A Systematic Meta-Analysis of Global Sarcocystis Infection in Sheep and Goats

Sarcocystosis is an intracellular parasitic disease caused by Sarcocystis spp. that has a worldwide prevalence. Symptoms of the disease include diarrhea and muscle pain. The disease poses a threat to the health of animals. The aim of this review is to investigate the global prevalence of Sarcocystis infection in sheep and goats during 2013–2022. We searched five databases: Web of Science, Science Direct, PubMed, Scopus, and Google Scholar. A total of 36 articles containing 44 datasets met the criteria and were included in the study. The total infection rates of Sarcocystis in sheep and goats were 66.3% (95% CI, 51.79–79.38%) and 52.1% (95% CI, 29.45–74.23%), respectively. It was found that Sarcocystis species tend to have a host species preference. Coinfection of S. tenella and S. arieticanis often occurred in sheep, and goats were frequently infected with S. capracanis. Age and sex were identified as risk factors for Sarcocystis infection in sheep and goats. The infection rates of female and male animals were significantly different, with females having a higher infection rate. Age-adjusted analysis showed that infection rates in animals older than one year were higher than in animals younger than one year. This study unveiled the global distribution of Sarcocystis and sheds light on its transmission in sheep and goats.


Introduction
Sarcocystosis is an intracellular parasitic disease with a worldwide distribution. Sarcocystis, the causative agent of the disease, is a two-host protozoan that can infect virtually all warm-blooded animals. There are more than 200 species of Sarcocystis, but only 26 species have known life cycles [1]. The definitive hosts of Sarcocystis are mostly predatory carnivores, while the intermediate hosts are mostly herbivores and omnivores. The intermediate hosts accidentally consume water or food contaminated with oocysts or sporocysts containing sporozoites. Upon digestion by gastric acid, the sporocysts containing sporozoites egress, migrate to the blood circulation, and travel to the muscles to form cysts. When the definitive hosts eat the intermediate hosts that harbor tissue cysts, the liberated bradyzoites migrate to the lamina propria of the small intestine and develop into male and female gametes. Male and female gametes fuse to develop into oocysts that are excreted in feces [2].
A variety of methods are now used to detect Sarcocystis, including visual inspection, microscopic examination of muscle squash, pepsin digestion, indirect immunofluorescence, histology, and polymerase chain reaction (PCR), including PCR, multiplex PCR, PCR-RFLP,

Inclusion and Exclusion Criteria
Studies were included if they were sheep-or goat-related, descriptive studies or analytic cross-sectional studies, published between 2013 and 2022, written in English, containing data obtained through microscopy, serology, and/or molecular tools, and containing sample size and number of positive animals. The studies were excluded if they were case reports, letters, or reviews. Studies that evaluated infections in humans or animals other than sheep and goats and those that involved experimental infections were excluded. Surveys lacking total sample size, prevalence, or full texts were all excluded.

Data Screening
After excluding duplicate articles and articles that had not been published in the last 10 years, the remaining articles were preliminarily scanned for titles and abstracts. Relevant data were extracted using an Excel spreadsheet containing the following items: first author, sampling time, sample size, infection rate, animal age, animal sex, data acquisition method, sampling site, and identified Sarcocystis species.

Inclusion and Exclusion Criteria
Studies were included if they were sheep-or goat-related, descriptive studies or analytic cross-sectional studies, published between 2013 and 2022, written in English, containing data obtained through microscopy, serology, and/or molecular tools, and containing sample size and number of positive animals. The studies were excluded if they were case reports, letters, or reviews. Studies that evaluated infections in humans or animals other than sheep and goats and those that involved experimental infections were excluded. Surveys lacking total sample size, prevalence, or full texts were all excluded.

Statistical Analysis
In this study, R software was used to calculate the infection rates in sheep and goats. The I 2 value was used to determine whether the meta-analysis should use a fixed or random effects model. The heterogeneity was divided into three grades of low, medium, and high, based on I 2 values of 25%, 50%, and 75%, respectively. A fixed effects model was used for low heterogeneity, and a random effects model was used for medium and high heterogeneity. The prevalence was analyzed according to sex and age.

