- Email: [email protected]
Prevalence and molecular characterization of Sarcocystis infections of retail beef products from central China
Acta Tropica 190 (2019) 339–343
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Prevalence and molecular characterization of Sarcocystis infections of retail beef products from central China
T
⁎
Rui Xue, Wenchao Yan , Weifeng Qian, Tianqi Wang, Min Zhang, Zhiguo Wei, Lifang Han, Bin He, Jichen Dou College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Retail beef Sarcocysts Sarcocystis cruzi S. heydorni 18S rRNA gene Co-infection
Cattle are the intermediate hosts for five Sarcocystis species including S. hominis and S. heydorni, which also infect humans. To investigate the prevalence of Sarcocystis infections in beef products from 17 cities in the Henan Province of central China, 62 raw beef samples from markets were collected and analyzed for Sarcocystis presence via muscle squashing microscopic observation, histological section examination, and molecular characterization with 18S rRNA gene sequencing. Sarcocystis were detected in a total of 20 of the meat samples. Four species were identified that comprised S. cruzi, S. rommeli, S. heydorni, and S. hirsuta, with S. cruzi as the dominant species. In addition, seven of the 20 infected samples were infected with two or three species. Analysis of the 18S rRNA sequences recovered from these samples suggested very little genetic diversity within each species. This study represents the first molecular identification of Sarcocystis species infection in retail beef products from China. These findings will provide valuable information for evaluating the potential public health risk of bovine Sarcocystis species infections and the control of sarcocystosis in cattle.
1. Introduction Sarcocystis is a food-borne zoonotic pathogen that infects a variety of animals and humans (Amairia et al., 2016; Fayer et al., 2015; Dubey, 2015a). In particular, Sarcocystis infections are globally ubiquitous in cattle and sheep (Dubey et al., 2016a; Hornok et al., 2015; Latif et al., 2015). Sarcocystis are protozoan parasites that exhibit an obligatory two-host life cycle. The life cycle first involves sexual development and oocyst formation in the intestinal mucosa of their definitive hosts. Following this stage, sequential asexual multiplication occurs in intermediate hosts within vascular endothelial cells (schizont stage), cardiac and striated muscle cells, and the central nervous system (sarcocyst stage) (Dubey et al., 2016a; Yan et al., 2013a). Cattle are intermediate hosts for five known species of Sarcocystis including S. cruzi, S. rommeli, S. hirsuta, S. hominis, and S. heydorni (Dubey et al., 2015; Hornok et al., 2015). The definitive hosts for S. cruzi are canids, felids for S. hirsuta and S. rommeli, humans and other primates for S. hominis, and S. heydorni (Dubey, 1982; Dubey et al., 2016b, Dubey et al., 2015; Hu et al., 2016). The sarcocysts from these species can either be thin-walled (S. cruzi and S. heydorni) or thickwalled (S. hirsuta, S. hominis, and S. rommeli) (Chen et al., 2011; Nourani et al., 2010). Of these, S. cruzi is the primary pathogen of
⁎
cattle, and acute infection can result in abortion, death, and economic losses for cattle farms (Dubey et al., 2016a; McCausland et al., 1984; More et al., 2009; Li et al., 2002). In contrast, S. hominis is a well-known human pathogen that causes nausea, vomiting and diarrhea (Fayer et al., 2015; Pena et al., 2001). Intermediate and final hosts can harbor more than one Sarcocystis species (Ahmed et al., 2016; Akhlaghi et al., 2016). Thus, the accurate identification of Sarcocystis infections in various hosts is essential to control sarcocystosis prevalence in humans and livestock, and especially cattle, because beef is one of the most important protein sources for people globally. Screening for sarcocysts is not mandatory for meat inspection in China (Chen and Li, 2002). Consequently, there is no report of Sarcocystis in beef that are sold in supermarkets in China. A system of differentiating among cyst types relies on ultrastructural microscopy but can only be achieved on fresh samples with high parasitic burdern (Claveria et al., 2001; Dubey et al., 2016a). Nested PCR that is based on genetic markers is more sensitive than transimission electronic microscopy for differentiating Sarcocystis species in meat samples. 18S rRNA, cox1 and ITS1 sequences are frequently used for molecular identification. Both cox1 and ITS1 markers are more variable and better genetic markers than 18S rRNA gene for genetic diversity among strains or isolates of one specific Sarcocystis species.
Corresponding author. E-mail address: [email protected] (W. Yan).
https://doi.org/10.1016/j.actatropica.2018.12.015 Received 17 February 2018; Received in revised form 6 December 2018; Accepted 8 December 2018 Available online 10 December 2018 0001-706X/ © 2018 Elsevier B.V. All rights reserved.
Acta Tropica 190 (2019) 339–343
R. Xue et al.
CGCAC3′) primers, as well as internal forward (SAR18SN2F: 5′ TACT ACATGGATAACCGT 3′) and reverse (SAR18SN2R: 5′ ATT CCTTTAAG TTTCAGC 3′) primers. The first PCR amplification was performed using the external primers SAR18SN1F/R. Reaction mixtures consisted of 1.0 μL of genomic DNA as template in 25 μL reaction volumes containing 12.5 μL of 2× EasyTaq PCR SuperMix (TransGen Biotech Co., Ltd, Beijing, China). The reactions were conducted in a TianLong thermal cycler (Xi’an TianLong Science and Technology Co., Ltd, China), using the following program: 5 min at 95 °C, followed by 35 cycles of denaturation at 94 °C for 40 s; annealing at 55 °C for 40 s; extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min. The parameters of the second PCR amplification step were the same as the first amplification, with the exception of the following steps: annealing at 56 °C for 40 s and extension at 72 °C for 90 s. One microliter of the amplicons from the first amplification were used as template for the second PCR steps. Negative controls were included for each nested PCR step. PCR products were verified via electrophoresis on 1% agarose gels using ethidium bromide for visualization.
However, so far cox1 and ITS1 sequences are not available for some of the Sarcocystis species in cattle. All 18S rRNA sequences of the five known species of Sarcocystis' in cattle are available in GenBank. Furthermore, 18S rRNA gene is variable enough for species discrimination in five known species infecting cattle (Gjerde et al., 2016; Marsh et al., 1999; More et al., 2014; Pritt et al., 2008). Here, 62 beef samples were collected from supermarkets, butcher shops, and retail meat booths in vegetable markets from 17 cities in the Henan Province of central China. Light microscopy was used to characterize samples, and molecular identification using 18S rRNA gene sequencing was used to confirm the presence of Sarcocystis species. Four bovine Sarcocystis species were detected from the samples, including S. heydorni which infects humans.S. cruzi was the most prevalent species among beef samples from this region. 2. Materials and methods 2.1. Beef samples A total of 62 beef samples (100–200 g each) were collected between February and September 2017 from different supermarkets, butcher shops, and retail meat booths in vegetable markets from 17 cities in the Henan Province of central China. The presence of sarcocysts in the samples was examined via muscle squashing microscopic observation. Positive samples were divided into two sections. The first section was fixed in 10% formalin for histopathological observation and the second was preserved at -20℃for molecular analyses. Samples shown to be negative for sarcocyst by microscopy were also preserved at -20℃ for molecular confirmation.
