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Mycoplasma hyopneumoniae is the primary etiologic agent of swine enzootic pneumonia (EP), in which the immune response is reduced, making pigs susceptible to secondary infections. We surveyed commercial pig herds in Brazil for viral and bacterial respiratory coinfections that could complicate EP. Over a 2-y period (2015-2016), we found that 854 of 2,206 pigs (38.7%) were positive for M. hyopneumoniae in herds from various production systems in 3 Brazilian regions (Central-West, Southeast, South). We collected samples of 321 lungs positive for M. hyopneumoniae from 169 farms to also screen for Pasteurella multocida, Actinobacillus pleuropneumoniae, Glaesserella parasuis, influenza A virus (IAV), and porcine circovirus 2 (PCV2) by real-time PCR. The prevalence of pathogens found in addition to M. hyopneumoniae varied: P. multocida (141; 43.9%), G. parasuis (71; 22.1%), PCV2 (50; 15.6%), IAV (23; 7.2%), and A. pleuropneumoniae (18; 5.6%). G. parasuis was more frequent in farrowing or nursery herds (48.7%) than in breeding and fattening herds (10% and 18.6%, respectively; p < 0.01); A. pleuropneumoniae was found only in herds on farrow-to-finish and fattening farms.

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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic spread rapidly and this scenario is concerning in South America, mainly in Brazil that presented more than 21 million coronavirus disease 2019 cases and 590 000 deaths. The recent emergence of novel lineages carrying several mutations in the spike protein has raised additional public health concerns worldwide. The present study describes the temporal spreading and evolution of SARS-CoV2 in the beginning of the second pandemic wave in Brazil, highlighting the fast dissemination of the two major concerning variants (P.1 and P.2). A total of 2507 SARS-CoV-2 whole-genome sequences (WGSs) with available information from the country (Brazil) and sampling date (July 2020-February 2021), were obtained and the frequencies of the lineages were evaluated in the period of the growing second pandemic wave. The results demonstrated the increasing prevalence of P.1 and P.2 lineages in the period evaluated. P.2 lineage was first detected in the middle of 2020, but a high increase occurred only in the last trimester of this same year and the spreading to all Brazilian regions. P.1 lineage emerged even later, first in the North region in December 2020 and really fast dissemination to all other Brazilian regions in January and February 2021. All SARS-CoV-2 WGSs of P.1 and P.2 were further separately evaluated with a Bayesian approach. The rates of nucleotide and amino acid substitutions were statistically higher in P.1 than P.2 (p < 0.01). The phylodynamic analysis demonstrated that P.2 gradually spread in all the country from September 2020 to January 2021, while P.1 disseminated even faster from December 2020 to February 2021. Skyline plots of both lineages demonstrated a slight rise in the spreading for P.2 and exponential growth for P.1. In conclusion, these data demonstrated that the P.1 (recently renamed as Gamma) and P.2 lineages have predominated in the second pandemic wave due to the very high spreading across all geographic regions in Brazil at the end of 2020 and beginning of 2021.

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Avian reovirus (ARV) is highly disseminated in commercial Brazilian poultry farms, causing arthritis/tenosynovitis, runting-stunting syndrome, and malabsorption syndrome in different meat- and egg-type birds (breeders, broilers, grillers, and layers). In Brazil, ARV infection was first described in broilers in the 1970s but was not considered an important poultry health problem for decades. A more concerning outcome of field infections has been observed in recent years, including condemnations at slaughterhouses because of the unsightly appearance of chicken body parts, mainly the legs. Analyses of the performance of poultry flocks have further evidenced economic losses to farms. Genetic and antigenic characterization of ARV field strains from Brazil demonstrated a high diversity of lineages circulating in the entire country, including four of the five main phylogenetic groups previously described (I, II, III, and V). It is still unclear if all of them are associated with different diseases affecting flocks' performance in Brazilian poultry. ARV infections have been controlled in Brazilian poultry farms by immunization of breeders and young chicks with classical commercial live vaccine strains (S1133, 1733, 2408, and 2177) used elsewhere in the Western Hemisphere. However, genetic and antigenic variations of the field isolates have prevented adequate protection against associated diseases, so killed autogenous vaccines are being produced from isolates obtained on specific farms. In conclusion, ARV field variants are continuously challenging poultry farming in Brazil. Epidemiological surveillance combined with molecular biological analyses from the field samples, as well as the development of vaccine strains directed toward the ARV circulating variants, are necessary to control this economically important poultry pathogen.