Results
Five international databases were searched, which yielded a total of 601 articles, among which 18 articles were retrieved from Science Direct, 108 from Scopus, 49 from PubMed, 212 from Web of Science, and 214 from Google Scholar (Figure 1). A total of 166 duplicate articles were deleted, including 139 that were removed using EndNote X9 software, and 27 were deleted manually. Based on our time scope (2013-2022), 222 articles were excluded. After checking the abstracts and titles, 116 irrelevant articles were weeded out. The full texts of the remaining 59 articles were downloaded and carefully read. In the end, only 35 articles were included in this study. Forty sets of data were obtained, including 30 sets on sheep [5,9, and 10 on goats ( Figure 2, Tables 1 and 2) [16,17,[36][37][38][41][42][43][44][45]. Meta analysis data can be downloaded from Supplementary Material.    Of the thirty studies in sheep, fifteen utilized the tissue squash method, nine used molecular techniques (PCR), two used histopathology, three used digestion, and one used Percoll gradient centrifugation. Sarcocystis tenella was identified in 18 studies, S. arieticanis infection in sheep was found in 13 studies, and 12 studies were mixed infections of S. tenella and S. arieticanis (Table 1). The infection rate of 201,603 sheep was 70.08% (95% CI, 0.5615-0.8234), and the infection rate of 29,078 goats was 52.1% (95% CI, 0.2945-0.7423), as shown in Figures 3 and 4 Of the thirty studies in sheep, fifteen utilized the tissue squash method, nine used molecular techniques (PCR), two used histopathology, three used digestion, and one used Percoll gradient centrifugation. Sarcocystis tenella was identified in 18 studies, S. arieticanis infection in sheep was found in 13 studies, and 12 studies were mixed infections of S tenella and S. arieticanis (Table 1).  [5,9,. The proportion column calculated the ratio of the number of positive samples (events) to the total number of samples (total). The weigh each study carried in the data analysis is listed in the last column.  [5,9,. The proportion column calculated the ratio of the number of positive samples (events) to the total number of samples (total). The weight each study carried in the data analysis is listed in the last column. Five of the ten studies on goats used tissue squash, four used PCR, and one used digestion. In six studies, the species was identified as S. capracanis (Table 2).
There were 12 sex-related datasets on sheep. Two were excluded due to a lack of male controls. In six of the remaining ten, females had higher infection rates than males (Table  3). A forest plot was drawn according to the odds ratio of infection in male and female sheep ( Figure 5). In the random effects model, OR = 0.36, 95% CI (0.14-0.97) (Figure 4), I 2 = 94%, and p < 0.01, indicating that there were significant differences in infection rates in sheep in different studies ( Figure 5). Of the three sex-related datasets on goats, two showed a higher infection rate in male goats than in female goats ( Table 4). As shown in Figure 6, a forest plot was drawn according to the odds ratio of infection in male and female goats. The random effects model showed that OR = 0.66, 95% CI (0.24-1.82), I 2 = 73%, and p = 0.02, indicating that the infection rates of goats varied significantly among studies ( Figure 6).  Five of the ten studies on goats used tissue squash, four used PCR, and one used digestion. In six studies, the species was identified as S. capracanis (Table 2).
There were 12 sex-related datasets on sheep. Two were excluded due to a lack of male controls. In six of the remaining ten, females had higher infection rates than males (Table 3). A forest plot was drawn according to the odds ratio of infection in male and female sheep ( Figure 5). In the random effects model, OR = 0.36, 95% CI (0.14-0.97) (Figure 4), I 2 = 94%, and p < 0.01, indicating that there were significant differences in infection rates in sheep in different studies ( Figure 5). Of the three sex-related datasets on goats, two showed a higher infection rate in male goats than in female goats ( Table 4). As shown in Figure 6, a forest plot was drawn according to the odds ratio of infection in male and female goats. The random effects model showed that OR = 0.66, 95% CI (0.24-1.82), I 2 = 73%, and p = 0.02, indicating that the infection rates of goats varied significantly among studies ( Figure 6).    There were ten age-related datasets on sheep, among which six showed that the infection rate increased with age (Table 5). A forest plot was drawn according to the ageadjusted infection rates. In the random effects model, OR = 0.66, 95% CI (0.32-1.39), I 2 = 73%, and p < 0.01, which means there was a significant difference in the rates of sheep infections among different studies (Figure 7). Goats had three age-related datasets, and the data were divided into two groups of ≤1-year-old and >1-year-old. Only two studies     There were ten age-related datasets on sheep, among which six showed that the infection rate increased with age (Table 5). A forest plot was drawn according to the ageadjusted infection rates. In the random effects model, OR = 0.66, 95% CI (0.32-1.39), I 2 = 73%, and p < 0.01, which means there was a significant difference in the rates of sheep infections among different studies (Figure 7). Goats had three age-related datasets, and the data were divided into two groups of ≤1-year-old and >1-year-old. Only two studies There were ten age-related datasets on sheep, among which six showed that the infection rate increased with age (Table 5). A forest plot was drawn according to the ageadjusted infection rates. In the random effects model, OR = 0.66, 95% CI (0.32-1.39), I 2 = 73%, and p < 0.01, which means there was a significant difference in the rates of sheep infections among different studies (Figure 7). Goats had three age-related datasets, and the data were divided into two groups of ≤1-year-old and >1-year-old. Only two studies met the criteria. The infection rate of goats older than one year of age was higher than that of those younger than one year of age (Table 6). In the random effects model, OR = 0.66 and 95% CI (0.32-1.39). A forest plot showed I 2 = 84% and p = 0.01, which means the age-adjusted infection rates varied significantly among studies (Figure 8). met the criteria. The infection rate of goats older than one year of age was higher than that of those younger than one year of age (Table 6). In the random effects model, OR = 0.66 and 95% CI (0.32-1.39). A forest plot showed I 2 = 84% and p = 0.01, which means the ageadjusted infection rates varied significantly among studies (Figure 8).  Figure 7. Forest plot of the age-adjusted infection rates in sheep from different studies [25,26,31,34,37,40]. The experimental group contained the number of positive samples and the total number of samples from sheep less than one year of age. In the control group, the number of positive samples and the total number of samples from sheep older than one year of age were included. OR (odds ratio) is the ratio of odds of infection in sheep less than and more than one year of age. OR = 1 is not statistically significant. The weight of each study is shown in the last column. Table 6. Age-adjusted infection rates in goats from different studies.  [25,26,31,34,37,40]. The experimental group contained the number of positive samples and the total number of samples from sheep less than one year of age. In the control group, the number of positive samples and the total number of samples from sheep older than one year of age were included. OR (odds ratio) is the ratio of odds of infection in sheep less than and more than one year of age. OR = 1 is not statistically significant. The weight of each study is shown in the last column.

