First report of Sarcocystis falcatula in naturally infected Razorbill auks (Alca torda) collected in Tunisian Mediterranean Sea shores.

Sarcocystis spp. are apicomplexan cyst-forming parasites that can infect numerous vertebrates, including birds. Sarcosporidiosis infection was investigated in three muscles (breast, right and left thigh muscle) and one organ (heart) of four Razorbill auks (Alca torda) stranded between November and December 2022 on the shores of the Mediterranean Sea in Nabeul and Bizerte governorates, Northern Tunisia. Two of the four tested A. torda were PCR positive for 18S rRNA Sarcocystis spp. gene. Among the examined 16 muscles/organs, only one breast and one right thigh were Sarcocystis spp. PCR-positive (12.5% ± 8.3, 2/16). Our results showed a relatively high molecular prevalence of Sarcocystis spp. in Razorbill auks (A. torda). Sarcocystis spp. sequence described in the present study (GenBank number: OR516818) showed 99.56-100% identity to Sarcocystis falcatula. In conclusion, our results confirmed the infection of Razorbill auks (A. torda) by S. falcatula. Further research is needed on different migratory seabirds' species in order to identify other Sarcocystis species.

Molecular identification of Sarcocystis species in wild boar (Sus scrofa) and pigs (Sus scrofa domesticus) in Brazil.

Sarcocystis spp. are protozoan parasites that form cysts in the organs and musculature of various animal species. The species Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs and wild boars (Sus scrofa), acting as intermediate hosts, while humans are the definitive host for S. suihominis. To date, there have been no reports of the identification of these coccidian species in Sus scrofa in Brazil. Therefore, in this study, we conducted the first molecular identification of Sarcocystis species using PCR-RFLP and sequencing. A total of 210 samples were analyzed, of this total, 67 tested positive for Sarcocystis spp., representing 31.9% of the total samples assessed. Out of the total positive samples, 55 (82.1%) were identified as S. miescheriana and 8 (11.9%) as S. suihominis, a zoonotic species. Additionally, other species related to bovines, such as S. cruzi and zoonotic S. hominis, were detected in 3.0% of the samples, serving as contaminants in the pork products. The presence of S. suihominis in swine and wild boar samples is concerning due to the zoonotic risk and potential environmental contamination, as humans act as definitive hosts, also for the presence of S. hominis as a bovine contaminant in pork sausages. Furthermore, we confirmed the efficacy of the PCR-RFLP technique as a reliable tool for the identification of Sarcocystis species, demonstrating its potential use in laboratories for molecular diagnosis and rapid identification of these parasites, aiming to protect public health and ensure food safety.

A cyst-forming coccidian with large geographical range infecting forest and commensal rodents: Sarcocystis muricoelognathis sp. nov.

BACKGROUND: The geographic distribution and host-parasite interaction networks of Sarcocystis spp. in small mammals in eastern Asia remain incompletely known. METHODS: Experimental infections, morphological and molecular characterizations were used for discrimination of a new Sarcocystis species isolated from colubrid snakes and small mammals collected in Thailand, Borneo and China. RESULTS: We identified a new species, Sarcocystis muricoelognathis sp. nov., that features a relatively wide geographic distribution and infects both commensal and forest-inhabiting intermediate hosts. Sarcocystis sporocysts collected from rat snakes (Coelognathus radiatus, C. flavolineatus) in Thailand induced development of sarcocysts in experimental SD rats showing a type 10a cyst wall ultrastructure that was identical with those found in Rattus norvegicus from China and the forest rat Maxomys whiteheadi in Borneo. Its cystozoites had equal sizes in all intermediate hosts and locations, while sporocysts and cystozoites were distinct from other Sarcocystis species. Partial 28S rRNA sequences of S. muricoelognathis from M. whiteheadi were largely identical to those from R. norvegicus in China but distinct from newly sequenced Sarcocystis zuoi. The phylogeny of the nuclear 18S rRNA gene placed S. muricoelognathis within the so-called S. zuoi complex, including Sarcocystis attenuati, S. kani, S. scandentiborneensis and S. zuoi, while the latter clustered with the new species. However, the phylogeny of the ITS1-region confirmed the distinction between S. muricoelognathis and S. zuoi. Moreover, all three gene trees suggested that an isolate previously addressed as S. zuoi from Thailand (KU341120) is conspecific with S. muricoelognathis. Partial mitochondrial cox1 sequences of S. muricoelognathis were almost identical with those from other members of the group suggesting a shared, recent ancestry. Additionally, we isolated two partial 28S rRNA Sarcocystis sequences from Low's squirrel Sundasciurus lowii that clustered with those of S. scandentiborneensis from treeshews. CONCLUSIONS: Our results provide strong evidence of broad geographic distributions of rodent-associated Sarcocystis and host shifts between commensal and forest small mammal species, even if the known host associations remain likely only snapshots of the true associations.

