Diversity of Sarcocystis parasites in southeastern Baltic Sea catchment ecosystems.

Currently, research on apicomplexan Sarcocystis parasites is mainly carried out by analyzing animal carcasses. However, environmental studies would not only allow faster detection of possible sources of infection but also avoid the use of animals for investigations. Therefore, in the current study, we aimed to identify tested Sarcocystis species in sediment collected from water bodies located in the southeastern Baltic countries. A total of 99 sediment samples were collected during the summer from different types of water bodies in Estonia, Latvia, Lithuania, and Poland. Species-specific nested PCR targeting cox1 gene was used for the detection of selected Sarcocystis species (S. cruzi, S. bovifelis, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. miescheriana, and S. bertrami) infecting livestock. The results showed a statistically lower (p < 0.05) occurrence of Sarcocystis parasites in Estonia (50%) compared to three countries, where the detection rate of Sarcocystis spp. DNA was remarkably higher, ranging from 88 to 100%. Among Sarcocystis species tested, S. cruzi (83.8%) and S. arieticanis (55.6%) using cattle and sheep as their intermediate hosts were most commonly identified. The detection rates of some of the analyzed Sarcocystis species were significantly different in southeastern Baltic countries. It is discussed that the detection rates of certain Sarcocystis species depend not only on the number of animals per 1 km2 but also on various ecological factors and farming practices that differ in the amount of contact domestic animals have with predators and the potential for animals to become infected through natural water or food sources.

Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples.

Sarcocystis parasites are among the most common parasitic protozoa in farm animals. So far, the diversity of these parasites has been mainly studied in animal carcasses by morphological or molecular methods. Research on parasitic protozoa in environmental samples is scarce due to the lack of an appropriate methodology and low concentrations of parasites. For these reasons, there is a paucity of validated methods for Sarcocystis identification from environmental samples. Therefore, the present study aims to investigate various molecular methods for Sarcocystis parasite identification in water samples. In the present study, the sample volume, sporocysts isolation, and various conventional PCR were evaluated, and species-specific primers for the identification of different Sarcocystis species have been developed. Of the methods studied, based on data the most appropriate method for the identification of analyzed Sarcocystis spp. in water bodies is nested PCR, using species-specific primers targeting the cox1 gene. Sarcocystis DNA was detected in 111 out of 114 (97.4%) samples. This paper represents the first identification of S. bovifelis, S. cruzi, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. bertrami, and S. miescheriana by PCR and sequencing in environmental water samples. Our pilot study is useful in developing techniques for the identification of Sarcocystis species from water samples.

The Bacterial Microbiota of Edible Insects Acheta domesticus and Gryllus assimilis Revealed by High Content Analysis.

In the concept of novel food, insects reared under controlled conditions are considered mini livestock. Mass-reared edible insect production is an economically and ecologically beneficial alternative to conventional meat gain. Regarding food safety, insect origin ingredients must comply with food microbial requirements. House crickets (Acheta domesticus) and Jamaican field crickets (Gryllus assimilis) are preferred insect species that are used commercially as food. In this study, we examined cricket-associated bacterial communities using amplicon-based sequencing of the 16S ribosomal RNA gene region (V3-V4). The high taxonomic richness of the bacterial populations inhabiting both tested cricket species was revealed. According to the analysis of alpha and beta diversity, house crickets and Jamaican field crickets displayed significantly different bacterial communities. Investigation of bacterial amplicon sequence variants (ASVs) diversity revealed cricket species as well as surface and entire body-associated bacterial assemblages. The efficiency of crickets processing and microbial safety were evaluated based on viable bacterial counts and identified bacterial species. Among the microorganisms inhabiting both tested cricket species, the potentially pathogenic bacteria are documented. Some bacteria representing identified genera are inhabitants of the gastrointestinal tract of animals and humans, forming a normal intestinal microflora and performing beneficial probiotic functions. The novel information on the edible insect-associated microbiota will contribute to developing strategies for cricket processing to avoid bacteria-caused risks and reap the benefits.

Molecular Identification of Protozoan Sarcocystis in Different Types of Water Bodies in Lithuania.

