1. Introduction
Members of the genus Sarcocystis are protozoans that are widely parasitic in reptiles, birds, rodents, livestock, fish, and occasionally in humans [1]. Sarcocystis spp. require two hosts to complete their life cycle, usually herbivores as intermediate hosts and carnivores as definitive hosts. Sarcocysts form in the tissues of intermediate hosts, whereas sporocysts develop in the small intestine of the definitive host [2]. Sarcocystosis can result in fever, lethargy, stunted growth, poor appetite, lameness, and abortion, in severe cases, lead to death [3]. Intermediate hosts acquire infection by ingesting food or water that are contaminated with Sarcocystis oocysts or sporocysts. Humans can be either definitive hosts or intermediate hosts [4]. Humans as definitive hosts can be infected with the Sarcocystis species by ingestion of contaminated food or water and can have symptoms such as abdominal pain, diarrhea, anorexia, vomiting, and in severe cases, anemia and necrotizing enterocolitis can occur [5,6]. Sarcocysts in humans have been found in skeletal muscle and cardiac muscle, and Sarcocystis spp. are considered one of the most important parasitic protozoans threatening human health globally [5,6].
Currently, more than 200 species of Sarcocystis have been recorded in different animals [7]. At least six species of Sarcocystis have been described in sheep (Ovis aries) as intermediate hosts, namely, S. arieticanis, S. tenella, S. gigantea, S. medusiformis, S. mihoensis, and S. microps [8]. Among them, S. arieticanis is slightly less pathogenic than S. tenella [9,10]. Sheep can be infected with these protozoans at any time under natural conditions [11]. The infection of Sarcocystis spp. in domestic sheep occurs worldwide, with prevalence ranging from 9 to 100% [5], which can lead to problems such as degradation of meat product quality, jeopardization of public health, and causation of economic losses to the livestock industry. Therefore, the prevention and control of Sarcocystis infection in humans and animals is very important [12].
Sheep and goats (Capra hircus) are the first livestock to be domesticated by humans [13], yielding a diverse range of invaluable products. They can provide meat, high-protein milk, and high-quality cashmere products for human beings [14]. Lamb meat can provide abundant protein, fatty acids, vitamins, and minerals to humans [15], thus sheep farming is considered an important sector of agriculture in China. As people’s quality of life improves, domestic demand for sheep products has also increased significantly [16].
Up to now, most molecular studies on Sarcocystis spp. have been based on the amplification and sequencing of the 18S ribosomal RNA (rRNA) gene, 28S rRNA gene, and mitochondrial cytochrome c oxidase subunit I (cox1) gene. According to several studies, it has been found that the 18S rRNA gene and 28S rRNA genes are not quite suitable for distinguishing closely related species, and its identification accuracy is inferior to that of the cox1 gene [17,18]. The cox1 gene is considered to be an ideal genetic marker in taxonomic identification and population genetics of Sarcocystis spp. [19,20]. In China, the annual production of sheep meat in 2017 was 4851 thousand tons [21]. However, surveys of Sarcocystis prevalence have been conducted only in a few Chinese provinces in the past [22], and there are no data on Sarcocystis spp. infection in sheep in Shanxi Province, which is an important province in sheep farming. Thus, the objectives of the present study were to examine whether sheep in Shanxi Province were infected with Sarcocystis spp. and to reveal the prevalence and geographical distribution of Sarcocystis spp. in sheep in Shanxi Province by PCR amplification and sequencing analyses of the mitochondrial cox1 gene, which will provide base-line data for executing intervention measures against Sarcocystis spp. infection in sheep in this province.
2. Materials and Methods
2.1. Sample Collection
From October to November 2023, fresh muscle tissue samples (3–5 g) of sheep were purchased from farmers’ markets from each animal. In total, 582 muscle samples (inner ridge, neck meat, front leg, and rear leg) of sheep were collected, among which 226, 124, and 232 samples were collected from Northern Shanxi Province (Youyu, Huairen, and Hunyuan), Central Shanxi Province (Qi, Taigu, and Pingyao), and Southern Shanxi Province (Fushan, Jishan, Xia, and Hejin), respectively (Table 1). Each sample was individually collected into a sample bag and labeled with the relevant details, such as types of tissues and geographic location, then kept in a foam box at a low temperature. After collection, the tissue samples were transported to the Laboratory of Parasitic Diseases at the College of Veterinary Medicine, Shanxi Agricultural University until further analysis and were frozen in a −20 °C freezer until the extraction of genomic DNA.