Morphological and molecular characterization of Sarcocystis spp. in pigs (Sus scrofa domestica) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study was to identify Sarcocystis spp. in pig muscles from Argentina, by light and transmission electron microscopy (TEM), and molecular studies. Muscles samples from 561 pigs (Sus scrofa domestica) were classified according to the breeding system in: intensive farming (IF, n = 295; animals kept in confinement during most of their productive cycle), or semi-extensive farming (SEF, n = 266; animals bred outdoors, generally family or backyard production). Results showed that 24.8% (139/561) were positive by light microscopy, with a significantly higher prevalence in the SEF (34.6%; 92/266) than the IF pigs (15.9%; 47/295) (p < 0.05). Of the 202 samples analyzed by PCR, 96 were positive (47.5%) for the 18S rRNA (18S ribosomal RNA) fragment. All samples analyzed by the S. suihominis specific coxI (mitochondrial cytochrome c oxidase subunit I) PCR (n = 235; 96 positives by 18S rRNA PCR and 139 positives by light microscopy) were negative. Fourteen individual cysts were positive for the 18S rRNA PCR and sequenced. Consensus sequences obtained from the 18S rRNA fragment PCR ranged from 613 to 880 bp and showed 100% of identity between them and with previously reported S. miescheriana sequences. In all the pig samples analyzed by TEM, cyst wall ultrastructure was compatible with S. miescheriana. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in pigs from Argentina.

Recent insights into the morphology, molecular characterization and tissue localization of the caprine Sarcocystis species infecting domestic goats (Capra hiricus): Sarcocystis moulei, Sarcocystis capracanis, and Sarcocystis hircicanis.

Ninety-seven (64.67%) out of 150 domestic goats (Capra hiricus) carcasses were found to be infected by Sarcocystis moulei, Sarcocystis capracanis, and Sarcocystis hircicanis sarcocysts. Sarcocystis moulei macrosarcocysts were detected in the cardiac, esophageal, skeletal, lingual, and diaphragmatic muscles of seven goats (4.67%) out of the 150 examined animals, whereas the microscopic Sarcocystis species were found in (90/150 = 60%). Two morphotypes of S. moulei were observed. Morphotype (I) macrosarcocysts were large-sized oval, ovoid, spherical, and measured 2-7 mm in length x 2-6 mm in width. Sarcocystis moulei morphotype (II) macrosarcocysts were spindle-shaped, spheroid, sometimes elongated, and measured 1.8-6 x 0.5-2 mm. By TEM, all S. moulei morphotypes were ultrastructurally the same and had a sarcocyst wall that was characterized by highly branched or cauliflower-like villar protrusions (VP) with dumbbell-like structures. The VP interior was packed with well-developed microtubules in longitudinal and cross arrangements. Sarcocystis moulei cyst wall was 3-6 µm thick. Sarcocystis capracanis microsarcocysts detected herein had a cyst wall that ranged from 4-8 µm in thickness. The VP was upright finger-like or cylindrical. The PVM had electron-dense corrugations in the region of the VP. Few amounts of microfilaments were detected inside the cores of VP. Sarcocystis hircicanis had a thinner cyst wall (~1-3 µm) with hairy long VP that ranged from 1 to 7.5 µm in length. Microtubules were missing inside the cores of the VP. The three caprine Sarcocystis species were molecularly characterized on the level of the 18S rRNA, 28S rRNA, and Cox1 genes.

Macroscopic sarcocystosis in a pig carcass from an Italian abattoir.

