João Rodrigo Mesquita

Vibrio cholerae and Vibrio parahaemolyticus are naturally occurring aquatic bacteria of public-health relevance that may contaminate seafood, including bivalve mollusks. This study characterized two Vibrio isolates recovered from commercially depurated mussels (Mytilus spp.) and clams (Spisula solida) marketed at retail in Portugal. Species identification, antimicrobial susceptibility testing, Oxford Nanopore whole-genome sequencing (WGS), average nucleotide identity analysis, antimicrobial resistance and virulence profiling, genoserotyping, prophage prediction, and core-genome phylogenomic analysis were performed. WGS confirmed the mussel-derived isolate as V. cholerae and the clam-derived isolate as V. parahaemolyticus. Phenotypic testing demonstrated meropenem resistance in the V. cholerae isolate, whereas several additional antimicrobial MICs could not be categorically interpreted because of panel concentration limits or the absence of Vibrio-specific clinical breakpoints. The V. cholerae isolate lacked ctxA, ctxB, the toxin-coregulated pilus cluster, ace, and zot, while its O1/O139 serogroup status remained unresolved because an O1-associated marker was absent and a complete but non-exact wbfZ-like sequence was detected. Its genome nevertheless contained an extensive Type VI secretion system repertoire together with several hemolysin- and toxin-associated determinants. Phylogenomically, the isolate was most closely related to the environmental strain RFB16 and was distinct from the pandemic reference lineages included in the analysis. The V. parahaemolyticus isolate was assigned to ST98 and OL4 and carried a divergent KL55-like K-antigen locus. Its virulence-associated repertoire was dominated by a well-conserved Type III secretion system 1, together with adhesion-, motility-, and hemolysin-associated determinants, whereas the major enteropathogenic markers tdh and trh were absent. It grouped most closely with strain vp-HL-202008, although this relationship received moderate phylogenetic support. Resistance-associated determinants were predominantly chromosomal, and prophage analysis identified two putative regions in V. cholerae and seven in V. parahaemolyticus. Overall, these findings characterize two Vibrio isolates recovered from commercially depurated bivalves marketed in Portugal. Their detection does not demonstrate depuration failure or an immediate consumer-health risk but highlights that conventional indicator-based compliance does not necessarily exclude autochthonous Vibrio spp. and supports the value of genomic surveillance in seafood safety.

Cycloviruses are circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses detected in a wide range of biological and environmental samples, although the natural hosts and biological significance of many members of the genus remain poorly understood. Information regarding their occurrence in cattle, particularly in fecal material, remains limited. In this study, 100 bovine fecal samples collected from 21 farms in northern Portugal were screened for circovirus- and cyclovirus-related DNA using a nested PCR assay targeting a conserved region of the replication-associated protein (rep) gene. Amplicons were characterized by bidirectional Sanger sequencing and maximum-likelihood phylogenetic analysis. Cyclovirus-related DNA was detected in 14/100 samples, corresponding to a molecular occurrence of 14.0% (cluster-bootstrap 95% CI: 5.6–26.5%), with positive animals originating from 7/21 farms. The obtained partial rep sequences showed 94.8–100% nucleotide identity with Crocidura russula feces-associated Cyclovirus MAVG5 (PQ576920), and five sequences were identical to the corresponding homologous region. Phylogenetic analysis grouped all bovine-derived sequences with MAVG5 within a broader lineage containing Human-associated Cyclovirus 6. In an exploratory GEE analysis accounting for within-farm clustering, no statistically significant association was observed between cyclovirus detection and diarrhea (GEE: OR = 0.76, 95% CI: 0.31–1.86; p = 0.549). These findings expand current knowledge of Cyclovirus-related DNA in livestock-associated fecal material and indicate the occurrence of closely related Cyclovirus sequences in distinct mammalian-associated matrices within the Iberian Peninsula. However, detection in feces alone does not establish cattle as the biological host or demonstrate active infection. Complete-genome characterization and broader host and environmental surveillance are required to clarify the taxonomy, host range, and ecological significance of these viruses.

Tick-associated viruses are an underexplored component of global viromes, and highly divergent RNA viruses often remain undetected due to low abundance and sequence divergence. Tacheng tick virus 8 (TcTV8) is a poorly characterized RNA flavivirus-like virus originally reported from Dermacentor-associated ticks in China, with few subsequent reports and no confirmed detections outside Asia. Here, we report the detection and partial genomic characterization of a TcTV8-like virus in ticks collected from Portugal. Sequence-independent (SISPA) nanopore sequencing of individual ticks recovered three partial fragments (~2.7 kb, ~14% of the reference genome; ~30% of the reference covered at ≥1×), which shared high amino-acid identity with the TcTV8 polyprotein, including a methyltransferase-region domain. Phylogenetic analysis placed the Portuguese sequence within the TcTV8 lineage. Read-level classification and coverage analysis further support the presence of this virus in the positive sample. These findings represent, to our knowledge, the first detection of a TcTV8-like virus in European ticks, extending its known geographic range beyond Asia. The detection of this cryptic viral lineage highlights the need for broader tick virome surveillance to better understand the diversity, evolution, and ecology of flavivirus-like viruses.

