A histopathological and ultrastructural study on experimental murine sarcocystosis

Wafaa M El Kersh; Amira F Afifi; Bahaa Eldeen W El Aswad; Noha M Abo Hussein

doi:10.4103/1110-2098.202523

ORIGINAL ARTICLE

Year : 2016 | Volume : 29 | Issue : 4 | Page : 862-867

A histopathological and ultrastructural study on experimental murine sarcocystosis

Wafaa M El Kersh, Amira F Afifi, Bahaa Eldeen W El Aswad, Noha M Abo Hussein MBBCh
Department of Medical Parasitology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission	03-Nov-2014
Date of Acceptance	16-Nov-2014
Date of Web Publication	21-Mar-2017

Correspondence Address:
Noha M Abo Hussein
Shebin Alkom City, Menoufia Governate, 32511
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/1110-2098.202523

Abstract

Objectives
To study the experimental induction of Sarcocystis spp. infection in rats and to identify the Sarcocystis spp. through parasitological, histopathological, and electron microscopic studies.
Background
Sarcocystis spp. is an obligatory intracellular parasite of humans and animals. The genus Sarcocystis comprises about 130 species with differences in their life cycle and pathogenicity and represents important members of the cyst-forming coccidian.
Materials and methods
Macroscopic sarcocysts were isolated manually from 80 selected buffaloes that were slaughtered in Sirs Ellian City, Menoufia Governorate. The extracted sarcocysts samples were used to infect rats and were also preserved and prepared for species identification by histopathological and electron microscopic studies. Sixty rats were classified into six groups, with 10 rats/group (one control not infected and five infected groups), according to the date they were killed. Every rat was infected by feeding on 10 g of sarcocysts containing about 20 visible intact sarcocysts. Stool examination every day until the time they were killed and histopathological and electron microscopic studies for intestinal tissue specimens were performed.
Results
Histological and electron microscopic identification of sarcocyst specimens extracted from infected buffaloes revealed Sarcocystis fusiformis(thick wall 3.5 μm and cauliflower villar protrusions). A parasitological study of the infected rats' stools revealed excretion of Sarcocystis sporocysts (measuring 9–11 × 7 μm) in 40% of the infected rats at 1 week postinfection (group IV), in 60% at 2 weeks postinfection (group V), and continuous shedding of sporulated oocysts occurred in 40% of the rats at 1 month postinfection (group VI). Histopathological and transmission electron microscope examination of the infected rats' intestinal tissues revealed dividing oocysts (16 × 12.5 μm) with two sporocysts (11 × 7 μm) in the lamina propria of the small intestine in group V.
Conclusion
Rats could be considered as an experimental definitive host for S. fusiformis and could play a role in Sarcocystis spp. transmission to domestic animals.

Keywords: rats, Sarcocystis fusiformis, sarcocystosis, transmission electron microscope

How to cite this article:
El Kersh WM, Afifi AF, El Aswad BW, Abo Hussein NM. A histopathological and ultrastructural study on experimental murine sarcocystosis. Menoufia Med J 2016;29:862-7

How to cite this URL:
El Kersh WM, Afifi AF, El Aswad BW, Abo Hussein NM. A histopathological and ultrastructural study on experimental murine sarcocystosis. Menoufia Med J [serial online] 2016 [cited 2023 Sep 29];29:862-7. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/862/202523

Introduction

Sarcocystis spp. represent one of the most frequently prevalent protozoans of livestock such as buffaloes, cattle, and pigs ^[1]. Sarcocystis spp. are cyst-forming coccidian parasites that need two obligatory hosts during their life cycle: sexual reproduction (gametogony followed by sporogony) in the intestine of a carnivore as a definitive host and asexual multiplication (schizogony) in the tissue of a herbivore as an intermediate host. Human beings may serve as both intermediate and definitive hosts ^[2]; both definitive and intermediate hosts may be infected and harbor more than one Sarcocystis spp. ^[3],[4]. The parasite sexually reproduces in the definitive host's intestinal epithelial cells forming thin-walled sporulated oocysts, which often rupture, releasing infective sporocysts; hence, the definitive host sheds both oocysts and sporocysts in the feces. When the intermediate host ingests the infective sporocysts with contaminated food or water, sporocysts excyst to invade the intestinal mucosa to reach its endothelial cells, where the asexual cycle begins, forming schizonts containing merozoites morphologically similar to sporozoites ^[5]. Subsequent generations of merozoites develop downstream in the direction of blood flow to arterioles, capillaries, and muscle cells, round up to form metrocytes (mother cells) and initiate sarcocyst formation ^[6]. In humans, sarcocystosis has two forms: the intestinal form presents with nausea, abdominal pain, and diarrhea, which may be occasionally severe or even life threatening, and the invasive form, presenting with vasculitis and myositis. Myositis is usually mild, but may involve a wide variety of tissues including lymph nodes, skeletal and cardiac muscles, and the larynx ^[7].