Representatives of the genus Sarcocystis are unicellular parasites having a two-host life cycle and infecting mammals, birds, and reptiles. Until now, Sarcocystis spp. have been mainly investigated in definitive and intermediate hosts. Only a few studies have been conducted on the detection of Sarcocystis parasites in water samples. The aim of this research was to examine whether the prevalence of Sarcocystis spp. parasitizing farm animals varies in different types of water bodies. Water samples (n = 150) were collected from the entire territory of Lithuania, dividing water bodies into five groups (lakes, rivers, ponds/canals, swamps, and the inshore zone of the territorial Baltic Sea area). One-liter samples were filtered and subsequently analyzed using nested PCR. At least one of the analyzed Sarcocystis spp. (S. arieticanis, S. bertrami, S. bovifelis, S. capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) was determined in all examined samples from water bodies. No significant difference in Sarcocystis spp. prevalence between different types of water sources was detected. Our research proved that selecting appropriate primers is important for the accurate identification of parasites in samples collected from water bodies.

Mycobiota in the Carposphere of Sour and Sweet Cherries and Antagonistic Features of Potential Biocontrol Yeasts.

Sour cherries (Prunus cerasus L.) and sweet cherries (P. avium L.) are economically important fruits with high potential in the food industry and medicine. In this study, we analyzed fungal communities associated with the carposphere of sour and sweet cherries that were freshly harvested from private plantations and purchased in a food store. Following DNA isolation, a DNA fragment of the ITS2 rRNA gene region of each sample was individually amplified and subjected to high-throughput NGS sequencing. Analysis of 168,933 high-quality reads showed the presence of 690 fungal taxa. Investigation of microbial ASVs diversity revealed plant-dependent and postharvest handling-affected fungal assemblages. Among the microorganisms inhabiting tested berries, potentially beneficial or pathogenic fungi were documented. Numerous cultivable yeasts were isolated from the surface of tested berries and characterized by their antagonistic activity. Some of the isolates, identified as Aureobasidium pullulans, Metschnikowia fructicola, and M. pulcherrima, displayed pronounced activity against potential fungal pathogens and showed attractiveness for disease control.

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

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.

Correction: Luksa, J., et al. Fungal Microbiota of Sea Buckthorn Berries at Two Ripening Stages and Volatile Profiling of Potential Biocontrol Yeasts. Microorganisms 2020, 8, 456.

The authors wish to make the following corrections to this paper [...].

Fungal Microbiota of Sea Buckthorn Berries at Two Ripening Stages and Volatile Profiling of Potential Biocontrol Yeasts.

Sea buckthorn, Hippophae rhamnoides L., has considerable potential for landscape reclamation, food, medicinal, and cosmetics industries. In this study, we analyzed fungal microorganism populations associated with carposphere of sea buckthorn harvested in Lithuania. An amplicon metagenomic approach based on the ITS2 region of fungal rDNA was used to reveal the ripening-affected fungal community alterations on sea buckthorn berries. According to alpha and beta diversity analyses, depending on the ripening stage, sea buckthorn displayed significantly different fungal communities. Unripe berries were shown to be prevalent by Aureobasidium, Taphrina, and Cladosporium, while ripe berries were dominated by Aureobasidium and Metschnikowia. The selected yeast strains from unripe and mature berries were applied for volatile organic compounds identification by gas chromatography and mass spectrometry techniques. It was demonstrated that the patterns of volatiles of four yeast species tested were distinct from each other. The current study for the first time revealed the alterations of fungal microorganism communities colonizing the surface of sea buckthorn berries at different ripening stages. The novel information on specific volatile profiles of cultivable sea buckthorn-associated yeasts with a potential role in biocontrol is important for the development of the strategies for plant cultivation and disease management, as well as for the improvement of the quality and preservation of the postharvest berries. Management of the fungal microorganisms present on the surface of berries might be a powerful instrument for control of phytopathogenic and potentially antagonistic microorganisms affecting development and quality of the berries.

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.

Sarcocystis morae (Apicomplexa) in Fallow Deer (Dama dama) from Spain: Ultrastructure and New Host Record.

Members of the genus Sarcocystis are frequently found infecting members of the family Cervidae. Although Sarcocystis species are generally host specific for their intermediate hosts, species in cervids appear to be less host specific. Here, we report fallow deer (Dama dama) as a new host for Sarcocystis morae, originally described from the red deer (Cervus elaphus). Tongues of 69 legally hunted animals in Spain were tested for sarcocysts, and the species were characterized by light microscopy, ultrastructurally and molecularly. Sarcocysts were identified in 66.7% of D. dama. Sarcocysts had thin (<2 µm thick) cyst wall with hair-like villar protrusions bifurcated at their tips resembling type 8a. Genetic sequences obtained for 18S rRNA and COI reached 99.6-100% and 97.9-98.7% similarity, respectively, to those of S. morae from the red deer. The present study provides new data concerning lower level of host specificity within Sarcocystis genus for cervids.