First Report of Sarcocystis Masoni in a Captive Alpaca (Vicugna Pacos) From China

Background: Sarcocystosis is a parasitic disease caused by intracellular protozoan parasite of the genus Sarcocystis. Tissue samples of alpacas (n = 4) from Henan province (China) were screened for Sarcocystis spp. infection by histological examination, pepsin digestion, and molecular assays. Results: Sarcocystis spp. was detected in heart, liver, spleen, lung, and kidney of an alpaca by molecular assays. Many sarcocysts with inflammation responses were observed in this alpaca myocardium, and they showed a high similarity to Sarcocystis masoni by sequence analysis. Conclusion: This study is the first to demonstrate Sarcocystis spp. infection in alpaca from China. The higher parasite load in the alpaca myocardium indicated that it had contact with an environment contaminated with sporocysts, and that the alpaca was susceptible to Sarcocystis spp.


INTRODUCTION
Sarcocystosis is a parasitic disease caused by intracellular protozoan parasite of the genus Sarcocystis. Its hosts include intermediate and definitive hosts. Intermediate hosts (birds, mammals, reptiles) are infected by ingesting sporozoites shed by a definitive host (typically carnivores) (1). More than 200 recognized Sarcocystis species worldwide can infect domestic and wild animals, causing a significant adverse impact on food health and economic development (1,2).
Most animals infected with Sarcocystis spp. have no clinical symptoms, except in the cases of high doses of infection or immunodeficiency of the host, which result in anorexia, fever, anemia, weakness, weight loss, neurological symptoms, diarrhea, and death (6,7,11). Gastroenteritis, nausea, diarrhea, colic, shivering, and breathing problems can occur in humans after eating raw or uncooked meat that contains some species of Sarcocystis, for example S. hommins or S. suihominnis (1). In this article, S. masoni was detected in the myocardium of an alpaca. This is the first report of a Sarcocystis spp. infection in alpaca from China.

Naturally Infected Alpacas and Sampling
Four dead alpacas (Vicugna pacos) from a zoo were collected in Zhengzhou city, Henan province (33 • N, 113.30 • E), China. Both of them were born in this zoo.
Case 1 was a 4-year-old female, breastfeeding a 3-month-old pup, displaying skin phyma and dyspnea, which suddenly died on June 20, 2020. Case 2 was an adult female with a systemic abscess and a stillborn fetus 1 month before death, which then showed neurological signs and died on August 6, 2020. Case 3 was a 1-year-old male with dyspnea and fever and died on June 4, 2021. Case 4 was an adult male, also displaying dyspnea and died on June 7, 2021. Treatments included azithromycin and multivitamins, which alleviated their clinical symptoms. However, the animals ultimately died. The tissues of case 1 (spleen, liver, lung, heart, kidney, or skin phyma), case 2 (kidney and brain), case 3 (blood, spleen, liver, lung, heart, kidney, and lymphonodus), and case 4 (blood, spleen, liver, lung, heart, kidney, intestine, and lymphonodus) were sent to the pathology laboratory of Henan Agricultural University for pathological diagnosis. These samples were also screened for T. gondii and Sarcocystis spp. infection. However, they all showed negative results for T. gondii by histological examination, pepsin digestion, and molecular assays. By serological assays, only case 3 showed anti-T. gondii antibody (modified agglutination test: 1:50 titer).

Light Microscopy Examination
The squashed myocardium sections of alpacas were directly examined for Sarcocystis spp. If sarcocysts were found, single cyst was separated from the slide. Three tissue pieces from each sample were processed using conventional paraffin histological methods, and 5-µm-thick sections were prepared and stained with hematoxylin and eosin. The tissue sections were examined for the presence of Sarcocystis spp. under a light microscope (Olympus Bx43, Tokyo, Japan).

Pepsin Digestion Examination
Myocardium (50 g) samples were homogenized and digested in acidic pepsin individually (12). The digested tissues were examined for Sarcocystis bradyzoites and cysts by light microscopy.

