SARCOCYSTIS HOMINIS CYST IN COMPARISON WITH OTHER SARCOCYSTIS SPECIES FOUND IN OCULAR MUSCLES OF CATTLE: A STUDY BY TRANSMISSION ELECTRON MICROSCOPE

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Dept. of Parasitology, Fac. of Medicine, Assiut University, Assiut

SARCOCYSTIS HOMINIS CYST IN COMPARISON WITH OTHER SARCOCYSTIS SPECIES FOUND IN OCULAR MUSCLES OF CATTLE: A STUDY BY TRANSMISSION ELECTRON MICROSCOPE

(With One Table and 4 Plates)

By FATMA G. SAYED; MAHA S.I. SHAHEEN; M.I. ARAFA* and HODA M. KORAA* * Animal Health Research Institute Assiut

(Received at 22/3/2006)

سارکوسيستس هومينس في مقارنة مع الانواع الاخرى من السارکوسيستس الموجودة في عضلات عيون الأبقار: دراسة بواسطة الميکرسکوب الالکتروني فاطمة جلال سيد،مها سيد ابراهيم شاهين،محسن ابراهیم عرفة،

هدی محمد محمد قراعة

تنتشر الإصابة بالسارکوسستس في الأبقار في جميع أنحاء العالم وبعض هذه الأنواع له أهمية طبية للأنسان. تم في هذا البحث دراسة أنواع السارکوسستس المختلفة التي تصيب الأبقار في محافظة أسيوط حيث تم فحص مائة عينة من عضلات عيون الأبقار وقد بلغت نسبة الإصابة فيها ۸۹% وتم تصنيف انواع السارکوسستس الموجودة في تلک العضلات بواسطة الميکرسکوب الألکتروني إلى أربعة أنواع. النوع الأول هو السارکوسستس هومینس وهو يتميز بوجود أمتدادات زغبية أسطوانية (شکل الأصبع) وتکون عمودية على السطح الخارجي بينما يکون له قمة عريضة أما النوع الثاني هو السارکوسسنس هیرسوتا والذي يتميز بوجود أمتدادات زغيبية لها شکل مضرب الکرة حيث له قمة مدببة وبعض قمم الزغبات تمتد لتکون بروزات قمعية والنوع الثالث هو سارکوسستی کروزای والذي يتميز بوجود جدار رفيع وله أمتدادات زغبية تشبه الشعيرات تنتشر على سطح الجدار وتکون تقريبا موازية له. أما النوع الرابع هو مشابه السارکوسستس فيوزيفورميس الموجود في الجاموس ويتميز بوجود أمتدادات زغبية عديدة التفرعات وله قاعدة متقلصة وتعد هذه أول مرة التسجيل هذا النوع في الأبقار. کما تعتبر هذه الدراسة أول مرة لتسجيل الأصابة بالسارکوسستس هومينس في عضلات عيون الأبقار في أسيوط.

SUMMARY

Sarcocystis species are widespread in cattle throughout the world. Some species are pathogenic and have medical importance. In the present

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work, one hundred sample from ocular muscles of cattle were examined where the infection rate was 89%. Ultra-structure studies of Sarcocystis species of cattle with transmission electron microscope revealed the presence of four species. The first species was Sarcocystis hominis with cylindrical (finger like) villar protrusions perpendicular on the cyst surface with broad bases. The second species was Sarcocystis hirsuta that showed club or bulb like villar protrusions with constricted base and tapered tips. Some of villar tips folded to form conical projections. The third one was Sarcocystis cruzi which was characterized by thin wall with hair like villar protrusions folded over the surface of the cyst and approximately parallel to the cyst surface. The fourth species was Sarcocystis fusiformis like cyst which had highly branched villar protrusions with constricted base. This is the first description of this species in cattle also it is the first time of description of Sarcocystis hominis in ocular muscles in Assiut.

Key words: Sarcocystis hominis, Sarcocystis species, Ocular muscles

INTRODUCTION

There are three species of Sarcocystis in cattle: Sarcocystis cruzi (Sarcocystis bovicanis), Sarcocystis hirsuta (Sarcocystis bovifelis) and Sarcocystis hominis (Sarcocystis bovihominis) (Heydorn et al., 1975; Dubey et al., 1989b). Human serves as the predator host for Sarcocystis hominis and also serves as accidental intermediate hosts for several unidentified species of Sarcocystis (Collier et al., 1998).

