Carrier state of enterotoxigenic Escherichia coli virulence markers in pigs: Effects on gut microbiota modulation and immune markers transcription.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs at early age, leading to high mortality rates and significant economic losses in the swine industry. ETEC effect on gut microbiota and immune system is mostly studied in diarrheic model under controlled laboratory conditions, however its impact on asymptomatic carriers remains unknown. Thus, we investigated whether ETEC can modulate gut microbiota or regulate the transcription of immune markers in asymptomatic pigs in farm environment. Stool samples from newborn piglets, nursery and growing pigs, and sows were screened for ETEC markers, then submitted to 16S-rDNA sequencing to explore gut microbiota composition in carriers (ETEC+) and non-carriers (ETEC-) animals. We observed a reduced α-diversity in ETEC+ animals (p < 0.05), while bacterial compositions were mostly driven by ageing (p > 0.05). Prevotella marked ETEC-carrier group, while Rikenellaceae RC9 gut group was a marker for a healthy gut microbiota, suggesting that they might be biomarker candidates for surveillance and supplementation purposes. Furthermore, we observed transcription regulation of il6 and tff2 genes in ETEC+ in newborn and nursery stages, respectively. Our findings indicate that ETEC presence modulate gut microbiota and the immune response in asymptomatic pigs; nevertheless, further studies using a probabilistic design must be performed to assess the effect of ETEC presence on gut imbalance in pigs despite the age bias.

Evolution and genomic profile of Salmonella enterica serovar Gallinarum biovar Pullorum isolates from Brazil.

Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is a pathogenic bacterium that causes Pullorum disease (PD). PD is an acute systemic disease that affects young chickens, causing white diarrhea and high mortality. Although many sanitary programs have been carried out to eradicate S. Pullorum, PD outbreaks have been reported in different types of birds (layers, broilers, breeders) worldwide. This study aimed to evaluate the evolution and genetic characteristics of S. Pullorum isolated from PD in Brazil. Phylogenetic analysis of S. Pullorum genomes sequenced in this study and available genomic databases demonstrated that all isolates from Brazil are from sequence type 92 (ST92) and cluster into two lineages (III and IV). ColpVC, IncFIC(FII), and IncFII(S) were plasmid replicons frequently found in the Brazilian lineages. Two resistance genes (aac(6')-Iaa, conferring resistance to aminoglycoside, disinfecting agents, and antiseptics (mdf(A)) and tetracycline (mdf(A)) were detected frequently. Altogether, these results are important to understand the circulation of S. Pullorum and, consequently, to develop strategies to reduce losses due to PD.

Evolución y perfil genómico de aislados de Salmonella enterica serovar Gallinarum biovar Pullorum de Brasil. Salmonella enterica subespecie enterica serovar Gallinarum biovar Pullorum (S. Pullorum) es una bacteria patógena que causa la enfermedad de Pullorum (EP). La EP es una enfermedad sistémica aguda que afecta a los pollos jóvenes causando diarrea blanca y alta mortalidad. Aunque se han llevado a cabo muchos programas sanitarios para erradicar S. Pullorum, se han informado brotes de EP en diferentes tipos de aves (ponedoras, pollos de engorde, reproductoras) en todo el mundo. Este estudio tuvo como objetivo evaluar la evolución y las características genéticas de S. Pullorum aislado de EP en Brasil. El análisis filogenético de los genomas de S. Pullorum secuenciados en este estudio y las bases de datos genómicas disponibles demostraron que todos los aislamientos de Brasil son del tipo de secuencia 92 (ST92) y se agrupan en dos linajes (III y IV). ColpVC, IncFIC (FII) e IncFII(S) fueron replicones de plásmidos frecuentemente encontrados en los linajes brasileños. Dos genes de resistencia (aac(6')-Iaa, que confiere resistencia a aminoglucósidos, desinfectantes y antisépticos (mdf(A)), y tetraciclina (mdf(A)) fueron detectados con frecuencia. En conjunto, estos resultados son importantes para comprender la circulación de S. Pullorum y, en consecuencia, desarrollar estrategias para reducir las pérdidas por EP.

Alternative amplicon-PCR protocol for maximizing bacterial and fungal sequencing in low-biomass samples.

