ABSTRACT
Sarcocystosis is an important zoonotic protozoal disease with worldwide distribution and wide range of hosts. The aim of the present study was to determine the intensity of Sarcocystis spp. infection and to show histopathological features of their cystic lesions in slaughtered cattle of Zabol- Iran.
From April to September 2018, 500 tissue samples from esophagus, heart, diaphragm, tongue and masticatory muscles were prepared from 100 slaughtered cattle. All samples were fixed in 10% neutral buffered formalin and routine tissue processing protocol was performed.
The microscopic results showed that 92.2% of specimens had thin-walled cysts of S. cruzi and 14% had thick-walled Sarcocystis (S. hirsuta and S. hominis) but macrocyst was only observed in one cattle. The positivity rate of thin walled cysts was 58.8% for heart, 13.9% for masticatory muscles, 10.2% for tongue, 9.3% for esophagus and 7.8% for diaphragm. The positivity rate of thick walled cysts was 32.8% for esophagus, 28.6% for tongue, 22.9% for heart, 15.7% for masticatory muscles and 0% for diaphragm, which could represent either S. hominis or S. hirsuta. The most infected tissue was heart and the least infected tissue was diaphragm. Thin walled cysts (S. cruzi) were mostly found in heart and were less found in diaphragm. However, thick-walled cysts (S. hirsuta and S. hominis) were mostly detected in esophagus. No thick-walled cysts were found in diaphragm muscle.
A high positivity rate of sarcocystosis in slaughtered cattle in Zabol abattoir revealed heavily environmental contamination of Sistan region by this important parasitic disease.
Keywords: Cattle, sarcocystosis, thin-walled cyst, thick-walled cyst
INTRODUCTION
Sarcocystosis is an important protozoan intracellular parasitic disease with global distribution in broad range of wild and domestic animal species as well as humans (1). Sarcocystis spp. have obligatory two-host life cycle and cause muscle cysts formation by merogony stage in herbivorous mammals (such as cattle, buffaloes, sheep and pigs) as intermediate hosts. Besides, sporogony and gamogony stages take place in carnivores and humans (by consumption of undercooked meat) as definitive hosts (2,3). In previous researches, three species of Sarcocystis have been reported in cattle: S. cruzi, S. hirsuta, and S. hominis, with canids, felids, and primates, as definitive hosts, respectively. Among these, S. cruzi with more than 90% prevalence in adult cattle cardiac muscle has the most occurrence reports in many countries in the world (4). S. hirsuta and S. hominis with less infection rates in comparison with S. cruzi, mostly have been found in esophagus and striated muscles (but less in cardiac muscle) (3,5); however, epidemiological reports have been scarce in the last few years. In Iran, there are only two reports about the occurrence of macroscopic type of sarcocystosis in cattle (S. hirsuta) (5,6).
Identification of Sarcocystis spp. is complicated. Although, molecular methods are insufficiently convenient for species identifications because of high genetic similarity; the ultrastructure observation of primary cyst wall technique is considered to be one possible reliable method for identifying Sarcocystis spp (6,7).
Based on the reports from different provinces of Iran, infection rate of sarcocystosis in adult bovine muscles is absolutely high (8-15), but there is no information about intensity of this parasitic disease in Sistan region. So, the present research aims to determine the intensity of infection and histopathological investigation of Sarcocystis spp. cystic lesions in slaughtered cattle of Zabol abattoir.
Collection of Samples
From April to September 2018, 100 slaughtered cattle in Zabol industrial slaughterhouse were randomly examined for the presence of Sarcocystis macroscopically and microscopically. A total of 500 tissue samples with the size of 1x1x0.5 cm from esophagus, heart, diaphragm, tongue and masticatory muscles of these cattle were obtained.
This manuscript was conducted on slaughtered cattle and does not contain clinical or experimental studies and patient data.
Microscopic Examination
All specimens were fixed in 10% neutral buffered formalin for 24 hours, then embedded in paraffin, sectioned at 5 µm with microtome, stained with hematoxylin and eosin staining according to routine protocol and finally submitted to light microscopy for examination of Sarcocystis spp. cystic lesions. Considering to the histologically differences of the wall thickness of S. cruzi (thin-walled sarcocyst) and S. hirsuta, or S. hominis (thick-walled sarcocyst), the found cysts were counted under light microscope and their wall thickness was measured by micrometry method using ocular micrometer (micro-scale).
Statistical Analysis
For statistical evaluation, data analysis was performed using SPSS version 20 (SPSS Inc., Chicago, IL, USA), and chi-square test. Significance level was considered as p<0.05.
