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.     

Ultrastructure of the cyst wall of Sarcocystis spp. from some rodents in Malaysia.

The cyst wall of Sarcocystis cysts from the skeletal muscles and subcutaneous tissues of 4 species of rats and 1 species of bandicoot in Malaysia was examined under the electron microscope. Three types of sarcosysts with morphologically distinct cyst walls were found in these rodents. These morphologically distinct types of sarcocysts occurred as single or mixed infections in their various rodent hosts, suggesting that these rodents are important, though non-specific, intermediate hosts in the life cycle of at least 3 species of Sarcocystis. The final hosts of these Sarcocystis species are unknown.     

Molecular identification of two Sarcocystis species in fallow deer (Dama dama) from Lithuania

Due to the lack of molecular research conducted, little is known about Sarcocystis species diversity in the fallow deer (Dama dama). Until now, Sarcocystis jorrini and Sarcocystis morae were described to form sarcocysts in the muscles of this host. In the present study diaphragm muscle samples of free-ranging fallow deer from Lithuania were investigated for Sarcocystis species. Sarcocysts were detected in 39 out of 48 (81.3%) fallow deer examined. Under a light microscope two types of sarcocysts having hair-like and finger-like protrusions were observed. Based on DNA sequence analysis of cox1 and 18S rDNA, two species, S. morae and Sarcocystis entzerothi were identified. In prior studies, the latter species was only detected in Lithuanian roe deer (Capreolus capreolus) and in sika deer (Cervus nippon). The haplotype network of S. morae sequences specified close relationships between haplotypes found in the same country. According to current knowledge, the fallow deer is characterised by low Sarcocystis species richness as compared with other cervid species from Europe.     

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.     

Sarcocystis eothenomysi n. sp. (Apicomplexa: Sarcocystidae) from the large oriental vole Eothenomys miletus (Thomas) (Cricetidae: Microtinae) from Anning,China

Fifty-six oriental voles, Eothenomys miletus (Thomas), were collected in Anning prefecture of Yunnan Province (China) between March 2012 and December 2013 and examined for the presence of sarcocysts. Sarcosysts of a new species, Sarcocystis eothenomysi n. sp., were found in 14 out of 56 E. miletus (25%); they possessed a striated cyst wall, c.1–2 μm thick. Under transmission electron microscopy the cysts of S. eothenomysi exhibited numerous small, irregular protrusions, which may appear T-shaped in some sections. A phylogenetic analysis based on 18S rRNA gene sequences indicated that S. eothenomysi shares closest affinity with those species of Sarcocystis Lankester, 1982, which use cobra or viperid snakes as definitive hosts. We therefore, hypothesise that a venomous snake may serve as the definitive host for S. eothenomysi. This is the first species of Sarcocystis reported from Eothenomys spp.     

Isolation and characterization of microsatellite markers from Sarcocystis neurona,a causative agent of equine protozoal myeloencephalitis

The population genetics and systematics of coccidian parasites of the genus Sarcocystis remain poorly defined, notwithstanding their relevency to veterinary and human health. Despite opportunities for sexual recombination, nonrecombinant parasite clones characterized by distinct transmission and pathogenesis traits persist in related parasites (i.e. Toxoplasma gondii). In order to determine whether this may be generally true for parasitic coccidia, and to address evolutionary and taxonomic problems within the genus Sarcocystis, we isolated 12 polymorphic microsatellite markers (four to 14 alleles) for Sarcocystis neurona, the major causative agent of equine protozoal myeloencephalitis (EPM).     

Molecular identification targeting cox1 and 18S genes confirms the high prevalence of Sarcocystis spp. in cattle in the Netherlands

The reported prevalence of Sarcocystis infection in cattle in Europe ranges between 66 and 94%. Although in the Netherlands a prevalence of 100% was reported in 1993, this study aimed to develop a method for sensitive and specific molecular detection and species identification of Sarcocystis spp., in order to provide more recent data on the prevalence and identification of these protozoa in cattle meat intended for human consumption in the Netherlands. For this purpose, 104 cattle samples were obtained from Dutch slaughterhouses. Genomic DNA was extracted, and analysed by 18S and cox1 PCR. Magnetic capture was used to extract and amplify 18S-specific DNA. Sarcocystis DNA was detected in 82.7% of the samples. PCR amplicons of both targets were sequenced, and sequence identities of ≥97% were observed for Sarcocystis cruzi (65.4%), Sarcocystis hominis (12.5%), Sarcocystis bovifelis (8.7%), Sarcocystis hirsuta and Sarcocystis heydorni (both 1.0%). Mixed infections were observed in 17.3% of the samples. The magnetic capture was not significantly more sensitive compared with standard DNA extraction, but magnetic capture did add to the overall sensitivity. Using cox1 sequencing, all species are clearly distinguished, whereas for 18S the variation between species is limited, which particularly hampers reliable identification of thick walled Sarcocystis spp. Furthermore, the detection of 12.5% S. hominis and 1% S. heydorni points towards an established transmission route between cattle and humans in the Netherlands. The availability of four additional well-identified and well-referenced S. hominis cox1 sequences in public databases enables development of species-specific diagnostic PCRs targeting cox1, which in combination with magnetic capture could provide the means to determine the prevalence of human sarcocystosis.     