Different food-safety institutions, including the European Food Safety Authority, encourage monitoring and characterising Sarcocystis spp. in animals and foodstuffs; among meat-producing animals, domestic pigs (Sus scrofa domesticus) can host two different Sarcocystis spp., that is Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis. Herein, we report for the first time the presence of macrocysts of Sarcocystis miescheriana in a domestic pig resulting in carcass condemnation. In North-West Italy, in June 2022 the carcass of a clinically healthy sow was condemned due to the detection of multifocal macroscopic whitish fusiform lesions. Affected muscle samples were submitted to histological and molecular analyses targeting the mtDNA cox1 and 18S rRNA genes. At gross examination and histology, well demarcated, oval or elongated macrocysts up to 8 mm in length characterized by a calcified central core surrounded by fibrosis were detected. The molecular amplification and sequencing of the cox1 mtDNA and 18S rRNA genes revealed the presence of Sarcocystis miescheriana DNA in all sampled macrocysts. Our study provides the first molecularly confirmed case of Sarcocystis miescheriana infection in a domestic pig in Italy. The present report highlights the need to increase data related to the occurrence and the prevalence of Sarcocystis spp. in meat-producing animals, and in wild and domestic pigs in particular, taking into account the zoonotic potential of Sarcocystis suihominis and the possible financial losses related to carcass discard due to macroscopic Sarcocystis spp. cysts.

Description of Sarcocystis platyrhynchosi n. sp. (Apicomplexa: Sarcocystidae) from domestic ducks Anas platyrhynchos (Anseriformes: Anatidae) in China.

BACKGROUND: Limited data are currently available on protozoan parasites of the genus Sarcocystis that infect their avian hosts within the order Anseriformes (waterfowl). To date, no Sarcocystis species has been recorded in ducks in China. METHODS: Leg muscles were sampled from 26 domestic ducks (Anas platyrhynchos) in China in 2021. Morphological characteristics of sarcocysts detected in the muscle tissue were described using light microscopy (LM) and transmission electron microscopy (TEM). Genomic DNA was extracted from single sarcocysts obtained from different ducks, and three genetic markers, 18S ribosomal DNA (18S rDNA), 28S ribosomal DNA (28S rDNA) and mitochondrial (mt) cytochrome oxidase subunit 1 (cox1), were amplified and cloned for sequence analyses. RESULTS: Sarcocysts were observed by LM in only three of the 28 samples (10.7%). These sarcocysts had a thick cyst wall with numerous brush-like villar protrusions (vps) of 3.8-4.3 µm in length (n = 30) on the cyst surface. TEM observation showed that the sarcocysts had lanceolated vps. Each vps narrowed in the stalk and contained a bundle of microtubules that extended into the ground substance. Comparisons of the new sequences with those deposited in GenBank showed that the most similar sequences were those of Sarcocystis halieti in the great cormorant Phalacrocorax carbo and European starling Sturnus vulgaris, and Sarcocystis calchasi in the domestic pigeon (Columba livia) at the 18S rDNA (99.1% identity); Sarcocystis wenzeli from the domestic chicken Gallus gallus at the 28S rDNA (95.9-96.0% identity); and Sarcocystis speeri from the opossum at the mtcox1 (98.2% identity). The new 18S rDNA, 28S rDNA and mitochondrial cox1 sequences shared up to 99.0%, 95.6% and 97.7% identity, respectively, with those of Sarcocystis spp. obtained from Anseriformes avian hosts. Phylogenetic analysis inferred from the sequences of the three genetic markers placed the organism within a group of Sarcocystis spp. obtained from avian or carnivorous intermediate hosts and avian, marsupial or carnivorous definitive hosts. Based on the morphological observation and molecular analyses, the organism found in the Chinese domestic ducks was regarded as a new species and named Sarcocystis platyrhynchosi n. sp. CONCLUSIONS: Based on morphology and sequence analyses, the microcysts diagnosed in the domestic ducks examined in this study were named as a new species. This is the first record of Sarcocystis spp. from waterfowl in China. Sarcocysts of similar morphology occur frequently in different Anseriformes birds, and the relationships among these species need to be further clarified in future studies using more molecular markers.

Sarcocyst Quantification and Viability: Freezing Treatment as an Alternative to Carcass Condemnation.