The clinical signs of infected cattle with Sarcocystis differ according to the amount of sporocysts inoculated (Dubey, 1982b). These signs included fever, anorexia, anemia, diarrhea, cachexia, weight loss, accelerated heart rate, abortion, myositis and neurological signs (Meads, 1976).

In humans, signs of intestinal sarcocystosis are nausea and diarrhea from three to six hours after ingesting infected beef with Sarcocystis, while the clinical signs of muscular sarcocystosis are fever, myalgias, bronchospasm, fleeting pruritic rashes, transient

lymphadenopathy, and subcutaneous nodules (Arness et al., 1999).

The cysts of Sarcocystis cruzi are thin - walled microcysts (Dubey, 1976), whereas those of Sarcocystis hirsuta and Sarcocystis hominis are thick - walled and are either microcysts or small developing macrocysts (Mehlhorn and Heydorn, 1978). The present work aimed at determination of Sarcocystis infection in cattle in Assiut

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through examination of their ocular muscles samples by naked eye and light microscope, in addition to study of their ultra-structure cyst wall for identification of the species.

MATERIALS and METHODS

A

I) Collection of samples:

In this study ocular muscle samples were collected from 100 cattle slaughtered in Assiut city abattoir, their ages were less than two years old. II) Examination of muscle samples: 1. Macroscopic examination:

Fresh muscle samples were examined by naked eye for the presence of macroscopic Sarcocystis cysts. 2. Microscopic examination:

For detection of microscopic Sarcocystis cysts, small pieces of fresh muscle were compressed between two slides and examined microscopically (Mowafy, 1993). 3. Preparation of samples for transmission electron microscope

examination: Small pieces (about 3 mm) of fresh infected muscles were fixed in 2.5% glutaraldehyde. Semi-thin sections were cut, stained with toluidine blue (Richardson et al., 1960) and examined by light microscope. Ultra-thin sections were stained with saturated aqueous uranyl acetate (Stempak and Ward, 1964) then examined under JEOL - JEM - 100C XII ELECTRON MICROSCOPE)

RESULTS

Examination of ocular muscles samples of 100 cattle in Assiut revealed that infection rate of was (89%). Four species of Sarcocystis were detected in present work:

1- Sarcocystis hominis

2-Sarcocystis hirsuta 3-Sarcocystis cruzi

4 -Sarcocystis fusiformis like cyst 1- Sarcocystis hominis Fresh cysts: It was detected as microscopic cysts in ocular muscles. Microscopic cyst appeared as spindle shaped, measured 1199.1u in length, 165.59u in breadth. The cysts were found parallel to muscle fibers and they appeared divided with fine striations into transverse sections (Plate I. Figl).

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Semi-thin sections: Examination of semi-thin sections of Sarcocystis hominis showed that their cyst wall was thick and the villar protrusions had palisade like appearance measuring 11.44u. Metrocytes were adhered to the inner surface of the cyst wall while bradyzoites were arranged in groups separated from each others by fine septa (Plate I. Fig. 2).

Ultrastructure of Sarcocystis hominis wall: Cyst wall of S. hominis consisted of cylindrical finger like villar protrusions (VP) not equal in length and their tips may be straight or slightly curved while their bases were broad. These protrusions were perpendicular on a thin basement membrane of the cyst. Eash protrusion contains a widely scattered of variable-sized electron dense granules (E). Few bundles of microfilaments (M) were scattered in between these granules (Plate I.

