Persistence of vaccine origin Salmonella Typhimurium through the poultry production continuum, and development of a rapid typing scheme for their differentiation from wild type field isolates.

Salmonella enterica serovar Typhimurium is one of the top Salmonella serovars annually linked to poultry production and corresponding human illnesses. Because of this, vaccination of commercial poultry against Salmonella Typhimurium has been a focal point in recent years. There are several commercially available Salmonella Typhimurium vaccines available for use in poultry production. Among these are modified live vaccines, including Poulvac ST (Zoetis), Megan Egg (AviPro), and Megan Vac 1 (AviPro). In this study, analyses of 27 field isolates of Salmonella Typhimurium from poultry sources indicated evidence for the persistence of some vaccine-origin strains through the commercial production cycle. Further analyses of 26,812 database isolates indicated vaccine-origin isolates are persisting frequently through processing, are present on retail meat products, and are even occasionally found in human patients. A novel polymerase chain reaction (PCR) was created and validated which enables simultaneous identification of Salmonella enterica sp., the Salmonella Typhimurium serovar, and differentiation of wild type Salmonella Typhimurium from live attenuated vaccines involving mutations in the cya/crp or aroA genes. The PCR was developed considering whole genome differences between the vaccines and wild type field isolates and was validated using different field isolates and recovered vaccine strains. This method enables poultry producers to rapidly determine if recovered field isolates have a vaccine origin.

Survey of clinical and commensal Escherichia coli from commercial broilers and turkeys, with emphasis on high-risk clones using APECTyper.

Molecular characterization of avian pathogenic Escherichia coli (APEC) is challenging due to the complex nature of its associated disease, colibacillosis, in poultry. Numerous efforts have been made toward defining APEC, and it is becoming clear that certain clonal backgrounds are predictive of an avian E. coli isolate's virulence potential. Thus, APEC can be further differentiated as high-risk APEC based upon their clonal background's virulence potential. However, less clear is the degree of overlap between clinical isolates of differing bird type, and between clinical and gastrointestinal isolates. This study aimed to determine genomic similarities and differences between such populations, comparing commercial broiler vs. turkey isolates, and clinical vs. gastrointestinal isolates. Differences were observed in Clermont phylogenetic groups between isolate populations, with B2 as the dominant group in turkey clinical isolates and G as the dominant group in broiler clinical isolates. Nearly all clinical isolates were classified as APEC using a traditional gene-based typing scheme, whereas 53.4% and 44.1% of broiler and turkey gastrointestinal isolates were classified as APEC, respectively. High-risk APEC were identified among 31.0% and 46.9% of broiler and turkey clinical isolates, compared with 5.7% and 2.9% of broiler and turkey gastrointestinal isolates. As found in previous studies, no specific known virulence or fitness gene sets were identified which universally differentiate between clinical and gastrointestinal isolates. This study further demonstrates the utility of a hybrid APEC typing approach, considering both plasmid content and clonal background, for the identification of dominant and highly virulent APEC clones in poultry production.

Refining the definition of the avian pathogenic Escherichia coli (APEC) pathotype through inclusion of high-risk clonal groups.

Colibacillosis in poultry is a unique disease manifestation of Escherichia coli in the animal world, as one of the primary routes of entry is via the respiratory tract of birds. Because of this, a novel extraintestinal pathogenic E. coli (ExPEC) subpathotype coined avian pathogenic E. coli (or APEC) has been described. Like other ExPEC, this pathotype has been challenging to clearly define, and in the case of APEC, its role as an opportunistic pathogen has further complicated these challenges. Using 3,479 temporally matched genomes of poultry-source isolates, we show that the APEC plasmid, previously considered a defining trait of APEC, is highly prevalent in clinical isolates from diseased turkeys. However, the plasmid is also quite prevalent among cecal E. coli isolates from healthy birds, including both turkeys and broilers. In contrast, we identify distinct differences in clonal backgrounds of turkey clinical versus cecal strains, with a subset of sequence types (STs) dominating the clinical landscape (ST23, ST117, ST131, ST355, and ST428), which are rare within the cecal landscape. Because the same clinical STs have also dominated the broiler landscape, we performed lethality assays using strains from dominant STs from clinical or cecal landscapes in embryonated turkey and chicken eggs. We show that, irrespective of plasmid carriage, dominant clinical STs are significantly more virulent than dominant cecal STs. We present a revised APEC screening tool that incorporates APEC plasmid carriage plus markers for dominant clinical STs. This revised APEC pathotyping tool improves the ability to identify high-risk APEC clones within poultry production systems, and identifies STs of interest for mitigation targets.