PURPOSE: The inspection of animal products is important for controlling parasitic zoonoses. Some processes that guarantee food safety to consumers such as carcass condemnation cause economic losses. This study aimed to detect Sarcocystis cysts in cattle hearts obtained from slaughterhouses and to evaluate sarcocyst viability after freezing treatment. METHODS: When myocardial tissues were minced and subjected to fresh examination, sarcocysts were observed in all analyzed tissues resulting in 21.73 cysts/g of tissue. Sarcocyst viability was verified after tissue freezing at 35 ± 2 °C and - 20 ± 2 °C for 0-12 h. After freezing, the tissues were minced, and sarcocysts were collected and stained with Tripan Blue. In addition, cysts were mechanically disrupted to check bradyzoite viability. RESULTS: Cysts and bradyzoites were unviable at - 35 °C for ≥ 3 h and - 20 °C for ≥ 8 h. CONCLUSION: These results suggest freezing treatment as an alternative to condemnation of cattle carcasses contaminated with Sarcocystis spp. Similar studies using freezing treatment with other animals infected by Sarcocystis must be conducted.

Intracranial medulloblastoma as the cause of progressive ataxia in a 6-month-old draft horse cross gelding.

We describe the unique clinical presentation of a central nervous system neoplasm in a 6-month-old draft horse cross gelding. Based on the neurologic examination at admission, neurolocalization was most consistent with a mildly asymmetric cervical, multifocal, or diffuse myelopathy. Mild vestibular involvement also was considered, but no cranial nerve deficits were observed. The gelding was negative for Sarcocystis neurona or Neospora hughesi based on paired serum and cerebrospinal fluid (CSF) samples analyzed, with no evidence of cervical compression based on contrast myelography. The horse was euthanized because of progression of clinical signs. At necropsy, a mass was identified associated with the cerebellum, and histopathology was consistent with medulloblastoma, which has not been reported previously in the horse.

Bovine sarcocystosis: Sarcocystis species, diagnosis, prevalence, economic and public health considerations, and association of Sarcocystis species with eosinophilic myositis in cattle.

Infections by Sarcocystis in cattle are ubiquitous worldwide. There is considerable debate concerning the identity of Sarcocystis spp. in cattle. Proper diagnosis of Sarcocystis spp. is important to assess their economic and public health importance. Currently there are seven named species: Sarcocystis hirsuta, Sarcocystis cruzi, Sarcocystis hominis, Sarcocystis bovifelis, arcocystis heydorni, Sarcocystis bovini and Sarcocystis rommeli. Additionally, there are unnamed Sarcocystis spp. Two species, S. hominis and S. heydorni, are zoonotic. One out of seven species (S. hirsuta, contracted from cats) forms macroscopic cysts which can be visible during carcass inspection. Current molecular characterization is based on DNA extracted from sarcocysts from naturally infected cattle because DNA was not characterized from tissues of experimentally infected cattle or feces of experimentally infected definitive hosts. Sarcocystis cruzi (transmitted via canids) is recognized as the most pathogenic species and it causes abortion, low milk yield, poor body growth, and outbreaks of clinical sarcocystosis and death. Additionally, Sarcocystis infections have been linked to an inflammatory condition of striated muscles termed bovine eosinophilic myositis (BEM). Cattle affected by BEM appear clinically normal. Diagnosis of BEM at slaughter occurs when inspecting the carcass surface, or once the carcass has been divided into prime cuts or quarters. Sex and breed have no apparent influence on prevalence of BEM. The condition evidently occurs with equal frequency in steers, cows, and heifers. Virtually all striated muscles can be affected including skeletal muscles, the muscles of the eye, larynx, and the heart. In the USA, regulations require condemnation of BEM-affected parts, or (in severe cases) the entire carcass. These aesthetic considerations result in economic losses. Cattle experimentally infected with Sarcocystis did not have BEM at slaughter. Here, we review the status of Sarcocystis spp. and BEM in cattle including prevalence, lesions, epidemiology, and association of BEM with different species of Sarcocystis.