Determining bacterial and fungal communities from low-biomass samples remains a challenge for high-throughput sequencing. Due to the low microbial load and host contamination, some sites, including the female upper reproductive tract and the lower respiratory tract, were even considered sterile until recent years. Despite efforts to improve sampling and DNA isolation protocols, some samples provide insufficient microbial DNA input for library preparation and sequencing. Herein, we propose an alternative amplicon-PCR protocol to be used in bacterial and fungal sequencing in low-biomass samples, targeting 16S-rDNA and the internal transcribed spacer region (ITS), respectively. Similar to a nested-PCR, we performed two sequential PCR reactions to maximise the target amplicon. We compared metagenomic results from the original Illumina protocol (Protocol 1 - P1) and the alternative one (Protocol 2 - P2), using a mock community and clinical samples with different microbial loads. Our findings showed no significant differences in data generated by P1 and P2, indicating that the second amplification round does not bias the microbiota diversity rates. Thus, the alternative protocol can be applied for low-biomass samples when the original protocol results in spurious output, preventing library preparation and sequencing.

A cross-sectional study on Campylobacter fetus subsp. venerealis prevalence and associated factors in Brazilian southern cattle farms.

Cattle farming is a major livestock activity with economic relevance in Rio Grande do Sul (RS), Brazil. However, this activity is still considered of intermediate to low technological level, and in this region, there are few epidemiologic reports of Campylobacter fetus subsp. venerealis (Cfv), the causative agent of bovine genital campylobacteriosis (BGC). Thus, we designed a cross-sectional study to assess the prevalence and Cfv-associated factors in cattle farms in RS, Brazil. In total, 99 farms were randomly selected to participate in the survey. Preputial mucus samples from selected bulls were collected twice (within a 15-day interval) and subjected to Cfv molecular detection. A farm was considered positive when at least one sample was positive for Cfv. Our findings indicate that the farm-level Cfv prevalence in RS is 67.67%. On average, the chance of a farm using natural service to be Cfv-positive increased approximately twice compared to farms that do not use natural service. We also determined that Cfv routine tests reduce the chance of a farm being positive by 92%. Therefore, both Cfv detection tests and the reduction of natural services decrease the chance of a farm being positive for Cfv. Finally, we conclude that Cfv is widely spread in Southern Brazil cattle farms and it is urgent the implementation of control measures to reduce Cfv prevalence in the target population.

Virulent potential of methicillin-resistant and methicillin-susceptible Staphylococcus pseudintermedius in dogs.

Staphylococcus pseudintermedius is a zoonotic pathogen responsible for several infectious diseases in pet animals, yet its pathogenic potential is not fully understood. Thus, this study aims to unravel the virulence profile of S. pseudintermedius from canine origin. Methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains were isolated from different infection sites and their genotypic and phenotypic features were compared to determine the clinical implications of MRSP and MSSP strains. Bacterial identification was performed using MALDI-TOF and 16S-rDNA sequencing. In addition, we used multilocus sequence typing (MLST) for strains' sequence type (ST) determination and phylogenetic relationship. The strains were screened for toxin genes, including cytotoxins (lukS, lukF), exfoliative toxin (siet), enterotoxins (sea, seb, sec, secCanine, sel, sem, and seq) and toxic shock syndrome toxin (tst-1). In vitro phenotypic analyses assessing antimicrobial susceptibility profile, biofilm formation ability, and expression of extracellular matrix components were performed. The investigated S. pseudintermedius strains belong to 17 unique ST, most of which were classified as ST71. MSSP and MRSP strains shared siet, lukS, and lukF virulence markers. Our findings showed that some MSSP strains also harbored sel, seq, and sem enterotoxin genes, suggesting a more diverse virulence profile. All MRSP strains and 77% of MSSP strains were classified as multidrug resistant (MDR). Moreover, all investigated S. pseudintermedius strains showed strong biofilm formation ability. In summary, our findings highlight the wide spread of highly virulent and drug-resistant zoonotic S. pseudintermedius strains, being a potential concern for One Health issues.

Characterization of the cervicovaginal microbiota of female beef cattle harboring Campylobacter fetus subsp. venerealis using 16S rDNA gene sequencing.