Molecular Analysis of Sarcocystis
DNA was extracted from the heart, liver, spleen, lung, kidney, brain, lymphonodus, intestine, skin phyma, myocardium digestive juices, and single cyst from the alpacas using a commercial DNA extraction kit (DP304; Tiangen Biotec Company, Beijing, China). PCR amplification of a 900-bp 18S rDNA segment was performed using the primer pair SarcoFext (GGTGATTCATAGTAACCGAACG) and SarcoRext (GATTTCTCATAAGGTGCAGGAG) to check the Sarcocystis spp. infection in alpacas (13). In addition, PCR was performed to amplify a segment of the mitochondrial cytochrome c oxidase subunit 1 gene (cox 1) using the specific primer pairs of SF1 (ATGGCGTACAACAATCATAAAGAA) and SR9 (ATATCCATACCRCCATTGCCCAT). The amplified PCR product was approximately 1038 bp (14,15), and both negative and positive controls were included. The PCR products were sent to Shanghai Sangon Company (China) for bi-directional sequencing on an ABI PRISM TM 3730 XL DNA Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The obtained sequences were analyzed by BLAST (GenBank database). The phylogenetic tree of the Sarcocystis spp. was constructed using the neighbor-joining method (Kimura two-parameter model).

Pathological Findings
No visible macroscopic sarcocyst was found in the tissues of four alpacas. Acute interstitial pneumonia, septicemia of the spleen, necrosis of hepatocytes, lipofuscin deposition, renal interstitial hemorrhage, acute glomerulonephritis, a large area of congestion in the lungs, and a large number of inflammatory cell infiltration in the myocardium were observed in case 1. Many fusiform or oval sarcocysts were observed in the myocardium and Purkinje fibers. An obvious inflammatory response (lymphocytes, macrophages, and plasma cell infiltration) was observed in the myocardium. Granulation tissue was observed in skin phyma. Pathological results showed that the infection of the Sarcocystis spp. is not the direct cause of the death of this alpaca, but respiratory failure due to acute interstitial pneumonia (Figure 1).
For case 2, tissues (kidney and brain) were stored at −20 • C for 2 weeks before being sent to the pathology laboratory of Henan Agricultural University. Thus, detailed pathological information could not be obtained. Both case 3 and case 4 showed acute interstitial pneumonia.

PCR Identification of Sarcocystis spp. Infection
DNA from the samples of heart, liver, spleen, lung, kidney, single cysts, and pepsin-digested myocardium of case 1 had amplified fragments of approximately 900 bp by primers of 18S rRNA (Supplementary Figure). By primers of cox1, except kidney, fragments of 1,038 bp was also amplified from these samples (Supplementary Figure), indicating the presence of Sarcocystis spp. The DNA from the samples of case 2, case 3, and case 4 was negative for Sarcocystis spp. Sequence analysis was performed on the PCR products from heart, liver, spleen, single cyst, and digestive fluid of case 1. The nucleotide sequences from the sarcocysts of the alpaca were submitted to GenBank (for 18S rRNA: accession number MW481703, MW481704, for cox 1: accession number PRJNA759055). BLAST results showed that the most similar sequences for MW481703, MW481704 in GenBank were S. masoni KU527113.1, KU527112.1, KU527111.1, KU527110.1, KU527109.1, and KU527108.1 (100% identity) (Figure 2). PRJNA759055 sequence had similarity to S. rommeli hosted by cattle, accession numbers KY120286.1, with only 87% pairwise identity.