Human intestinal sarcocystosis is caused by two Sarcocystis spp.: Sarcocystis fusiformis (Sarcocystis bovihominis and Sarcocystis hominis) due to consumption of raw infected beef and Sarcocystis meischeriana (Sarcocystis suihominis) due to consumption of infected raw pork ^[8].

In animals, infection by some of the Sarcocystis spp. can lead to anemia, loss of weight, abortion, and even death in cases of severe infection ^[9].

Egyptian domestic buffaloes are widely infected with Sarcocystis spp. (82.3%). This high infection rate was attributed to the abundance of final hosts, especially dogs and cats, which encourage the spreading of infection ^[10].

Rats survive and reproduce in close association with humans in households and in agricultural and commercial places. Wild rodents act as definitive and/or intermediate hosts of many endoparasites including parasites with zoonotic potential: for example – Sarcocystis spp. such as Sarcocystis cymruensis ^[11], Sarcocystis gigantea ^[12], Capillaria heaptica, and Angiostrongylus cantonensis ^[13].

The aim of the present work was to study the experimental induction of Sarcocystis spp. infection in rats and to identify the Sarcocystis spp. through parasitological, histopathological, and electron microscopic examinations.

Materials and Methods

This study was carried out in the Parasitology Department, Faculty of Medicine, Menoufia University, during the period from 2011 to 2013.

Sample collection

Macroscopic examination of infected buffaloes' muscles was performed by veterinary doctors in a slaughter house in Sirs Ellian City, Menoufia Governate, Egypt. Fresh muscle samples (esophagi) of 80 buffaloes were collected and preserved in properly labeled ice bags during the transporting process, and then they were kept refrigerated until usage. The sarcocyst specimens were extracted and collected from each buffalo for experimental animals' infection as fresh samples.

Experimental animals

Sixty male albino laboratory-bred rats were used in this study, weighing about 180–200 g. They were parasitologically free as confirmed by stool examination over 3 successive days before Sarcocystis spp. infection. Animals were divided into six groups (10 rats/each); group I: control, not infected; group II: infected and killed 24 h postinfection; group III: infected and killed 48 h postinfection; group IV: infected and killed 1 week postinfection; group V: infected and killed 2 weeks postinfection; and group VI: infected and killed 1 month postinfection.

Experimental infection

Rats were infected with Sarcocystis spp. by feeding each rat with 10 g of sarcocysts containing about 20 visible intact sarcocysts once after they were fasted for 12 h, and the rat that did not eat was discarded from the experiment ^[14]; this dose was selected after primary experiments using different infective doses of Sarcocystis spp. (2, 5, 7, 9, and 10 g) as it was found that the optimum dose for infection was 10 g.

Parasitological examination for oocysts and sporocysts

Stool analysis was performed for each rat every day after infection until the time they were killed by the direct smear method ^[15], the formal ether concentration method ^[16], the zinc sulfate floatation method ^[17], and the Geimsa stain method ^[18].

Histopathological examination

Sarcocysts collected from buffalo muscles and the small parts from the small intestine of the infected rats from each group were fixed in 10% formaldehyde, and mounted and stained by hematoxylin and eosin stains. The stained slides of sarcocysts were examined microscopically for the histological identification of species (S. fusiformis), and the stained slides of rats' intestinal tissues were examined to detect the pathological changes produced by the infection; this was performed in the Pathology Department, Faculty of Medicine, Menoufia University ^[19].

Ultarstructural study

Sarcocysts collected from buffalo muscles and small parts from the small intestine of infected rats from each group were immersed in a glutraldehyde fixative, referred and examined by a transmission electron microscope (TEM) in the electron microscopic unit, Faculty of Medicine, Tanta University ^[20],[21].

Results

It was found that 77.5% (62/80) of the samples collected were infected by Sarcocystis spp. cysts. The esophagus was more infected with macroscopic sarcocysts (S. fusiformis) at a rate of 72.5% (58/80), whereas microscopic infection in the esophagi was 5% (4/80).

Sarcocystis spp. cysts were spindle or fusiform in shape, milky white in color, with variable sizes (7–15 × 5–9 mm), lying between muscle bundles parallel to the longitudinal axis of the muscle [Figure 1]a.