2.4. Amplicon sequencing and phylogenetic analysis PCR products were ligated into the pEASY T1 cloning vector (TransGen Biotech Co., Ltd, Beijing, China) and transformed into DH5α competent cells. After cultivation of transformed cells, ten colonies carrying the insert were selected for propagation. After PCR confirmation of the presence of insert, six to eight positive recombinant clones for each sample were sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing in both directions, using the plasmid-specific forward and reverse primers M13 F/R (Yan et al., 2013b). 18S rRNA gene sequences were assembled using the ChromasPro 1.7.7 (http://technelysium.com.au/wp/chromas-pro/) software package and edited in BioEdit 7.04 (www.mbio.ncsu.edu/BioEdit/ bioedit.html). The 18S rRNA gene sequences generated here were aligned with others that were retrieved from the GenBank database using the BLAST software program. To better discern the taxonomic relationships among Sarcocystis isolated from beef samples in the Henan Province and those represented by entries in GenBank, a phylogenetic analysis was conducted based on the 18S rRNA gene dataset using neighbor-joining methods with Tamura 3-parameter model in the MEGA6 software program (http://www.megasoftware.net/). Toxoplasma gondii was used as the outgroup. In addition, genetic distances among the various bovine Sarcocystis isolates and phylotypes were computed using MEGA6. Finally, the MegAlign program within DNASTAR 7.0 was used to calculate the sequence identities among the bovine Sarcocystis species.
2.2. Light microscopy The sarcolemma and adipose tissues adhered to each sample were removed for microscopic analyses. Each sample was then randomly cut into 20 rice-sized pieces along the direction of muscle fibers for muscle squashing microscopic examination. A ToupView U3CCD06000KPA Microscopic Digital Camera system (ToupTek Photonics Co. Ltd, Hangzhou, China) was used to identify and measure sarcocyst sizes in the samples. The diagnostic criteria for trichinosis (Wang et al., 2012), with slight modifications, were used to identify sarcocysts by examining 20 squashed slides with rice-sized pieces of beef for each sample. Sarcocysts were then counted individually using a BK6000 series microscope (Optec®, Chongqing, China). The presence of one to three cysts was considered a mild infection, four to seven as a moderate infection, and eight or more as a severe infection. A piece of muscle was processed using the conventional histological method, and 6 μm thick sections were prepared and stained with hematoxylin and eosin (HE), according to previously described methods (Yin et al., 1997). The tissue sections were then examined for parasites and presence of myositis caused by Sarcocystis cysts.
2.5. Nucleotide sequence accession numbers Nucleotide sequences of the 18S rRNA gene sequences generated in this study were deposited in GenBank under the accession numbers MG787077-MG787082 and MH681963-MH681985.
2.3. Genomic DNA extraction and nested PCR amplification
3. Results
Genomic DNA was extracted from a small piece of each sample using an animal tissue genomic DNA isolation kit (Beijing Tiangen Biotech Co., Ltd, China), following the manufacturer’s instructions. Extracted DNAs were then stored at −20 °C prior to further use. To design Sarcocystis-specific primers, 18S rRNA gene sequences for Sarcocystis species that infect cattle, pigs, and other hosts were retrieved from the Genbank database and aligned using the MegAlign program within the DNASTAR 7.0 package (http://www.dnastar.com/t-dnastarlasergene.aspx). The Oligo 6 software package (http://www.oligo.net/) was then used to design nested PCR primers for the genus Sarcocystis based on conserved 18S rRNA gene sequences (Yang et al., 2001). The primers included the external forward (SAR18SN1F: 5′ GGTAAGCTTT TATACGGCGA 3′) and reverse (SAR18SN1R: 5′ CAAAACTTTCCATTC
3.1. Microscopic observations Obvious cysts were not macroscopically visible in any of the 62 beef samples. Muscle squashing microscopic examination revealed the presence of shuttle or club-like Sarcocystis cysts in 18 of the beef samples. The length of most cysts ranged from 158.8 to 384.8 μm, with a few measuring as long as 1028 μm. Histological investigation revealed numerous cysts within muscular fibers, with many crescent-like bradyzoites present within the cysts. However, it was not possible to distinguish cyst wall thicknesses for some samples using HE staining. Two samples from Nanyang (NY04) and Kaifeng (KF01) were 340
Acta Tropica 190 (2019) 339–343
R. Xue et al.
Table 1 Infection prevalence of Sarcocystis in beef products from the Henan province, China. City
Prevalence rate (positive samples/total sample numbers)
Infection intensity per samplea Mild infection
Moderate infection
Severe infection
Luoyang Zhengzhou
0% (0/4) 37.5% (3/8)
ND 3
ND 0
ND 0
Anyang Zhoukou Nanyang
33.3% (1/3) 33.3% (1/3) 50% (4/8)
1 1 3
0 0 0
0 0 1
Hebi Xinyang Xinxiang Kaifeng
0% (0/3) 0% (0/3) 0% (0/3) 66.7% (2/3)
ND ND ND 1
ND ND ND 0
ND ND ND 1
Zhumadian
66.7% (2/3)
2
0
0
Xuchang
100% (3/3)
2
1
0
Luohe Pingdingshan Sanmenxia Puyang Jiaozuo Shangqiu
0% (0/3) 33.3% (1/3) 0% (0/3) 33.3% (1/3) 0% (0/3) 66.7% (2/3)
ND 1 ND 1 ND 2
ND 0 ND 0 ND 0
ND 0 ND 0 ND 0
Total
32.3% (20/62)
17
1
2
Sample IDb
Molecular identification based on 18S rRNA genes
ND ZZ01 ZZ02 ZZ03d AY01 ZK01 NY01 NY02 NY03 NY04 ND ND ND KF01 KF02 ZMD01 ZMD02 XC01 XC02 XC03 ND PDS01 ND PY01 ND SQ01 SQ02d
ND S. cruzi S. cruzi S. cruzi S. rommeli + S. hirsutac S. cruzi S. cruzi S. rommeli S. rommeli + S. heydornic S. cruzi+ S. heydorni+ S. hirsutac ND ND ND S. cruzi S. cruzi S. cruzi + S. rommelic S. cruzi + S. rommelic S. cruzi+ S. heydornic S. rommeli S. cruzi ND S. rommeli + S. heydornic ND S. cruzi ND S. cruzi S. cruzi
Annotations: ND: no data; a Twenty muscle squashing slides were prepared for each sample and microscopically examined individually, one to three cysts among 20 slides from each sample were designated as a mild infection, four to seven cysts as a moderate infection, and more than eight cysts as a severe infection. b Samples positive for sarcocyst presence were assigned an identification number. c One sample was infected with two or three Sarcocystis species. d Samples considered positive based on nested PCR analysis, but not microscopical observations.
4. Discussion
classified as exhibiting severe infections. One sample from Xuchang (XC01) exhibited a moderate infection, and the remaining samples had mild infections (Table 1). Together, these observations indicate that infections of Sarcocystis within beef is common in supermarket meat products in the Henan Province.
