The Distribution of Sarcocsytis Species Described by Ungulates-Canids Life Cycle in Intestines of Small Predators of the Family Mustelidae.

PURPOSE: Using molecular techniques, we have previously shown that carnivorous mammals of the family Mustelidae might be common definitive hosts for various protozoan Sarcocystis species. In the present study we aimed to unravel whether Sarcocystis species using ungulates as intermediate hosts and canids or felids as definitive hosts can be found in intestine of mustelids. METHODS: Small intestine samples of 93 individual mustelids of five different species from Lithuania were examined. Sarcocystis species were identified based on species-specific PCR and subsequent cox1 sequencing. RESULTS: Six Sarcocystis species (S. arieticanis, S. bertrami, S. capracanis, S. capreolicanis, S. linearis and S. morae) defined by ungulate-canid life cycle were detected for the first time in small intestines of mustelids. By contrast, the prevalence of Sarcocystis characterised by ungulate-felid life cycle was low (3.2%). Overall, 76% of the examined animals were positive for at least one of the studied Sarcocystis species. Four species, S. arieticanis, S. bertrami, S. capracanis and S. morae were most commonly found, with the detection rate of about 40%. CONCLUSIONS: The current finding, in addition to our previous studies, suggests that mustelids play an important role in the spread of various Sarcocystis species.

First report of Sarcocystis halieti (Apicomplexa) in bearded vulture (Gypaetus barbatus).

At least three Sarcocystis species (S. falcatula, S. halieti and S. wobeseri-like) have been detected infecting raptorial birds. By histopathology and PCR-sequencing of the ITS1 marker, S. halieti was detected in a bearded vulture (Gypaetus barbatus) and a black kite (Milvus migrans) from the Catalonia region in North Spain. The 241 bp-long sequences obtained from the Sarcocystis organisms detected in both raptors showed 97.5-99.6% and 97.9-100% similarity with those of previously identified S. halieti; also, the phylogenetic trees generated placed the identified sequences together with other sequences of S. halieti available in GenBank. In sum, the description of the bearded vulture as a new intermediate host for S. halieti adds new insights on the complex epidemiology of the genus involving avian hosts.

Dynamics of Polyamines, Proline, and Ethylene Metabolism under Increasing Cold in Winter Oilseed Rape.

Cold stress is among the most important environmental factors reducing the yield of crops. The present study aimed to investigate the impact of increasing cold stress conditions on winter oilseed rape polyamines, proline, and ethylene metabolism in acclimated and non-acclimated winter oilseed rape. This study was carried out under controlled conditions in the laboratory. The winter oilseed rape hybrid 'Visby' was used in the experiment. Acclimated and non-acclimated plants were subjected to a two-day-long increasing cold (from -1 °C to -3 °C) treatment. HPTLC, RT-qPCR, spectral analysis, and gas chromatography methods were used to analyse the levels of polyamines, gene expression, proline, and ethylene, respectively. This study showed a decrease in putrescine, spermidine, and spermine content during cold acclimation and a decrease in putrescine and spermidine levels at sub-zero temperatures. There were intensive changes in ADC2 gene expression, proline, and ethylene levels in non-acclimated plants: a substantial increase after exposure to -1 °C temperature and a sharp decrease after exposure to -3 °C temperature. The changes in these parameters were lower or absent in acclimated plants. The phenomena observed in this study add new insights to the knowledge about the plant stress response and suggest questions to be answered in the future.

Protozoan Parasites of Sarcocystis spp. in Rodents from Commercial Orchards.

Small mammals are an important group of wildlife that can transmit pathogens to humans and animals. There is a lack of comprehensive studies on the protozoan parasites of the genus Sarcocystis in agricultural areas. The aim of the current research was to evaluate the prevalence of Sarcocystis spp., and to identify the parasite species found in the skeletal muscles of rodents and insectivores from commercial orchards. A total of 679 muscle samples from small mammals, mainly rodents (n = 674), belonging to eight species were examined. Muscle samples were pooled into groups, then digested, and the presence of the Sarcocystis species was confirmed by molecular methods. The examined parasites were determined in five rodent species, Apodemus agrarius, A. flavicollis, Clethrionomys glareolus, Microtus arvalis, and M. oeconomus. The prevalence of Sarcocystis spp. was low: 2.23% in voles and 0.79% in mice. Based on a sequence comparison of cox1 and 28S rDNA, four species were identified: S. myodes, Sarcocystis cf. strixi, Sarcocystis sp. Rod1, and Sarcocystis sp. Rod2. This is the first report of S. myodes in A. agrarius, A. flavicollis, and M. arvalis. The identified species were most closely related to Sarcocystis spp., and were transmitted by predatory mammals and birds. Future studies are needed to describe the species morphologically, as well as to define the host spectrum and to evaluate their possible pathogenicity.

Soil Fungistasis against Fusarium Graminearum under Different tillage Systems.

