Identification of Sarcocystis spp. in Slaughtered Sheep from Spain and Evaluation of Bradyzoite Viability after Freezing.

Sarcocystis spp. are complex apicomplexan parasites that cause a substantial economic impact on livestock used for meat production. These parasites are present worldwide. Our study aimed to identify Sarcocystis species affecting sheep meat in southern-central Spain and to evaluate the effectiveness of freezing for parasite inactivation. A total of 210 condemned samples of sheep meat were thoroughly assessed grossly and microscopically; the presence of macro- and microcysts was confirmed. The samples were then frozen at -20 °C for various time intervals (24, 48, 72, 96, 120, and 144 h) and compared with untreated samples. Bradyzoites were isolated through pepsin digestion for subsequent molecular analysis and viability assessment, employing trypan blue and double fluorescence staining techniques. Our measurements confirmed the presence of S. tenella, S. gigantea, and S. medusiformis in Spanish domestic sheep. Freezing for 96 to 144 h resulted in a significant reduction in parasite viability, with a robust correlation observed between the two staining methods. Both stains effectively measured the viability of Sarcocystis, thereby promising future advances in meat safety.

Antioxidant activity of exo-metabolites produced by Fusarium oxysporum: An endophytic fungus isolated from leaves of Otoba gracilipes.

Tropical ecosystems hold an extremely diverse array of endophytic fungi, but their potential use still remains to be explored. In this study, we isolated an endophytic fungus from the leaves of Otoba gracilipes, a medicinal tree from a tropical rainforest in Colombia. Following isolation and cultivation, we evaluated its extracellular crude extract for antioxidant activity. Using traditional and molecular methods (ITS1, NL1 regions), the endophyte was identified as Fusarium oxysporum. Fresh spores from the fungal isolate were inoculated in liquid media (potato dextrose broth [PDB] and potato dextrose-yeast extract broth [PDYB]) and centrifuged for recovering extracellular polysaccharides from the exhausted medium after 30 days of cultivation. Crude extracts were recovered, purified, lyophilized, and evaluated for their ability to inactivate the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). The extracts obtained from PDB culture media had a 51.5% of scavenging effect on DPPH after 5 min of reaction compared with the extracts from PDBY (26.4%), which suggests a high antioxidant potential of these fungal extracts. Thus, our results suggest other fungi from tropical ecosystems should be explored as potential sources of novel enzymes and other metabolites with bioactivity.

Prevalence of intestinal parasites, with emphasis on the molecular epidemiology of Giardia duodenalis and Blastocystis sp., in the Paranaguá Bay, Brazil: a community survey.

BACKGROUND: Intestinal protozoan parasites are major contributors to the global burden of gastrointestinal disease causing significant socioeconomic consequences. Children living in resource-poor settings with restricted access to water and sanitary services are particularly at risk of these infections. METHODS: A prospective, community-based, cross-sectional survey was conducted in Paraná (southern Brazil) between May 2015 and May 2016. A total of 766 stool samples were individually collected from volunteers (male/female ratio: 0.99; age range: 0-76 years) and used for investigating the presence of intestinal helminth and protozoan species by routine microscopic procedures including the Kato-Katz and modified Ritchie concentration methods and the Ziehl-Neelsen stain technique. Quantitative real-time PCR confirmed microscopy-positive samples for Giardia duodenalis and the assemblages and sub-assemblages determined by multilocus sequence-based genotyping of the glutamate dehydrogenase (gdh) and ß-giardin (bg) genes of the parasite. Identification of Blastocystis subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene (SSU rDNA) of this heterokont microorganism. RESULTS: Overall, 46.1% (353/766) of the participants were infected/colonised by at least one intestinal parasite/commensal species. Protozoan and helminth species were detected in 42.7% and 10.1% of the surveyed population, respectively. Blastocystis sp. (28.2%), Endolimax nana (14.9%), and Giardia duodenalis (11.0%) were the most prevalent species found among protozoans and Ascaris lumbricoides (5.0%), Trichuris trichiura (4.6%) and hookworms (1.0%) among helminths. A total of 38 G. duodenalis-positive samples were genotyped at gdh and bg markers, revealing the presence of the sub-assemblages AII (47.4%), AII/AIII (2.6%), BIII (5.3%), BIV (26.3%) and BIII/BIV (13.1%). Two samples (5.3%) were only identified as assemblage B. AII was predominantly found in females aged 5-9 years and was associated with a higher likelihood of reporting gastrointestinal symptoms. A total of 102 Blastocystis-positive samples were successfully subtyped at the SSU rRNA gene revealing the presence of ST1 (36.3%), ST2 (15.7%), ST3 (41.2%), ST4 (2.9%), ST6 (1.0%) and ST8 (2.9%). CONCLUSIONS: Data presented here indicate that enteric parasites still represent a pressing health concern in Paraná, Brazil, probably due to sub-optimal water, sanitation and hygiene conditions. A mostly anthroponotic origin is suspected for G. duodenalis and Blastocystis sp. infections.

Occurrence and molecular epidemiology of Giardia duodenalis infection in dog populations in eastern Spain.

BACKGROUND: Giardia duodenalis is one of the most common enteric parasites in domestic animals including dogs. Young animals are more prone to the infection, with clinical manifestations ranging from asymptomatic to acute or chronic diarrhoea. Dogs are primarily infected by canine-specific (C-D) assemblages of G. duodenalis. However, zoonotic assemblages A and B have been increasingly documented in canine isolates, raising the question of whether and to which extent dogs can act as natural reservoirs of human giardiosis. METHODS: In this cross-sectional epidemiological survey we assessed the molecular diversity of G. duodenalis in dogs in the province of Castellón, Eastern Spain. A total of 348 individual faecal samples from sheltered (n = 218), breeding (n = 24), hunting (n = 68), shepherd (n = 24), and pet (n = 14) dogs were collected between 2014 and 2016. Detection of G. duodenalis cysts in faecal material was carried out by direct fluorescence microscopy as a screening test, whereas a qPCR targeting the small subunit ribosomal RNA gene of the parasite was subsequently used as a confirmatory method. RESULTS: Giardia duodenalis was detected in 36.5% (95% CI: 31.6-41.7%) of dogs. No significant differences in prevalence rates could be demonstrated among dogs according to their sex and geographical origin, but breeding (45.8%; 95% CI: 27.9-64.9%) and sheltered (40.4%; 95% CI: 34.1-47.0%) dogs harboured significantly higher proportions of G. duodenalis. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and ß-giardin genes of G. duodenalis allowed the characterization of 35 canine isolates that were unambiguously assigned to assemblages A (14.3%), B (22.9%), C (5.7%), and D (37.1%). A number of inter-assemblage mixed infections including A + B (11.4%), A + D (2.9%), and A + B + D (5.7%) were also identified. CONCLUSIONS: Data presented here are strongly indicative of high infection pressures in kennelled animals. Zoonotic sub-assemblages AII, BIII, and BIV were responsible for a considerable proportion of the G. duodenalis infections detected, but very few of the genotypes identified have been previously documented in Spanish human populations. Although possible, zoonotic transmission between dogs and humans seems an infrequent event in this Spanish region.