Molecular differentiation of cattle Sarcocystis spp. by multiplex PCR targeting 18S and COI genes following identification of Sarcocystis hominis in human stool samples

Rubiola, Selene; Civera, Tiziana; Ferroglio, E.; Zanet, Stefania; Zaccaria, Teresa; Brossa, S.; Cipriani, R.; Chiesa, Francesco

doi:10.1016/j.fawpar.2020.e00074

Cited by 23 publications

(31 citation statements)

References 31 publications

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“…The majority of the 18S rRNA sequences formerly presented in GenBank as S. hominis actually belonged to S. bovini, S. bovifelis or S. sinensis infecting the water buffalo [8]. There is a lack of detailed S. hominis molecular data [49] and the first cox1 sequence of this species became available in the GenBank database only in 2018. The prevalence of S. hominis infection in different countries based on molecular methods is poorly studied.…”

Section: Discussionmentioning

confidence: 99%

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Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method

et al. 2020

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Background Six Sarcocystis species are known to use cattle (Bos taurus) as the intermediate host, two of which, S. hominis and S. heydorni, are zoonotic. There is a need for a method that will enable rapid identification of the Sarcocystis species in cattle. Methods The diaphragm muscles of 102 cattle from Lithuania were examined for the presence of Sarcocystis spp., using two different methods for species identification. Individual sarcocysts were isolated from squash preparations of the diaphragm muscle under the light microscope, followed by genetic characterisation of excised cysts using sequence analysis of the 18S rRNA (18S rRNA) and cytochrome c oxidase subunit I (cox1) genes. The same cattle muscle samples were digested and species-specific PCR analyses targeting cox1 were developed to identify the Sarcocystis isolates to the species level. Results Under the light microscope, sarcocysts were detected in 87.3% of animals, and Sarcocystis infection was verified in all digested samples. Three species, namely S. cruzi (n = 20), S. bovifelis (n = 23) and S. hirsuta (n = 6), were identified by DNA sequence analysis of isolated sarcocysts. Based on sequence analysis of cox1, the level of genetic variability depended on Sarcocystis species and geographical location. Four Sarcocystis species, S. cruzi (96.1%), S. bovifelis (71.6%), S. hirsuta (30.4%) and S. hominis (13.7%), were confirmed in the digested samples. In individual samples, the most common finding was two species of Sarcocystis (44.1%), followed by three species (26.5%), a single species (24.5%) and four species (4.9%). Conclusions Although examination of tissue preparations under the light microscrope did not detect any sarcocysts belonging to S. hominis, this species was identified in the digested samples subjected to a cox1-specific PCR analysis. These results demonstrate the need for effective molecular diagnosis techniques to detect Sarcocystis spp., which may be present at a lower prevalence and not detectable among the limited number of sarcocysts identified individually under the light microscope.

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Section: Discussionmentioning

confidence: 99%

“…There is a lack of detailed S . hominis molecular data [ 49 ] and the first cox 1 sequence of this species became available in the GenBank database only in 2018. The prevalence of S .…”

Section: Discussionmentioning

confidence: 99%

Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method

et al. 2020

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“…The identi cation of different Sarcocystis spp. was performed through the application of the multiplex-PCR assay described by Rubiola et al [14] targeting the 18S rDNA gene and the mtDNA cox1 gene. The multiplex-PCR contained 2.5 μl of template DNA (5-20 ng/μl), 0,5 mM of each primer, Sarco Rev, Sar F, Hirsuta, Cruzi, COI HB, COI H and COI B, 2 mM MgCl2, 0.2 mM of each dNTP, 1 U Platinum Taq DNA polymerase, 10 x PCR Buffer and RNasefree water to a total volume of 25 μl.…”

Section: Sample Collection and Processingmentioning

confidence: 99%

“…The ampli cation was performed in an Applied Biosystems 2720 Thermal Cycler (AppliedBiosystems, CA, USA) with the following cycling pro le: a denaturation step at 95°C for 3 min, followed by 35 cycles at 95 °C for 60 s, 58 °C for 60 s and 72 °C for 30 s and nal extension 72 °C for 3 min. In each PCR run, 2.5 μl of DNA from a collection of Sarcocystis positive samples isolated from cattle striated muscle in the Department of Veterinary Science of Turin University [9,14,15] were used as positive controls while extracted DNA from negative cattle muscles as well as reagent blanks were included as negative controls. PCR products were observed in 2% agarose gel stained with SYBR safe stain (Invitrogen, Carlsbad, CA) and observed in a blue light transilluminator (Invitrogen, Groningen, The Netherlands).…”

Section: Sample Collection and Processingmentioning

confidence: 99%

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Molecular Detection of Cattle Sarcocystis Spp. In North-West Italy Targeting Cox1 And 18S Genes Highlights Its Association With Bovine Eosinophilic Myositis.

Rubiola

Civera

Panebianco

et al. 2021

Preprint

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Background: Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa due to the evidences supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based either on morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis.Methods: To reach our aim, individual cattle samples from BEM condemned carcasses (N=54) and randomly sampled carcasses (N=59) were obtained from Piedmont slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S and cox1 genes. PCR products amplified using the genus specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis, were sequenced to achieve species identification.Results: Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in 2 carcasses.Conclusions: Our study contributes to update the data on the prevalence of the different Sarcocystis spp. in cattle in Italy and emphasize the role of S. hominis and S. bovifelis as the major sarcosporidian species involved.

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Cystoisospora , Cyclospora , and Sarcocystis

Qvarnstrom,

Arrowood

2023

ClinMicroNow

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The parasites traditionally referred to as coccidia that develop in the intestines of humans— Cystoisospora belli , Cyclospora cayetanensis , and Sarcocystis spp.—are in the Stramenopiles, Alveolates, Rhizaria supergroup in the current taxonomic scheme used by protozoologists. Oocysts of these coccidia are found in the feces of humans, and diagnosis is based ultimately on demonstrating oocysts in human stool samples. The use of molecular methods to evaluate the phylogenetics and genetic diversity of parasites has resulted in a revision of the coccidian parasites. In vitro culture of intestinal coccidian parasites is most often used as a tool to study developmental biology or to identify active chemotherapeutic agents. Differentiation of the intestinal coccidia to the subspecies/strain level—genotyping—can be important for epidemiologic reasons, for example to link cases in outbreak investigations or to better understand clinical outcome differences.

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Molecular differentiation of cattle Sarcocystis spp. by multiplex PCR targeting 18S and COI genes following identification of Sarcocystis hominis in human stool samples

Cited by 23 publications

References 31 publications

Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method

Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method

Molecular Detection of Cattle Sarcocystis Spp. In North-West Italy Targeting Cox1 And 18S Genes Highlights Its Association With Bovine Eosinophilic Myositis.

Cystoisospora , Cyclospora , and Sarcocystis

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