The establishment of the harmful pathogen Fusarium graminearum in different agroecosystems may strongly depend on the ability of the soils to suppress its development and survival. This study aimed to evaluate the influence of different soil tillage systems (i.e., conventional tillage, reduced tillage and no-tillage) on soil fungistasis against F. graminearum. Soil samples were collected three times during the plant growing season in 2016 and 2017 from a long-term, 20-year soil tillage experiment. The F. graminearum in the soil samples was quantified by real-time qPCR. The soil fungistasis was evaluated by the reduction in the radial growth of F. graminearum in an in vitro assay. The antagonistic activity of the soil bacteria was tested using the dual culture method. The F. graminearum DNA contents in the soils were negatively correlated with soil fungistasis (r = -0.649 *). F. graminearum growth on the unfumigated soil was reduced by 70-87% compared to the chloroform fumigated soil. After the plant vegetation renewal, the soil fungistasis intensity was higher in the conventionally tilled fields than in the no-tillage. However, no significant differences were obtained among the tillage treatments at the mid-plant growth stage and after harvesting. 23 out of 104 bacteria isolated from the soil had a moderate effect, and only 1 had a strong inhibitory effect on the growth of F. graminearum. This bacterium was assigned 100% similarity to the Bacillus amyloliquefaciens Hy7 strain (gene bank no: JN382250) according to the sequence of the 16S ribosome subunit coding gene. The results of our study suggest that the presence of F. graminearum in soil is suppressed by soil fungistasis; however, the role of tillage is influenced by other factors, such as soil biological activity, type and quantity of plant residues and environmental conditions.

Molecular Identification of Sarcocystis rileyi and Sarcocystis sp. (Closely Related to Sarcocystis wenzeli) in Intestines of Mustelids from Lithuania.

The genus Sarcocystis is a group of numerous protozoan parasites having a two-host life cycle. Based on laboratory experiments and/or phylogenetic analysis results it was shown that seven Sarcocystis spp. producing sarcocsyts in bird tissues are transmitted via predatory placental mammals. To date the role of small mammals of the family Mustelidae in the distribution of avian Sarcocystis spp. have not been studied. During the current investigation, intestinal mucosa scrapings of 115 mustelids belonging to five species were tested for S. albifronsi, S. anasi, S. rileyi, and S. wenzeli infecting anseriforms and chickens. Microscopically, free sporocysts, sporulating oocysts, and loose oocysts were found in 61 samples (53.0%). Using nested PCR targeting the ITS1 region and sequencing, S. rileyi was confirmed in eight American minks, two European polecats and single European badger. Sarcocystis sp. was identified in one American mink and one European pine marten. Based on the partial ITS1 region this parasite showed that 100% identity to pathogenic Sarcocystis sp. caused a fatal infection in backyard chickens from Brazil. Phylogenetically, the Sarcocystis sp. identified in our study was most closely related to S. wenzeli parasitising domestic fowl (Gallus domesticus).

A novel RFLP method for identification of morphologically similar avian Sarcocystis species.

Protozoans of genus Sarcocystis are widespread parasites infecting mammals, birds, and reptiles. Morphology of their sarcocysts is an important criterion for species identification. However, as more and more morphologically similar Sarcocystis species are being found and described, additional methods for their routine diagnostics are needed. We investigated restriction fragment length polymorphism (RFLP) as potential alternative to sequencing data analysis for the identification of Sarcocystis species using birds as an intermediate host. The internal transcribed spacer 1 (ITS1) region sequences of seventeen Sarcocystis species (S. albifronsi, S. anasi, S. calchasi, S. columbae, S. cornixi, S. corvusi, S. cristata, S. halieti, S. falcatula, S. fulicae, S. kutkienae, S. lari, S. lindsayi, S. rileyi, S. turdusi, S.wenzeli, and S. wobeseri) of interest were analysed and five best-fitting endonucleases generating most informative restriction fragments were selected for routine testing. In general, RFLP analyses are always inconclusive as they target very short DNA sequences. However, it can be an irreplaceable technique when fast and cheap identification and discrimination of known species are required, which was our main goal and preliminary results indicate that RFLP could be successfully used when identifying closely related avian Sarcocystis species with just two nucleases.

The Role of Birds of the Family Corvidae in Transmitting Sarcocystis Protozoan Parasites.