Faecalibacterium prausnitzii Strain HTF-F and Its Extracellular Polymeric Matrix Attenuate Clinical Parameters in DSS-Induced Colitis

A decrease in the abundance and biodiversity of intestinal bacteria within the Firmicutes phylum has been associated with inflammatory bowel disease (IBD). In particular, the anti-inflammatory bacterium Faecalibacterium prausnitzii, member of the Firmicutes phylum and one of the most abundant species in healthy human colon, is underrepresented in the microbiota of IBD patients. The aim of this study was to investigate the immunomodulatory properties of F. prausnitzii strain A2-165, the biofilm forming strain HTF-F and the extracellular polymeric matrix (EPM) isolated from strain HTF-F. For this purpose, the immunomodulatory properties of the F. prausnitzii strains and the EPM were studied in vitro using human monocyte-derived dendritic cells. Then, the capacity of the F. prausnitzii strains and the EPM of HTF-F to suppress inflammation was assessed in vivo in the mouse dextran sodium sulphate (DSS) colitis model. The F. prausnitzii strains and the EPM had anti-inflammatory effects on the clinical parameters measured in the DSS model but with different efficacy. The immunomodulatory effects of the EPM were mediated through the TLR2-dependent modulation of IL-12 and IL-10 cytokine production in antigen presenting cells, suggesting that it contributes to the anti-inflammatory potency of F. prausnitzii HTF-F. The results show that F. prausnitzii HTF-F and its EPM may have a therapeutic use in IBD.     

Infection survey and morphological characteristics of Sarcocystis spp. in naturally infected Tibetan sheep from Qinghai in northwestern China

Sarcocystosis is a parasitic disease caused by intracellular coccidian protozoans that belong to the genus Sarcocystis. These parasites can cause diseases of the nervous system, abortion and economically significant losses in host animals. Previous studies have reported that Sarcocystis is found in mammals, birds and reptiles, while molecular and morphological studies of infected Tibetan sheep have not been performed in the Qinghai region. The aim of this study was to determine the prevalence of Sarcocystis spp. in Tibetan sheep in Qinghai, northwestern China. The results showed that in 1155 samples, sarcocysts from unspecified species were found in 50% (577/1155) of the sheep tissues by microscopy detection. The positive rates of sarcocysts in the diaphragmatic, esophageal and cardiac muscles were 78.4% (175/223), 29.1% (207/711), and 88.2% (195/221), respectively. Ultrastructural features were exclusively observed in Sarcocystis gigantea in the esophageal tissues. The specific architecture was characterized as a space between the two layers of the original capsule wall, which was filled with fiber bundles and tissue fluid. Cauliflower-like protrusions of the original capsule wall were observed toward the outer surface of the capsule. Prominent protrusions contained fibers and matrix. In addition, the Sarcocystis 18S rRNA genes from 6 esophageal tissue samples were cloned, sequenced, and aligned to related sequences from GenBank. All 5 S. gigantea sequences examined in this study were grouped into the same cluster and belonged to the same genotype. The other 5 Sarcocystis tenella sequences were obtained from cardiac muscle and diaphragm muscle and belonged to the same clade. Overall, this study revealed a high infection rate of Sarcocystis in Tibetan sheep in the region. The results of this study may provide a reference for further research investigating the sarcocystosis epidemic in Qinghai, China.     

High prevalence of Sarcocystis spp. infections in cattle (Bos taurus) from central China

Myocardium and diaphragm samples of cattle (n?=?521) from HeNan Province (China) were screened for Sarcocystis sarcocysts by histological examination, pepsin digestion, and molecular assays. Morphology and molecular assays were used for identification. The prevalence of Sarcocystis infection in cattle was 41.5% (216/521). Histological examination identified sarcocysts in the myocardium (49.4%, 200/405) and diaphragm (13.8%, 16/116) of cattle. Two species were identified, namely S. cruzi (41.3%, 215/521) and S. hominis (0.2%, 1/521). The findings of the present study indicate a high prevalence of S. cruzi infection in cattle from central China.     