Figure 1: (a) Fresh meat sample from the esophagus of a buffalo infected with Sarcocystis spp. showed visible white cylindrical-shaped sarcocysts (yellow arrows). They were distributed mainly under the serosal membrane. (b) A section of the Sarcocystis fusiformis cyst isolated from an infected buffalo's esophagus showed a thick cyst wall of about 1.9 mm (H&E) ( × 200). (c) A section of S. fusiformis cyst isolated from an infected buffalo's esophagus showed a thick cyst wall of about 3.5 mm (black arrows) (H&E) (×400). (d) A section of an S. fusiformis cyst isolated from an infected buffalo's esophagus showed that the cyst wall contained long palisade-shaped villar protrusions (H&E) (×100). (e) A section of an S. fusiformis cyst isolated from an infected buffalo's esophagus showed an interconnecting septal meshwork (SE) and the septae divided the cyst into compartments, each of them containing numerous crowded merozoites (ME) (H&E) (×1000).

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Histological examination showed that the cyst wall ranged from 1.9 to 3.5 mm, with long villar protrusions that are characteristic of S. fusiformis [Figure 1]b,[Figure 1]c,[Figure 1]d. The interconnecting septal meshwork encases a single or a cluster of metrocytes and the septae divide the cyst into compartments, each of them containing numerous crowded merozoites. The metrocytes ranged in size from 4.1 × 0.7 to 7.3 × 2.5 mm on the basis of the cyst size, the wall thickness, and the metrozoite size [Figure 1]e.

TEM study of the macroscopic Sarcocystis spp. cyst from the esophageal wall of the examined specimens revealed that the cyst wall had cauliflower villar protrusions and electron-dense granules in the ground substance [Figure 2]a. The large elongated mertozoites had a nucleus in the posterior portion and numerous polysaccharide granules with a diameter of 0.2 mm in the middle portion. The anterior part of the merozoites was rich in micronemes and rhobtries. A conoid structure was present at the anterior end. The developing merozoites were seen with the internal structures attaching directly to the septal meshwork [Figure 2]b.

Figure 2: (a) A TEM photo of the macroscopic Sarcocystis fusiformis cyst wall revealed villar protrusions (VP), electron-dense granules (g) within the ground substance (GS) and cyst merozoites (CM). (b) A TEM photo of the macroscopic S. fusiformis cyst from the esophageal wall of a buffalo showed dense granules forming the cyst covering and septal meshwork (M). The large elongated merozoites had a nucleus (N) in the posterior portion and numerous polysaccharide granules (P) with a diameter of 0.2 mm in the middle portion. The anterior part of the merozoites was rich in micronemes (m) and rhobtries (r). A conoid (C) structure was present at the anterior end. The developing merozoites were seen with the internal structures attaching directly to the septal meshwork (black arrow). (c) An infected rat's stool (1 week postinfection) stained by iodine showed oval-shaped sporocysts (11×7 mm) (SPC) (group IV) (×1000). (d) Wet mounts of a rat's stool (2 weeks postinfection) showed an oocyst (16×12.5 mm) with the oocyst wall (O) and contained two sporocysts (SPC) (g roup V) (×1000). (e) An infected rat's stool (1 month postinfection) stained by Geimsa stain showed the oocyst with the oocyst wall (OW), containing two sporocysts (SPC) (group VI) ( × 1000). TEM, transmission electron microscope.

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Regarding rats infected with Sarcocystis spp., disruption in the rats' normal activities, physiological states, and weight reduction were noted on the 5th day postinfection in 40% of the infected rats.

In addition, the stool consistency was watery and filled with mucous in 20% of the rats on the 7th day postinfection. Sporocyst (9–11 × 7.0 mm) shedding started at 1 week postinfection (group IV) in 40% of the rats [Figure 2]c; oocysts (16 × 12.5 mm) were detected in 60% of the rats 2 weeks postinfection (group V) [Figure 2]d, whereas 40% of the infected rats continued shedding oocysts up to 1 month postinfection (group VI) [Figure 2]e.

Histopathological changes were noticed on H&E-stained intestinal tissues 1 week postinfection (group IV), including broadening and destruction of intestinal villi, dilated congested capillaries, extravasated RBCs, a marked increase in the number of goblet cells, intense eosinophilic infiltrate, moderate lymphocytic infiltrate, and interstitial edema [Figure 3]a and [Figure 3]b. However, TEM examination of the intestinal tissue of group IV showed loss of a part of the surface epithelial lining and a corrupt brush border [Figure 3]c.