Discussion
Sarcocystis is an intracellular protozoan with a global distribution. Infection is often asymptomatic in ruminants, but in severe cases, infection can lead to loss of appetite, anemia, abortion, premature birth, or difficulty breathing and death [13]. Sarcocystosis causes hair loss and a reduction in milk production in sheep. Sarcocystis costs the Spanish sheep industry an estimated EUR 20 million per year [46]. As a result, epidemiological studies of Sarcocystis in sheep and goats provide guidance to the farming industry and public health safety. Recently, two meta-analysis studies have been conducted on Sarcocystis infection in ruminants in China [47] and Iran [48]. The infection rates of Sarcocystis in ruminants in China and Iran were reported to be 65% and 74.4%, respectively. In another metaanalysis study, the global prevalence of Sarcocystis in cattle was reported to be 62.7% [49]. In our study, we focused on sheep and goats and compiled data obtained from 14 countries worldwide (Figure 2). This article compiled and analyzed data from 30 studies on Sarcocystis in sheep from 13 countries (three from Italy, one from Malaysia, one from Tunisia, nine from Iran, three from China, one from Brazil, five from Egypt, two from Iraq, and one from each of the following countries: Saudi Arabia, Ethiopia, Lithuania, Turkey, and Algeria) [5,9,. In addition, we included ten studies on Sarcocystis in goats from eight countries (two from Malaysia, two from Iraq, and one from each of the following countries: Tunisia, China, Brazil, South Korea, Saudi Arabia, and Ethiopia) [16,17,36,37,[41][42][43][44][45]. The highest (100%) infection rate in sheep was found in Italy, Lithuania, and Iran [15,18,21,39], and the lowest (1.25%) infection rate was found in Iran [50]. Infection rates were associated with the sampling site and detection method. The highest (90.48%) infection rate in goats was reported in Malaysia [41], and the lowest (2.91%) infection rate was found in South Korea [45]. In Iran, one study that took samples from the diaphragm and esophagus of sheep found the  [37,44]. The experimental group contained the number of positive samples and the total number of samples from goats less than one year of age. The control group contained the number of positive samples and the total number of samples from goats older than one year of age. OR (odds ratio) is the ratio of odds of infection in goats less than and more than one year of age. OR = 1 is not statistically significant. The weight of each study is shown in the last column.