Prevalence of Sarcocystis in Grackles in Maryland

SYNOPSIS. One hundred and fifty-nine grackles (Quiscalus quiscula) in Maryland were examined grossly and microscopically for Sarcocystis sp. Of 98 mature birds, 91 had Sarcocystis. None of 51 juvenile birds and none of 10 birds hatched in the laboratory had Sarcocystis. More mature females had Sarcocystis than did mature males. However, the number of intramuscular cysts (per bird) was greater in male grackles than in females. Both male and female grackles had more infections in the leg and thigh muscles than in breast muscles. No tongue or heart muscles contained Sarcocystis. No seasonal fluctuation in the number of infected birds was apparent.     

Epimorphin Regulates Bile Duct Formation via Effects on Mitosis Orientation in Rat Liver Epithelial Stem-Like Cells

Understanding how hepatic precursor cells can generate differentiated bile ducts is crucial for studies on epithelial morphogenesis and for development of cell therapies for hepatobiliary diseases. Epimorphin (EPM) is a key morphogen for duct morphogenesis in various epithelial organs. The role of EPM in bile duct formation (DF) from hepatic precursor cells, however, is not known. To address this issue, we used WB-F344 rat epithelial stem-like cells as model for bile duct formation. A micropattern and a uniaxial static stretch device was used to investigate the effects of EPM and stress fiber bundles on the mitosis orientation (MO) of WB cells. Immunohistochemistry of liver tissue sections demonstrated high EPM expression around bile ducts in vivo. In vitro, recombinant EPM selectively induced DF through upregulation of CK19 expression and suppression of HNF3α and HNF6, with no effects on other hepatocytic genes investigated. Our data provide evidence that EPM guides MO of WB-F344 cells via effects on stress fiber bundles and focal adhesion assembly, as supported by blockade EPM, β1 integrin, and F-actin assembly. These blockers can also inhibit EPM-induced DF. These results demonstrate a new biophysical action of EPM in bile duct formation, during which determination of MO plays a crucial role.     

Endospore surface properties of commonly used Bacillus anthracis surrogates vary in aqueous solution

The hydrophobic character and electrophoretic mobility (EPM) of microorganisms are vital aspects of understanding their interactions with the environment. These properties are fundamental in fate-and-transport, physiological, and virulence studies, and thus integral in surrogate selection. Hydrophobic and electrostatic forces are significant contributors to particle and microorganism mobility in the environment. Herein, the surface properties of commonly used Bacillus anthracis surrogate endospores were tested under comparable conditions with respect to culture, endospore purification, buffer type and strength. Additionally, data is presented of endospores suspended in dechlorinated tap water to evaluate the surrogates in regard to a breach of water infrastructure security. The surface properties of B. anthracis were found to be the most hydrophobic and least electronegative among the six Bacillus species tested across buffer strength. The effect of EPM on hydrophobicity varies in a species-specific manner. This study demonstrates that surrogate surface properties differ and care must be taken when choosing the most suitable surrogate. Moreover, it is shown that Bacillus thuringensis best represents Bacillus anthracis-Sterne with respect to both EPM and hydrophobicity across all test buffers.     

Pythons,parasites, and pests: anthropogenic impacts on Sarcocystis (Sarcocystidae) transmission in a multi‐host system

Parasites are essential components of ecosystems and can be instrumental in maintaining host diversity and populations; however, their role in trophic interactions has often been overlooked. Three apicomplexan parasite species of Sarcocystis (S. singaporensis, S. zamani, and S. villivillosi) use the reticulated python as their definitive hosts and several species within the Rattus genus as intermediate hosts, and they form a system useful for studying interactions between host–parasite and predator–prey relationships, as well as anthropogenic impacts on parasite transmission. Based on predictions from a 1998 survey, which detected an inverse relationship between urban development and Sarcocystis infection in Rattus, we tested the hypothesis that Sarcocystis transmission in Singapore will decrease over time due to anthropogenic activities. Despite a large proportion of the reticulated python diet consisting of Rattus species at all sizes of pythons, Sarcocystis infection rates decreased from 1998 to 2010. Pythons found in industrial areas had lower Sarcocystis infection rates, particularly in the western industrial area of Singapore Island. Average python size also decreased, with implications that we predict may disrupt host–parasite relationships. Anthropogenic activities such as habitat modification, fragmentation, and systematic removal and translocation of pythons have negative impacts on Sarcocystis transmission in Singapore, which in turn may augment pest rat populations. Trends observed may ultimately have negative impacts on human health and biodiversity in the region.     