Reovirus aviares en granjas avícolas de Brasil. El reovirus aviar (ARV) está muy diseminado en las granjas avícolas comerciales brasileñas y causa artritis/tenosinovitis viral, síndrome de retraso de enanismo infeccioso y síndrome de malabsorción en diferentes tipos de aves de carne y huevos (reproductoras, pollos de engorde, parrillas y ponedoras). En Brasil, la infección por reovirus aviares se describió por primera vez en pollos de engorde en la década de 1970, pero no se consideró un problema importante de salud avícola durante décadas. En los últimos años se ha observado un resultado más preocupante de las infecciones de campo, incluidos los decomisos en las plantas de procesamiento debido a la apariencia antiestética de las partes del cuerpo de los pollos, principalmente las patas. Los análisis del desempeño de las parvadas avícolas han evidenciado pérdidas económicas adicionales para las granjas. La caracterización genética y antigénica de las cepas de campo de reovirus aviares de Brasil demostró una gran diversidad de linajes que circulan en todo el país, incluidos cuatro de los cinco grupos filogenéticos principales descritos anteriormente (I, II, III y V). Todavía no está claro si todos ellos están asociados con diferentes enfermedades que afectan el rendimiento de las parvadas en las aves de corral brasileñas. Las infecciones por reovirus aviares se han controlado en granjas avícolas brasileñas mediante la inmunización de reproductores y pollitos jóvenes con cepas vacunales vivas comerciales clásicas (S1133, 1733, 2408 y 2177) utilizadas en otras partes del hemisferio occidental. Sin embargo, las variaciones genéticas y antigénicas de los aislamientos de campo han impedido una protección adecuada contra enfermedades asociadas, por lo que se están produciendo vacunas autógenas inactivadas a partir de aislamientos obtenidos en granjas específicas. En conclusión, las variantes de campo de ARV son un desafío continuo para la avicultura en Brasil. La vigilancia epidemiológica combinada con análisis de biología molecular de las muestras de campo, así como el desarrollo de cepas de vacunas dirigidas a las variantes circulantes de los reovirus aviares, son necesarias para controlar este patógeno avícola económicamente importante.

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Salmonella enterica serovar Heidelberg is isolated from poultry-producing regions around the world. In Brazil, S. Heidelberg has been frequently detected in poultry flocks, slaughterhouses, and chicken meat. The goal of the present study was to assess the population structure, recent temporal evolution, and some important genetic characteristics of S. Heidelberg isolated from Brazilian poultry farms. Phylogenetic analysis of 68 S. Heidelberg genomes sequenced here and additional whole-genome data from NCBI demonstrated that all isolates from the Brazilian poultry production chain clustered into a monophyletic group, here called S. Heidelberg Brazilian poultry lineage (SH-BPL). Bayesian analysis defined the time of the most recent common ancestor (tMRCA) as 2004, and the overall population size (Ne) was constant until 2008, when an ∼10-fold Ne increase was observed until circa 2013. SH-BPL presented at least two plasmids with replicons ColpVC (n = 68; 100%), IncX1 (n = 66; 97%), IncA/C2 (n = 65; 95.5%), ColRNAI (n = 43; 63.2%), IncI1 (n = 32; 47%), ColMG828, Col156, IncHI2A, IncHI2, IncQ1, IncX4, IncY, and TrfA (each with n < 4; <4% each). Antibiotic resistance genes were found, with high frequencies of fosA7 (n = 68; 100%), mdf(A) (n = 68; 100%), tet(34) (n = 68; 100%), sul2 (n = 64; 94.1%), and blaCMY-2 (n = 56; 82.3%), along with an overall multidrug resistance (MDR) profile. Ten Salmonella pathogenicity islands (SPI1 to SPI5, SPI9, and SPI11 to SPI14) and 139 virulence genes were also detected. The SH-BPL profile was like those of other previous S. Heidelberg isolates from poultry around the world in the 1990s. In conclusion, the present study demonstrates the recent introduction (2004) and high level of dissemination of an MDR S. Heidelberg lineage in Brazilian poultry operations. IMPORTANCES. Heidelberg is the most frequent serovar in several broiler farms from the main Brazilian poultry-producing regions. Therefore, avian-source foods (mainly chicken carcasses) commercialized in the country and exported to other continents are contaminated with this foodborne pathogen, generating several national and international economic losses. In addition, isolates of this serovar are usually resistant to antibiotics and can cause human invasive and septicemic infection, representing a public health concern. This study demonstrates the use of whole-genome sequencing (WGS) to obtain epidemiological information for one S. Heidelberg lineage highly spread among Brazilian poultry farms. This information will help to define biosecurity measures to control this important Salmonella serovar in Brazilian and worldwide poultry operations.