Sarcocystis is one of the most globally important food-borne zoonotic parasites (Dubey, 2015a). Here, we investigated the prevalence of Sarcocystis infection of raw beef products in supermarkets, butcher shops, and retail meat booths in vegetable markets in 17 cities of Henan Province, central China. A total of 62 samples were examined using light microscopical observations and molecular characterization based on the presence of Sarcocystis 18S rRNA gene sequences. Positive identification of sarcocysts occurred in 32.3% of the raw beef samples, which comprised four Sarcocystis species: S. cruzi, S. rommeli, S. heydorni, and S. hirsuta. Of these, S. cruzi was the dominant species identified in the samples. These results highlight the public health risks of bovine Sarcocystis in the Henan Province and the prevalence of sarcocystosis in cattle farms of the region. S. cruzi is the most pathogenic species of Sarcocystis, and natural outbreaks or cases of acute sarcocystosis of this species can lead to fever, eosinophilic myositis, abortions, neonatal mortality, necrotizing encephalitis, decreased milk yield, and death in both beef cattle and dairy cows (Dubey et al., 2016a; Vangeel et al., 2013; Wouda et al., 2006; Mumba et al., 2012). Recent surveys have indicated that S. cruzi is the most globally prevalent species of bovine Sarcocystis (Gjerde et al., 2016; More et al., 2011). The results reported here demonstrated that 15 of the 62 beef products that were sampled were positive for S. cruzi presence, and the parasite was the most dominant species among Sarcocystis infections within the Henan Province. These results suggest that sarcocystosis caused by S. cruzi is an increasingly important threat to the health of beef cattle and dairy cows in the Henan region. The definitive hosts for S. cruzi are canids (Dubey, 1982), and farm and stray dogs are the main source of infection in cattle. Therefore, to
3.2. Molecular identification of Sarcocystis presence All the 18 samples that were confirmed to have Sarcocystis infections via microscopy and two other samples, yielded amplifications of Sarcocystis species 18S rRNA genes, as shown by the presence of amplicon products of about 1.1 kbp length after agarose gel electrophoresis. After cloning and sequencing of PCR products, four Sarcocystis species were detected within the 18S rRNA gene dataset including S. cruzi, S. rommeli, S. heydorni, and S. hirsuta. Seven of the 20 beef samples were infected with two or three Sarcocystis species (Table 1). Fifteen of the 20 samples were infected with S. cruzi, seven with S. rommeli, four with S. heydorni, and two with S. hirsuta (Fig. 1). Nucleotide sequences of the 18S rRNA gene fragments from the Henan beef sample S. cruzi phylotypes were 98.9%–99.9% similar to entries in the Genbank database. The genetic distance among the 15 S. cruzi Henan phylotypes was 0.007. The 18S rRNA gene sequences of the seven S. rommeli Henan phylotypes were 99.3%–100% similar to a S. rommeli isolate reported from the Yunnan Province, China. The genetic distance among the seven Henan S. rommeli phylotypes was 0.008. The genetic distance among the four S. heydorni phylotypes was 0.002. Lastly, 18S rRNA gene sequences from the two phylotypes of S. hirsuta (AY01 and NY04) were identical. Phylogenetic analysis confirmed that there was limited genetic diversity within each of the bovine Sarcocystis species detected in the Henan Province beef samples. 341
Acta Tropica 190 (2019) 339–343
R. Xue et al.
Fig. 1. Phylogenetic relationships among Sarcocystis species phylotypes identified in raw beef samples from China with other Sarcocystis species present in the GenBank database, as based on 18S rRNA gene sequences. The phylogenetic tree was constructed using neighbor-joining methods with Tamura 3-parameter model in the MEGA6 software package. Numbers on branches represent bootstrap values (> 50) based on 1000 bootstrap replicates. Toxoplasma gondii was used as the out-group. ●: phylotypes identified in beef samples from various cities in the Henan province of China. The seven samples that were co-infected with > 1 species of Sarcocystis are indicated in pink.
Light microscopy techniques including muscle squashing examination and histological section analysis are able to confirm the presence of sarcocysts in skeletal and cardiac muscles, whereas light microscopy examination cannot distinguish morphological similar species of Sarcocystis in a given host (Dubey et al., 2016a). However, ultrastructural observations of sarcocyst walls and molecular genetic analysis are reliable methods for identifying Sarcocystis species (Dubey et al., 2016a; Yan et al., 2013a; Yang et al., 2001). The investigation of sarcocyst ultrastructures was attempted in this study, but was not successful due to tissue degradation. To determine the prevalence of multi-species infections in beef samples, PCR primers specific for the genus Sarcocystis were designed based on conserved 18S rRNA gene sequences for the genus. Cloning of successful PCR amplification products and subsequent sequencing indicated the presence of six samples (out of 20) that were co-infected with two species. In addition, three Sarcocystis species were identified in one sample. These results confirm that the experimental design described above and implemented here can accurately identify multiple Sarcocystis infections in actual samples. In conclusion, this is the first molecular survey and characterization of Sarcocystis infections within raw beef products in China. Four Sarcocystis species, including the human pathogen S. heydorni, were identified in beef samples from supermarkets across the Henan Province of central China. S. cruzi was the most prevalent species among the infections, and co-infection was observed with two or three species of Sarcocystis in some samples. These results indicate that it is necessary to further investigate the prevalence of Sarcocystis infections in beef products within other provinces of China in order to fully evaluate the potential public health risk from bovine Sarcocystis infections in China.
effectively control sarcocystosis in cattle, dogs should be kept as far away as possible from cattle farms. Moreover, sulfonamide and pyrimethamine should be regularly administered to dogs to treat parasite infections (Dubey et al., 2016a). S. rommeli and S. heydorni were recently identified in cattle, although their final hosts are felids and humans, respectively (Dubey et al., 2016b, Dubey et al., 2015; Hu et al., 2017). S. rommeli infection has been reported in Argentina, Germany, Hungary, Austria, Vietnam, and China (Dubey et al., 2016b; Chen et al., 2011). S. heydorni infection was first reported in a human volunteer that ate raw minced beef in Turkey (Dubey et al., 2015) and was later identified in humans within the Yunnan Province of China (Hu et al., 2016). In the Henan beef samples investigated here, seven of the 62 samples were infected with S. rommeli, and four were infected with S. heydorni. Since S. heydorni can infect humans, the beef products identified here as containing sarcocysts of this parasite could represent a potential health risk to people. These findings suggest that the drinking water or food stock of the cattle was likely contaminated with the feces of felids or humans that contained oocysts or sporocysts of S. rommeli and S. heydorni. Considerably low genetic diversity has been previously reported among various isolates of S. cruzi from different countries and regions (Rosenthal et al., 2008). Consistent with this observation, the withinspecies genetic diversity of S. cruzi, S. rommeli, and S. heydorni phylotypes identified here was very low based on the 18S rRNA gene, despite that they originated from different cities. Indeed, the 18S rRNA gene sequences were identical between two phylotypes of S. hirsuta from different cities. These data support the observation that there is little to no genetic diversity within bovine Sarcocystis species. In addition to single-species infections, 7 of the 20 Sarcocystis-positive samples from the Henan Province were infected with two or three species. Multispecies infections have also been previously reported in other investigations of bovine Sarcocystis infections (Akhlaghi et al., 2016; Dubey et al., 2016a). These multi-species infections could be due to the lack of cross-protective immunity in cattle towards various species of bovine Sarcocystis.
Conflict of interest The authors state that there are no competing interests.
