Silvia De Carli

Virulence gene content in Escherichia coli isolates from poultry flocks with clinical signs of colibacillosis in Brazil

Escherichia coli is a commensal bacterium of the birds intestinal tract, but it can invade different tissues resulting in systemic symptoms (colibacillosis). This disease occurs only when the E. coli infecting strain presents virulence factors (encoded by specific genes) that enable the adhesion and proliferation in the host organism. Thus, it is important to differentiate pathogenic (APEC, avian pathogenic E. coli) and non-pathogenic or fecal (AFEC, avian fecal E. coli) isolates. Previous studies analyzed the occurrence of virulence factors in E. coli strains isolated from birds with colibacillosis, demonstrating a high frequency of the bacterial genes cvaC, iroN, iss, iutA, sitA, tsh, fyuA, irp-2, ompT and hlyF in pathogenic strains. The aim of the present study was to evaluate the occurrence and frequency of these virulence genes in E. coli isolated from poultry flocks in Brazil. A total of 138 isolates of E. coli was obtained from samples of different tissues and/or organs (spleen, liver, kidney, trachea, lungs, skin, ovary, oviduct, intestine, cloaca) and environmental swabs collected from chicken and turkey flocks suspected to have colibacillosis in farms from the main Brazilian producing regions. Total DNA was extracted and the 10 virulence genes were detected by traditional and/or real-time PCR. At least 11 samples of each gene were sequenced and compared to reference strains. All 10 virulence factors were detected in Brazilian E. coli isolates, with frequencies ranging from 39.9% (irp-2) to 68.8% (hlyF and sitA). Moreover, a high nucleotide similarity (over 99%) was observed between gene sequences of Brazilian isolates and reference strains. Seventy-nine isolates were defined as pathogenic (APEC) and 59 as fecal (AFEC) based on previously described criteria. In conclusion, the main virulence genes of the reference E. coli strains are also present in isolates associated with colibacillosis in Brazil. The analysis of this set of virulence factors can be used to differentiate between APEC and AFEC isolates in Brazil.

Oseltamivir-resistant influenza A(H1N1)pdm09 virus in southern Brazil.

The neuraminidase (NA) genes of A(H1N1)pdm09 influenza virus isolates from 306 infected patients were analysed. The circulation of oseltamivir-resistant viruses in Brazil has not been reported previously. Clinical samples were collected in the state of Rio Grande do Sul (RS) from 2009-2011 and two NA inhibitor-resistant mutants were identified, one in 2009 (H275Y) and the other in 2011 (S247N). This study revealed a low prevalence of resistant viruses (0.8%) with no spread of the resistant mutants throughout RS.

Draft Genome Sequence of a Salmonella enterica subsp. enterica Serovar Gallinarum bv. Gallinarum Isolate Associated with Fowl Typhoid Outbreaks in Brazil

ABSTRACT Salmonella enterica subsp. enterica serovar Gallinarum bv. Gallinarum strains are bird pathogens causing fowl typhoid (FT). Isolate BR_RS12 was obtained from a poultry flock with FT in 2014. The sequencing of this genome will enable to track the origin of the recent outbreaks in Brazil.

Performance of direct immunofluorescence assay for the detection of human metapneumovirus under clinical laboratory settings

INTRODUCTION Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. METHODS Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. CONCLUSIONS DIF is not very sensitive under clinical laboratory settings.

Taqman Real-Time PCR Assays for Rapid Detection of Avian Pathogenic Escherichia coli Isolates

