The Occurrence and Meta-Analysis of Investigations on Sarcocystis Infection among Ruminants (Ruminantia) in Mainland China

Simple Summary Sarcocystis can infect almost all warm-blooded animals, including humans. Ruminants (Ruminantia) are the intermediate hosts for Sarcocystis, representing a potential risk for public health. In China, there are many studies on the prevalence of Sarcocystis. However, the overall prevalence of Sarcocystis infection among ruminants in mainland China remains unclear and relevant risk factors need to be assessed to develop preventive measures. Therefore, we conducted this systematic review and meta-analysis to collect data on the prevalence and risk factors of Sarcocystis in ruminants in mainland China. Based on the results, we discussed the potential factors that may affect the prevalence of Sarcocystis in ruminants and the limitations in the current epidemiological investigation. Abstract Sarcocystis is a zoonotic pathogen that threatens public health and the quality of food safety. To determine the Sarcocystis spp. prevalence in ruminants (Ruminantia) in China, we conducted a systematic review and meta-analysis. Data were collected from English databases (PubMed and Web of Science) and Chinese databases (Chinese Web of knowledge (CNKI), Database for Chinese Technical Periodicals (VIP) and Wan Fang databases). A total of 20,301 ruminants from 54 publications were evaluated. The pooled prevalence of Sarcocystis spp. among ruminants in mainland China was 65% (95% CI: 57–72%). Our results indicate that sarcocystosis is prevalent in ruminants, which show significant geographical differences. Therefore, it there is a need for continuous monitoring of infections of Sarcocystis spp. in ruminants to reduce the threat to human health and economic losses to the animal industry.


Introduction
Sarcocystis is a food-borne zoonotic pathogen that can infect humans and a variety of animals, there are 196 valid Sarcocystis species [1]. Sarcocystis has an obligate two-host life cycle, the intermediate hosts are usually herbivores, and the definitive hosts are usually carnivores or omnivores. Sarcocystis can form sarcocysts in the striated muscles and central nervous system of domestic animals such as sheep (Ovis aries Linnaeus, 1758), cattle (Bos taurus Linnaeus, 1758), and yaks (Poephagus grunniens Linnaeus, 1766) [1]. At present, a variety of Sarcocystis has been confirmed to have strong pathogenicity. Five [2,3]. Among them, humans are the definitive host of S. hominis, which can cause nausea, vomiting, and diarrhea [4]. S. cruzi is the most pathogenic species in cattle, and acute infection can lead to weakness, reduced milk yield, and economic losses in cattle farms [5]. Animals can be infected with Sarcocystis spp. by ingesting water or food contaminated with sporocysts or oocysts [1]. Humans become infected with S. hominis, S. heydorni and Sarcocystis suihominis tadros and Laarman, 1976 by eating undercooked meat containing mature sarcocysts.
Three validated species of Sarcocystis have been described in sheep in China: the pathogenic microscopic Sarcocystis tenella Railliet, 1886, Sarcocystis arieticanis Heydorn, 1985 and the non-pathogenic macroscopic Sarcocystis gigantea Railliet, 1886 [6]. Depending on the number and types of animals infected with Sarcocystis, it can cause anorexia, fever, muscle inflammation, abortion, premature birth, and even death [1]. Furthermore, the formation of sarcocysts in the muscles can affect the appearance and quality of meat, cause waste of meat products, and bring economic losses to animal husbandry. However, diagnosis is difficult due to the lack of specific clinical symptoms of Sarcocystis.
The animal industry is important in China, the annual production of meat from sheep in 2017 was 4851 thousand tons [7], and 66,728,000 tons of beef and 32,012,400 tons of milk in 2020 [8]. The economic significance of Sarcocystis in ruminants and its threat to human health via consumption of infected raw and undercooked meat (e.g., hot pots, roasts) highlights the importance of research on Sarcocystis. In addition, the relatively large number of surveys on the prevalence of Sarcocystis in China makes it necessary to conduct a systematic study to collect these data and to analyze the limitations and strengths of these studies. Meta-analysis and meta-regression are techniques that help to gain insight into the causes of such differences and to describe current knowledge in an evidence-based manner. Therefore, the aim of this study was to analyze the prevalence of Sarcocystis in ruminants and the factors that may influence the prevalence of Sarcocystis in ruminants in mainland China through meta-analysis and meta-regression.