2.2. DNA Extraction and PCR Amplification
For each sheep tissue sample, approximately 200 mg of muscle fiber was used to extract the genomic DNA by using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s specifications, and the DNA samples were stored in a freezer at −20 °C until further analysis.
The cox1 gene was amplified from each DNA sample by PCR using the primers SF1 [23] and SR9 [24], and the length of the amplified products was approximately 1100 bp. PCR reactions were carried out in volumes of 25 µL mixture containing 2.5 µL of 10 × PCR buffer (Mg2+ plus), 2 µL of DNA samples, 2 µL of dNTP, 1 µL of each primer, 0.5 µL of Ex Taq DNA polymerase (5 U/µL), and 16 µL of ddH2O. The PCR reaction conditions were as follows: initial denaturation (95 °C for 5 min); followed by 35 cycles denaturation (94 °C for 50 s), annealing (52 °C for 1 min), and extension at (72 °C for 1 min); and a final extension (72 °C for 10 min). To ensure the reliability of the results, the positive control (verified DNA of Sarcocystis spp. by sequencing), Sarcocystis-negative sheep muscle DNA and negative control (reagent-grade water) were added to each PCR assay. All PCR products were examined by gel electrophoresis using 1% agarose with ethidium bromide. The positive PCR products were sent to Sangon Biotech Ltd. (Shanghai, China) for bidirectional sequencing.
2.3. Sequence and Phylogenetic Analyses
In order to determine the Sarcocystis species, the obtained cox1 sequences were compared with those of Sarcocystis spp. deposited previously in GenBank using BLAST. Ambiguous sequences were excluded, since they may represent sequencing errors or mixed infections of Sarcocystis spp. Only those pure sequences with no double peaks or indels were further analyzed. The representative sequences of Sarcocystis spp. from sheep detected in this study were deposited in the GenBank with accession numbers PQ189447, PQ189448, and PQ165949. The phylogenetic tree was re-constructed using the neighbor-joining (NJ) method and Kimura two-parameter (K2P) model in the MEGA 7.0 software [22]. One thousand replicates (bootstrap value) were selected to determine the support for the clades generated. Toxoplasma gondii (GenBank accession No. JX473253) was used as the outgroup.
2.4. Histopathology
The positive mutton samples (verified by sequencing of cox1 gene) were fixed in 10% formalin buffer and embedded in paraffin wax. Sections (0.5 μm thick) were prepared. The tissue was fixed on glass slides for hematoxylin and eosin staining. The histological sections were scanned for Sarcocystis, and all the pathological changes were thoroughly evaluated with a light microscope (Olympus BX43, Olympus, Japan).
2.5. Statistical Analysis
In this study, the prevalence of Sarcocystis spp. infection among different regions was statistically analyzed by the chi-square test using SPSS V20.0 (IBM, Chicago, IL, USA). The difference is considered significant when the p-value is <0.05. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) based on likelihood ratio statistics were also estimated to explore the strength of the association between prevalence and test conditions [25].
3. Results
3.1. Prevalence of Sarcocystis spp. in Sheep in Shanxi Province
In this study, based on the amplification of the cox1 gene, 197 out of 582 sheep tissue samples were detected as positive for Sarcocystis, with an overall prevalence of 33.85% (197/582) (Table 1). The prevalence of Sarcocystis spp. in sheep ranged from 19.67% to 58.97% in ten counties located in Shanxi Province. Among the 197 positive samples, 56 samples were from Northern Shanxi, 48 samples were from Central Shanxi, and 93 samples were from Southern Shanxi. Sarcocystis prevalence in sheep among the ten areas was significantly different (χ2 = 32.63, p < 0.001). The PCR results of some representative sheep muscle samples are shown in Figure 1, and the original gel image is available in the Supplementary Materials (Figure S1).
3.2. Sarcocysts in the Muscle of Sheep
The cysts were blue-purple in color and were parallel to the muscles, which were pink in color. The cyst wall was smooth and thin, and the cyst cavity was full of crescent-shaped bradyzoites (Figure 2).