Bovine genital campylobacteriosis (BGC) is a leading cause of return to estrus in cows. The etiologic agent, Campylobacter fetus subsp. venerealis (Cfv) is transmitted by venereal route. Hence, the surrounding reproductive tract microenvironment could play a role in return to estrus in cows. The presence of Cfv in cervicovaginal mucus of Angus breed females provide three experimental groups, which were subject to bacteriome analyses: 10 Cfv-positive cows (CVP), 10 Cfv-negative cows (CVN), and 10 nonsexually active heifers (NSA). Cows with return to estrus showed higher bacterial richness than NSA. Beta diversity analysis showed a significant difference (P = 0.006) in bacterial composition among the three groups analyzed (CVP, CVN, and NSA). However, no significant difference was found when comparing the CVP versus CVN groups. Ureaplasma and Pseudomonas were the genera most frequently observed in NSA, being Ureaplasma the predictor genus to that group, whereas Alistipes, Bacteroides, Rikenellaceae_RC9_gut_group, UCG-005, and UCG-10 were both significantly more abundant and predictors genera in cows with return to estrus. Our results provide an overview of the cervicovaginal bacterial microbiota in cows harboring Cfv and improve the knowledge of the pathogenesis of BGC.

Emergence, Dissemination and Antimicrobial Resistance of the Main Poultry-Associated Salmonella Serovars in Brazil.

Salmonella infects poultry, and it is also a human foodborne pathogen. This bacterial genus is classified into several serovars/lineages, some of them showing high antimicrobial resistance (AMR). The ease of Salmonella transmission in farms, slaughterhouses, and eggs industries has made controlling it a real challenge in the poultry-production chains. This review describes the emergence, dissemination, and AMR of the main Salmonella serovars and lineages detected in Brazilian poultry. It is reported that few serovars emerged and have been more widely disseminated in breeders, broilers, and layers in the last 70 years. Salmonella Gallinarum was the first to spread on the farms, remaining as a concerning poultry pathogen. Salmonella Typhimurium and Enteritidis were also largely detected in poultry and foods (eggs, chicken, turkey), being associated with several human foodborne outbreaks. Salmonella Heidelberg and Minnesota have been more widely spread in recent years, resulting in frequent chicken/turkey meat contamination. A few more serovars (Infantis, Newport, Hadar, Senftenberg, Schwarzengrund, and Mbandaka, among others) were also detected, but less frequently and usually in specific poultry-production regions. AMR has been identified in most isolates, highlighting multi-drug resistance in specific poultry lineages from the serovars Typhimurium, Heidelberg, and Minnesota. Epidemiological studies are necessary to trace and control this pathogen in Brazilian commercial poultry production chains.

Multidrug-resistant Escherichia coli from free-living pigeons (Columba livia): Insights into antibiotic environmental contamination and detection of resistance genes.

Bacterial resistance is a public and one health problem. Free-living birds can be reservoirs of multidrug-resistant bacteria and resistance genes. This study aimed to characterize the antimicrobial resistance of Escherichia coli isolated from free-living urban pigeons (Columba livia) in South Brazil. Ninety-two animals were sampled, and one isolate was obtained from each one. The isolates were characterized, and the antimicrobial resistance profile and beta-lactam and colistin resistance genes were investigated. The isolates were classified as phylogroups B1 (35%), B2 (33%), A (16%) and D (16%), and 14% of the strains had the eae virulence gene. All isolates were resistant to at least one antimicrobial, and 63% of them were multidrug-resistant. Geographical location where the pigeons were captured and presence of the eae gene were associated with multidrug resistance. blaVIM and mcr-1 genes were detected in one and two isolates, respectively. This is the first report of these genes in E. coli of pigeons. The blaVIM -positive isolate was classified as Shiga toxin-producing E. coli, and the isolates with mcr-1 were classified as Enterohaemorrhagic E. coli and Enteropathogenic E. coli, which raise additional concerns related to public health since these are zoonotic pathotypes. The results reveal that pigeons carry multidrug-resistant pathogenic E. coli, which may interest public health. Nonetheless, further studies on whether these animals are sources of contamination for humans must be performed to understand their role in spreading antimicrobial resistance.

Wild capybaras as reservoir of shiga toxin-producing Escherichia coli in urban Amazonian Region.