Figure 3: (a) The small-intestinal section of a rat examined 1 week postinfection (group IV) showed intense eosinophilic infiltrate (E), lymphocytic infiltrate (L), goblet cells (G), and broadening of intestinal villi (black arrow) (H&E, ×200). (b) A small-intestinal section of a rat examined 1 week postinfection (group IV) showed dilated congested capillaries (C) with intense eosinophilic (E) infiltrate, moderate lymphocytic (L) infiltrate, and interstitial edema (ed) (H&E, ×1000). (c) A TEM photo of a rat's intestinal tissue (1 week postinfection) showed the loss of the epithelial lining with a corrupt brush border (black arrow) in group IV. TEM, transmission electron microscope.

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The intestinal tissue of group V showed loss of the intestinal surface epithelial lining, inflammatory edema, and subepithelial dense inflammatory infiltrate in the form of lymphocytes, eosinophils, and abundant plasma cells [Figure 4]a and [Figure 4]b. Also, intracellular sporulated oocysts were detected with a thin cyst wall (10 × 7 mm), and they contained two sporocysts with apparent sporozoites [Figure 4]c. TEM examination of the intestinal tissue of group V showed abnormal epithelial cells with rarified cytoplasm [Figure 4]d. Also, a dividing oocyst was detected, which was subspherical or ovoid in shape and had a thin wall and contained two ellipsoidal sporocysts with sporozoites inside [Figure 4]e.

Figure 4: (a) A small-intestinal section of a rat examined 2 weeks postinfection (group V) showed destruction of the surface epithelial lining (black arrows) with subepithelial dense inflammatory infiltrate, mainly eosinophils (E), with lymphocytes (L) and abundant goblet cells (G), and there was inflammatory edema (ed) (H&E, × 200). (b) A small-intestinal section of a rat examined 2 weeks postinfection (group V) showed loss of the intestinal surface epithelial lining (black arrows) and subepithelial dense inflammatory infiltrate, mainly lymphocytes (L) and a divided oocyst (yellow arrow) (H&E, ×200). (c) A small-intestinal section of a rat examined 2 weeks postinfection (group V) showed a dividing oocyst (10×7 mm) with the oocyst wall (OW) and two sporocysts (SPC) (H&E, × 1000). (d) A TEM photo of a ratfs intestinal tissue (2 weeks postinfection) showed abnormal epithelial cells with the nucleus (N), abnormal mitochondria (M), and rarified cytoplasm (C) in group V. (e) A TEM photo of a ratfs intestinal tissue (2 weeks postinfection) showing a dividing oocyst with the oocyst wall (OW), sporocyst (SPC), and sporozoites (SPR) in group V.

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Histopathological changes 1 month postinfection (group VI) showed extravasated RBCs, eosinophilic and lymphocytic infiltrate, and interstitial edema. Also, dividing oval oocysts and sporulated oocysts in the lamina propria were detected [Figure 5]a and [Figure 5]b. TEM examination of the intestinal tissue of group VI showed eosinophils [Figure 5]c.

Figure 5: (a) A small-intestinal section of a rat examined 1 month postinfection (group VI) showing the eosinophilic (E) and lymphocytic (L) infiltrate (H&E, ×200). (b) A small-intestinal section of a rat examined 1 month postinfection (group VI) showing a dividing oocyst with two sporocysts (black arrow) (H&E, ×1000). (c) A TEM photo of a rat's intestinal tissue showing the eosinophil in group VI.

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Discussion

In the present study, 77.5% (62/80) of the collected buffalo samples were infected by Sarcocystis spp. This high infection rate was nearly similar to that obtained in Beni-Suef, Egypt abattoirs buffaloes. The authors concluded that Egyptian domestic buffaloes were widely infected with Sarcocystis spp., with an infection rate of 78.9% ^[10].

It was found that 77.5% (62/80) of the samples collected were infected by Sarcocystis spp. cysts. The esophagus was more infected with macroscopic sarcocysts (S. fusiformis) at a rate of 72.5% (58/80), whereas microscopic infection in the esophagi was 5% (4/80). These results were also recorded by El-Dakhly et al. ^[10] who recorded that out of 379 buffaloes examined in abattoirs in Beni-Suef, 299 were found to be infected, with an overall prevalence of 78.9%. Depending on the age, three categorized groups of naturally infected buffaloes were examined: male buffalo calves aged 1.5–2 years, adult female buffaloes aged 2–5 years, and female buffaloes older than 5 years. Among these groups, infection rates were 74.5, 82.3, and 81.2%, respectively. In contrast, Abdel-Ghaffar et al. ^[14] reported microscopic sarcocyst infection in various organs as follows: esophagus, diaphragm, tongue, skeletal, and heart muscles were 60, 50, 40, 40, and 10%, respectively.