Fig. 3, 4). 2-Sarcocystis hirsuta: Fresh cysts: Both macroscopic and microscopic cysts were detected. Macroscopic cysts appeared as fusiform shaped cysts, white in colour measured 1000.0 - 7000.0 u m in length, 1000.0 - 1500 u m in breadth (4000x1250 um). The cysts were found parallel to muscle fibers (Plate II. Fig. 1). Semithin sections: Sarcocystis hirsuta cyst in semithin section showed thick wall cys with sloping pattern villar protrusions. Cyst wall (W) was thick measured 7.41u (mean thickness). Metrocytes (Me) were close to the inner surface of the cyst wall followed by bradyzoites (Bz) in groups separated from each others with fine septa (Plate II. Fig 2). Ultrastructure of Sarcocystis hirsuta wall: Cyst wall of Sarcocystis hirsuta consisted of villar protrusions (VP) which were club or bulb shaped in outline with constricted base and tapered tips. Near the tips of these protrusions there were small papillary projections (PP). Some of villar tips were folded to form conical projections (CP).The villar protrusion contain microfilaments (M) and widely scattered electron dense granules (Plate II Fig. 3, 4). 3- Sarcocystis cruzi: Fresh cysts: It was detected as a microscopic cyst only. It appears as a small spindle shaped cyst. It measured 114.2 - 643.05 u in length and 45.68 - 200.06 u in breadth, (378.63 x 122.87 u m) (Plate III Fig. 1). Semithin sections: Examination of cross section of Sarcocystis cruzi cyst in ocular muscle of cattle showed that it has thin cyst wall Less than 0.57 um (Plate III Fig 2).

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Ultrastructure of Sarcocystis cruzi wall: Cyst wall consisted of a parasitophorous vacuolar membrane (PVM) which is characterized by irregular undulations (irregularly spaced pits interrupted at irregular intervals) (Plate III Fig 3). This membrane is provided with hair like villar protrusions (VP) folded over the surface of the cyst. These protrusions were rod, round, oval or irregular in shape according their sections and they were approximately parallel to the cyst surface (Plate III Fig 4). Each protrusion contains coarse or fine electron dense granules (E) with no fibrillar structure. Groups of bradyzoites (Bz) and metrocytes were found in inner side of ground substanse (GS) separated by fine septa. 4- Sarcocystis fusiformis like cyst Fresh cyst: It was detected as a macroscopic cyst only. It appears as spindle shaped cyst, creamy white in colour measured 2226.9x473.93 um (Plate VI Fig 1). Semithin section: Semithin section of this species showed that the cyst wall was thick measured 10.725 u. Behind the wall, metrocytes (Me) were arranged in a thin layer and take ovoid shape while bradyzoites (Bz) found in a large separated groups (Plate VI Fig 2). Ultrastructure of Sarcocystis fusiformis like cyst wall: The cyst wall consisted of a parasitophorous vacuolar membrane (PVM) lined with electron dense layer which is wavy. This membrane was provided with highly branched villar protrusions (VP) with constricted base. Inside the cyst wall found metrocytes (Me) peripherally (Plate VI Fig 3). These protrusions were branched in different directions making the wall spongy in appearance, some of these protrusions were provided with small papillary protrusions (PP) along the parasitophorous vacuolar membrane. Each protrusion has filamentous core (c) with widely scattered electron dense granules (E) (Plate VI Fig 4). Inner to metrocytes located bradyzoites groups (Bz) separated by thick dark septa (S). Higher magnification of bradyzoites clear their characteristic organelles as conoid (Co) with preconoidal rings, micronemes (m), amylopectin (A) and protein granules.

Table 1: Comparative morphology of Sarcocystis cysts obtained from

ocular muscles of cattle:

Species

Microtubules

Sarcocystis hominis |

Sarcocystis hirsuta Sarcocystis cruzi

Sarcocystis fusiformis like cyst

Mean cyst dimension(um) 1199.1x165.59

4000x1250 378.63x122.87

Cyst wall Thicknes (u m)

11.44

7.41 Less than 0.57

10.725

Villar protrusion

shape Cylindrical shape Club or bulb like

Hair like

Branched (irregular shape)

Present Present Not found

2226.9x473.93

Present

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ht

IN

LO

UN

COM

WoW

S

WE

Men

ON

WAT

G

WE

MW

NA

Wed

UN

Plate I:

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ETITUTI

401 761

UNCU

GEWO

WWW

be

SWIM

W

M

E

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IMGERA

Me

28

PELE

LA

AT

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W

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2017692

Plate II:

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M

WEN

M

WO

Plate III:

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15

RAVO

HOMME

NG

M

NO

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A

WA

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Plate IV:

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Plate I: Sarcocyst hominis: 1- Fresh cyst parallel to muscle fibers. X100 2- Semithin sections of Sarcocystis hominis: Showing longitudinal

section of the thick-walled cyst. Toluidine blue stain X100 3-TEM of Cyst wall of S. hominis: Showing unequal cylindrical finger

like villar protrusions (VP) has straight or slightly curved tips with widely scattered electron dense granules (E) and bundles of microfilaments (M). Then layer of ground substance (GS) while bradyzoites separated from each others with fine septa (s) contains micronemes (m) and amylopectin granules (A) X 14.000. 4- High magnification of villar protrusions (VP) of S. hominis. X 20.000. Plate II: Sarcocyst hirsuta: 1- fresh white fusiform macroscopic cyst of Sarcocyst hirsuta. X100 2- Semithin sections of Sarcocystis hirsuta cyst: Showing thick wall

cyst with sloping pattern villar protrusions. Metrocytes (Me) and bradyzoites (Bz) were adhered to the inner surface of the cyst wall.

Toluidine blue stain. X 100 3- TEM of Cyst wall of S. hirsuta: Showing charactarestic unequal club

or bulb -shaped villar protrusions (VP), small papillary projections (PP), conical projections (CP) at their teps. Microfilaments (M) and electron dense granules widely scattered inside of each Villar protrusion. While bradyzoites (Bz) adhered to the inner surface of the ground substance (GS) separated from each others with fine septa (S)

and contain micronemes (m) and amylopectin granules (A) X 4.000. 4- High magnification of cyst wall of S. hirsuta: Showing small papillary projections (PP), conical projections (CP) of villar protrusions, microfilaments (M) and electron dense granules in eash

one. X 8.000. Plate III: Sarcocyst cruzi: 1- spindle shaped microscopic cyst of S. cruzi X 200. 2-Semithin section of S. cruzi cyst: Showing thin cyst wall Toluidine

blue stain X 400. 3- TEM of S. cruzi cyst: Showing variable shape of hair like villar protrusions (VP) and cross section of meny bradyzoites (Bz) at the inner side of ground substanse (GS) of the cyst while host cells (HC) surrounding it X 2.700. 4- High magnification of cyst wall of S. cruzi: Showing irregular

undulations of a parasitophorous vacuolar membrane (PVM), different sections of villar protrusions (VP) and meny bradyzoites (Bz)

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separated by thin septa (s) at the inner side of ground substanse (GS) X 10.000. Plate VI: Sarcocystis fusiformis-like cyst: 1- Fresh S. fusiformis like cyst: Showing as macroscopic spindle

shaped, creamy white cyst 2- Semithin section of S. fusiformis like cyst: Showing thick cyst wall (arrow) behind the wall, thin layer of ovoid shape metrocytes (Me) followed by large groups bradyzoites (Bz).X 1000 3- TEM of S. fusiformis like cyst: Showing thick cyst wall consisting of highly branched villar protrusions (VP) overlapping each other, wavy parasitophorous vacuolar membrane of ground substance (GS)

while metrocytes (Me) found peripherally X 10.000. 4- High magnification of cyst wall of S. fusiformis like cyst: Showing villar protrusions (VP) branched in different directions, small papillary protrusions (PP) along the parasitophorous vacuolar membrane, filamentous core (c) with widely scattered electron dense

granules (E) in each villar protrusion X 20.000.

DISCUSSION

In Egypt, records on high incidence of Sarcocystis species of cattle were reported by El-Afifi (1958) 84% and 100% in adult healthy and emaciated cattle respectively, Abdel Rahman (1975) 39.2%, Ali (1985) 58.02%, Mohamed (1996) 30 % El-Saieh (1998) 65.46% and Abdel Rahman (2001) 41.4 %. Records on low incidence were reported by Soliman et al. (1964) 1.6%.

The high rate of infection by Sarcocystis in the present work might be due to presence of cattle in close association with dogs and cats which contaminate the soil and animal's food with large number of Sarcocystis sporocysts. In addition to that the sporocysts are mostly already infective when passed in the faeces, so these factors play an important role in the high prevalence of Sarcocystis in cattle. This opinion agrees with (Dubey, 1976) who mentioned that close association of cattle with dogs in pasture at European countries play an important role in the epidemiology of sarcocystosis.

In present work the infection rate with Sarcocystis species in ocular muscles was (89%). Juyal et al. (1982) found that the heavy sarcocystosis in ocular musculature of cattle was 71.5. Mohanty et al. (1995) found that the highest incidence of sarcocystosis in ocular muscles of cattle (86.20%).