Capybaras are rodent widely distributed in South America, which inhabit lakeside areas including ecological parks and urban sites. Due to anthropological interaction, monitoring zoonotic pathogens in wildlife is essential for One Health. We investigated faecal samples from capybaras living in an urban area in Rio Branco (Acre, Brazil) for the presence diarrhoeagenic E. coli. Virulence factors from shiga toxin-producing E. coli (STEC), enterohaemorrhagic E. coli (EHEC), and enteropathogenic E. coli (EPEC) were screened by PCR. We detected at least one virulence factor in 81% of the animals, being classified as STEC and EHEC pathotypes. The presence of zoonotic E. coli in capybaras is a warning due to the highly frequent anthropological interactions with wild animals in this area. Our findings highlight the importance of investigating wild animals as carriers of zoonotic E. coli, requiring further investigations into wildlife surveillance and epidemiological monitoring.

Genetic and molecular Omp25 analyses from worldwide Brucella canis strains: Possible mutational influences in protein function.

Brucella canis is responsible for canine brucellosis, a neglected zoonotic disease. The omp25 gene has been described as an important marker for Brucella intra-species differentiation, in addition to the ability to interact with the host immune system. Therefore, this study investigated the omp25 sequence from B. canis strains associated to a phylogenetic characterization and the unveiling of the molecular structure. In vitro analyses comprised DNA extraction, PCR, and sequencing of omp25 from 19 B. canis strains. Moreover, in silico analyses were performed at nucleotide level for phylogenetic characterization and evolutionary history of B. canis omp25 gene; and in amino acid level including modeling, dynamics, and epitope prediction of B. canis Omp25 protein. Here, we identified a new mutation, L109P, which diverges the worldwide omp25 sequences in two large branches. Interestingly, this mutation appears to have epidemiology importance, based on a geographical distribution of B. canis strains. Structural and molecular dynamics analyses of Omp25 revealed that Omp25L109P does not sustain its native ß-barrel. Likewise, the conformation of B-cell epitope on the mutated region was changed in Omp25L109P protein. Even without an evolutive marker, the new identified mutation appears to affect the basic function of B. canis Omp25 protein, which could indicate virulence adaptation for some B. canis strains in a context of geographical disposition.

Cows' reproductive performances and parity order influences the cervicovaginal fungal community.

Fungi have already been described as etiological agents of reproductive diseases such as endometritis and infertility in cows. However, few studies have been developed to elucidate the entire cervicovaginal fungal communities in cows. Therefore, our study aimed to characterize the fungal community present in the cervix of cows with different reproductive performances. Cervicovaginal mucus was collected from 36 Angus breed cows (1.5-12 years old) on a commercial beef cattle ranch. Twenty-one cows had a history of infertility in the year prior to the collection, showing early return to estrus. Ten cows were sampled at 60-70 days postpartum being considered fertile cows. Additionally, five non-sexually active heifers were employed as control group. Ascomycota and Basidiomycota were the predominant fungal phyla in the analyzed animals. Diversity metrics of the cervicovaginal fungal community revealed statistical differences in the composition of the fungal community among infertile cows, fertile cows and non-sexually active heifers. In addition, the cervicovaginal fungal microbiota had significative increased richness and evenness in nulliparous cows and non-sexually active heifers, while in multiparous cows a decreased richness and evenness of the fungal microbiota were identified. These results provide an unprecedented understanding of the cervicovaginal fungal structure associated with infertility and parity order.

Avian Reoviruses in Poultry Farms from Brazil.

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.

Pet Pyometra: Correlating Bacteria Pathogenicity to Endometrial Histological Changes.

Pyometra is a life-threatening infectious disease that frequently affects bitches and queens. Although histopathological patterns of pyometra have been extensively explored, the microbiological aspects, such as bacteria pathogenicity, have not been correlated to microscopy endometrial lesions so far. In this study, these two pathological aspects of pyometra were analysed and correlated. Uterus fragments and intrauterine content samples were collected from pets diagnosed with pyometra (30) and submitted to histopathology analysis and bacterial culture, respectively. The degree of endometrial histopathological lesions in pyometra cases were classified as mild, moderate and severe. Thirty different bacteria isolates were identified from intrauterine content culture. Escherichia coli (E. coli) was pure isolated in 57.7% and highly related to severe endometrial lesions. Immunohistochemistry assay revealed the adhesion and invasion of this bacteria agent to the injured endometrium. Virulence aspects of these E. coli strains were explored, demonstrating biofilm formation ability and a set of virulence genes in most isolates. These results support the adaptive genetic and phenotypic advantages of E. coli for uterus infection, and justify the high frequency of this agent involved in pyometra cases.