SUMMARY Avian pathogenic Escherichia coli (APEC) isolates are currently differentiated from nonpathogenic strains by classical PCR of virulence genes. This study improves the detection of the five main virulence genes used for APEC detection with the development of duplex and single Taqman real-time PCR to these targets. Primers and probes targeted to ompT, hlyF, iroN, iutA, and iss genes were designed and used in the implementation of single (iss) and duplex (hlyF/ompT and iroN/iutA) Taqman PCR assays. All five virulence genes of E. coli strains were successfully detected by classical and Taqman real-time (single and duplex) PCR. A panel of 111 E. coli isolates, obtained from avian samples collected in different Brazilian regions between 2010 and 2011, were further tested by both assays. Complete agreement was observed in the detection of four genes, ompT, hlyF, iron, iutA, but not for iss. This issue was addressed by combining the forward primer of the classical PCR to the new iss reverse primer and probe, resulting in complete agreement for all five genes. In total, 61 (55%) Brazilian E. coli isolates were detected as APEC, and the remaining 50 (45%) as avian fecal E. coli (AFEC). In conclusion, classical and Taqman real-time PCR presented exactly the same analytical performance for the differentiation of APEC and AFEC isolates. The developed real-time Taqman PCR assays could be used for the detection and differentiation of APEC isolates. RESUMEN Nota de Investigación- Métodos de PCR en tiempo real TaqMan para la detección rápida de aislamientos de Escherichia coli patógenos para las aves. Los aislamientos de Escherichia coli patógenos para las aves (con las siglas en inglés APEC) actualmente se pueden diferenciar de cepas no patógenas mediante métodos de PCR clásicos para genes de virulencia. Este studio actual mejoró la detección de los cinco genes de virulencia principales utilizados para la detección de APEC con el desarrollo métodos de PCR en tiempo real TaqMan de tipo dúplex y sencillo para estas secuencias objetivo. Los iniciadores y sondas específicas para ompT, hlyF, iroN, iutA, y los genes iss fueron diseñados y utilizados en la ejecución de un ensayo de PCR en tiempo real TaqMan simple (iss) y dúplex (hlyF/ompT and iroN/iutA). Los cinco genes de virulencia de cepas de E. coli se detectaron con éxito por PCR clásico y en tiempo real TaqMan (simple y dúplex). Un panel de 111 aislamientos de E. coli, obtenidos a partir de muestras de aves, recolectadas de diferentes regiones de Brasil entre los años 2010 y 2011, se analizaron mediantes ambos ensayos. Se observó concordancia completa en la detección de cuatro genes, ompT, hlyF, iroN, iutA, pero no para iss. Este problema fue solucionado mediante la combinación del iniciador delantero de PCR clásico con el iniciador de reversa y la sonda del gene iss, lo que resulta en una concordancia completa para los cinco genes. En total, 61 (55%) de aislamientos brasileños de E. coli se detectaron como APEC y los 50 restantes (45%) se identificaron con cepas de E. coli aviares de origen fecal (AFEC). En conclusión, el método de PCR clásico y en tiempo real TaqMan presentan exactamente el mismo rendimiento analítico para la diferenciación de aislamientos de E. coli patógenos para aves y de origen fecal. El ensayo de PCR en tiempo real TaqMan podría ser utilizado para la detección y diferenciación de aislamientos de E. coli patógenos para aves.

Molecular and phylogenetic analyses of Salmonella Gallinarum trace the origin and diversification of recent outbreaks of fowl typhoid in poultry farms

Fowl typhoid (FT) and pullorum disease (PD) are two important poultry infections caused by Salmonella enterica subsp. enterica serotype Gallinarum (S. Gallinarum). S. Gallinarum strains are adapted to birds and classified into biovars Gallinarum (bvGA) and Pullorum (bvPU) as they are the causative agent of FT and PD, respectively. In Brazil, FT/PD outbreaks have been reported along the last 50 years, but there was a recent increase of FT field reports with the suspicion it could be due to virulence reversion of the attenuated live vaccine SG9R. In this study, we applied molecular biology assays and phylogenetic methods to detect and investigate S. Gallinarum isolates from commercial poultry flocks in order to understand the evolutionary history and origin of the recent FT outbreaks in Brazil. S. Gallinarum isolates were obtained from thirteen different poultry flocks with clinical signs of FT/PD from 2013 to 2015. These isolates were serotyped, tested with three specific PCR (for the detection of bvGA, bvPU and live vaccine strain SG9R) and submitted to sequencing of a variable genome region (ISR analysis). The complete genome of one bvGA strain (BR_RS12) was also compared to other S. Gallinarum complete genomes (including other two Brazilian ones: bvGA 287/91 and bvPU FCVA198). PCR detected all thirteen isolates as S. Gallinarum (eight bvGA and five bvPU), none positive for SG9R strain. ISR analysis revealed that all eight bvGA isolates showed exactly the same nucleotide sequences with 100% similarity to reference strains, while two patterns were observed for bvPU. Genome phylogeny demonstrated distinct clades for bvGA and bvPU, with the bvGA clade showing a clear subdivision including three genomes: SG9R vaccine, the respective SG9 parent strain and one SG9R revertant field isolate (MB4523). The evolutionary rate of the total S. Gallinarum genome was calculated at 6.15×10-7 substitutions/site/year, with 2.8 observed substitutions per year per genome (1 SNP per 4292 bases). Phylodynamics analysis estimated that at least two introductions of S. Gallinarum bvGA happened in Brazil, the first in 1885 and the second in 1950. The Brazilian bvGA genomes 287/91 and BR_RS12 analyzed here were related to the early and the late introductions, respectively. In conclusion, these results indicate the occurrence of S. Gallinarum strains associated with FT outbreaks that have been circulating for more than 50 years in Brazil and are not originated from virulence reversion of the SG9R vaccine.

Corrigendum to “Molecular and phylogenetic analyses of Salmonella Gallinarum trace the origin and diversification of recent outbreaks of fowl typhoid in poultry farms” [Vet. Microbiol. 212 (2017) 80–86]

Corrigendum to “Molecular and phylogenetic analyses of Salmonella Gallinarum trace the origin and diversification of recent outbreaks of fowl typhoid in poultry farms” [Vet. Microbiol. 212 (2017) 80–86] Silvia De Carli, Tiago Gräf, Diéssy Kipper, Fernanda Kieling Moreira Lehmann, Nathalie Zanetti, Franciele Maboni Siqueira, Samuel Cibulski, André Salvador Kazantzi Fonseca, Nilo Ikuta, Vagner Ricardo Lungea,e,

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.