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Salmonella enterica serovar Enteritidis is frequently isolated from animal-source foods associated with human salmonellosis outbreaks. This serovar was spread to animal (mainly poultry) farms worldwide in the 1980s, and it is still detected in foods produced in many countries, including Brazil. The present study reports a retrospective genome-wide comparison of S. Enteritidis from foodborne outbreaks in Southern Brazil in the last two decades. Fifty-two S. Enteritidis isolates were obtained from foodborne outbreaks occurring in different cities of the Brazilian southernmost State, Rio Grande do Sul (RS), from 2003 to 2015. Whole-genome sequences (WGS) from these isolates were obtained and comparatively analyzed with 65 additional genomes from NCBI. Phylogenetic and Bayesian analyses were performed to study temporal evolution. Genes related to antibiotic resistance and virulence were also evaluated. The results demonstrated that all S. Enteritidis isolates from Southern Brazil clustered in the global epidemic clade disseminated worldwide originally in the 1980s. Temporal analysis demonstrated that all Brazilian isolates had a tMRCA (time to most recent common ancestor) in 1986 with an effective population size (Ne) increase soon after until 1992, then becoming constant up to now. In Southern Brazil, there was a significant decrease in the spreading of S. Enteritidis in the last decade. In addition, three antibiotic resistance genes were detected in all isolates: aac(6')-Iaa, mdfA, and tet(34). These results demonstrate the high frequency of one only specific S. Enteritidis lineage (global epidemic clade) in foodborne outbreaks from Southern Brazil in the last two decades.

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In this study, we performed phylogenetic and evolutionary analysis on bovine viral diarrhea virus 1 (BVDV-1) sequences to investigate the origin and temporal diversification of different BVDV-1 subtypes. Dated phylogenies using the complete polyprotein sequence were reconstructed, and the time of the most recent common ancestor (tMRCA) was estimated. The results demonstrated that BVDV-1 subtypes clustered into two phylogenetic clades, where the predominant subtypes worldwide grouped together. In the temporal analysis, the tMRCA of BVDV-1 was 1336, and the diversification into different subtypes appears to have occurred around 363 years ago. The present results help to elucidate the origins of BVDV-1 subtypes and the dynamics of ruminant pestiviruses.