Search Strategy and Inclusion Criteria
To evaluate the prevalence of Sarcocystis infection in ruminants, we performed a nonregistered systematic review and meta-analysis of the literature published online. This system review and meta-analysis was conducted according to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [9]. We performed the PRISMA 2020 checklist (Table S1) to ensure the relevant information included was consistent with the study criteria. The meta-analysis was conducted by searching English databases (PubMed, Web of Science) and Chinese databases (Chinese Web of knowledge (CNKI), Database for Chinese Technical Periodicals (VIP), and Wan Fang databases) for publications related to Sarcocystis infection in ruminants in China, from 30 March 1983 to 1 January 2022. We searched databases using the following MeSH (Medical Subject Heading) terms alone or in combination: "Sarcocystis" or "Sarcocystis spp." And "China" and "cattle" or "yak" or "sheep" or "goats" or "camelus" or "deer" or "ruminants" or "water buffaloes". Literature inclusion criteria: (1) the study population was limited to Chinese ruminants; (2) the types of studies were epidemiological and/or molecular studies of Sarcocystis spp. in ruminants; (3) the study must clearly report information on sources of samples, number of samples, infection prevalence of Sarcocystis, and detection methods for Sarcocystis; (4) the languages included in the study were limited to Chinese and English.
Literature exclusion criteria: (1) the source of the reported samples and animal species was unclear, and the data were incorrect or incomplete; (2) samples were not randomly selected; (3) duplicate publications, conference publications, articles given to the editor, review articles, no full text or insufficient information, etc.

Data Extraction
Data were extracted independently by two investigators (Zhu Zifu and Feng Zixuan) and checked by other authors. From all included publications, we extracted the following information: first author, year of publication, year of sampling, geographical area of study, host, total number of samples examined, number of Sarcocystis spp. positive samples, diagnostic method, and age, gender, or identified species (if reported) ( Table 1).

Statistical Analysis
This study was performed using R software for Meta-Analysis [63]. We used four methods to transform the original data to make them conform to the normal distribution: logarithmic transformation (PLN), logit transformation (PLOGIT), arcsine transformation (PAS), and double arcsine transformation (PFT). Statistical heterogeneity between studies was evaluated using the Cochran's Q statistics, p-value, and I 2 statistics. The heterogeneity of included studies was used as the basis for selecting the effect model, and heterogeneity was considered insignificant when p > 0.1 and I 2 < 50%, when p < 0.05 and I 2 > 50%, the heterogeneity was considered significant. When the heterogeneity was not significant, a fixed-effect model was selected; otherwise, a random-effect model was used [64]. We conducted a sensitivity analysis in which one study was removed and the remaining studies were analyzed to assess the potential influence of the presence of outliers on each model per species. To account for sources of heterogeneity, we performed subgroup analyses based on region, host type, year of publication, and diagnostic method. The publication bias was evaluated by trim and fill analysis.

Study Selection and Data Extraction
The search of five databases identified 332 records. After removing duplicate studies and preliminary screening, 244 papers remained. After screening titles and abstracts, 121 papers remained. We were unable to evaluate the full text of one article. After reading the full text, a total of 66 articles were excluded for the following reasons: review articles, non-ruminant animals, lack of Sarcocystis prevalence, and samples not randomly selected. Finally, a meta-analysis of 54 publications was performed, including 73 studies (Figure 1).

Prevalence of Sarcocystis in Ruminants in China
In this study, we used the arcsine transformation (PAS) conversion (the value of W was closer to 1, p > 0.05) for rate conversion (Table 2). High heterogeneity was shown in the included studies (χ 2 = 8755. 27, d.f. = 72, p < 0.001, I 2 = 99.2%), the pooled infection rate of Sarcocystis in ruminants in mainland China was 65% (95% CI: 57-72%), as shown in Figure 2. A total of 20,301 ruminant samples were detected, of which 11,555 were positive for Sarcocystis. The detailed Sarcocystis prevalence in ruminants in different articles ranged from 7% to 100%. The presence of publication bias in the included articles could not be directly determined from the funnel plot ( Figure S2). Begg's and Egger's tests showed that there was no publication bias in the included studies (p = 0.1514 > 0.05, Figure S3). Sensitivity analysis showed that no study had a significant effect on pooled prevalence ( Figure  S4). The included studies were conducted in 9 provinces in China, including 4 provinces in northwest China, 2 provinces in southwest China, 2 provinces in central China, and 1 province in northeast China (Table 1, Figure S1).

Prevalence of Sarcocystis in Ruminants in China
In this study, we used the arcsine transformation (PAS) conversion (the value of W was closer to 1, p > 0.05) for rate conversion (Table 2). High heterogeneity was shown in the included studies (χ 2 = 8755.27, d.f. = 72, p < 0.001, I 2 = 99.2%), the pooled infection rate of Sarcocystis in ruminants in mainland China was 65% (95% CI: 57-72%), as shown in Figure 2. A total of 20,301 ruminant samples were detected, of which 11,555 were positive for Sarcocystis. The detailed Sarcocystis prevalence in ruminants in different articles ranged from 7% to 100%. The presence of publication bias in the included articles could not be directly determined from the funnel plot ( Figure S2). Begg's and Egger's tests showed that there was no publication bias in the included studies (p = 0.1514 > 0.05, Figure S3).
Sensitivity analysis showed that no study had a significant effect on pooled prevalence ( Figure S4).

