M and Tarantini, S (1978) - All SOC Ital Sci
Vet 31: 754
Ashford, R W (1977) - Ann Trop Med Parasitol 71: 29
Atkinson, E M and Collins, G H (1981) - Syst Purasitol 2: 213
Awad, F J (1958) - A m J Vet Res 19: 1010
Bierschenck, A (1979) - Das Vorkommen von Sarkosporidien bei
Schlachtschafen in Siiddeutschland, Inaug Diss, Univ Munich
278
Boch, J , Bierschenck, A, Erber, M and Weiland, G (1979) - Berl
Munch Tierarztl Wschr 92: 137
Breuer, H J (1966) - Schlacht - Viehhztg 66: I19
Cerna, Z and Merhautova, V (1981) - Fol Parasitol (Praha) 28: 125
Cerva, L and Gut, J (1983) - Fol Parasitol (Prahu) 30: 223
Cochran, W G (1963) - Sampling Techniques John Wiley and Sons
Inc, New York
Collins. G H, Atkinson, E M and Charleston, W A G (1979) - NZ
Vet J 27: 204
Diez-Banos, P (1978) - An Fac Vet Leon 24: 195
Elias, H, Hennig, A and Schwartz, D E (1971) - Physiol Rev 51:
158
Erber, M (1982) - Z Parasitenkd 68: 171
Fassi-Fehri, N, Cabaret, J , Amaqdouf, A and Dardar, R (1978) -
Ann Rech Vet 9: 409
Ford, G E (1974) - Aust Vet J 50: 38
Gut, J (1982) - Fol Parasitol (Praha) 29: 289
Hartley, W J and Blakemore, W F (1974) - Vet Path 11: I
Heydorn, A 0, Gestrich, R, Mehlhorn, H and Rommel. M (1975)
- Z Parasitenkd 48: 73
Kruijf, J M de and Bibo, T M (1976) - Tijdschr Diergeneesk 101:
I093
Leek, R G and Fayer, R (1978) - Cornell Vet 68: 108
Leek, R G and Fayer, R (1983) - J Parasitol 69: 271
Mehlhorn, H and Scholtyseck, E (1973) - Z Parasitenkd 41: 291
Mehlhorn, H, Heydorn, A 0 and Gestrich, R (1975) - Z Parasitenkd
48: 83
Meshkov, S (1973) - Vet Med Nuuki 10: 73
Meshkov, S and Kotomindev, S (1976) - Vet Med Nauki 13: 72
Moore, S (1980) - N Z Vet J 28: 101
Morgan, G, Terlecki, S and Bradley, R (1984) - Brit Vet J 140: 64
MoulC, L (1886) - Bull et Mern SOC Centr Med Vet 40: 125
Munday, B L and Corbould, A (1971) - Aust J Med Tech 2: 3
Munday, B L and Corbould, A (1974) - Brit Vet J 130: 9
Munday, B L and Rickard, M D (1974) - Aust Vet J 50: 558
Munday, B L (1975) - Aust Vet J 51: 478
Munday, B L, Barker, I K and Rickard, M D (1975) - Z Parasitenkd
46: I l l
Munday, B L (1979) - Vet Parusitol 5: 129
Munday, B L (1984) - Vet Parasitol 15: 91
Munday, B L and Obendorf, D L (1984) - Vet Parasitol 16: 193
O’Donoghue, P J and Weyreter, H (1984) - Zhl Bakt Hyg I Abt
Orig A 251: 168
O’Donoghue, P J, Adams, M, Dixon, B R, Ford, G E and Baverstock,
P R (1986) - J Protozool 33: I14
Phillips, P H (1982) - The Pathophysiology of Sarcocystis tenella
Infections in Specific-pathogen-free (Sporozoa) Sheep, Ph D Thesis,
Univ Adelaide
Piekarski, 0, Schafer, E and Niederlander, R (1961) - Z Parasitenkd
20: 479
Railliet, A (1886a) - Bull el Mern SOC Centr Med Vet 40: 130
Railliet, A (1886b) - Bull el Mern SOCCentr Med Vet 40: 130
Reiter, I, Weiland, 0, Roscher, B, Meyer, J and Frahm, K (1981)
- Berl Miinch Tierarztl Wschr 94: 425
Retzlaff, M and Ludwig, A (1965) - Fleischwirtschafr 45: 345
Rommel, M, Heydorn, A 0 and Gruber, F (1972) - Berl Munch
Tierarztl Wschr 85: 101
Roscher, B (1980) - Die Erarbeitung serologischer Verfahren (IFAT,
IHA) zum Nachweis einer Sarcocystis-lnfektion beim Schaf, Inaug
Diss, Univ Munich
Seneviratna, P, Edward, A G and DeGiusti, D L (1975) - A m J
Vet Res 36: 337
Stubbings, D P and Jeffrey M (1985) - Vet Rec 116: 373
Suzuki, K, Suto, T and Fujita, J (1965) - Nut lnst Anim Hlth Quart
5: 73
Trajstman, A C (1979) - Aust Vet J 55: 501
Weibel, E R and Gomez, D M (1962) - J Appl fhysiol 17: 343
Weibel, E R (1972) - J Microscopy 95: 229
Weibel, E R (1979) - Stereological Methods Academic Press London
Weiland, G , Reiter, I and Boch, J (1982) - Berl Munch Tierarztl
Wschr 95: 387
Whiting, R H (1972) - Aust Vet J 48: 449
(Accepted for publication 14 March 1986)
Australian Veterinary Journal, Vol. 63, No. 9, September, 1986