Temporal evolution and global spread of hepatitis B virus genotype G.

Hepatitis B virus (HBV) infection is considered a major health problem in the world. HBV is classified into genotypes A to J disseminated worldwide. Genotypes A, D and F are the most frequent in the Western World, B and C are predominant in the East, and E, F, H and J are infrequent and restricted to specific regions. HBV-G is a rare genotype, but it has been detected in different continents. This study aimed to report the temporal evolution and global spread of HBV-G comparing whole-genome sequences of this genotype from different regions in the world. Bayesian coalescent analysis was performed to estimate the time to the most recent common ancestor (tMRCA) and the population dynamics in the last decades. The results demonstrated that tMRCA of all HBV-Gs dated back to 1855 (95% highest posterior density interval [HPD 95%]: 1778 - 1931). This genotype has a possible origin in North America and it was disseminated to other continents (South and Central America, Europe, Asia and Africa) more than one century later (around the 1970s). The viral population demonstrated constant spreading from 1855 to the 1980s, followed by an increase in the 1990s and reached a plateau after the 2000s. Wide spreading at the beginning of the 1990s was probably associated with the dissemination by highly sexual active groups and injecting drug users. In conclusion, the present study demonstrated that HBV-G was originated in the 19th century with main events of spread at the end of the 20th century.

Frozen bovine preputial mucus as a suitable sample for the direct molecular diagnosis of Campylobacter fetus subsp. venerealis.

Long transportation times remain a challenge to the satisfactory diagnosis of Campylobacter fetus subsp. venerealis (Cfv). Here we demonstrated that samples of frozen bovine preputial mucus maintained at -20 °C for 10 days can be used as an alternative source for molecular diagnosis of Cfv. This approach will improve the analysis of this bacterium.

Genotypic characterization and molecular evolution of avian reovirus in poultry flocks from Brazil.

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.

Tracing back hepatitis B virus genotype D introduction and dissemination in South Brazil.

Hepatitis B virus (HBV) infection is widespread and it is considered a major health problem in the world. HBV is classified into genotypes and subgenotypes. HBV genotype D (HBV-D) has been detected worldwide with high prevalence in some specific regions from Europe and South America. In Brazil, this genotype is very frequent in the South region and its introduction and dissemination have been associated with European immigration (mainly Italian). The present study aimed to trace back the introduction and dissemination of HBV-D in South Brazil. Fifty-two chronic hepatitis B patients from two cities with an early history of Italian immigration in South Brazil were selected for the present study. HBV-DNA was detected, quantified and a partial genomic region (S/P overlapped genes) was amplified by PCR and sequenced for the determination of HBV genotypes/subgenotypes. HBV complete genome sequences of some selected samples were further obtained. Bayesian coalescent analyses were performed to estimate the HBV-D evolutionary dynamics. Phylogenetic analysis demonstrated the occurrence of three genotypes according the tree topology: HBV-D (n = 49; 94.2%), HBV-A (n = 2; 3.9%) and HBV-G (n = 1; 1.9%). In addition, HBV-D presented three subgenotypes: HBV-D3 (n = 39; 79.6%), HBV-D2 (n = 8; 16.3%), and HBV-D1 (n = 2; 4.1%). The Bayesian coalescent analysis demonstrated that the HBV-D was introduced in the 20th century. HBV-D3 was the first to be introduced in South Brazil, probably between 1904 and 1942. HBV-D2 and HBV-D1 were introduced later; HBV-D2 between 1946 and 1953 and HBV-D1 between 1954 and 1969. HBV-D3 spread at a high rate from the 1920s to the 1980s, while HBV-D2 showed a slower growth from the 1960s to the 1990s and HBV-D1 infections demonstrated low and constant population size across time. After the 2000s, a stationary growth was detected for all these three-D subgenotypes. HBV-D showed a high prevalence in South Brazil and this is possibly associated with the first introduction and dissemination of HBV-D3 at the beginning of the 20th century.