Identification of Sarcocystis spp. in wild boars (Sus scrofa) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study is to identify Sarcocystis spp. in wild boar muscles from Argentina by light and transmission electron microscopy and molecular characterization. Muscle samples from diaphragm, tongue, masseter, intercostals, heart, and forelimbs of 240 wild boars were analyzed. Of the animals, 48.3% (116/240) were positive for sarcocysts by light microscopy, whereas 45.8% (110/240) were positive for Sarcocystis spp. by PCR targeting 18S rRNA fragment. These samples were subjected to a specific PCR for S. suihominis coxI gene, 3.6% (4/110) of which were weak positives. Unfortunately, sequence analysis was inconclusive. This could be related to a potentially low S. suihominis cyst load in the samples, or to an incomplete primer matching with the South American S. suihominis sequences. Seventeen individual sarcocysts were positive by PCR for the 18S rRNA fragment, whose sequences showed 99.75-100% identity with each other and with previously reported S. miescheriana sequences. A total of 21 cysts collected from 11 muscle samples and analyzed by TEM presented a cyst wall type compatible with S. miescheriana, and one cyst presented an ultrastructure compatible with S. suihominis. The latter came from a sample that also contained S. miescheriana cysts, indicating that the animal was co-infected. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in wild boars from South America.

Epidemiological aspects of the occurrence of anti-Sarcocystis spp. andanti-Toxoplasma gondii antibodies in dogs in the Northern region of Brazil / Aspectos epidemiológicos da ocorrência de anticorpos anti-Sarcocystisspp. e anti-Toxoplasma gondii em cães da região norte do Brasil

We conducted aseroepidemiological study on the occurrence of anti-Sarcocystisspp. and anti-Toxoplasma gondii antibodies in dogs from family farming properties in the municipality of Ji-Paraná, Rondônia.Blood samples were collected from apparently healthy dogs between September 2012 and November 2013. In total, 181 blood serum samples were analyzed using an indirect immunofluorescence assay, among which 57 (31.49%) and 20 (11.04%) were positive for anti-T. gondii and anti-Sarcocystis spp., respectively. Statistical analyses showed that the type of food fed to the dogs was associated with the occurrence of anti-Sarcocystisspp. antibodies. In contrast, age and access to bovine carcasses were the risk factors for anti-T. gondii.The high occurrence of seropositive dogs for Sarcocystis spp. and T. gondii evidences the wide distribution of these agents in the studied area, possibly due to human and animal exposure to these protozoan species. In addition, anti-T. gondii antibodies were directly proportional to dog age. The increase in the number of positive animals with age was statistically significant. Furthermore, high antibody titers (up to 800) against Sarcocystis spp. in dogs suggest the possibility of recent exposure, in addition to environmental contamination by oocysts/sporocysts eliminated by the feces of these animals.

Conduzimos um estudo soroepidemiológico sobre a ocorrência de anticorpos anti- Sarcocystis spp. e anti-Toxoplasma gondiiem cães de propriedades de agricultura familiar no município de Ji-Paraná, Rondônia. Amostras de sangue foram coletadas de cães aparentemente saudáveis, entre setembro de 2012 e novembro de 2013. Ao todo, foram analisados 181 soros sanguíneos por meio do ensaio de imunofluorescência indireta, sendo positivas 57 (31,49%) e 20 (11,04%) amostras para anticorpos anti-T. gondii e anti-Sarcocystis spp., respectivamente. As análises estatísticas demonstraram que o tipo de alimentação fornecida aos cães esteve associado à ocorrência de anticorpos anti-Sarcocystis spp. Em contraste a idade e o acesso à carcaça bovina foram fatores de risco para a presença de anticorpos anti-T. gondii. A alta ocorrência de cães soropositivos para Sarcocystis spp. e T. gondii evidencia a ampla distribuição desses agentes na área estudada, possivelmente devido à exposição humana e animal a essas espécies de protozoários. Além disso, o resultado dos anticorpos anti-T. gondii relacionados a idade do cão mostraram diferença estatística, com aumento significativo no número de animais positivos com a idade. Além disso, altos títulos de anticorpos (até 800) contra Sarcocystis spp. em cães sugerem a possibilidade de exposição recente, além da contaminação ambiental por oocistos/esporocistos eliminados pelas fezes desses animais.

Molecular detection of Sarcocystis cruzi in three beef carcases with eosinophilic myositis lesions and in unaffected beef from animals in the same herd.