Discussion
Sarcocystis is an intracellular protozoan with a global distribution. Infection is often asymptomatic in ruminants, but in severe cases, infection can lead to loss of appetite, anemia, abortion, premature birth, or difficulty breathing and death [13]. Sarcocystosis causes hair loss and a reduction in milk production in sheep. Sarcocystis costs the Spanish sheep industry an estimated EUR 20 million per year [46]. As a result, epidemiological studies of Sarcocystis in sheep and goats provide guidance to the farming industry and public health safety. Recently, two meta-analysis studies have been conducted on Sarcocystis infection in ruminants in China [47] and Iran [48]. The infection rates of Sarcocystis in ruminants in China and Iran were reported to be 65% and 74.4%, respectively. In another meta-analysis study, the global prevalence of Sarcocystis in cattle was reported to be 62.7% [49]. In our study, we focused on sheep and goats and compiled data obtained from 14 countries worldwide ( Figure 2). This article compiled and analyzed data from 30 studies on Sarcocystis in sheep from 13 countries (three from Italy, one from Malaysia, one from Tunisia, nine from Iran, three from China, one from Brazil, five from Egypt, two from Iraq, and one from each of the following countries: Saudi Arabia, Ethiopia, Lithuania, Turkey, and Algeria) [5,9,. In addition, we included ten studies on Sarcocystis in goats from eight countries (two from Malaysia, two from Iraq, and one from each of the following countries: Tunisia, China, Brazil, South Korea, Saudi Arabia, and Ethiopia) [16,17,36,37,[41][42][43][44][45]. The highest (100%) infection rate in sheep was found in Italy, Lithuania, and Iran [15,18,21,39], and the lowest (1.25%) infection rate was found in Iran [50]. Infection rates were associated with the sampling site and detection method. The highest (90.48%) infection rate in goats was reported in Malaysia [41], and the lowest (2.91%) infection rate was found in South Korea [45]. In Iran, one study that took samples from the diaphragm and esophagus of sheep found the infection rate to be 100% [18], while another study that took brain samples found the infection rate to be 1.25% [50]. In a study published by Kutty et al. in 2015, the same samples were subjected to different detection methods [41]. The infection rates were 52.38% (55/105), 43.8% (46/105), and 90.48% (95/105) using microscopic examination of muscle squash, H&E staining, and PCR, respectively [41].
Among the included articles, 13 reported the infection rate of sarcocystosis by sex (Tables 3 and 4). Seven of the thirteen studies showed that females had higher infection rates than males, while the other six studies showed the opposite. Modiri et al. (2014) found that female sheep (98.34%, 237/241) had a higher infection rate than male sheep (37.03%, 30/81) [23]. Studies that showed a higher infection rate in male animals often had a larger sampling size of males than females. Salam et al. (2021) found that the infection rate difference between male goats (50.7%, 491/17809) and female goats (49.3%, 478/9911) [51] was not significant. However, with the same sampling size of female and male animals, Gerab et al. (2022) found that female sheep (93%, 93/100) had a higher infection rate than male sheep (68%, 68/100) [30]. Some scholars believed that the higher infection rate in females was because female animals often had compromised immunity during gestation and delivery, which increased their risk of infection [11].
Among the included studies, we found that in most articles, the infection rate was higher in animals older than one year (59.42%) as compared to those younger than one year (26.82%) ( Tables 5 and 6). This result is related to the increase in the probability of infection as animals age [52]. While a few studies have shown higher rates of infection in younger animals, this may be due to smaller sample sizes of adult animals.
The current study is subject to a few limitations. First, we are unclear as to how the animals were raised. Animals raised in different environments will likely have different infection rates. Second, different detection methods offer varying sensitivity and specificity, which will affect the infection rate [44]. The Percoll gradient centrifugation method was less widely employed in the detection of Sarcocystis. Third, the obtained data resulted from one-time surveys and may not represent the infection rates at other times. Fourth, the infection rates of Sarcocystis in sheep and goats were not reported in many other countries around the world.
The PCR detection method is fast, accurate, and suitable for routine laboratory practice. Compared with conventional PCR, nested PCR and PCR-RFLP have more steps and are more suitable for distinguishing species [14]. Microscopic examination of tissue pellets is among the most commonly used detection techniques, and complete cysts can be visualized under a microscope. However, it requires trained hands and is time-consuming and laborintensive, which limits the number of samples that can be processed. The accuracy is in question because sometimes cysts are easy to miss [2].
The histopathological method is time-consuming and laborious, but the internal structure of the cyst can be clearly seen. This method is more suitable for the study of the entire cyst structure. In the digestion method, the cysts in the tissue are digested, and the release of bradyzoites from the cysts facilitates the detection of the parasite. However, because the cyst wall is dissolved, the species of Sarcocystis cannot be distinguished, and a complete cyst cannot be obtained [2]. The Percoll gradient centrifugation method has a high cost and is not suitable for epidemiological detection, but it is more suitable for purifying cysts. With this method, the morphology of the obtained cysts will not change [40].
This study is the first statistical analysis of the global prevalence of Sarcocystis in sheep and goats in the past 10 years (2013-2022). We investigated risk factors, including age and sex, for Sarcocystis infection in sheep and goats. The data analysis provided here enriches our understanding of the distribution and transmission of Sarcocystis in sheep and goats on a global scale.

Conclusions
The total infection rates of Sarcocystis in sheep and goats worldwide were 70.08% (95% CI, 0.5615-0.8234) and 52.1% (95% CI, 0.2945-0.7423), respectively. Age and sex were identified as risk factors for Sarcocystis infection. Female sheep and goats had higher infection rates than male sheep and goats. In terms of age, animals older than one year of age had higher infection rates than those younger than one year of age.