Discussion
This review describes the current knowledge on the prevalence of Sarcocystis among ruminants in mainland China in a systematic manner. All 54 publications included in this study were from naturally infected ruminants in China, including sheep, yaks, cattle, goats, water buffalos, and camels. The included studies were published from 1983 to 2021 and reported data on 20,301 ruminants. The pooled prevalence of Sarcocystis in ruminants was 65% (95% CI: 57-72%), which was lower than the overall prevalence of sarcocystosis in domestic ruminants in Iran (74.40%, 95% CI: 64.01-83.56%) [65]. Although pooled prevalence estimates from different animal species may be considered of limited value, it enables us to statistically define differences in the prevalence of Sarcocystis spp. and may provide information for taking appropriate measures to improve public health.
In this meta-analysis, based on the I² test, we observed a high degree of heterogeneity regarding the prevalence of Chinese ruminants among eligible studies. This may be related to detection method, age, region, year of sampling, year of publication, and host.
At present, there is no standard serological diagnostic method for the detection of Sarcocystis, which is usually detected by muscle squashing microscopic observation, histological examination, HCl-pepsin digestion method, or molecular detection with specific primers [66]. Most of the studies in this review used morphological observation as the detection method, and the detection rate of this method is closely related to the sample type, sample size, and infection density of Sarcocystis, so the detection rate may be lower than that of the real infection. In addition, PCR identification was performed only on a