The Risk of Highly Pathogenic Influenza A Virus Transmission to Turkey Hen Flocks Through Artificial Insemination.

Artificial insemination is a routine practice for turkeys that can introduce pathogens into breeder flocks in a variety of ways. In this manuscript, a risk analysis on the potential transmission of highly pathogenic avian influenza (HPAI) to naïve hens through artificial insemination is presented. A case of HPAI on a stud farm where the potential transmission of the virus to susceptible hens in the 2015 H5N2 HPAI outbreak in Minnesota is described along with documentation of known and potential transmission pathways from the case. The pathways by which artificial insemination might result in the spread of HPAI to susceptible hens were determined by considering which could result in the 1) entry of HPAI virus onto a premises through semen movement; and 2) exposure of susceptible hens to HPAI as a result of this movement. In the reported case, HPAI virus was detected in semen from infected toms, however, transmission of HPAI to naïve hens through semen is unclear since the in utero infectious dose is not known. This means that the early detection of infection might limit but not eliminate the risk of hen exposure. Because of the numerous potential pathways of spread and the close contact with the birds, it is highly likely that if semen from an HPAI-infected tom flock is used, there will be spread of the virus to naïve hens through insemination. If insemination occurs with semen from stud farms in an HPAI control area, receiving hen farms should have restricted movements to prevent outbreak spread in the event that they become infected.

Artículo regular­Riesgo de transmisión del virus de la influenza A altamente patógeno a parvadas de pavos hembras mediante inseminación artificial. La inseminación artificial es una práctica de rutina para los pavos que puede introducir patógenos en las parvadas de reproductores de diversas formas. En este manuscrito, se presenta un análisis de riesgo sobre la posible transmisión de la influenza aviar altamente patógena a gallinas susceptibles mediante inseminación artificial. Un caso de influenza aviar altamente patógena en una granja de machos sementales donde se describe la posible transmisión del virus a gallinas susceptibles en el brote de influenza aviar altamente patógena H5N2 del año 2015 en Minnesota, junto con la documentación de las vías de transmisión conocidas y potenciales del caso. Las vías por las cuales la inseminación artificial podría resultar en la propagación de la influenza aviar altamente patógena a las gallinas susceptibles se determinaron considerando cuáles podrían resultar en 1) la entrada del virus de la influenza aviar altamente patógena en una granja a través del movimiento del semen; y 2) exposición de gallinas susceptibles a la influenza aviar altamente patógena como resultado de este movimiento. Sin embargo, se demostró la detección del virus de la influenza aviar altamente patógena en el semen de machos infectados. Debido a que se desconoce la dosis infecciosa del virus de la influenza aviar administrada en el útero necesaria para transmitir la influenza aviar altamente patógena a las gallinas susceptibles, está claro que la detección de la infección no puede ser la única estrategia de contención. La detección temprana de la infección puede limitar, pero no eliminar, el riesgo de exposición de las gallinas. Debido a las numerosas vías potenciales de propagación y al estrecho contacto con las aves, es muy probable que si se usa semen de una parvada de machos infectados con influenza aviar de alta patogenicidad, se propague el virus a gallinas susceptibles a través de la inseminación. Si la inseminación ocurre con semen de granjas de sementales en un área de control de influenza aviar de alta patogenicidad, las granjas de gallinas receptoras deben tener movimientos restringidos para prevenir la propagación del brote en caso de que se infecten.