RESULTS
Out of 100 cattle examined, only one cattle (1%) showed macroscopic type of Sarcocystis spp. In this case, numerous milky-white colored spindle-shaped macrocysts of S. hirsuta were observed in the tunica muscularis of masticatory muscle (Figure 1). In microscopic evaluation, spherical shape cysts with a mean 14 µ in diameter impacted with bradyzoites were observed. The cysts wall thickness was more than 2µ (Figure 2). Totally, 531 cysts were observed in 500 tissue samples and all 100 cattle (100%) were involved with Sarcocystis spp. at least in one of their organs, histologically. Thin-walled Sarcocystis corresponding with S. cruzi were found in 92.2% of specimens, while 14% had thick-walled cysts (S. hirsuta or S. hominis). The distribution of thin-walled and thick-walled microscopic cysts in different muscles of cattle is summarized in Table 1. The chi-square test showed significant differences between frequencies of Sarcocystis in different muscles (p<0.001). The highest incidence of thin-walled cysts (S. cruzi) was in heart muscles, whereas thick-walled cysts (S. hirsuta and S. hominis) were mostly observed in the esophagus. In addition, thin-walled sarcocysts have a lower frequency (7.8%) in the diaphragm muscle compared to other muscles.
In histopathological examination, the different shapes of microcysts (spherical, oval or elongated) with thin or thick distinct walls were observed in over mentioned muscles but no inflammatory reaction was found around the cysts or surround the infected muscle fibers (Figures 3-4-5).
DISCUSSION
Sarcocystosis is an important zoonotic disease with economic impact and worldwide distribution in various animal species and human being. Many considerable researches have been conducted about the importance and prevalence of this disease in different intermediate hosts and various countries (1,2,16-21). Although, there are many reports of sarcocystosis occurrence in domestic animals (especially cattle) in Iran (9,10,13-15), but most studies have not attempted to identify the species of the parasite in cattle. Moreover, considering the popularity of cattle meat consumption in Sistan region, up to now, there has not been any study about intensity of this disease in Zabol city.
In histopathological investigation, thin-walled sarcocysts (<1 µm) identify as S. cruzi and Thick-walled sarcocysts (2-7µm) identify as S. hirsuta or S. hominis using micrometry method (3,22). Based on the abovementioned method, the results of the present study revealed that all cattle and all tissue samples were infected with Sarcocystis spp. (92.2% of tissue samples with S. cruzi and 14% with S. hirsuta or S. hominis). These findings are in accordance to some investigations which have found in other countries: 100% in Brazil (23), 100% in Argentina (4), 100% in the United States (3), 97.4% in Belgium (24), 97.8% in Iraq (25) and 93.4% in Turkey (Iran border countries) (26). Whereas, the infection rate in the present study is considerable higher than Egypt, 7.45 (20), Malaysia, 36.2% (2), Philippines, 55% (27) and Ethiopia 82% (28). Besides, in comparison to other areas of Iran, the frequency of sarcocystosis in cattle in the present study (100%) is consistent with Shiraz, 100% (9), Kerman, 100% (11), Hamadan, 100% (14), Bojnurd, 100% (13), Tabriz, 100% (5), Ahvaz, 100% (15) and higher than Isfahan, 89% (12), Shahrekord, 91% (10) and Karaj, 96/8% (29). Aforementioned researches which are conducted in various parts of Iran demonstrate that cattle contamination to Sarcocystis spp. is notable. The first detection of macroscopic type of sarcocystosis in cattle (S. hirsuta) in Iran was reported by Shekarforoush et al. (6). In this case report, several spindle-shaped macrocysts were observed in diaphragmatic muscle. In a related study, the incidence of macroscopic sarcocysts in cattle slaughtered in Tabriz was declared 8.2%, predominantly in esophagus muscle (5).
In the present study macro-sarcocysts in cattle (S. hirsuta) were observed in one case (1%) and in the masticatory muscle. In this study the shape and size of macrocysts were come in agree with over mentioned studies. In other studies, in Iran, no macroscopic cysts were found in cattle which were examined for Sarcocystis infection (11-14,17,29). The results of the microscopic examination in the current study revealed that the frequency of thin-walled sarcocysts (S. cruzi) and thick-walled sarcocysts (S. hirsuta or S. hominis) were 92.2% and 14%, respectively and all cattle were infected with sarcocystosis at least in one of their organs. The most infected tissue was cardiac muscle (58.8% for thin-walled cysts) and esophagus (32.8% for thick-walled cysts) and the less was diaphragm (for both of thin and thick-walled cysts, 7.8% and 0% respectively). According to these findings, previous researches have demonstrated that S. cruzi is predominantly occurred in cardiac muscle (23,29,30). The frequency of thick-walled sarcocysts in the present study (14%) is lower than two other studies (12,29), 21% and 34/4%, respectively. Additionally, opposite to the results of Nourollahi Fard et al. (29) which reported that thick-walled sarcocyst mostly were detected in diaphragm, in our study diaphragm showed no thick-walled cyst. It seems useful to do accurate recognition of S. hirsuta and S. hominis thick-walled cyst but it was not possible for us considering some problems in ultrastructure observation of primary cysts wall via electron microscopy which is too expensive and difficult to access (30).
In present histopathological investigation, opposite to some studies which reported the existence of inflammatory reactions around some sarcocysts continuing to degeneration (18,21,29), no inflammatory reaction was found.
CONCLUSION
The results of present study demonstrate high frequency of S. cruzi in comparison with low frequency of S. hirsuta and S. hominis. Considering to this fact that the Sarcocystis parasite can cause economic losses in livestock animals and could be a threat to the human community health, future investigation about parasite phylogeny and prevention procedures seems to be useful.