Genomic comparison of Clostridium chauvoei isolates from classical and visceral clinical manifestation.

Clostridium chauvoei is the etiological agent of blackleg, an infectious disease affecting cattle and small ruminants worldwide. This disease can manifest as classical blackleg, a condition in which skeletal muscles are affected and visceral blackleg, which affects the heart, sublingual muscles, and the diaphragm. The pathogenesis of the visceral form of the disease is poorly understood. The objective of this study is to determine and analyze complete genomic sequences of six C. chauvoei strains, five isolates from skeletal muscle and one isolate from a visceral case of blackleg in Brazil, to provide insights into the differences in pathogenic profiles of strains causing the different forms of disease. The full genomes of the six C. chauvoei strains were sequenced and comparative analyses were performed among these genomes and the C. chauvoei reference strain JF4335. The results of this study revealed that the genomes of the C. chauvoei strains analyzed are highly conserved; no particular differences were noted that could be associated with the two different clinical manifestations of the disease.

Salmonella serotype assignment by sequencing analysis of intergenic regions of ribosomal RNA operons.

Salmonella laboratorial detection is usually carried out by bacteriological culture and serological methods. Salmonella isolates are then classified into more than 2,650 serotypes with different somatic (O) and flagellar (H) antigenic combinations. More recently, DNA analysis methods were developed and applied for the identification of Salmonella serotypes, including intergenic spacer regions (ISRs) that separates DNA-encoding ribosomal subunits (rRNA gene) in Salmonella genomes. The present study aimed to evaluate the nucleotide diversity of the ISRs in 2 rRNA operons (rrnB and rrnH) for the assignment of Salmonella serotypes. A total of 63 Salmonella isolates (bacterial cultures) from 21 serotypes were obtained in the period of 2014 to 2017. DNA was extracted, and PCRs were used to detect the genus Salmonella and 4 important serotypes: Enteritidis, Gallinarum, Heidelberg, and Typhimurium. ISRs of the operons rrnB and rrnH were amplified by PCR and then sequenced. All sequence results were submitted to BLASTn search and were aligned in comparison to 72 Salmonella reference nucleotide sequences. The results demonstrated that 60 (95.2%) samples returned a sequence of the same serotype (determined by the traditional serological procedure) after searching in BLASTn and/or in the alignment with the reference sequences for both operons (rrnB and rrnH). These PCR-sequencing procedures had a general agreement index of 0.792 based on the Kappa analysis, 98.7% sensitivity value, 100% specificity, and 98.4% accuracy. Three different phylogenetic trees were generated from the alignments with the sequences of rrnH, rrnB, and rrnH plus rrnB and isolates clustered in specific branches according to the serotypes.

Pathological and molecular findings of avian reoviruses from clinical cases of tenosynovitis in poultry flocks from Brazil.

Avian reoviruses (ARVs) can infect a variety of species worldwide. Birds can present stunting syndrome, respiratory and/or enteric diseases, immunosuppression, malabsorption, viral arthritis/tenosynovitis, and even secondary infections by other microorganisms. Flaws in conventional vaccines and the increase in the diagnostic rate of disease in the last 5 yr suggest the emergence of pathogenic ARVs in the poultry flocks worldwide. This study aimed to characterize birds lesions and to detect/genotype ARVs from a severe outbreak of tenosynovitis in broiler poultry flocks from Brazil. Tissue samples of lesions on pelvic limbs of broiler chicken were collected in poultry flocks with a high condemnation rate of carcasses due to lesions and submitted to histological and molecular analysis. Major gross pathological lesions included marked swelling, edema, and hemorrhages. Serous exudate was present between the tendons and hock joint. Histological examination demonstrated necrosis and inflammation of muscle fibers, mixed inflammatory infiltrate was observed in subcutaneous tissue and tendon sheaths. ARVs RNA was detected in 5 samples tested by polymerase chain reaction. These samples were also genotyped and demonstrated the occurrence of strains of the ARVs lineages II and V in the flocks. These results suggest that theses field ARVs, genetically distant from previously characterized strains, are associated to tenosynovitis and present in commercial Brazilian poultry flocks.