342
Acta Tropica 190 (2019) 339–343
R. Xue et al.
Acknowledgements
Latif, B., Kannan, K.M., Muslim, A., Hussaini, J., Omar, E., Heo, C.C., Rossle, N.F., Abdullah, S., Kamarudin, M.A., Zulkarnain, M.A., 2015. Light microscopy and molecular identification of Sarcocystis spp. in meat producing animals in Selangor, Malaysia. Trop. Biomed. 32, 444–452. Li, Q.Q., Yang, Z.Q., Zuo, Y.X., Attwood, S.W., Chen, X.W., Zhang, Y.P., 2002. A PCRbased RFLP analysis of Sarcocystis cruzi (Protozoa: Sarcocystiidae) in Yunnan province, PR China, reveals the water buffalo (Bubalus bubalis) as a natural intermediate host. J. Parasitol. 88, 1259–1261. Marsh, A.E., Barr, B.C., Tell, L., Bowman, D.D., Conrad PA Ketcherside, C., Green, T., 1999. Comparison of the internal transcribed spacer, ITS-1, from Sarcocystis falcatula isolates and Sarcocystis neurona. J. Parasitol. 85 (4), 750–757. McCausland, I.P., Badman, R.T., Hides, S., Slee, K.J., 1984. Multiple apparent Sarcocystis abortion in four bovine herds. Cornell Vet. 74, 146–154. More, G., Bacigalupe, D., Basso, W., Rambeaud, M., Beltrame, F., Ramirez, B., Venturini, M.C., Venturini, L., 2009. Frequency of horizontal and vertical transmission for Sarcocystis cruzi and Neospora caninum in dairy cattle. Vet. Parasitol. 160, 51–54. More, G., Abrahamovich, P., Jurado, S., Bacigalupe, D., Marin, J.C., Rambeaud, M., Venturini, L., Venturini, M.C., 2011. Prevalence of Sarcocystis spp. in Argentinean cattle. Vet. Parasitol. 177, 162–165. More, G., Pantchev, A., Skuballa, J., Langenmayer, M.C., Maksimov, P., Conraths, F.J., Venturini, M.C., Schares, G., 2014. Sarcocystis sinensis is the most prevalent thickwalled Sarcocystis in beef for consumers in Germany. Parasitol. Res. 113, 2223–2230. Mumba, C., Pandey, G.S., Kakandelwa, C., 2012. Sarcocystosis in an adult Brahman cow. Int. J. Livest. Res. 2, 271–275. Nourani, H., Matin, S., Nouri, A., Azizi, H., 2010. Prevalence of thin-walled Sarcocystis cruzi and thick-walled Sarcocystis hirsuta or Sarcocystis hominis from cattle in Iran. Trop. Anim. Health Prod. 42, 1225–1227. Pena, H.F., Ogassawara, S., Sinhorini, I.L., 2001. Occurrence of cattle Sarcocystis species in raw kibbe from Arabian food establishments in the city of Sao Paulo, Brazil, and experimental transmission to humans. J. Parasitol. 87, 1459–1465. Pritt, B., Trainer, T., Simmons-Arnold, L., Evans, M., Dunams, D., Rosenthal, B.M., 2008. Detection of Sarcocystis parasites in retail beef: a regional survey combining histological and genetic detection methods. J. Food Prot. 71, 2144–2147. Rosenthal, B.M., Dunams, D.B., Pritt, B., 2008. Restricted genetic diversity in the ubiquitous cattle parasite, Sarcocystis cruzi. Infect. Genet. Evol. 8, 588–592. Vangeel, L., Houf, K., Geldhof, P., De Preter, K., Vercruysse, J., Ducatelle, R., Chiers, K., 2013. Different Sarcocystis spp. are present in bovine eosinophilic myositis. Vet. Parasitol. 197, 543–548. Wang, Z., Cui, J., Xu, B., Zhang, H., Guan, Y., Tang, L., 2012. Diagnosis of trichinellosis. Standardization Adminstration of The People’s Republic of China, Beijing. Wouda, W., Snoep, J.J., Dubey, J.P., 2006. Eosinophilic myositis due to Sarcocystis hominis in a beef cow. J. Comp. Pathol. 135, 249–253. Yan, W., Qian, W., Li, X., Wang, T., Ding, K., Huang, T., 2013a. Morphological and molecular characterization of Sarcocystis miescheriana from pigs in the central region of China. Parasitol. Res. 112, 975–980. Yan, W., Wang, W., Wang, T., Suo, X., Qian, W., Wang, S., Fan, D., 2013b. Simultaneous identification of three highly pathogenic Eimeria species in rabbits using a multiplex PCR diagnostic assay based on ITS1-5.8S rRNA-ITS2 fragments. Vet. Parasitol. 193, 284–288. Yang, Z.Q., Zuo, Y.X., Yao, Y.G., Chen, X.W., Yang, G.C., Zhang, Y.P., 2001. Analysis of the 18S rRNA genes of Sarcocystis species suggests that the morphologically similar organisms from cattle and water buffalo should be considered the same species. Mol. Biochem. Parasitol. 115, 283–288. Yin, P., Kong, L., Zhao, Y., 1997. Experimental Guideline of Clinical Pathology. Zhong Yuan Nong Min Press, Zhengzhou, China.
This study was supported by the Natural Science Foundation of Henan Province (182300410088) and the Science and Technology Breakthrough Project of Henan Province (172102110017). We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript. References Ahmed, A.M., Elshraway, N.T., Youssef, A.I., 2016. Survey on Sarcocystis in bovine carcasses slaughtered at the municipal abattoir of El-Kharga, Egypt. Vet. World 9, 1461–1465. Akhlaghi, M., Razavi, M., Hosseini, A., 2016. Molecular differentiation of bovine sarcocysts. Parasitol. Res. 115, 2721–2728. Amairia, S., Amdouni, Y., Rjeibi, M.R., Rouatbi, M., Awadi, S., Gharbi, M., 2016. First molecular detection and characterization of Sarcocystis species in slaughtered cattle in North-West Tunisia. Meat Sci. 122, 55–59. Chen, L.W., Li, K.P., 2002. The careless omission about Sarcocystosis in animal epidemic prevention regulations. Guangxi J. Anim. Husbandry Vet. Med. 18, 35–37. Chen, X., Zuo, Y., Rosenthal, B.M., He, Y., Cui, L., Yang, Z., 2011. Sarcocystis sinensis is an ultrastructurally distinct parasite of water buffalo that can cause foodborne illness but cannot complete its life-cycle in human beings. Vet. Parasitol. 178, 35–39. Claveria, F.G., San-Pedro, L.R., Cruz-Flores, M.J., Nagasawa, H., Suzuki, N., 2001. Ultrastructural studies of Sarcocystis cruzi (Hasselmann, 1926) Wenyon, 1926 infection in cattle (Bos taurus): Philippine cases. Parasites Vectors 251–254. Dubey, J.P., 1982. Development of ox-coyote cycle of Sarcocystis cruzi. J. Protozool. 29, 591–601. Dubey, J.P., 2015a. Foodborne and waterborne zoonotic sarcocystosis. Food Waterborne Parasitol. 1, 2–11. Dubey, J.P., van Wilpe, E., Calero-Bernal, R., Verma, S.K., Fayer, R., 2015. Sarcocystis heydorni, n. sp. (Apicomplexa: Sarcocystidae) with cattle (Bos taurus) and human (Homo sapiens) cycle. Parasitol. Res. 114, 4143–4147. Dubey, J.P., Calero-Bernal, R., Rosenthal, B.M., Speer, C.A., Fayer, R., 2016a. Sarcocystosis of Animals and Humans, 2nd edition. CRC Press, Boca Raton, Florida. Dubey, J.P., More, G., van Wilpe, E., Calero-Bernal, R., Verma, S.K., Schares, G., 2016b. Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from cattle (Bos taurus) and its differentiation from Sarcocystis hominis. J. Eukaryot. Microbiol. 63, 62–68. Fayer, R., Esposito, D.H., Dubey, J.P., 2015. Human infections with Sarcocystis species. Clin. Microbiol. Rev. 28, 295–311. Gjerde, B., Hilali, M., Abbas, I.E., 2016. Molecular differentiation of Sarcocystis buffalonis and Sarcocystis levinei in water buffaloes (Bubalus bubalis) from Sarcocystis hirsuta and Sarcocystis cruzi in cattle (Bos taurus). Parasitol. Res. 115, 2459–2471. Hornok, S., Mester, A., Takacs, N., Baska, F., Majoros, G., Fok, E., Biksi, I., Nemet, Z., Hornyak, A., Janosi, S., Farkas, R., 2015. Sarcocystis-infection of cattle in Hungary. Parasites Vectors 8, 69. Hu, J.J., Wen, T., Chen, X.W., Liu, T.T., Esch, G.W., Huang, S., 2016. Prevalance, morphology, and molecular characterization of Sarcocystis heydorni Sarcocysts from cattle (Bos Taurus) in China. J. Parasitol. 102, 545–548. Hu, J.J., Huang, S., Wen, T., Esch, G.W., Liang, Y., Li, H.L., 2017. Morphology, molecular characteristics, and demonstration of a definitive host for Sarcocystis rommeli from cattle (Bos taurus) in China. J. Parasitol. 103, 471–476.