A total of 100 fecal samples collected from cattle in the district of Viana do Castelo, northern Portugal, were molecularly screened for Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. This study represents the first molecular survey of enteric protozoa in cattle from the district of Viana do Castelo, northern Portugal. Blastocystis sp. was the most frequently detected protozoan and exhibited considerable subtype diversity, including the zoonotic subtype ST3 and the potentially zoonotic subtypes ST10a and ST14, a circumstance highlighting the occurrence in cattle of Blastocystis subtypes with potential public health relevance and the need to further investigate their cross-host transmission dynamics. Giardia duodenalis was detected at a relatively low occurrence, although unsuccessful multilocus sequence typing (MLST) amplification prevented assessment of its assemblages and zoonotic potential. Cryptosporidium spp. were not detected, likely reflecting the predominance of adult cattle in the sampled population. Overall, these findings contribute to the understanding of the molecular epidemiology of enteric protozoa in Portuguese cattle and provide updated baseline geographical data for northern Portugal. Continued molecular surveillance using optimized genotyping approaches and larger, more representative sample sets will be essential to better characterize the diversity, epidemiology, and zoonotic importance of these parasites within a One Health framework.

We present a genome assembly of Thelazia callipaeda (nematode; Nematoda; Chromadorea; Rhabditida; Thelaziidae). The genome sequence has a total length of 117.59 megabases. Most of the assembly (75.82%) is scaffolded into 4 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has been assembled, with a length of 13.66 kilobases.

Filter-feeding bivalves can accumulate fecal bacteria and antimicrobial resistance determinants, yet routine regulatory monitoring relies mainly on quantitative Escherichia coli criteria. This study provides a whole-genome characterization of an ExPEC-like E. coli ST536 (O21) isolate recovered from commercially depurated retail mussels originating from a Class B harvesting area in Portugal. The isolate was phenotypically identified by MicroScan WalkAway Plus and subjected to antimicrobial susceptibility testing and Oxford Nanopore long-read sequencing, followed by genome assembly, annotation, antimicrobial resistance and virulence screening, plasmid and prophage detection, and phylogenomic analysis. Phenotypically, only resistance to tobramycin was detected using the MicroScan WalkAway system. Genomic analysis revealed a complex mobile element-rich architecture, including multiple resistance-associated determinants, 116 virulence-associated loci, prophage regions, and a mobilizable IncY plasmid carrying CTX-like class A β-lactamase-associated hits detected by ABRicate. Despite its ST536 assignment and ExPEC-associated traits, phylogenomic analysis placed the isolate within a phylogroup A/commensal-like background rather than among classical B2 ExPEC lineages, suggesting acquisition of pathoadaptive modules by an atypical environmental lineage. These findings highlight the potential utility of WGS as a complementary tool and suggest that depurated bivalves may serve as sentinels, warranting further investigation.

Tularemia is a zoonotic disease caused by the highly contagious bacterium Francisella tularensis, with rabbits, hares, and rodents considered as primary reservoirs. Clinical manifestations in humans can range from mild to life-threatening, depending on the mode of exposure (ingestion, inhalation, skin contact, injection, or tick bite), with high fever being a common feature. The primary aim of the present research was to evaluate the circulation of F. tularensis and the potential infection among wild boar (Sus scrofa) and red deer (Cervus elaphus) from the central region of Portugal. A total of 368 samples (including serum and organ samples) were collected from 184 wild boar and 184 red deer. For serological analysis, two commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to detect immunoglobulin M (IgM) and G (IgG) antibodies to F. tularensis. Antibodies to F. tularensis (IgG and IgM) were detected in one adult male wild boar, corresponding to a seroprevalence of 0.54% (1/184, 95% confidence interval [95% CI]: 0.01–3.0%). No antibodies to F. tularensis were detected in red deer. Molecular detection by PCR was negative in the seropositive animal, in which submandibular lymph node, liver, and spleen samples were analysed targeting the 16S rRNA gene. These findings indicate exposure of wild boar to F. tularensis in central Portugal, suggesting a sporadic presence of the pathogen. Although no evidence of active infection was detected in the analysed tissues, the presence of seropositive individuals highlights the need for further investigation. Despite the very low seroprevalence observed, the zoonotic potential of F. tularensis supports the importance of continued surveillance within a One Health perspective.