3.3. Phylogenetic Analysis
The obtained 197 cox1 sequences were aligned and compared with the corresponding cox1 gene sequences of Sarcocystis spp. available in the GenBank. Among them, 196 cox1 sequences shared the highest nucleotide similarities with those of S. tenella, ranging from 98.56% to 99.81%, followed by S. capracanis (OP470343 and OP470344) from Capra hircus (93.36–93.46%, on average 93.41% identity), and S. heydorni (KX057995 and KX057994) from Bos taurus (90.10–90.29% identity, on average 90.20% identity). The remaining one cox1 sequence showed the highest nucleotide similarity (99.71%) with that of S. arieticanis, followed by S. hircicanis (KU820975 and KU820976) from Capra hircus (92.28–92.57%, on average 92.43% identity), and S. grueneri (KC209615–KC209623) from Rangifer tarandus (83.83–84.47% identity, on average 84.15% identity). The obtained 197 cox1 sequences of S. tenella in this study showed nucleotide similarities ranging from 98.12% to 100.00%. Notably, S. tenella were detected in sheep in all cities, whereas S. arieticanis was identified only in one county, namely, Hejin County, suggesting that the infection of sheep in Shanxi Province was mainly dominated by S. tenella.
For phylogenetic analysis of Sarcocystis spp., two representative cox1 sequences of S. tenella (accession nos. PQ189447 and PQ189448) and one cox1 sequence of S. arieticanis (accession no. PQ165949) obtained from the present study were used to re-construct the phylogenetic tree with other relevant Sarcocystis spp. In the phylogenetic tree inferred from cox1 sequences (Figure 3), the newly sequenced S. tenella isolates (PQ189447 and PQ189448) clustered with S. tenella (KC209725) from Ovis aries and S. tenalla (MK419984) from Ovis aries, which then formed a clade with S. capracanis (MF039322) from goat. The newly sequenced S. arieticanis isolate (PQ165949) clustered with S. arieticanis (MK419975) from Ovis aries, which then formed a clade with S. hircaicanis (OP470341) from goat.
4. Discussion
Sarcocystosis is a zoonotic parasitic disease. Sarcocystis spp. infections can lead to a decline in the quality of livestock products, causing economic losses to the livestock industry and public health problems. Sarcocystis spp. in sheep have been reported both domestically and internationally and are widely distributed in Oceania (93.31%, 990/1061) [5], Europe (23.28%, 179/769) [26], Africa (54.67%, 621/1136) [5], South America (95.83%, 115/120) [27], North America (67.40%, 1141/1693) [5], and Asia (48.02%, 26061/54270) [28]. In the present study, the overall Sarcocystis spp. prevalence in sheep in Shanxi Province was 33.85% (197/582), which was lower than that in Iran (63.83%) [3], Brazil (95.80%) [27], Mongolia (96.90%) [28], and some provinces of China, such as Qinghai Province (49.96%) [22]. However, the Sarcocystis spp. prevalence in sheep in Shanxi Province was higher than that in Gansu Province (7.74%), Henan Province (11.60%), and Liaoning Province (27.83%) [29].
In this study, the highest Sarcocystis spp. prevalence in sheep was detected in Qi County (58.97%, 23/39), followed by Fushan County (54.39%, 31/57) and Jishan County (37.93%, 33/87) in Shanxi Province. Qi County is in close proximity to Taiyuan city, the capital of Shanxi Province, which has a huge population and convenient transportation, which may facilitate the spread of pathogens, and these may have contributed to the highest rate of Sarcocystis infection in sheep in Qi County. We found that the prevalence of Sarcocystis spp. in sheep gradually decreased with increasing latitude. This is probably because mild and warm climate could be conducive to the survival and dissemination of Sarcocystis. spp. Fushan County and Jishan County is closer to the equator than other cities, which may contribute to their relatively high rates of Sarcocystis spp. infection in sheep.
Notably, only two Sarcocystis species, namely, S. tenella and S. arieticanis, were identified in sheep in Shanxi Province in the present study. Both Sarcocystis species form microcysts in muscle tissues and are transmitted by canids, i.e., sporulated oocysts shed in the feces of infected canids [5]. In Shanxi Province, domestic dogs are raised frequently to safeguard the sheepfold, which greatly increases opportunities for the livestock to contact the feces of domestic dogs; this could facilitate the efficient circulation of S. tenella and S. arieticanis between domesticated livestock (intermediate) hosts and domestic canines (definitive) hosts. Thus, we speculate that this is the primary factor for the popularity of S. tenella and S. arieticanis in sheep in Shanxi Province.
Traditional morphological methods for the identification of Sarcocystis species have limitations, such as their inability to distinguish between species with similar morphology and structure, and the process is also time-consuming and labor-intensive, whereas molecular approaches are able to rapidly and accurately differentiate Sarcocystis species compared to morphological methods [30]. The mitochondrial genome has a small molecular mass and a relatively stable structure. The cox1 gene is relatively conserved in the mitochondrial genome within a species and has been widely used for Sarcocystis species identification and genetic evolutionary studies in recent years [31,32]. The PCR method using cox1 as the target gene has excellent specificity and is suitable for the identification of Sarcocystis spp. in animals [33].