Eosinophilic myositis in bovine striated muscle thought to be caused by a hypersensitivity reaction to the degradation of Sarcocystis tissue cysts, is a rare reason for carcase condemnation in the United Kingdom. This paper describes the identification of Sarcocystis cruzi associated with lesions of generalised eosinophilic myositis in three English beef carcases, by gross and histopathological examination followed by PCR with subsequent sequencing. Samples from two unaffected animals were also examined. Although sarcocystosis caused by S.cruzi is not considered a public health risk, the clinically affected carcases were deemed unfit for human consumption due to the extensive lesions affecting meat quality. We believe this to be the first report from the UK describing the molecular-based identification of Sarcocystis cruzi in meat affected and unaffected with eosinophilic myositis.

Molecular characterization of Sarcocystis spp. in seabirds from southern Brazil.

Sarcocystis spp. are cyst forming apicomplexan parasites that infect many vertebrates including birds. Sarcocystis spp. infection was investigated in tissue samples (pectoral muscles, heart, and brain) of 47 dead seabirds collected from the coastline of Santa Catarina State SC - Brazil, between August 2019 and March 2020. A portion of each tissue was fixed in 10% buffered formalin for histopathologic analysis while DNA was extracted from another portion and screened using nested-PCR targeting ITS1. Based on molecular analysis, Sarcocystis spp. were identified in 15/47 (31.9%) seabirds of five species, kelp gull (Larus dominicanus), manx shearwater (Puffinus puffinus), neotropic cormorant (Phalacrocorax brasilianus), brown booby (Sula leucogaster) and great skua (Stercorarius skua). Microscopically visible sarcocysts were observed only in the pectoral muscle of four seabirds 8.5% (4/47), while in one brown booby, sarcocysts were seen in both pectoral and cardiac muscles. Two types of sarcocysts, thin walled (≤1 µm) and thick-walled (≥ 2 µm) were identified. Based on ITS1 sequence comparison, S. halieti, S. falcatula and three not yet described Sarcocystis spp. were detected. Phylogenetically, S. falcatula isolates were classified as two distinct clusters. This is the first confirmation of S. halieti in seabird's species in South America and S. falcatula in birds of the order Charadriiformes. Further molecular studies are needed to understand the epidemiology of the Sarcocystis spp. infection and its impact on the health of seabirds.

Investigations on Sarcocystis species in the leg muscles of the bird family Corvidae in Lithuania.

Although three species of Sarcocystis, S. cornixi, S. corvusi and S. kutkienae, have been described in corvids, molecular studies of sarcocysts isolated from these birds are incomplete. Leg muscles of 83 corvids, 35 hooded crows (Corvus cornix), 21 western jackdaws (Coloeus monedula), 11 rooks (Corvus frugilegus), 9 common ravens (Corvus corax), 4 common magpies (Pica pica) and 3 Eurasian jays (Garrulus glandarius), from Lithuania were examined for the presence of Sarcocystis spp. in the present study. In methylene blue-stained squashed samples, sarcocysts were detected in 26 birds (31.0%). Under a light microscope, two morphological types of sarcocysts were distinguished (type A and type B). Sarcocysts of type A had a smooth and thin (about 1 µm) cyst wall, while cysts of type B were characterised by a thicker (1.4-2.5 µm) cyst wall. Based on ITS1 sequence comparison, sarcocysts of type A were identified as S. halieti and Sarcocystis sp. ex Corvus corax, whereas cysts of type B belonged to S. kutkienae and S. cornixi. Furthermore, it was demonstrated that a single bird could host two different Sarcocystis spp. Sarcocystis halieti was detected in corvids for the first time in the common raven and the hooded crow. Also, this study presents the first evidence of S. kutkienae in the hooded crow and the common magpie, and S. cornixi in the western jackdaw. Sarcocystis sp. ex Corvus corax was genetically characterised using almost complete 18S rDNA, partial 28S rDNA and complete ITS1 sequences. Sarcocystis sp. ex Corvus corax clustered together with S. columbae, S. corvusi and S. halieti in phylogenetic trees reconstructed using 28S rDNA and ITS1 sequences.

Infection of the Asian gray shrew Crocidura attenuata (Insectivora: Soricidae) with Sarcocystis attenuati n. sp. (Apicomplexa: Sarcocystidae) in China.