Also, in another study conducted by Latif et al. ^[22] in cattle, skeletal muscles and the diaphragm were highly infected organs (27%), followed by the tongue and the esophagus (24.3%), and lastly the heart (8%). However, in water buffaloes, the heart was the most often infected organ (66.7%), followed by the esophagus and skeletal muscles at 50 and 33.3%, respectively.

In the present study, the sarcocyst criteria supported the species identification of S. fusiformis, which was in accordance with a study was conducted by Bunyaratvej et al. ^[23] who recorded human infection by S. fusiformis (S. bovihominis and S. hominis)produced due to consumption of infected beef and S. meisheriana (S. suihominis) due to consumption of infected pork.

Also, Al-Hyali et al. ^[12] described histological forms of the different Sarcocystis spp. They concluded that sarcocysts that had a thin wall (<1 mm) were related to Sarcocystis murensis in infected rats. Also, More et al. ^[24] reported that thin-walled sarcocysts corresponding to Sarcocystis cruzi, whereas thick-walled sarcocysts (>3 mm) were related to Sarcocystis hirsuta and S. hominis. A TEM study also proved that thick-walled sarcocysts with cauliflower villar protrusions and electron-dense granules in the ground substance were nearly similar to features described by Jehle et al. ^[25] and these were the characteristic morphology of S. fusiformis.

In the current study, the change in rats' behavior and weight reduction were close to that reported by Al-Hyali et al. ^[12]; they reported a significant weight reduction of rats inoculated with a lysate of S. gigantea. They attributed this reduction to the release of tumor necrotic factor-a, which is responsible for the occurrence of anorexia and cachexia due to the reduction of gastric motility and suppression of food intake. Also, they reported that tumor necrotic factor-a blocks myogensis, preventing the skeletal muscle from competing for amino acids and ultimately inhibiting muscle growth.

In the present work, sporocysts (9–11 × 7 mm) started to be shed 7 days postinfection (group III) and continued to be shed 1 month postinfection (group VI). This was in harmony with a study that was conducted in Mansoura, Egypt: Sarcocystis muris sporocysts were detected in the lamina propria of the small intestine of infected cats 6–7 days postinfection ^[26], whereas in cats infected with S. cruzi ^[27] and Sarcocystis chalcidicolubris ^[28], they were detected 15 and 21 days postinfection, respectively.

Abdel-Ghaffar et al. ^[14] recorded a shorter period of sporocyst and oocyst shedding, wherein the shedding began 12 h to 3 days postinfection in Sarcocystis camelicanis-infected dogs. However, Mckenna and Charleston ^[29] recorded a longer period of sporocyst shedding; the infected cats began sporocyst shedding of S. gigantea 10–11 days postinfection; the peak of production occurred between 13 and 22 days postinfection and shedding continued to 60 days postinfection. Also, a longer period of oocyst shedding extended to 35 days postinfection was detected in S. camelicanis-infected dogs ^[14].

In the present study, histopathological changes of the intestinal tissue 1 week postinfection (group IV) were in agreement with a study that was conducted in Egypt by Morsy et al. ^[30] to examine the susceptibility of dogs to be a definitive host for Sarcocystis capracanis. Dogs were allowed to eat cysts of S. capracanis isolated from goats. Their intestinal parts showed bloody coagulates and many ulcers at sites of parasite development. Also, a marked increase in the number of goblet cells was observed near the infected sites of dogs' intestines.

Histopathological examination of the rats' intestinal tissue in group V were in harmony with a study conducted for experimental infection of dogs by S. cruzi. Histopathological examination of puppies' intestinal tissue after 12 days showed edema and plasma cell infiltrations in addition to sporulated sporocysts with sporozoites under the mucosa ^[31].

In the current study, TEM changes of rats' intestinal tissue in GV were in agreement with another electron microscopic study conducted by Zaman and Colley ^[32] on Malaysian reticulated python (Python reticulatus) intestinal tissue. Oocystsof Sarcocystis orientalis were observed; they were subspherical or ovoid. Authors recorded that the oocyst wall was composed of a single colorless membrane tightly applied to the two sporocysts, so that the oocyst sometimes had an hourglass shape, and sporocysts were ellipsoidal in shape.

Conclusion

Rats should be considered as an experimental definitive host for S. fusiformis, and further studies are recommended for molecular identification of macroscopic and microscopic cysts in infected buffaloes. In addition, testing other animals such as cats or dogs could be useful in further experimental studies.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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