DISCUSSION
Sarcocystis infection is common in many species of animals worldwide, including China. Sarcocysts could be found in skeletal muscles and myocardium (1,16), smooth muscles, and the central nervous system (1,17). They have an adverse effect on the livestock industry, food health, and animal reproduction. However, there is no vaccine to protect animals against clinical sarcocystosis.
Studies have reported antibodies against T. gondii in 30% and 44% of llamas from Peru and the USA (18)(19)(20) and in 9% of alpacas from China (21). In this study, antibodies to T. gondii (modified agglutination test: 1:50 titer) were found in case 3 alpaca.
Alpacas are raised on a large scale in South America, Africa, Australia, Europe and the United States. Their meat and wool have economic value (10,22,23). Macroscopic (S. aucheniae) and microscopic (S. masoni) sarcocysts were identified in alpacas. Canids are potential definitive hosts (1,5). S. aucheniae appeared in skeletal muscles, and S. masoni were commonly found in the cardiac muscle of alpaca. However, little is known of clinical sarcocystosis in alpaca. Severe myositis was reported in an alpaca (6). Human consumption of Sarcocystis-infected meat (raw or undercooked beef or pork) may lead to gastroenteritis and diarrhea (1,22).
Compared to 18S rRNA, cox 1 was found better in differentiation of closely related but morphologically indistinguishable Sarcocystis spp. from cattle (24,25). In this study, BLAST results showed that the most similar sequences for Sarcocystis spp. from alpaca were S. masoni (100% identity) by 18S rRNA and S. rommeli (87% identity) by cox 1 gene. Further, recent research found that cox 1 gene from camels in Saudi Arabia showed low similarity to two Sarcocystis spp. (S. aucheniae or S. masoni) from camels and suggests identification of Sarcocystis spp. with more taxa and different molecular markers, including 18S rRNA and cox 1 (15). Mohamed et al. (26) also suggested using the hybrid approach to characterize the microbe, including Next-Generation Sequencing and conventional PCR combined with Sanger sequencing, and also using RFLP-PCR for characterization of Sarcocystis isolated from camel (27).
Alpacas in China are mainly imported from Australia. Then, most of them are bred in zoos, while some are kept at home as pets. In this study, all the four cases of alpacas were bred in zoo. According to the sarcocyst's morphology, location, host, and molecular characteristics, the sarcocysts in cardiac muscle of alpaca maybe S. masoni. S. masoni nucleotides (18S rRNA) were amplified in the heart, liver, spleen, lung, and kidney of the case 1 alpaca (Supplementary Figure). This indicated that except for the striated muscles, S. masoni could grow or exist in other tissues, providing molecular evidence of the S. masoni infection in other tissues. However, the life cycle of S. masoni in the intermediate host and definitive host is unknown.
Both case 1 and case 2 alpacas showed skin granulation tissue or systemic abscess. These findings agree with a previous report of an alpaca with a case history of multiple subcutaneous abscesses (6). More investigation is needed to confirm whether sarcocystosis is associated with multiple abscesses in alpacas.
Although parasite loads from different hosts are not comparable. The average myocardial sarcocyst load in alpaca was 79.00 per square centimeter, which is much higher than in beef (1.40 cysts/cm 2 ) and feral pigs (3.03 cysts/cm 2 ) from the United States and mutton (7.84 cysts/cm 2 ), sheep (18.07 cysts/cm 2 ), and cattle (7.1 cysts/cm 2 ) from China (28)(29)(30)(31)(32). In this study, the higher parasite load in the alpaca indicated that it had contact with an environment contaminated with sporocysts, and that the alpaca was susceptible to S. masoni. Poor sanitation and contact with dogs are considered risk factors for Sarcocystis spp. infection in alpacas (10). For alpaca meat, boiling (100 • C for 10 min), baking (105 • C for 65 min), and freezing (−20 • C for 10 days) could inactivate the Sarcocystis spp. and reduce its risk for other animals (33). This work is the first report of Sarcocystis spp. infection in alpaca from China. It is necessary to further explore the natural clinical cases of sarcocystosis to determine the infection source and the definitive host.

CONCLUSION
This study is the first to demonstrate Sarcocystis spp. infection in alpaca from China. The higher parasite load in the alpaca myocardium indicated that it had contact with an environment contaminated with sporocysts, and that the alpaca was susceptible to Sarcocystis spp. Further study will focus the possible route of infection of these animals, ultrastructure of the cysts (walls and protrusions), and molecular characteristics with more markers of these parasites.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/genbank/, MW481703, MW481704.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Use Protocol Committee of the Henan Agricultural University, China.