Convergence of the turkey gut microbiota following cohabitation under commercial settings.

BACKGROUND: Microbiota development is a critical aspect of turkey poult maturation, and the succession of microbes in the turkey gut has been shown to correlate with poult performance. The purpose of this study was to determine the fate of the microbiota in turkey poults after movement of birds first raised in an isolated hatch brood system into a more traditional commercial brood facility with pre-existing birds. Turkey poults were first divided into groups raised in conventional brood pens from day-of-hatch and those raised in an experimental hatch brood system. After 11 days of growth, hatch brood birds were moved into pens within the conventional brood barn and monitored for an additional 18 days. Sampling of both hatch brood and conventional pen birds was performed at multiple timepoints throughout the study, and cecal content was used to analyze the bacterial microbiota using 16S rRNA gene amplicon sequencing. RESULTS: Alpha diversity tended to be higher in samples from conventional pen birds compared to those from hatch brood birds prior to the day 11 move, but the difference between systems was not observed post-move. Using beta diversity metrics, bacterial community succession appeared delayed in the hatch brood system birds pre-move, but post-move community composition quickly converged with that of the conventional pen birds. This was validated through assessment of significantly different genera between hatch brood system and conventional pen birds, where numbers of significantly different taxa quickly decreased following the move. Some key taxa previously associated with poult performance were delayed in their appearance and relative abundance in hatch brood birds. CONCLUSIONS: Overall, this study demonstrates that the use of isolated hatch brood systems has an impact on the poult gut microbiota, but its impact is resolved quickly once the birds are introduced into a conventional brood environment. Therefore, the benefits of pathogen reduction with hatch brood systems may outweigh negative microbiota impacts due to isolation.

Emergence of a Novel Salmonella enterica Serotype Reading Clonal Group Is Linked to Its Expansion in Commercial Turkey Production, Resulting in Unanticipated Human Illness in North America.

Two separate human outbreaks of Salmonella enterica serotype Reading occurred between 2017 and 2019 in the United States and Canada, and both outbreaks were linked to the consumption of raw turkey products. In this study, a comprehensive genomic investigation was conducted to reconstruct the evolutionary history of S. Reading from turkeys and to determine the genomic context of outbreaks involving this infrequently isolated Salmonella serotype. A total of 988 isolates of U.S. origin were examined using whole-genome-based approaches, including current and historical isolates from humans, meat, and live food animals. Broadly, isolates clustered into three major clades, with one apparently highly adapted turkey clade. Within the turkey clade, isolates clustered into three subclades, including an "emergent" clade that contained only isolates dated 2016 or later, with many of the isolates from these outbreaks. Genomic differences were identified between emergent and other turkey subclades, suggesting that the apparent success of currently circulating subclades is, in part, attributable to plasmid acquisitions conferring antimicrobial resistance, gain of phage-like sequences with cargo virulence factors, and mutations in systems that may be involved in beta-glucuronidase activity and resistance towards colicins. U.S. and Canadian outbreak isolates were found interspersed throughout the emergent subclade and the other circulating subclade. The emergence of a novel S Reading turkey subclade, coinciding temporally with expansion in commercial turkey production and with U.S. and Canadian human outbreaks, indicates that emergent strains with higher potential for niche success were likely vertically transferred and rapidly disseminated from a common source.IMPORTANCE Increasingly, outbreak investigations involving foodborne pathogens are difficult due to the interconnectedness of food animal production and distribution, and homogeneous nature of industry integration, necessitating high-resolution genomic investigations to determine their basis. Fortunately, surveillance and whole-genome sequencing, combined with the public availability of these data, enable comprehensive queries to determine underlying causes of such outbreaks. Utilizing this pipeline, it was determined that a novel clone of Salmonella Reading has emerged that coincided with increased abundance in raw turkey products and two outbreaks of human illness in North America. The rapid dissemination of this highly adapted and conserved clone indicates that it was likely obtained from a common source and rapidly disseminated across turkey production. Key genomic changes may have contributed to its apparent continued success in commercial turkeys and ability to cause illness in humans.