Discussion
This review describes the current knowledge on the prevalence of Sarcocystis among ruminants in mainland China in a systematic manner. All 54 publications included in this study were from naturally infected ruminants in China, including sheep, yaks, cattle, goats, water buffalos, and camels. The included studies were published from 1983 to 2021 and reported data on 20,301 ruminants. The pooled prevalence of Sarcocystis in ruminants was 65% (95% CI: 57-72%), which was lower than the overall prevalence of sarcocystosis in domestic ruminants in Iran (74.40%, 95% CI: 64.01-83.56%) [65]. Although pooled prevalence estimates from different animal species may be considered of limited value, it enables us to statistically define differences in the prevalence of Sarcocystis spp. and may provide information for taking appropriate measures to improve public health.
In this meta-analysis, based on the I 2 test, we observed a high degree of heterogeneity regarding the prevalence of Chinese ruminants among eligible studies. This may be related to detection method, age, region, year of sampling, year of publication, and host.
At present, there is no standard serological diagnostic method for the detection of Sarcocystis, which is usually detected by muscle squashing microscopic observation, histological examination, HCl-pepsin digestion method, or molecular detection with specific primers [66]. Most of the studies in this review used morphological observation as the detection method, and the detection rate of this method is closely related to the sample type, sample size, and infection density of Sarcocystis, so the detection rate may be lower than that of the real infection. In addition, PCR identification was performed only on a small number of samples identified as positive by morphological observation, which could not truly reflect the infected species of Sarcocystis in all positive samples.
The age of the animals is considered to be an important factor because the likelihood of animal exposure to parasites increases with age [67]. In this review, only two studies investigated the prevalence of Sarcocystis at different ages. One study reported a significantly higher prevalence of Sarcocystis infection in adult sheep (91.1%, 1912/2108) than in 5-8 months old lambs (68.2%, 101/148) [26]. Another study found that the infection rate (75%, 15/20) and intensity of infection (9.35 sarcocysts/cm 2 ) were significantly higher in adult yaks than in young yaks (10%, 2/20) and (0.15 sarcocysts/ cm 2 ), respectively, indicating an increase in infection rate with the age of the animals [32].
According to the subgroup analysis, there were significant differences in the infection rate of Sarcocystis among ruminants in different regions. The data showed that northwest China had the highest infection rate among the five regions in China, with the research mainly concentrated in Qinghai Province. There was only one study in northeast China, and the results of this study could not truly reflect the infection of Sarcocystis in this area. In addition, in this review, only 9 provinces have reported infection with Sarcocystis in ruminants, and many provinces have not yet reported it.
The infection of ruminants with Sarcocystis is related to their living environment and feeding management. Strengthening the breeding management and reducing the exposure of ruminants to potential sources of Sarcocystis such as wild animals, cats and dogs can greatly reduce the probability of infection in ruminants [68]. According to our analysis, ruminants had the highest rate of Sarcocystis infection before 2005, which might be related to the continuous improvement of feeding management in recent years. However, in this review, there is no information on the feeding management of ruminants and the existence of definitive hosts.
Sarcocystis is a food-borne pathogen which can infect a variety of animals and humans [1]. In this review, the overall prevalence of camels (Camelus dromedarius and Camelus bactrianus, Linnaeus, 1758), cattle, goats (Capra hircus, Linnaeus, 1758), sheep, water buffalo (Bubalus bubalis, Linnaeus, 1758), and yaks was generally higher, at 59%, 56%, 71%, 69%, 24%, and 64%, respectively. Among them, cattle are the intermediate hosts of Sarcocystis spp. Five species of Sarcocystis have been identified in beef, namely S. cruzi, S. hirsuta, S. hominis, S. rommeli, and S. heydorni [2,3]. Humans are the final hosts of S. hominis and S. heydorni, which can cause gastrointestinal distress in humans [4]. In addition to the possibility of zoonosis, there is evidence that these protozoa are related to bovine eosinophilic myositis (BEM), a specific inflammatory myopathy with gray-green lesions that causes carcass necrosis and considerable economic losses [69]. However, there are only nine studies on sarcocystosis in cattle and beef in this systematic review.
This meta-analysis showed that sheep are the most common animal studied in the investigation of sarcocystosis in China, and 34 studies have reported Sarcocystis infection in sheep. The results showed that the pooled infection rate of Sarcocystis spp. was 69% (95% CI: 59-78%) in sheep, which is second only to goats among ruminants in China. Of these, only 3 articles identified infected Sarcocystis species in sheep, which are S. gigantea, S. tenella, and S. arieticanis. Sarcocystis in sheep is widely distributed in the world. The prevalence of Sarcocystis in sheep was reported to be 63.83% in Iran [65], 96.9% in Mongolia [70], 95.8% in Brazil [71], and 13.20% in Egypt [72].
In this systematic review, after sheep, yak became the most common target of sarcocystosis studies in China. Yak meat contains abundant trace elements and essential fatty acids beneficial to human body [73]. Yak meat products are welcomed by consumers because of their excellent quality. There are about 1.3 million yaks in China, accounting for 90% of the world's yak population [73]. This may reveal why there are many studies on Sarcocystis among yaks in China. The results of this study showed that the prevalence of Sarcocystis spp. was 64% (95% CI: 50-78%), with significant differences in the infection rate of Sarcocystis in yaks in different studies (p < 0.01), which may be related to a variety of factors, such as age of animals, number of samples, and detection methods.
In this systematic review, only 6, 2, and 1 studies were available for goats, water buffalo, and camels, respectively. Although only a few studies were available, it shows that Sarcocystis spp. infection occurs in these animals and deserves further attention. The pooled infection rate of Sarcocystis in goats was 71% (95% CI: 40-93%), which was highest among ruminants. The prevalence of Sarcocystis among goats in China was lower than that reported in Bahia, Brazil (91.6%) [71], and Iran (82.36%, 95% CI: 54.74-98.35%) [65]. Only one study identified infected species of Sarcocystis in goats, S. capracanis and S. hircanis [26]. The pooled infection of water buffalo was 24% (95% CI: 0-72%). The two studies on Sarcocystis of water buffalo were nearly 30 years apart, which showed high heterogeneity (I 2 = 96%, p < 0.01) [18,43]. As camels live mainly in desert areas, they are mainly used as vehicles and are not a major source of meat products [74]. In this review, there is only one study on Sarcocystis of camel published in 1994, with an infection rate of 59% (95% CI: 51-65%) [14].
Our meta-analysis has several limitations: (1) the prevalence of Sarcocystis spp. in ruminants has not been reported in many regions of China; (2) the number of eligible studies is limited; (3) most of the studies were of medium or low quality, mainly because few risk factors (e.g., age, sex, and the presence of the final host) were available for analysis of Sarcocystis infection; (4) data on the ruminant living environment are insufficient and there may be different degrees of environmental pollution, which may explain the reason for the differences in the prevalence of Sarcocystis spp.; (5) the detection methods used were relatively simple, and only a few studies reported the infection of Sarcocystis species.

Conclusions
This meta-analysis determined the pooled infection rate of Sarcocystis in ruminants, indicating that Sarcocystis is widely present in many regions and animals in China. However, information on risk factors associated with Sarcocystis infection is very limited, and highquality investigations are needed to further determine Sarcocystis infections in ruminants in China.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani13010149/s1, Table S1: PRISMA checklist 2020; Figure S1: Forest plot of Sarcocystis infection according to region. Figure S2: Publications bias of studies using trim and fill analysis. Figure S3: Egger's test for publication bias; Figure S4: Sensitivity analysis of any one included study. Figure S5: Forest plot of Sarcocystis infection according to host. Figure  S6: Forest plot of Sarcocystis infection according publication years. Reference [75] are cited in the supplementary materials.

Conflicts of Interest:
The authors state that there is no conflict of interest.