BACKGROUND: Data on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae in South Asia and Southeast Asia. To our knowledge, species of Sarcocystis have never been recorded previously in this host. METHODS: Tissues were obtained from 42 Asian gray shrews caught in 2017 and 2018 in China. Sarcocysts were observed using light microscopy (LM) and transmission electron microscopy (TEM). To describe the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes Elaphe taeniura. Individual sarcocysts from different Asian gray shrews, and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes, were selected for DNA extraction, and seven genetic markers, namely, two nuclear loci [18S ribosomal DNA (18S rDNA) and internal transcribed spacer region 1 (ITS1)], three mitochondrial genes [cytochrome oxidase subunit 1 (cox1), cox3 and cytochrome b], and two apicoplast genes (RNA polymerase beta subunit and caseinolytic protease C), were amplified, sequenced and analyzed. RESULTS: Sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts showed saw- or tooth-like protrusions measuring 3.3-4.5 µm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to those described for type 9h of the common cyst wall classification. The experimental beauty rat snakes shed oocysts/sporocysts measuring 11.9-16.7 × 9.2-10.6 µm with a prepatent period of 10-11 days. Comparison of the newly obtained sequences with those previously deposited in GenBank revealed that those of 18S rDNA and cox1 were most similar to those of Sarcocystis scandentiborneensis recorded in the tree shrews Tupaia minor and Tupaia tana (i.e., 97.6-98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA or ITS1 sequences placed this parasite close to Sarcocystis spp. that utilize small animals as intermediate hosts and snakes as the known or presumed definitive host. On the basis of morphological and molecular characteristics and host specificity, the parasite was proposed as a new species, named Sarcocystis attenuati. CONCLUSIONS: Sarcocysts were recorded in Asian gray shrews, to our knowledge for the first time. Based on morphological and molecular characterization, a new species of parasite is proposed: Sarcocystis attenuati. According to the LM and TEM results, S. attenuati sarcocysts are distinct from those of Sarcocystis spp. in other insectivores and those of S. scandentiborneensis in tree shrews. The 18S rDNA or cox1 sequences of Sarcocystis attenuati shared high similarity with those of Sarcocystis scandentiborneensis, Sarcocystis zuoi, Sarcocystis cf. zuoi in the Malayan field rat, and Sarcocystis sp. in the greater white-toothed shrew. Therefore, we suggest that more research on the relationships of these closely related taxa should be undertaken in the future.

Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages / Soroprevalência de Sarcocystis spp. e Toxoplasma gondii em suínos e detecção do DNA desses protozoários em tecidos e embutidos

Abstract The seroprevalence of Sarcocystis spp. and Toxoplasma gondii was researched in swine raised in Santa Maria, RS, Brazil. Serum samples from 84 pigs from 31 farms were tested using indirect immunofluorescence assay (IFA) for both agents. Additionally, 53 samples of pork sausages and tissues destined for human consumption, including: salami, sausage, black pudding, heart, tongue, brain, and rib muscle, were submitted to PCR to detect DNA for each agent. The frequency of anti-Sarcocystis spp. antibodies was 36.9% (31/84), with titers ranging from 32 to 1024, and 25% (21/84) for anti-T. gondii antibodies, with titers ranging from 64 to 2048. Sarcocystis spp. and T. gondii DNA were detected in 67.9% (36/53) and 13.2% (7/53) of samples, respectively. The presence of antibodies and the detection of DNA from Sarcocystis spp., and T. gondii suggests that the pigs were infected and may serve as an important reservoir for both parasites. The infection by these protozoa in the swine population is relevant to public health due to their zoonotic potential.

Resumo A soroprevalência de Sarcocystis spp. e Toxoplasma gondii foi pesquisada em suínos criados em Santa Maria, RS, Brasil. Amostras de soro de 84 suínos de 31 fazendas foram testadas pela reação deimunofluorescência indireta (IFA) para ambos os agentes. Adicionalmente, 53 amostras de embutidos suínos e tecidos cárneos destinados ao consumo humano, incluindo: salame, linguiça, morcela, coração, língua, cérebro e músculo da costela foram submetidas à PCR para detecção de DNA para cada agente. A frequência de anticorpos anti-Sarcocystis spp. foi de 36,9% (31/84), com títulos variando de 32 a 1.024; e 25% (21/84) para anticorpos anti-T. gondii, com títulos variando de 64 a 2048. A presença de DNA de Sarcocystis spp. e T. gondii foi detectada em 67,9% (36/53) e 13,2% (7/53) das amostras avaliadas, respectivamente. A detecção de anticorpos e DNA de Sarcocystis spp. e T. gondii sugere que os suínos foram infectados e podem servir como um importante reservatório de ambos os parasitas. A circulação desses agentes na população suína é relevante para a saúde pública devido ao seu potencial zoonótico.