Members of the family Corvidae are ecologically flexible omnivorous birds, particularly adaptive to urban habitats, and living in proximity to humans; these birds may serve as definitive hosts (DH) for Sarcocystis spp., but research about this is lacking. In the present study, intestinal samples from 91 corvids collected in Lithuania were molecularly tested by species-specific PCR targeting the ITS1 and cox1 genes and subsequently sequenced for the presence of Sarcocystis spp. Under a light microscope, oocysts of Sarcocystis spp. were observed in 43 samples (47.3%), while molecular methods, detected Sarcocystis spp. in 77 birds (84.6%). Eleven Sarcocystis spp. (S. columbae, S. cornixi, potentially pathogenic S. halieti, S. kutkienae, S. lari, S. turdusi, S. wobeseri, S. arctica, S. lutrae, S. ovalis, and S. oviformis) were identified in the intestinal samples from six corvid species from Lithuania. Infections with multiple Sarcocystis spp. were detected in 79.2% of the infected corvid birds. Three of the identified Sarcocystis spp. use corvids as intermediate hosts (IH); therefore, corvids may serve as IH and DH of the same Sarcocystis species. Based on molecular results and on corvid diet, omnivorous corvids may play an important role in transmitting Sarcocystis spp.

Molecular Detection and Characterization of Intestinal and Blood Parasites in Wild Chimpanzees (Pan troglodytes verus) in Senegal.

Wild chimpanzee populations in West Africa (Pan troglodytes verus) have dramatically decreased as a direct consequence of anthropogenic activities and infectious diseases. Little information is currently available on the epidemiology, pathogenic significance, and zoonotic potential of protist species in wild chimpanzees. This study investigates the occurrence and genetic diversity of intestinal and blood protists as well as filariae in faecal samples (n = 234) from wild chimpanzees in the Dindefelo Community Nature Reserve, Senegal. PCR-based results revealed the presence of intestinal potential pathogens (Sarcocystis spp.: 11.5%; Giardia duodenalis: 2.1%; Cryptosporidium hominis: 0.9%), protist of uncertain pathogenicity (Blastocystis sp.: 5.6%), and commensal species (Entamoeba dispar: 18.4%; Troglodytella abrassarti: 5.6%). Entamoeba histolytica, Enterocytozoon bieneusi, and Balantioides coli were undetected. Blood protists including Plasmodium malariae (0.4%), Trypanosoma brucei (1.3%), and Mansonella perstans (9.8%) were also identified. Sanger sequencing analyses revealed host-adapted genetic variants within Blastocystis, but other parasitic pathogens (C. hominis, P. malariae, T. brucei, M. perstans) have zoonotic potential, suggesting that cross-species transmission between wild chimpanzees and humans is possible in areas where both species overlap. Additionally, we explored potential interactions between intestinal/blood protist species and seasonality and climate variables. Chimpanzees seem to play a more complex role on the epidemiology of pathogenic and commensal protist and nematode species than initially anticipated.

Invasive American mink (Neovison vison) as potential definitive host of Sarcocystis elongata, S. entzerothi, S. japonica, S. truncata and S. silva using different cervid species as intermediate hosts.

Canids and scavenger birds were shown to act as definitive hosts of numerous Sarcocystis species using members of the Cervidae family as an intermediate host, whereas definitive hosts spreading closely related S. elongata, S. entzerothi, S. japonica, S. matsuoae, S. rangiferi, S. truncata, S. silva and S. tarandi remain unknown. In the current study, the intestine samples of 40 American minks (Neovison vison) were molecularly tested for the presence of the above-mentioned Sarcocystis spp. Species-specific PCR of cytochrome c oxidase subunit I (cox1) fragments and subsequent sequencing revealed the presence of sporocysts/oocysts of five species, S. elongata (n=2), S. entzerothi (n=10), S. japonica (n=4), S. silva (n=13) and S. truncata (n=21) in the analysed samples. Sarcocystis infection was confirmed in 32/40 (80%) examined samples. In addition, half of the infected animals (50%) were infected with multiple Sarcocystis species suggesting that American minks had access to meat of different deer species, such as roe deer, red deer and sika deer. This causes concern about compliance of hunters and game processing companies with game waste management rules. Further research on the involvement of mustelids in the transmission of various Sarcocystis spp. from different geographical locations is needed.

Population Structure of Fusarium graminearum Isolated from Different Sources in One Area over the Course of Three Years.

Fusarium head blight (FHB) is an important crop disease worldwide and is mainly caused by members of the Fusarium graminearum species complex. F. graminearum sensu stricto is the most common cosmopolitan and predominant FHB causal agent in Europe. Thus far, the majority of studies have focused on the primary hosts (wheat and barley) of this pathogen, while the relationships between other sources of infection remain unclear. We monitored and sampled test fields over the course of 3 years and acquired 804 F. graminearum isolates from different sources: primary hosts and other plant species included in the crop rotations, weeds from the test fields, decaying plant residue, soil samples, and crop seed. Of these isolates, 73.3% had the 15-acetyldeoxynivalenol genotype and 26.7% had the 3-acetyldeoxynivalenol genotype. F. graminearum isolation rates from weeds (>50%) were much higher than from soil (< 10%) or decaying plant matter (4%). Variable number of tandem repeat markers were used for population analysis. Noticeable genetic differentiation was detected between isolates from living plants and soil biome. In contrast, absence of any noticeable division between primary and alternative plant host communities indicates the importance of weeds and other segetal plants for FHB control and prevention.