Genomic Landscape of Ornithobacterium rhinotracheale in Commercial Turkey Production in the United States.

Ornithobacterium rhinotracheale is a causative agent of respiratory tract infections in avian hosts worldwide but is a particular problem for commercial turkey production. Little is known about the ecologic and evolutionary dynamics of O. rhinotracheale, which makes prevention and control of this pathogen a challenge. The purpose of this study was to gain insight into the genetic relationships between O. rhinotracheale populations through comparative genomics of clinical isolates from different U.S. turkey producers. O. rhinotracheale clinical isolates were collected from four major U.S. turkey producers and several independent turkey growers from the upper Midwest and Southeast, and whole-genome sequencing was performed. Genomes were compared phylogenetically using single nucleotide polymorphism (SNP)-based analysis, and then assembly and annotations were performed to identify genes encoding putative virulence factors and antimicrobial resistance determinants. A pangenome approach was also used to establish a core set of genes consistently present in O. rhinotracheale and to highlight differences in gene content between phylogenetic clades. A total of 1,457 nonrecombinant SNPs were identified from 157 O. rhinotracheale genomes, and four distinct phylogenetic clades were identified. Isolates clustered by company on the phylogenetic tree, however, and each company had isolates in multiple clades with similar collection dates, indicating that there are multiple O. rhinotracheale strains circulating within each of the companies examined. Additionally, several antimicrobial resistance proteins, putative virulence factors, and the pOR1 plasmid were associated with particular clades and multilocus sequence types, which may explain why the same strains seem to have persisted in the same turkey operations for decades.IMPORTANCE The whole-genome approach enhances our understanding of evolutionary relationships between clinical Ornithobacterium rhinotracheale isolates from different commercial turkey producers and allows for identification of genes associated with virulence, antimicrobial resistance, or mobile genetic elements that are often excluded using traditional typing methods. Additionally, differentiating O. rhinotracheale isolates at the whole-genome level may provide insight into selection of the most appropriate autogenous vaccine strain, or groups of strains, for a given population of clinical isolates.

Antimicrobial Usage Factors and Resistance Profiles of Shiga Toxin-Producing Escherichia coli in Backyard Production Systems From Central Chile.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen and important cause of foodborne disease worldwide. Many animal species in backyard production systems (BPS) harbor STEC, systems characterized by low biosecurity and technification. No information is reported on STEC circulation, antimicrobial resistance (AMR) and potential drivers of antimicrobial usage in Chilean BPS, increasing the risk of maintenance and transmission of zoonotic pathogens and AMR generation. Thus, the aim of this study was to characterize phenotypic and genotypic AMR and to study the epidemiology of STEC isolated in BPS from Metropolitana region, Chile. A total of 85 BPS were sampled. Minimal inhibitory concentration and whole genome sequencing was assessed in 10 STEC strain isolated from BPS. All strains were cephalexin-resistant (100%, n = 10), and five strains were resistant to chloramphenicol (50%). The most frequent serotype was O113:H21 (40%), followed by O76:H19 (40%), O91:H14 (10%), and O130:H11 (10%). The stx1 type was detected in all isolated strains, while stx2 was only detected in two strains. The Stx subtype most frequently detected was stx1c (80%), followed by stx1a (20%), stx2b (10%), and stx2d (10%). All strains harbored chromosomal bla AmpC. Principal component analysis shows that BPS size, number of cattle, pet and horse, and elevation act as driver of antimicrobial usage. Logistic multivariable regression shows that recognition of diseases in animals (p = 0.038; OR = 9.382; 95% CI: 1.138-77.345), neighboring poultry and/or swine BPS (p = 0.006; OR = 10.564; 95% CI: 1.996-55.894), visit of Veterinary Officials (p = 0.010; OR = 76.178; 95% CI: 2.860-2029.315) and close contact between animal species in the BPS (p = 0.021; OR = 9.030; 95% CI: 1.385-58.888) increase significantly the risk of antimicrobial use in BPS. This is the first evidence of STEC strains circulating in BPS in Chile, exhibiting phenotypic AMR, representing a threat for animal and public health. Additionally, we identified factors acting as drivers for antimicrobial usage in BPS, highlighting the importance of integration of these populations into surveillance and education programs to tackle the potential development of antimicrobial resistance and therefore the risk for ecosystemic health.