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Examination of semithin sections of infected samples revealed that some bovine ocular Sarcocystis cysts possessed thick cyst wall approximately 7.41um in thickness and radially striated and others have thin wall provided with hair like villar protrusions. According to available literature, species of Sarcocystis provided with thick wall might be Sarcocystis hirsuta or Sarcocystis hominis while thin wall cysts might be Sarcocystis cruzi. Dubey (1982a) mentioned that thickness of the cyst wall of Sarcocystis depend on the stage of development of the cyst, therefore an overlap may occur when light microscope is used in identification of Sarcocystis species. The accurate diagnosis is only possible through transmission electron microscopy (Vercruysee et al., 1989 and Savini et al., 1992). Concerning the ultrastructural studies of the different Sarcocystis species, the results of the present work agree with the opinion of Mandour (1974) who mentioned that the cyst wall and morphological characters of villar protrusions are considered the main criteria of differentiation between species of Sarcocystis.

Ultrastructural studies of Sarcocystis hominis in the present work revealed that they are thick walled with villar protrusions cylindrical in shape, nearly perpendicular to the cyst surface. The villar protrusions had broad base. These characters agree with that of Sarcocystis hominis mentioned by (Odening et al., 1995; Saito et al., 1996; Pena et al., 2001).

Ultrastructural studies of Sarcocystis hirsuta of cattle in the present study revealed thick walled cyst with villar protrusions of club or bulb shape. These villar protrusions were sloping and had constricted base with papillary protrusions near the tips. Some tips of villar protrusions were folded to form conical projections. This description is closely similar to that of: (Dubey et al., 1989a; Odening et al., 1995; Wang et al., 2000; Pena et al., 2001).

The ultrastructural studies of Sarcocystis cruzi cysts in the present study showed that they have thin wall provided with hair-like villar protrusions parallel to cyst surface and this villar protrusions had electron dense granules and no fibrillar structure. The parasitophorous vacular membrane of ground substance had irregular undulations (irregularly spaced pits interrupted at irregular intervals). These morphological characters were similar with those mentioned by (Claveria et al. 2001; Pena et al., 2001).

Bottner et al. (1987a) isolated macroscopically visible Sarcocystis species cysts from the skeletal muscle of slaughtered cattle. They found the cyst wall thickness 3.3 - 7.0 um in thickness.

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VIT.

Transmission electron microscopy revealed previously undescribed features of the cyst wall. It appeared that, with increasing age, the cyst wall protrusions become larger and develop a highly irregular surface. Their attachment to the cyst wall are slender and widely spaced indicating that growth of the cyst continues without the formation of new protrusions.

Odening et al. (1995) found fourth species of Sarcocystis in addition to the three named bovine Sarcocystis species (Sarcocystis cruzi, Sarcocystis hominis and Sarcocystis hirsuta) in musculature of a dwarf zebu. They described this species by light and electron microscopy. They found that this species was not previously reported

from cattle. It mostly resemble Sarcocystis gracilis from roe deer.

Saito et al. (2000) detected a Sarcocystis species from cattle slaughtered in Japan, the cyst wall of which was morphologically different from that of the species recorded from cattle. The cyst wall measured 7 - 10 um thick and provided with finger – like villar

protrusions. The protrusions had microtubule in the core.

In the present work, fourth Sarcocystis species was found, the cyst was macroscopic and had the thicker cyst wall than all the above three bovine species. The ultrastructure of their villar protrusion by transmission electron microscopy were more useful in distinguishing the present species from the other species. The villar protrusions branched in different direction with cauliflower like appearance, some of these protrusions provided with small papillary protrusions along the parasitophorous vacular membrane. Each protrusion has filamentous core with widely scattered electron dense granule.

From the above morphological characteristics of the cyst wall, especially of their villar protrusions, the present species could be discriminated from all the known Sarcocystis species parasitic in cattle. It was suggested that this species resembles Sarcocystis fusiformis in buffalo which had cauliflower like appearance villar protrusions. Because all Sarcocystis species are considered host specific (Dubey et al., 1989b), and differences in the structure of the cyst wall alone are insufficient to recognize the present species to be new we propose its name to be Sarcocystis fusiformis - like cyst until its life cycle will be clarified.

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