343
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Prevalence and molecular characterization of Sarcocystis infections of retail beef products from central China
T
⁎
Rui Xue, Wenchao Yan , Weifeng Qian, Tianqi Wang, Min Zhang, Zhiguo Wei, Lifang Han, Bin He, Jichen Dou College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Retail beef Sarcocysts Sarcocystis cruzi S. heydorni 18S rRNA gene Co-infection
Cattle are the intermediate hosts for five Sarcocystis species including S. hominis and S. heydorni, which also infect humans. To investigate the prevalence of Sarcocystis infections in beef products from 17 cities in the Henan Province of central China, 62 raw beef samples from markets were collected and analyzed for Sarcocystis presence via muscle squashing microscopic observation, histological section examination, and molecular characterization with 18S rRNA gene sequencing. Sarcocystis were detected in a total of 20 of the meat samples. Four species were identified that comprised S. cruzi, S. rommeli, S. heydorni, and S. hirsuta, with S. cruzi as the dominant species. In addition, seven of the 20 infected samples were infected with two or three species. Analysis of the 18S rRNA sequences recovered from these samples suggested very little genetic diversity within each species. This study represents the first molecular identification of Sarcocystis species infection in retail beef products from China. These findings will provide valuable information for evaluating the potential public health risk of bovine Sarcocystis species infections and the control of sarcocystosis in cattle.
1. Introduction Sarcocystis is a food-borne zoonotic pathogen that infects a variety of animals and humans (Amairia et al., 2016; Fayer et al., 2015; Dubey, 2015a). In particular, Sarcocystis infections are globally ubiquitous in cattle and sheep (Dubey et al., 2016a; Hornok et al., 2015; Latif et al., 2015). Sarcocystis are protozoan parasites that exhibit an obligatory two-host life cycle. The life cycle first involves sexual development and oocyst formation in the intestinal mucosa of their definitive hosts. Following this stage, sequential asexual multiplication occurs in intermediate hosts within vascular endothelial cells (schizont stage), cardiac and striated muscle cells, and the central nervous system (sarcocyst stage) (Dubey et al., 2016a; Yan et al., 2013a). Cattle are intermediate hosts for five known species of Sarcocystis including S. cruzi, S. rommeli, S. hirsuta, S. hominis, and S. heydorni (Dubey et al., 2015; Hornok et al., 2015). The definitive hosts for S. cruzi are canids, felids for S. hirsuta and S. rommeli, humans and other primates for S. hominis, and S. heydorni (Dubey, 1982; Dubey et al., 2016b, Dubey et al., 2015; Hu et al., 2016). The sarcocysts from these species can either be thin-walled (S. cruzi and S. heydorni) or thickwalled (S. hirsuta, S. hominis, and S. rommeli) (Chen et al., 2011; Nourani et al., 2010). Of these, S. cruzi is the primary pathogen of
⁎
cattle, and acute infection can result in abortion, death, and economic losses for cattle farms (Dubey et al., 2016a; McCausland et al., 1984; More et al., 2009; Li et al., 2002). In contrast, S. hominis is a well-known human pathogen that causes nausea, vomiting and diarrhea (Fayer et al., 2015; Pena et al., 2001). Intermediate and final hosts can harbor more than one Sarcocystis species (Ahmed et al., 2016; Akhlaghi et al., 2016). Thus, the accurate identification of Sarcocystis infections in various hosts is essential to control sarcocystosis prevalence in humans and livestock, and especially cattle, because beef is one of the most important protein sources for people globally. Screening for sarcocysts is not mandatory for meat inspection in China (Chen and Li, 2002). Consequently, there is no report of Sarcocystis in beef that are sold in supermarkets in China. A system of differentiating among cyst types relies on ultrastructural microscopy but can only be achieved on fresh samples with high parasitic burdern (Claveria et al., 2001; Dubey et al., 2016a). Nested PCR that is based on genetic markers is more sensitive than transimission electronic microscopy for differentiating Sarcocystis species in meat samples. 18S rRNA, cox1 and ITS1 sequences are frequently used for molecular identification. Both cox1 and ITS1 markers are more variable and better genetic markers than 18S rRNA gene for genetic diversity among strains or isolates of one specific Sarcocystis species.
Corresponding author. E-mail address: [email protected] (W. Yan).
https://doi.org/10.1016/j.actatropica.2018.12.015 Received 17 February 2018; Received in revised form 6 December 2018; Accepted 8 December 2018 Available online 10 December 2018 0001-706X/ © 2018 Elsevier B.V. All rights reserved.
Acta Tropica 190 (2019) 339–343
R. Xue et al.
CGCAC3′) primers, as well as internal forward (SAR18SN2F: 5′ TACT ACATGGATAACCGT 3′) and reverse (SAR18SN2R: 5′ ATT CCTTTAAG TTTCAGC 3′) primers. The first PCR amplification was performed using the external primers SAR18SN1F/R. Reaction mixtures consisted of 1.0 μL of genomic DNA as template in 25 μL reaction volumes containing 12.5 μL of 2× EasyTaq PCR SuperMix (TransGen Biotech Co., Ltd, Beijing, China). The reactions were conducted in a TianLong thermal cycler (Xi’an TianLong Science and Technology Co., Ltd, China), using the following program: 5 min at 95 °C, followed by 35 cycles of denaturation at 94 °C for 40 s; annealing at 55 °C for 40 s; extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min. The parameters of the second PCR amplification step were the same as the first amplification, with the exception of the following steps: annealing at 56 °C for 40 s and extension at 72 °C for 90 s. One microliter of the amplicons from the first amplification were used as template for the second PCR steps. Negative controls were included for each nested PCR step. PCR products were verified via electrophoresis on 1% agarose gels using ethidium bromide for visualization.
However, so far cox1 and ITS1 sequences are not available for some of the Sarcocystis species in cattle. All 18S rRNA sequences of the five known species of Sarcocystis' in cattle are available in GenBank. Furthermore, 18S rRNA gene is variable enough for species discrimination in five known species infecting cattle (Gjerde et al., 2016; Marsh et al., 1999; More et al., 2014; Pritt et al., 2008). Here, 62 beef samples were collected from supermarkets, butcher shops, and retail meat booths in vegetable markets from 17 cities in the Henan Province of central China. Light microscopy was used to characterize samples, and molecular identification using 18S rRNA gene sequencing was used to confirm the presence of Sarcocystis species. Four bovine Sarcocystis species were detected from the samples, including S. heydorni which infects humans.S. cruzi was the most prevalent species among beef samples from this region. 2. Materials and methods 2.1. Beef samples A total of 62 beef samples (100–200 g each) were collected between February and September 2017 from different supermarkets, butcher shops, and retail meat booths in vegetable markets from 17 cities in the Henan Province of central China. The presence of sarcocysts in the samples was examined via muscle squashing microscopic observation. Positive samples were divided into two sections. The first section was fixed in 10% formalin for histopathological observation and the second was preserved at -20℃for molecular analyses. Samples shown to be negative for sarcocyst by microscopy were also preserved at -20℃ for molecular confirmation.