Environmental Sampling for Influenza A Viruses in Turkey Barns.

We have examined a variety of sampling strategies for detecting pathogens in turkey flocks undergoing infections with low pathogenicity avian influenza virus (LPAIV). We found that viral RNA was widely distributed in the barn environment of turkey flocks undergoing an active LPAIV infection and was in both water and drinker biofilm samples. Viral RNA was concentrated in drinker biofilm and sediment and was detectable using real-time reverse-transcription polymerase chain reaction (RRT-PCR) and by virus isolation. Drinker biofilm sample results correlated with concurrently collected oropharyngeal (OP) sample results from flocks on a farm with LPAI in which the two sampling strategies were directly compared. To evaluate the utility of biofilm sampling for the detection of highly pathogenic avian influenza virus (HPAIV), biofilm and OP swabs from mortality pools were collected daily from negative turkey flocks on an HPAI-positive premise. The biofilm swabs were positive 1-2 days prior to positives appearing in the OP sample pools. The drinker biofilm sampling strategy overcame the difficulty of finding a subclinical infectious bird in a population by collecting material from a large number of individuals and testing a sample in which a positive signal persists for several days to weeks. The sampling method is convenient for use in turkey barns and has been reliably used in both active and passive surveillance programs for LPAIV and HPAIV using RRT-PCR.

Muestreo ambiental para el virus de influenza A en casetas de pavos. Se han examinado una variedad de estrategias de muestreo para detectar patógenos en parvadas de pavos que sufren infecciones con el virus de la influenza aviar de baja patogenicidad (con las siglas en inglés LPAIV). Se encontró que el ARN viral se distribuyó ampliamente en el ambiente de las casetas con parvadas de pavos con infección activa por el virus de la influenza aviar de baja patogenicidad y se determinó tanto en muestras de agua como en muestras de la biopelícula de bebederos. El ARN viral se concentró en la biopelícula y en el sedimento de bebederos y se detectó mediante transcripción reversa y reacción en cadena de la polimerasa en tiempo real (RRT-PCR) y mediante el aislamiento del virus. Los resultados de la muestra de la biopelícula del bebedero se correlacionaron con los resultados de la muestra orofaríngea (OP) colectada de forma simultánea de parvadas en una granja con influenza aviar de baja patogenicidad en las que se compararon directamente las dos estrategias de muestreo. Para evaluar la utilidad del muestreo de la biopelícula para la detección del virus de la influenza aviar altamente patógena (HPAIV), se recolectaron diariamente biopelículas e hisopos orofaríngeos de grupos de mortalidad de parvadas de pavos negativas en una granja positiva para la influenza aviar de alta patogenicidad. Los hisopos de biopelículas fueron positivos de uno a dos días antes de que aparecieran resultados positivos en las muestras orofaríngeas agrupadas. La estrategia de muestreo de la biopelícula del bebedero eliminó la dificultad de encontrar un ave infectada subclínicamente en una población al recolectar material de un gran número de individuos y analizar una muestra en la que persiste una señal positiva durante varios días o semanas. El método de muestreo es adecuado para su uso en casetas de pavos y se ha utilizado de manera confiable en los programas de vigilancia activa y pasiva para el virus de influenza aviar tanto de baja como de alta patogenicidad utilizando transcripción reversa y reacción en cadena de la polimerasa en tiempo real.

Phylogenomic Analysis of Extraintestinal Pathogenic Escherichia coli Sequence Type 1193, an Emerging Multidrug-Resistant Clonal Group.