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Salmonella serotype Minnesota has been increasingly detected in Brazilian poultry farms and food products (chicken meat, eggs) in recent years. In addition, S. Minnesota isolates from poultry are generally resistant to several antibiotics and persistent in farm environments. The present study aimed to assess phylogenomic diversity of S. Minnesota isolates from the poultry production chain in Brazil. In total, 107 worldwide S. Minnesota whole genomes (including 12 from Brazil) were analyzed using a comparative approach. Phylogenetic analysis demonstrated two clades more related to poultry production in Brazil: S. Minnesota poultry lineages I and II (SM-PLI and SM-PLII). Phylodynamic analysis demonstrated that SM-PLI had a common ancestor in 1915, while SM-PLII originated circa 1971. SM-PLII encompassed a higher number of isolates and presented a recent increase in effective population size (mainly from 2009 to 2012). Plasmids IncA/C2 and ColRNA, antimicrobial resistance genes (aph(3')-Ia, blaCMY-2, qnrB19, sul2, and tet(A)) and mainly a virulence genetic cluster (including the yersiniabactin operon) were detected in isolates from SM-PLI and/or SM-PLII. This study demonstrates the dissemination of two distinct S. Minnesota lineages with high resistance to antibiotics and important virulence genetic clusters in Brazilian poultry farms.

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Avian reovirus (ARV) is one of the main causes of infectious arthritis/tenosynovitis and malabsorption syndrome (MAS) in poultry. ARVs have been disseminated in Brazilian poultry flocks in the last years. This study aimed to genotype ARVs and to evaluate the molecular evolution of the more frequent ARV lineages detected in Brazilian poultry-producing farms. A total of 100 poultry flocks with clinical signs of tenosynovitis/MAS, from all Brazilian poultry-producing regions were positive for ARV by PCR. Seventeen bird tissues were submitted to cell culture and ARV RNA detection/genotyping by two PCRs. The phylogenetic classification was based on σC gene alignment using a dataset with other Brazilian and worldwide ARVs sequences. ARVs were specifically detected by both PCRs from the 17 cell cultures, and σC gene partial fragments were sequenced. All these sequences were aligned with a total of 451 ARV σC gene data available in GenBank. Phylogenetic analysis demonstrated five well-defined clusters that were classified into lineages I, II, III, IV, and V. Three lineages could be further divided into sub-lineages: I (I vaccine, Ia, Ib), II (IIa, IIb, IIc) and IV (IVa and IVb). Brazilian ARVs were from four lineages/sub-lineages: Ib (48.2%), IIb (22.2%), III (3.7%) and V (25.9%). The Bayesian analysis demonstrated that the most frequent sub-lineage Ib emerged in the world around 1968 and it was introduced into Brazil in 2010, with increasing spread soon after. In conclusion, four different ARV lineages are circulating in Brazilian poultry flocks, all associated with clinical diseases. RESEARCH HIGHLIGHTS One-hundred ARV-positive flocks were detected in all main poultry-producing regions from Brazil. A large dataset of 468 S1 sequences was constructed and divided ARVs into five lineages. Four lineages/sub-lineages (Ib, IIb, III and V) were detected in commercial poultry flocks from Brazil. Brazilian lineages shared a low identity with the commercial vaccine lineage (I vaccine). Sub-lineage Ib emerged around 1968 and was introduced into Brazil in 2010.

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Abstract Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously known as canine astrovirus or astrovirus-like particles, has been reported in several countries to be associated with viral enteric disease in dogs since the 1980s. Astroviruses have been detected in fecal samples from a wide variety of mammals and birds that are associated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCR was used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 was detected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derived from dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses were simultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5 a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed. Phylogenetic analyses based on the ORF2 amino acid sequences, samples described herein grouped into the putative genotype 'a' closed related with Chinese samples. Other studies are required to attempt the clinical and antigenic implications of these astrovirus genotypes in dogs.

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Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously known as canine astrovirus or astrovirus-like particles, has been reported in several countries to be associated with viral enteric disease in dogs since the 1980s. Astroviruses have been detected in fecal samples from a wide variety of mammals and birds that are associated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCR was used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 was detected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derived from dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses were simultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus, canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5 a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed. Phylogenetic analyses based on the ORF2 amino acid sequences, samples described herein grouped into the putative genotype a' closed related with Chinese samples. Other studies are required to attempt the clinical and antigenic implications of these astrovirus genotypes in dogs.(AU)

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