2.4. Amplicon sequencing and phylogenetic analysis PCR products were ligated into the pEASY T1 cloning vector (TransGen Biotech Co., Ltd, Beijing, China) and transformed into DH5α competent cells. After cultivation of transformed cells, ten colonies carrying the insert were selected for propagation. After PCR confirmation of the presence of insert, six to eight positive recombinant clones for each sample were sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing in both directions, using the plasmid-specific forward and reverse primers M13 F/R (Yan et al., 2013b). 18S rRNA gene sequences were assembled using the ChromasPro 1.7.7 (http://technelysium.com.au/wp/chromas-pro/) software package and edited in BioEdit 7.04 (www.mbio.ncsu.edu/BioEdit/ bioedit.html). The 18S rRNA gene sequences generated here were aligned with others that were retrieved from the GenBank database using the BLAST software program. To better discern the taxonomic relationships among Sarcocystis isolated from beef samples in the Henan Province and those represented by entries in GenBank, a phylogenetic analysis was conducted based on the 18S rRNA gene dataset using neighbor-joining methods with Tamura 3-parameter model in the MEGA6 software program (http://www.megasoftware.net/). Toxoplasma gondii was used as the outgroup. In addition, genetic distances among the various bovine Sarcocystis isolates and phylotypes were computed using MEGA6. Finally, the MegAlign program within DNASTAR 7.0 was used to calculate the sequence identities among the bovine Sarcocystis species.
2.2. Light microscopy The sarcolemma and adipose tissues adhered to each sample were removed for microscopic analyses. Each sample was then randomly cut into 20 rice-sized pieces along the direction of muscle fibers for muscle squashing microscopic examination. A ToupView U3CCD06000KPA Microscopic Digital Camera system (ToupTek Photonics Co. Ltd, Hangzhou, China) was used to identify and measure sarcocyst sizes in the samples. The diagnostic criteria for trichinosis (Wang et al., 2012), with slight modifications, were used to identify sarcocysts by examining 20 squashed slides with rice-sized pieces of beef for each sample. Sarcocysts were then counted individually using a BK6000 series microscope (Optec®, Chongqing, China). The presence of one to three cysts was considered a mild infection, four to seven as a moderate infection, and eight or more as a severe infection. A piece of muscle was processed using the conventional histological method, and 6 μm thick sections were prepared and stained with hematoxylin and eosin (HE), according to previously described methods (Yin et al., 1997). The tissue sections were then examined for parasites and presence of myositis caused by Sarcocystis cysts.
2.5. Nucleotide sequence accession numbers Nucleotide sequences of the 18S rRNA gene sequences generated in this study were deposited in GenBank under the accession numbers MG787077-MG787082 and MH681963-MH681985.
2.3. Genomic DNA extraction and nested PCR amplification
3. Results
Genomic DNA was extracted from a small piece of each sample using an animal tissue genomic DNA isolation kit (Beijing Tiangen Biotech Co., Ltd, China), following the manufacturer’s instructions. Extracted DNAs were then stored at −20 °C prior to further use. To design Sarcocystis-specific primers, 18S rRNA gene sequences for Sarcocystis species that infect cattle, pigs, and other hosts were retrieved from the Genbank database and aligned using the MegAlign program within the DNASTAR 7.0 package (http://www.dnastar.com/t-dnastarlasergene.aspx). The Oligo 6 software package (http://www.oligo.net/) was then used to design nested PCR primers for the genus Sarcocystis based on conserved 18S rRNA gene sequences (Yang et al., 2001). The primers included the external forward (SAR18SN1F: 5′ GGTAAGCTTT TATACGGCGA 3′) and reverse (SAR18SN1R: 5′ CAAAACTTTCCATTC
3.1. Microscopic observations Obvious cysts were not macroscopically visible in any of the 62 beef samples. Muscle squashing microscopic examination revealed the presence of shuttle or club-like Sarcocystis cysts in 18 of the beef samples. The length of most cysts ranged from 158.8 to 384.8 μm, with a few measuring as long as 1028 μm. Histological investigation revealed numerous cysts within muscular fibers, with many crescent-like bradyzoites present within the cysts. However, it was not possible to distinguish cyst wall thicknesses for some samples using HE staining. Two samples from Nanyang (NY04) and Kaifeng (KF01) were 340
Acta Tropica 190 (2019) 339–343
R. Xue et al.
Table 1 Infection prevalence of Sarcocystis in beef products from the Henan province, China. City
Prevalence rate (positive samples/total sample numbers)
Infection intensity per samplea Mild infection
Moderate infection
Severe infection
Luoyang Zhengzhou
0% (0/4) 37.5% (3/8)
ND 3
ND 0
ND 0
Anyang Zhoukou Nanyang
33.3% (1/3) 33.3% (1/3) 50% (4/8)
1 1 3
0 0 0
0 0 1
Hebi Xinyang Xinxiang Kaifeng
0% (0/3) 0% (0/3) 0% (0/3) 66.7% (2/3)
ND ND ND 1
ND ND ND 0
ND ND ND 1
Zhumadian
66.7% (2/3)
2
0
0
Xuchang
100% (3/3)
2
1
0
Luohe Pingdingshan Sanmenxia Puyang Jiaozuo Shangqiu
0% (0/3) 33.3% (1/3) 0% (0/3) 33.3% (1/3) 0% (0/3) 66.7% (2/3)
ND 1 ND 1 ND 2
ND 0 ND 0 ND 0
ND 0 ND 0 ND 0
Total
32.3% (20/62)
17
1
2
Sample IDb
Molecular identification based on 18S rRNA genes
ND ZZ01 ZZ02 ZZ03d AY01 ZK01 NY01 NY02 NY03 NY04 ND ND ND KF01 KF02 ZMD01 ZMD02 XC01 XC02 XC03 ND PDS01 ND PY01 ND SQ01 SQ02d
ND S. cruzi S. cruzi S. cruzi S. rommeli + S. hirsutac S. cruzi S. cruzi S. rommeli S. rommeli + S. heydornic S. cruzi+ S. heydorni+ S. hirsutac ND ND ND S. cruzi S. cruzi S. cruzi + S. rommelic S. cruzi + S. rommelic S. cruzi+ S. heydornic S. rommeli S. cruzi ND S. rommeli + S. heydornic ND S. cruzi ND S. cruzi S. cruzi
Annotations: ND: no data; a Twenty muscle squashing slides were prepared for each sample and microscopically examined individually, one to three cysts among 20 slides from each sample were designated as a mild infection, four to seven cysts as a moderate infection, and more than eight cysts as a severe infection. b Samples positive for sarcocyst presence were assigned an identification number. c One sample was infected with two or three Sarcocystis species. d Samples considered positive based on nested PCR analysis, but not microscopical observations.
4. Discussion
classified as exhibiting severe infections. One sample from Xuchang (XC01) exhibited a moderate infection, and the remaining samples had mild infections (Table 1). Together, these observations indicate that infections of Sarcocystis within beef is common in supermarket meat products in the Henan Province.
