The sharp increase of COVID-19 cases in late 2020 has made Brazil the new epicenter of the ongoing SARS-CoV-2 pandemic. The novel viral lineages P.1 (Variant of Concern Gamma) and P.2, respectively identified in the Brazilian states of Amazonas and Rio de Janeiro, have been associated with potentially higher transmission rates and antibody neutralization escape. In this study, we performed the whole-genome sequencing of 185 samples isolated from three out of the five Brazilian regions, including Amazonas (North region), Rio Grande do Norte, Paraíba and Bahia (Northeast region), and Rio de Janeiro (Southeast region) in order to monitor the spread of SARS-CoV-2 lineages in Brazil in the first months of 2021. Here, we showed a widespread dispersal of P.1 and P.2 across Brazilian regions and, except for Amazonas, P.2 was the predominant lineage identified in the sampled states. We estimated the origin of P.2 lineage to have happened in February, 2020 and identified that it has differentiated into new clades. Interstate transmission of P.2 was detected since March, but reached its peak in December, 2020 and January, 2021. Transmission of P.1 was also high in December and its origin was inferred to have happened in August 2020. We also confirmed the presence of lineage P.7, recently described in the southernmost region of Brazil, to have spread across the Northeastern states. P.1, P.2 and P.7 are descended from the ancient B.1.1.28 strain, which co-dominated the first phase of the pandemic in Brazil with the B.1.1.33 strain. We also identified the occurrence of a new lineage descending from B.1.1.33 that convergently carries the E484K mutation, N.9. Indeed, the recurrent report of many novel SARS-CoV-2 genetic variants in Brazil could be due to the absence of effective control measures resulting in high SARS-CoV2 transmission rates. Altogether, our findings provided a landscape of the critical state of SARS-CoV-2 across Brazil and confirm the need to sustain continuous sequencing of the SARS-CoV-2 isolates worldwide in order to identify novel variants of interest and monitor for vaccine effectiveness.

Quantitative real-time polymerase chain reactions (qPCRs) of the most prevalent bacteria causing foodborne diseases worldwide, such asSalmonellaspp.,Escherichia coli, andStaphylococcus aureus,can be an important tool for quantitative microbial risk assessment, which requires numerical data to determine the level of contamination at a specific stage of food production. However, most of qPCR assays described in the literature for these pathogens are qualitative; their objective is pathogen detection and not pathogen quantification. Thus, the aim of our work was to develop a qPCR for the simultaneous quantification ofSalmonellaspp.,E. coli, andS. aureusand to propose its use in the analysis of foods, as a tool for microbiological quality monitoring. For this, a multiplex qPCR was standardized for the simultaneous quantification of specific fragments of target genes (ssf,phoA, andnuc) corresponding to each one of the mentioned bacteria. The limit of detection of the technique was 13, 10, and 12 gene copies forssf,phoA, andnuc, respectively; standard curves showed R²> 0.99, with efficiencies ranging from 99 to 110%, and inter- and intraexperiment reproducibility presented a low coefficient of variation in all trials. This methodology was applied in different food matrices (milk, ground beef, and oyster meat), and the results were compared with official microbiological culture methodology and with ready-to-use test. Advantages and disadvantages of each methodology used in this study are pointed out. We suggest that this multiplex qPCR can be used as a rapid screening technique for the analysis of food microbiological quality.

The objective of this study was to verify whether Toxoplasma gondii is excreted in the milk of naturally infected sheep. In order to accomplish this, 275 lactating ewes were used; these were bred extensively in 17 estates distributed across nine cities. Polymerase chain reaction amplification was used to detect T. gondii DNA in milk samples, and the indirect immunofluorescence test was employed for the detection of anti-T. gondii IgG antibodies in the sera, with a cut-off value of 1:64. It was possible to verify the presence of the parasite DNA in 6.5% (18/275) of the studied animals. Anti-T. gondii antibodies were present in 41.5% of the animals studied (114/275). There was no correlation between parasite excretion in milk and the presence of IgG in 38.9% of the studied animals (7/18). The high seropositivity and the presence of parasite DNA in the milk led to the conclusion that T. gondii infection is present in the sheep population in southern and southwestern Bahia, and that there is a risk of the human population becoming infected due to the consumption of raw, in natura milk.

The aim of this study was to detect evidence of Toxoplasma gondii using polymerase chain reaction (PCR)-based techniques in oysters (Crassostrea rhizophorae) obtained from the southern coastal region of Bahia, Brazil. A total of 624 oysters were collected, and the gills and digestive glands were dissected. Each tissue sample was separated into pools containing tissues (of the same type) from three animals, leading to a total of 416 experimental samples for analysis (208 samples each from the gills and digestive glands). Molecular analysis using PCR-based detection of the T. gondii AF 146527 repetitive fragment yielded negative results for all samples. However, when nested-PCR was used for detection of the T. gondii SAG-1 gene, 17 samples were positive, with the gills being the tissue with maximal detection of the parasite. These positive results were confirmed by sample sequencing. It is therefore suggested that C. rhizophorae oysters are capable of filtering and retaining T. gondii oocysts in their tissue. This represents a risk to public health because they are traditionally ingested in natura.

Wild animals have an ecological function and can serve as sentinels to identify infectious agents and as indicators of environmental health. Among the zoonotic pathogens, Salmonella spp. deserve special attention due to their high worldwide prevalence and their ubiquity of hosts. With the aim of investigating the presence of Salmonella spp. in wild birds from the Atlantic Forest in southern Bahia, Brazil, we collected 114 fecal samples of wild birds (14 families) between 2016 and 2017. Fecal samples were collected by means of cloacal swab and subjected to microbiological culture to isolate and serotype Salmonella spp. specifically. Antibiotic susceptibility was determined using the disk diffusion test protocol. Only one bird, Ceratopipra rubrocapilla, tested positive for Salmonella enterica subsp. enterica serotype Agona, which is the first record for this bird species. This isolate exhibited intermediate sensitivity to amikacin and gentamicin and sensitivity to the other 13 antibiotics tested. Results may indicate environmental preservation since the studied areas had minimal human activity and good sanitary quality. Despite the low prevalence, it is necessary to monitor wildlife and establish disease control and surveillance systems, especially for zoonotic diseases.