The prevention and control of Sarcocystis spp. in sheep is of importance. Sarcocystis spp. in sheep can cause economic losses to the livestock industry, reduce meat quality, and threaten public health [12]. Therefore, the relevant units should strengthen the feeding management of sheep, ensure forage and drinking water are clean, disinfect farms on a regular basis, strengthen the management of stray dogs and cats, and reduce the contact between these stray animals and sheep, as well as strengthen the relevant quarantine and pollution control [34].
5. Conclusions
The present study revealed, for the first time, an overall 33.85% prevalence of Sarcocystis spp. infection in sheep in Shanxi Province based on the amplification and sequencing of the mitochondrial cox1 gene. Two Sarcocystis species were identified, namely, S. tenella and S. arieticanis, with S. tenella being the predominant species. These results extend the geographical distribution and provide base-line data for executing intervention measures against Sarcocystis spp. in sheep in Shanxi Province and elsewhere.
X.-Q.Z. and J.-J.H. conceived and designed the study. Y.K., X.-S.L. and Y.-H.H. performed the experiments and analyzed the data. Y.K. wrote the manuscript. C.W., Z.-X.W., L.W. and X.-J.W. participated in the implementation of the study. J.-J.H. and X.-Q.Z. critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The data sets supporting the results of this article have been submitted to GenBank, and the accession numbers are shown in the article.
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 1. PCR products amplified from sheep muscle samples targeting the mitochondrial cytochrome c oxidase subunit I (cox1) gene of Sarcocystis spp, with template DNA from reference samples of S. tenella (P1) and S. arieticanis (P2). Lanes 1–12 represent the PCR results of DNA samples extracted from representative sheep muscle samples. Lane N1 represents negative control, N2 represents Sarcocystis-negative sheep as a control, and lane M represents DNA size markers.
Enlarge this image.Figure 2. HE staining of Sarcocystis in sheep muscle tissue. (a) Sheep muscle tissue not infected by Sarcocystis. (b) The sarcocysts from Sarcocystis in the infected sheep muscle tissue. Scale bar: 50 µm.
Enlarge this image.Figure 3. Phylogenetic relationships of Sarcocystis spp. based on cox1 gene sequences using the neighbor-joining (NJ) method. Toxoplasma gondii was used as the outgroup. Representative Sarcocystis cox1 sequences obtained by this study are labeled with black triangle (▲). The bootstrap value is shown when >50%.
Prevalence of Sarcocystis spp. in sheep in Shanxi Province, China.
| Geographical Location | Category (County) | No. Tested | No. Positive | Prevalence % (95% CI) | Odds Ratio (95% CI) | p-Value |
|---|---|---|---|---|---|---|
| Northern Shanxi | Youyu | 80 | 21 | 26.25 (16.61–35.89) | 1.45 (0.65–3.25) | <0.001 |
| Huairen | 85 | 23 | 27.06 (17.61–36.50) | 1.52 (0.69–3.35) | ||
| Hunyuan | 61 | 12 | 19.67 (9.70–29.65) | 1 | ||
| Central Shanxi | Qi | 39 | 23 | 58.97 (43.54–74.41) | 5.87 (2.39–14.40) | |
| Pingyao | 50 | 15 | 30.00 (17.30–42.70) | 1.75 (0.73–4.20) | ||
| Taigu | 35 | 10 | 28.57 (13.61–43.54) | 1.63 (0.62–4.30) | ||
| Southern Shanxi | Jishan | 87 | 33 | 37.93 (27.74–48.13) | 2.50 (1.16–5.37) | |
| Fushan | 57 | 31 | 54.39(41.46–67.32) | 4.87 (2.15–11.04) | ||
| Hejin | 55 | 19 | 34.55 (21.98–47.11) | 2.16 (0.93–5.00) | ||
| Xia | 33 | 10 | 30.30 (14.62–45.98) | 1.78 (0.67–4.71) | ||
| Total | 582 | 197 | 33.85 (30.00–37.69) |
Supplementary Materials
The following supporting information can be downloaded at:
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; Xin-Sheng, Lu 1 ; Yuan-Hui, He 1 ; Wang, Chen 1 ; Wu, Ze-Xuan 1
; Wang, Lu 1 ; Xiao-Jing, Wu 1 ; Jun-Jie Hu 2
; Xing-Quan, Zhu 1
1 Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030801, China;
2 School of Ecology and Environmental Sciences, Yunnan University, Kunming 650091, China