Sarcocystis is one of the most globally important food-borne zoonotic parasites (Dubey, 2015a). Here, we investigated the prevalence of Sarcocystis infection of raw beef products in supermarkets, butcher shops, and retail meat booths in vegetable markets in 17 cities of Henan Province, central China. A total of 62 samples were examined using light microscopical observations and molecular characterization based on the presence of Sarcocystis 18S rRNA gene sequences. Positive identification of sarcocysts occurred in 32.3% of the raw beef samples, which comprised four Sarcocystis species: S. cruzi, S. rommeli, S. heydorni, and S. hirsuta. Of these, S. cruzi was the dominant species identified in the samples. These results highlight the public health risks of bovine Sarcocystis in the Henan Province and the prevalence of sarcocystosis in cattle farms of the region. S. cruzi is the most pathogenic species of Sarcocystis, and natural outbreaks or cases of acute sarcocystosis of this species can lead to fever, eosinophilic myositis, abortions, neonatal mortality, necrotizing encephalitis, decreased milk yield, and death in both beef cattle and dairy cows (Dubey et al., 2016a; Vangeel et al., 2013; Wouda et al., 2006; Mumba et al., 2012). Recent surveys have indicated that S. cruzi is the most globally prevalent species of bovine Sarcocystis (Gjerde et al., 2016; More et al., 2011). The results reported here demonstrated that 15 of the 62 beef products that were sampled were positive for S. cruzi presence, and the parasite was the most dominant species among Sarcocystis infections within the Henan Province. These results suggest that sarcocystosis caused by S. cruzi is an increasingly important threat to the health of beef cattle and dairy cows in the Henan region. The definitive hosts for S. cruzi are canids (Dubey, 1982), and farm and stray dogs are the main source of infection in cattle. Therefore, to
3.2. Molecular identification of Sarcocystis presence All the 18 samples that were confirmed to have Sarcocystis infections via microscopy and two other samples, yielded amplifications of Sarcocystis species 18S rRNA genes, as shown by the presence of amplicon products of about 1.1 kbp length after agarose gel electrophoresis. After cloning and sequencing of PCR products, four Sarcocystis species were detected within the 18S rRNA gene dataset including S. cruzi, S. rommeli, S. heydorni, and S. hirsuta. Seven of the 20 beef samples were infected with two or three Sarcocystis species (Table 1). Fifteen of the 20 samples were infected with S. cruzi, seven with S. rommeli, four with S. heydorni, and two with S. hirsuta (Fig. 1). Nucleotide sequences of the 18S rRNA gene fragments from the Henan beef sample S. cruzi phylotypes were 98.9%–99.9% similar to entries in the Genbank database. The genetic distance among the 15 S. cruzi Henan phylotypes was 0.007. The 18S rRNA gene sequences of the seven S. rommeli Henan phylotypes were 99.3%–100% similar to a S. rommeli isolate reported from the Yunnan Province, China. The genetic distance among the seven Henan S. rommeli phylotypes was 0.008. The genetic distance among the four S. heydorni phylotypes was 0.002. Lastly, 18S rRNA gene sequences from the two phylotypes of S. hirsuta (AY01 and NY04) were identical. Phylogenetic analysis confirmed that there was limited genetic diversity within each of the bovine Sarcocystis species detected in the Henan Province beef samples. 341
Acta Tropica 190 (2019) 339–343
R. Xue et al.
Fig. 1. Phylogenetic relationships among Sarcocystis species phylotypes identified in raw beef samples from China with other Sarcocystis species present in the GenBank database, as based on 18S rRNA gene sequences. The phylogenetic tree was constructed using neighbor-joining methods with Tamura 3-parameter model in the MEGA6 software package. Numbers on branches represent bootstrap values (> 50) based on 1000 bootstrap replicates. Toxoplasma gondii was used as the out-group. ●: phylotypes identified in beef samples from various cities in the Henan province of China. The seven samples that were co-infected with > 1 species of Sarcocystis are indicated in pink.
Light microscopy techniques including muscle squashing examination and histological section analysis are able to confirm the presence of sarcocysts in skeletal and cardiac muscles, whereas light microscopy examination cannot distinguish morphological similar species of Sarcocystis in a given host (Dubey et al., 2016a). However, ultrastructural observations of sarcocyst walls and molecular genetic analysis are reliable methods for identifying Sarcocystis species (Dubey et al., 2016a; Yan et al., 2013a; Yang et al., 2001). The investigation of sarcocyst ultrastructures was attempted in this study, but was not successful due to tissue degradation. To determine the prevalence of multi-species infections in beef samples, PCR primers specific for the genus Sarcocystis were designed based on conserved 18S rRNA gene sequences for the genus. Cloning of successful PCR amplification products and subsequent sequencing indicated the presence of six samples (out of 20) that were co-infected with two species. In addition, three Sarcocystis species were identified in one sample. These results confirm that the experimental design described above and implemented here can accurately identify multiple Sarcocystis infections in actual samples. In conclusion, this is the first molecular survey and characterization of Sarcocystis infections within raw beef products in China. Four Sarcocystis species, including the human pathogen S. heydorni, were identified in beef samples from supermarkets across the Henan Province of central China. S. cruzi was the most prevalent species among the infections, and co-infection was observed with two or three species of Sarcocystis in some samples. These results indicate that it is necessary to further investigate the prevalence of Sarcocystis infections in beef products within other provinces of China in order to fully evaluate the potential public health risk from bovine Sarcocystis infections in China.
effectively control sarcocystosis in cattle, dogs should be kept as far away as possible from cattle farms. Moreover, sulfonamide and pyrimethamine should be regularly administered to dogs to treat parasite infections (Dubey et al., 2016a). S. rommeli and S. heydorni were recently identified in cattle, although their final hosts are felids and humans, respectively (Dubey et al., 2016b, Dubey et al., 2015; Hu et al., 2017). S. rommeli infection has been reported in Argentina, Germany, Hungary, Austria, Vietnam, and China (Dubey et al., 2016b; Chen et al., 2011). S. heydorni infection was first reported in a human volunteer that ate raw minced beef in Turkey (Dubey et al., 2015) and was later identified in humans within the Yunnan Province of China (Hu et al., 2016). In the Henan beef samples investigated here, seven of the 62 samples were infected with S. rommeli, and four were infected with S. heydorni. Since S. heydorni can infect humans, the beef products identified here as containing sarcocysts of this parasite could represent a potential health risk to people. These findings suggest that the drinking water or food stock of the cattle was likely contaminated with the feces of felids or humans that contained oocysts or sporocysts of S. rommeli and S. heydorni. Considerably low genetic diversity has been previously reported among various isolates of S. cruzi from different countries and regions (Rosenthal et al., 2008). Consistent with this observation, the withinspecies genetic diversity of S. cruzi, S. rommeli, and S. heydorni phylotypes identified here was very low based on the 18S rRNA gene, despite that they originated from different cities. Indeed, the 18S rRNA gene sequences were identical between two phylotypes of S. hirsuta from different cities. These data support the observation that there is little to no genetic diversity within bovine Sarcocystis species. In addition to single-species infections, 7 of the 20 Sarcocystis-positive samples from the Henan Province were infected with two or three species. Multispecies infections have also been previously reported in other investigations of bovine Sarcocystis infections (Akhlaghi et al., 2016; Dubey et al., 2016a). These multi-species infections could be due to the lack of cross-protective immunity in cattle towards various species of bovine Sarcocystis.
Conflict of interest The authors state that there are no competing interests.