The fluoroquinolone-resistant sequence type 1193 (ST1193) of Escherichia coli, from the ST14 clonal complex (STc14) within phylogenetic group B2, has appeared recently as an important cause of extraintestinal disease in humans. Although this emerging lineage has been characterized to some extent using conventional methods, it has not been studied extensively at the genomic level. Here, we used whole-genome sequence analysis to compare 355 ST1193 isolates with 72 isolates from other STs within STc14. Using core genome phylogeny, the ST1193 isolates formed a tightly clustered clade with many genotypic similarities, unlike ST14 isolates. All ST1193 isolates possessed the same set of three chromosomal mutations conferring fluoroquinolone resistance, carried the fimH64 allele, and were lactose non-fermenting. Analysis revealed an evolutionary progression from K1 to K5 capsular types and acquisition of an F-type virulence plasmid, followed by changes in plasmid structure congruent with genome phylogeny. In contrast, the numerous identified antimicrobial resistance genes were distributed incongruently with the underlying phylogeny, suggesting frequent gain or loss of the corresponding resistance gene cassettes despite retention of the presumed carrier plasmids. Pangenome analysis revealed gains and losses of genetic loci occurring during the transition from ST14 to ST1193 and from the K1 to K5 capsular types. Using time-scaled phylogenetic analysis, we estimated that current ST1193 clades first emerged approximately 25 years ago. Overall, ST1193 appears to be a recently emerged clone in which both stepwise and mosaic evolution have contributed to epidemiologic success.

Quantifying the effect of swab pool size on the detection of influenza A viruses in broiler chickens and its implications for surveillance.

BACKGROUND: Timely diagnosis of influenza A virus infections is critical for outbreak control. Due to their rapidity and other logistical advantages, lateral flow immunoassays can support influenza A virus surveillance programs and here, their field performance was proactively assessed. The performance of real-time polymerase chain reaction and two lateral flow immunoassay kits (FluDETECT and VetScan) in detecting low pathogenicity influenza A virus in oropharyngeal swab samples from experimentally inoculated broiler chickens was evaluated and at a flock-level, different testing scenarios were analyzed. RESULTS: For real-time polymerase chain reaction positive individual-swabs, FluDETECT respectively detected 37% and 58% for the H5 and H7 LPAIV compared to 28% and 42% for VetScan. The mean virus titer in H7 samples was higher than for H5 samples. For real-time polymerase chain reaction positive pooled swabs (containing one positive), detections by FluDETECT were significantly higher in the combined 5- and 6-swab samples compared to 11-swab samples. FluDETECT detected 58%, 55.1% and 44.9% for the H7 subtype and 28.3%, 34.0% and 24.6% for the H5 in pools of 5, 6 and 11 respectively. In our testing scenario analysis, at low flock-level LPAIV infection prevalence, testing pools of 11 detected slightly more infections while at higher prevalence, testing pools of 5 or 6 performed better. For highly pathogenic avian influenza virus, testing pools of 11 (versus 5 or 6) detected up to 5% more infections under the assumption of similar sensitivity across pools and detected less by 3% when its sensitivity was assumed to be lower. CONCLUSIONS: Much as pooling a bigger number of swab samples increases the chances of having a positive swab included in the sample to be tested, this study's outcomes indicate that this practice may actually reduce the chances of detecting the virus since it may result into lowering the virus titer of the pooled sample. Further analysis on whether having more than one positive swab in a pooled sample would result in increased sensitivity for low pathogenicity avian influenza virus is needed.

Environmental Sampling Survey of H5N2 Highly Pathogenic Avian Influenza-Infected Layer Chicken Farms in Minnesota and Iowa.