Sarcocystis calchasi in a captive Patagonian conure (Cyanoliseus patagonus) in Finland.

Sarcosystis calchasi is an emerging pathogen causing encephalitis in many avian species and has been documented in North America, Germany and Japan. In November 2019, a captive Patagonian conure (Cyanoliseus patagonus), kept in a zoological aviary in Finland, was euthanized due to acute respiratory distress. At necropsy, histopathological examination revealed numerous parasitic tissue cysts in the skeletal muscles and myocardium, chronic moderate multifocal lymphoplasmacytic and histiocytic meningoencephalitis and acute moderate multifocal purulent pneumonia caused by aspiration of foreign material. By light and transmission electron microscopy, tissue cysts had structures typical of Sarcocystis organisms. The ultrastructure of the cyst wall was compatible with S. calchasi and Sarcocystis columbae. S. calchasi-specific semi-nested polymerase chain reaction testing resulted in amplification of the internal transcribed spacer (ITS) gene, which had 100% identity with S. calchasi ITS sequences. This is the first report of S. calchasi in Fennoscandia and of a naturally-occurring S. calchasi infection in a captive psittacine bird in Europe. Our finding suggests that captive psittacine birds kept in outdoor facilities may be at risk of S. calchasi infection throughout the Holarctic.

Equine Protozoal Myeloencephalitis associated with Neospora caninum in a USA captive bred zebra (Equus zebra).

A 6-year-old female captive zebra (Equus zebra) had a three-year history of slow progressive neurologic signs that recently worsened with hind limb ataxia, head tilt, and circling. Gross examination including the brain and spinal cord were unremarkable. On histopathology, the brain and brainstem had multiple random areas of severe lymphoplasmacytic meningoencephalitis associated with numerous 15-25 µm in diameter protozoal cysts with a discernible outer wall containing numerous 2 × 4 µm oval to crescent-shaped organisms. Immunohistochemistry and PCR identified the presence of Neospora organisms associated with the lesions. Equine protozoal myeloencephalitis (EPM) is generally associated with Sarcocystis neurona or less commonly Neospora hughesi. Molecular characterization revealed the first case of EPM associated with Neospora caninum in an equid as confirmed by DNA analysis.

Clinical signs, treatment, and outcome for California sea lions (Zalophus californianus) with Sarcocystis-associated polyphasic rhabdomyositis.

OBJECTIVE: To describe clinical signs, treatment, and outcome for California sea lions (Zalophus californianus) with Sarcocystis-associated polyphasic rhabdomyositis. ANIMALS: 38 free-ranging juvenile to adult California sea lions examined at a rehabilitation center in California between September 2015 and December 2017. PROCEDURES: Medical records at The Marine Mammal Center were reviewed to identify sea lions in which sarcocystosis had been diagnosed. RESULTS: Clinical signs were highly variable and associated with polyphasic rhabdomyositis attributed to Sarcocystis neurona infection. Generalized severe muscle wasting, respiratory compromise, and regurgitation secondary to megaesophagus were the most profound clinical findings. Respiratory compromise and megaesophagus were associated with a poor prognosis. Eight of the 38 sea lions were treated and released to the wild, and 2 subsequently restranded and were euthanized. Two additional animals received no targeted treatment and were released. The remaining 28 animals were either euthanized or died during treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that unlike other marine mammals, which typically develop encephalitis, California sea lions with sarcocystosis often have polyphasic rhabdomyositis with highly variable clinical signs and that extensive diagnostic testing may be required to confirm the diagnosis. Treatment with an antiprotozoal drug in combination with corticosteroids may resolve clinical disease, but the prognosis is guarded.