342
Acta Tropica 190 (2019) 339–343
R. Xue et al.
Acknowledgements
Latif, B., Kannan, K.M., Muslim, A., Hussaini, J., Omar, E., Heo, C.C., Rossle, N.F., Abdullah, S., Kamarudin, M.A., Zulkarnain, M.A., 2015. Light microscopy and molecular identification of Sarcocystis spp. in meat producing animals in Selangor, Malaysia. Trop. Biomed. 32, 444–452. Li, Q.Q., Yang, Z.Q., Zuo, Y.X., Attwood, S.W., Chen, X.W., Zhang, Y.P., 2002. A PCRbased RFLP analysis of Sarcocystis cruzi (Protozoa: Sarcocystiidae) in Yunnan province, PR China, reveals the water buffalo (Bubalus bubalis) as a natural intermediate host. J. Parasitol. 88, 1259–1261. Marsh, A.E., Barr, B.C., Tell, L., Bowman, D.D., Conrad PA Ketcherside, C., Green, T., 1999. Comparison of the internal transcribed spacer, ITS-1, from Sarcocystis falcatula isolates and Sarcocystis neurona. J. Parasitol. 85 (4), 750–757. McCausland, I.P., Badman, R.T., Hides, S., Slee, K.J., 1984. Multiple apparent Sarcocystis abortion in four bovine herds. Cornell Vet. 74, 146–154. More, G., Bacigalupe, D., Basso, W., Rambeaud, M., Beltrame, F., Ramirez, B., Venturini, M.C., Venturini, L., 2009. Frequency of horizontal and vertical transmission for Sarcocystis cruzi and Neospora caninum in dairy cattle. Vet. Parasitol. 160, 51–54. More, G., Abrahamovich, P., Jurado, S., Bacigalupe, D., Marin, J.C., Rambeaud, M., Venturini, L., Venturini, M.C., 2011. Prevalence of Sarcocystis spp. in Argentinean cattle. Vet. Parasitol. 177, 162–165. More, G., Pantchev, A., Skuballa, J., Langenmayer, M.C., Maksimov, P., Conraths, F.J., Venturini, M.C., Schares, G., 2014. Sarcocystis sinensis is the most prevalent thickwalled Sarcocystis in beef for consumers in Germany. Parasitol. Res. 113, 2223–2230. Mumba, C., Pandey, G.S., Kakandelwa, C., 2012. Sarcocystosis in an adult Brahman cow. Int. J. Livest. Res. 2, 271–275. Nourani, H., Matin, S., Nouri, A., Azizi, H., 2010. Prevalence of thin-walled Sarcocystis cruzi and thick-walled Sarcocystis hirsuta or Sarcocystis hominis from cattle in Iran. Trop. Anim. Health Prod. 42, 1225–1227. Pena, H.F., Ogassawara, S., Sinhorini, I.L., 2001. Occurrence of cattle Sarcocystis species in raw kibbe from Arabian food establishments in the city of Sao Paulo, Brazil, and experimental transmission to humans. J. Parasitol. 87, 1459–1465. Pritt, B., Trainer, T., Simmons-Arnold, L., Evans, M., Dunams, D., Rosenthal, B.M., 2008. Detection of Sarcocystis parasites in retail beef: a regional survey combining histological and genetic detection methods. J. Food Prot. 71, 2144–2147. Rosenthal, B.M., Dunams, D.B., Pritt, B., 2008. Restricted genetic diversity in the ubiquitous cattle parasite, Sarcocystis cruzi. Infect. Genet. Evol. 8, 588–592. Vangeel, L., Houf, K., Geldhof, P., De Preter, K., Vercruysse, J., Ducatelle, R., Chiers, K., 2013. Different Sarcocystis spp. are present in bovine eosinophilic myositis. Vet. Parasitol. 197, 543–548. Wang, Z., Cui, J., Xu, B., Zhang, H., Guan, Y., Tang, L., 2012. Diagnosis of trichinellosis. Standardization Adminstration of The People’s Republic of China, Beijing. Wouda, W., Snoep, J.J., Dubey, J.P., 2006. Eosinophilic myositis due to Sarcocystis hominis in a beef cow. J. Comp. Pathol. 135, 249–253. Yan, W., Qian, W., Li, X., Wang, T., Ding, K., Huang, T., 2013a. Morphological and molecular characterization of Sarcocystis miescheriana from pigs in the central region of China. Parasitol. Res. 112, 975–980. Yan, W., Wang, W., Wang, T., Suo, X., Qian, W., Wang, S., Fan, D., 2013b. Simultaneous identification of three highly pathogenic Eimeria species in rabbits using a multiplex PCR diagnostic assay based on ITS1-5.8S rRNA-ITS2 fragments. Vet. Parasitol. 193, 284–288. Yang, Z.Q., Zuo, Y.X., Yao, Y.G., Chen, X.W., Yang, G.C., Zhang, Y.P., 2001. Analysis of the 18S rRNA genes of Sarcocystis species suggests that the morphologically similar organisms from cattle and water buffalo should be considered the same species. Mol. Biochem. Parasitol. 115, 283–288. Yin, P., Kong, L., Zhao, Y., 1997. Experimental Guideline of Clinical Pathology. Zhong Yuan Nong Min Press, Zhengzhou, China.
This study was supported by the Natural Science Foundation of Henan Province (182300410088) and the Science and Technology Breakthrough Project of Henan Province (172102110017). We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript. References Ahmed, A.M., Elshraway, N.T., Youssef, A.I., 2016. Survey on Sarcocystis in bovine carcasses slaughtered at the municipal abattoir of El-Kharga, Egypt. Vet. World 9, 1461–1465. Akhlaghi, M., Razavi, M., Hosseini, A., 2016. Molecular differentiation of bovine sarcocysts. Parasitol. Res. 115, 2721–2728. Amairia, S., Amdouni, Y., Rjeibi, M.R., Rouatbi, M., Awadi, S., Gharbi, M., 2016. First molecular detection and characterization of Sarcocystis species in slaughtered cattle in North-West Tunisia. Meat Sci. 122, 55–59. Chen, L.W., Li, K.P., 2002. The careless omission about Sarcocystosis in animal epidemic prevention regulations. Guangxi J. Anim. Husbandry Vet. Med. 18, 35–37. Chen, X., Zuo, Y., Rosenthal, B.M., He, Y., Cui, L., Yang, Z., 2011. Sarcocystis sinensis is an ultrastructurally distinct parasite of water buffalo that can cause foodborne illness but cannot complete its life-cycle in human beings. Vet. Parasitol. 178, 35–39. Claveria, F.G., San-Pedro, L.R., Cruz-Flores, M.J., Nagasawa, H., Suzuki, N., 2001. Ultrastructural studies of Sarcocystis cruzi (Hasselmann, 1926) Wenyon, 1926 infection in cattle (Bos taurus): Philippine cases. Parasites Vectors 251–254. Dubey, J.P., 1982. Development of ox-coyote cycle of Sarcocystis cruzi. J. Protozool. 29, 591–601. Dubey, J.P., 2015a. Foodborne and waterborne zoonotic sarcocystosis. Food Waterborne Parasitol. 1, 2–11. Dubey, J.P., van Wilpe, E., Calero-Bernal, R., Verma, S.K., Fayer, R., 2015. Sarcocystis heydorni, n. sp. (Apicomplexa: Sarcocystidae) with cattle (Bos taurus) and human (Homo sapiens) cycle. Parasitol. Res. 114, 4143–4147. Dubey, J.P., Calero-Bernal, R., Rosenthal, B.M., Speer, C.A., Fayer, R., 2016a. Sarcocystosis of Animals and Humans, 2nd edition. CRC Press, Boca Raton, Florida. Dubey, J.P., More, G., van Wilpe, E., Calero-Bernal, R., Verma, S.K., Schares, G., 2016b. Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from cattle (Bos taurus) and its differentiation from Sarcocystis hominis. J. Eukaryot. Microbiol. 63, 62–68. Fayer, R., Esposito, D.H., Dubey, J.P., 2015. Human infections with Sarcocystis species. Clin. Microbiol. Rev. 28, 295–311. Gjerde, B., Hilali, M., Abbas, I.E., 2016. Molecular differentiation of Sarcocystis buffalonis and Sarcocystis levinei in water buffaloes (Bubalus bubalis) from Sarcocystis hirsuta and Sarcocystis cruzi in cattle (Bos taurus). Parasitol. Res. 115, 2459–2471. Hornok, S., Mester, A., Takacs, N., Baska, F., Majoros, G., Fok, E., Biksi, I., Nemet, Z., Hornyak, A., Janosi, S., Farkas, R., 2015. Sarcocystis-infection of cattle in Hungary. Parasites Vectors 8, 69. Hu, J.J., Wen, T., Chen, X.W., Liu, T.T., Esch, G.W., Huang, S., 2016. Prevalance, morphology, and molecular characterization of Sarcocystis heydorni Sarcocysts from cattle (Bos Taurus) in China. J. Parasitol. 102, 545–548. Hu, J.J., Huang, S., Wen, T., Esch, G.W., Liang, Y., Li, H.L., 2017. Morphology, molecular characteristics, and demonstration of a definitive host for Sarcocystis rommeli from cattle (Bos taurus) in China. J. Parasitol. 103, 471–476.
343