Respiratory secretions, feces, feathers, and eggs of avian influenza-infected hens provide ample sources of virus which heavily contaminate barn and farm environments during a disease outbreak. Environmental sampling surveys were conducted in the Midwestern United States on affected farms during the 2015 H5N2 highly pathogenic avian influenza (HPAI) outbreak to assess the degree of viral contamination. A total of 930 samples were obtained from various sites inside and outside layer barns housing infected birds and tested with real-time reverse transcriptase PCR. The distribution and load of viral RNA in barns in which most birds were dead at the onset of depopulation efforts (high-mortality barns) were compared with those of barns in which birds were euthanatized before excess mortality occurred (normal-mortality barns). A statistically significant difference was seen between cycle threshold (Ct) values for samples taken of fans, feed troughs, barn floors, barn walls, cages, manure-associated locations, barn doors, egg belts, and the exterior of high-mortality vs. normal-mortality barns. In high-mortality barns, sample sites were found to be the most to least contaminated in the following order: cages, manure-associated locations, barn floors, egg belts, feed troughs, barn doors, barn walls, fans, exterior, and egg processing. Significant changes in Ct values over time following HPAI detection in a barn and depopulation of birds on an infected farm were observed for the manure-associated, barn floor, barn wall, and fan sampling sites. These results show that high mortality in a flock as a result of HPAI will increase contamination of the farm environment. The results also suggest optimal sampling locations for detection of virus; however, the persistence of RNA on highmortality farms may delay the determination that adequate sanitization has been performed for restocking to take place.

Estudios de muestreo ambiental de granjas de gallinas de postura infectadas con influenza aviar altamente patógena H5N2 en Minnesota y Iowa. Las secreciones respiratorias, las heces, las plumas y huevos de gallinas infectadas con influenza aviar brindan amplias fuentes de virus para contaminar las casetas y el ambiente de la granja durante un brote de la enfermedad. Se realizaron estudios de muestreo ambiental en el medio oeste de los Estados Unidos en granjas afectadas durante el brote de influenza aviar altamente patógena H5N2 del año 2015 para evaluar el grado de contaminación viral. Se obtuvieron un total de 930 muestras de varios sitios dentro y fuera de las casetas de gallinas de postura que albergaron aves infectadas y se analizaron mediante pruebas de transcripción reversa y PCR en tiempo real. La distribución y la carga de ARN viral en casetas en las que la mayoría de las aves estaban muertas al inicio de los esfuerzos de despoblación (casetas de alta mortalidad) se compararon con los de las casetas en los que las aves se sacrificaron antes de que se produjera un exceso de mortalidad (casetas de mortalidad normal). Se observó una diferencia estadísticamente significativa entre los valores de ciclos umbrales (Ct) para muestras tomadas de ventiladores, comederos, pisos de casetas, paredes de casetas, jaulas, sitios asociados con gallinaza, puertas de casetas, bandas transportadoras de huevos y el exterior de las casetas con alta mortalidad en comparación con las casetas con mortalidad normal. En las casetas de alta mortalidad, se encontró que los sitios donde se recolectaron muestras presentaron contaminación de mayor grado a menor grado en el siguiente orden: jaulas, lugares asociados con gallinaza, pisos de casetas, bandas de huevos, comederos, puertas de casetas, paredes de casetas, ventiladores, exteriores y locales para el tratamiento del huevo. Se observaron cambios significativos en los valores de Ct a lo largo del tiempo después de la detección de la influenza aviar de alta patogenicidad en una caseta y de la despoblación de aves en una granja infectada en los sitios de muestreo asociados con gallinaza, en el piso de la caseta, en las paredes y en los ventiladores. Estos resultados muestran que la alta mortalidad en una parvada como resultado de influenza aviar de alta patogenicidad aumentará la contaminación del entorno de la granja. Los resultados también sugieren ubicaciones de muestreo óptimas para la detección de virus; sin embargo, la persistencia del ARN en las granjas de alta mortalidad puede retrasar la determinación de que se haya realizado un saneamiento adecuado para que se lleve a cabo la repoblación.

Separate F-Type Plasmids Have Shaped the Evolution of the H30 Subclone of Escherichia coli Sequence Type 131.

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131's evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage's success.