Molecular identification of seven Sarcocystis species in red deer (Cervus elaphus) from Lithuania

The diaphragm muscles of 77 free-ranging red deer (Cervus elaphus) were examined for Sarcocystis species in Lithuania. Sarcocysts were detected in 61 out of 77 (79.2%) animals investigated. A total of 60 isolated sarcocysts were identified to species using subunit I of cytochrome c oxidase (cox1) sequence analysis. Overall, seven species, S. entzerothi, S. hjorti, S. iberica, S. linearis, S. pilosa, S. truncata and S. venatoria, were confirmed in Lithuanian red deer. Sarcocystis entzerothi was reported in red deer for the first time. Previously this species was shown to use sika deer as well as roe deer and fallow deer as an intermediate host. Based on cox1, with the addition of the current data, altogether 13 Sarcocystis species have so far been shown to use red deer as an intermediate host. Species detected in red deer demonstrated considerable differences in intraspecific genetic variation at cox1. Genetic distances between different samples of S. hjorti and S. linearis were calculated using principal coordinates analysis (PCoA), implying molecular divergence of same Sarcocystis species using different hosts in the same geographical area and divergence of those employing same intermediate host species from different areas.     

Effect of Resveratrol on Behavioral Performance of Streptozotocin-induced Diabetic Mice inAnxiety Tests

The aim of this study was to evaluate with anxiety tests the effect of resveratrol (RSV) on streptozotocin (STZ)-induced diabetic mouse behavioral performance at the second and fourth week of treatment. Confirmed diabetic mice (>250 mg/dl of glucose in blood after STZ injection) were treated with RSV (RDM, n=12) or control treated (DM, n=12) for 4 weeks. DM and RDM were tested in the Open Field Test (OFT) and Elevated Plus Maze (EPM). In the second week of RSV treatment, a higher grooming frequency (P<0.05) and a lower defecation and rearing frequency (P<0.05) were detected in the OFT in the RDM group compared with the DM. There was a higher grooming frequency (P<0.05) and higher percentage of entries in open arms (P<0.05) in the RDM group than in the DM group in the EPM. However, in the fourth week of RSV treatment, the only effect observed was a higher grooming frequency in the RDM group than in the DM group (P<0.05) in the EPM. In conclusion, RSV treatment in diabetic mice provoked anxiolytic-like effects in both tests (OFT and EPM), and these effects were observed in a short time window (2 weeks). It is suggested that RSV may help diabetic animals to adapt to new stressing and anxiety situations and thus to improve their welfare.     

Recolonizing gray wolves increase parasite infection risk in their prey

The recent recolonization of Central Europe by the European gray wolf (Canis lupus) provides an opportunity to study the dynamics of parasite transmission for cases when a definitive host returns after a phase of local extinction. We investigated whether a newly established wolf population increased the prevalence of those parasites in ungulate intermediate hosts representing wolf prey, whether some parasite species are particularly well adapted to wolves, and the potential basis for such adaptations. We recorded Sarcocystis species richness in wolves and Sarcocystis prevalence in ungulates harvested in study sites with and without permanent wolf presence in Germany using microscopy and DNA metabarcoding. Sarcocystis prevalence in red deer (Cervus elaphus) was significantly higher in wolf areas (79.7%) than in control areas (26.3%) but not in roe deer (Capreolus capreolus) (97.2% vs. 90.4%) or wild boar (Sus scrofa) (82.8% vs. 64.9%). Of 11 Sarcocystis species, Sarcocystis taeniata and Sarcocystis grueneri occurred more often in wolves than expected from the Sarcocystis infection patterns of ungulate prey. Both Sarcocystis species showed a higher increase in prevalence in ungulates in wolf areas than other Sarcocystis species, suggesting that they are particularly well adapted to wolves, and are examples of “wolf specialists”. Sarcocystis species richness in wolves was significantly higher in pups than in adults. “Wolf specialists” persisted during wolf maturation. The results of this study demonstrate that (1) predator–prey interactions influence parasite prevalence, if both predator and prey are part of the parasite life cycle, (2) mesopredators do not necessarily replace the apex predator in parasite transmission dynamics for particular parasites of which the apex predator is the definitive host, even if meso‐ and apex predators were from the same taxonomic family (here: Canidae, e.g., red foxes Vulpes vulpes), and (3) age‐dependent immune maturation contributes to the control of protozoan infection in wolves.     

Growth of <Emphasis Type="Italic">Theileria parva</Emphasis>-infected Bovine Lymphoid Cells in Irradiated Mice

SEVERAL attempts to establish Theileria parva, the causative organism of East Coast fever of cattle, in laboratory animals^1–4 have failed. The recent successful establishment of T. parva macroschizont-infected bovine lymphoid cells in tissue culture⁵ provided a concentrated source of material which could be used in further attempts to infect laboratory animals.     

Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

The electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates was measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here provide new insight for Bacillus anthracis surrogate selection and for attachment/detachment and transport studies.