Long-term evaluation of anterior thalamic deep brain stimulation for epilepsy in the European MORE registry.

OBJECTIVE: Short-term outcomes of deep brain stimulation of the anterior nucleus of the thalamus (ANT-DBS) were reported for people with drug-resistant focal epilepsy (PwE). Because long-term data are still scarce, the Medtronic Registry for Epilepsy (MORE) evaluated clinical routine application of ANT-DBS. METHODS: In this multicenter registry, PwE with ANT-DBS were followed up for safety, efficacy, and battery longevity. Follow-up ended after 5 years or upon study closure. Clinical characteristics and stimulation settings were compared between PwE with no benefit, improvers, and responders, that is, PwE with average monthly seizure frequency reduction rates of ≥50%. RESULTS: Of 170 eligible PwE, 104, 62, and 49 completed the 3-, 4-, and 5-year follow-up, respectively. Most discontinuations (68%) were due to planned study closure as follow-up beyond 2 years was optional. The 5-year follow-up cohort had a median seizure frequency reduction from 16 per month at baseline to 7.9 per month at 5-year follow-up (p < .001), with most-pronounced effects on focal-to-bilateral tonic-clonic seizures (n = 15, 77% reduction, p = .008). At last follow-up (median 3.5 years), 41% (69/170) of PwE were responders. Unifocal epilepsy (p = .035) and a negative history of epilepsy surgery (p = .002) were associated with larger average monthly seizure frequency reductions. Stimulation settings did not differ between response groups. In 179 implanted PwE, DBS-related adverse events (AEs, n = 225) and serious AEs (n = 75) included deterioration in epilepsy or seizure frequency/severity/type (33; 14 serious), memory/cognitive impairment (29; 3 serious), and depression (13; 4 serious). Five deaths occurred (none were ANT-DBS related). Most AEs (76.3%) manifested within the first 2 years after implantation. Activa PC depletion (n = 37) occurred on average after 45 months. SIGNIFICANCE: MORE provides further evidence for the long-term application of ANT-DBS in clinical routine practice. Although clinical benefits increased over time, side effects occurred mainly during the first 2 years. Identified outcome modifiers can help inform PwE selection and management.

Molecular Characterization of the Chicken Parvovirus Based on VP1 Gene Circulating in Brazilian Chicken Flocks.

Parvovirus infection affects several animal species, especially young animals. In birds, parvovirus infection has been described in Muscovy ducks, turkeys, and chickens, all of which had enteric diseases characterized by diarrhea. Chicken parvovirus (ChPV) has been detected in poultry around the world in animals affected by enteric problems, showing dwarfism, cloacal pasting, and diarrhea. In Brazil, ChPV was detected in chickens affected by diarrhea fifteen years ago. However, the genetic characteristics of ChPV circulating in chicken flocks were not determined. Therefore, the aim of the present investigation was to determine the genetic characteristics of the VP1 gene from ChPV detected in chickens affected by enteric diseases in Brazil. For this purpose, a molecular approach was used. Specific primers were designed to flank the complete VP1 gene of ChPV and amplify it using PCR. The amplified products from samples of chickens with enteric diseases were sequenced, and 22 complete CDs of the VP1 gene were obtained. These samples, compared to the ABU-P1 sequence, showed 17 sequences with high nucleotide (NT) similarity of 92.7-97.4% and amino acid (AA) similarity of 94.8-99.5% associated with Runting and Stunting syndrome (RSS); there were also five samples associated with hens with diarrhea with unusual jejunal dilatation (JD) that had less similarity than the RSS sequences (NT of 86.5% and AA of 93-93.1%). The phylogenetic analysis determined four groups. Group I had sequences from Korea. The second group included sequences from Korea, China, and Brazil (not included in this work). The third group had studied RSS sequences grouped with the ABU-P1 strain and sequences from China and the United States. Finally, the sequences from JD were clustered in a separate group with a bootstrap of 100%, a group that was denoted as group IV, and included sequences from China. RDP4 and SimPlot analysis showed one point of recombination with the sequences of group III ChPV in the JD sequences. Herein, we show that circulating strains of ChPV exhibit genetic differences in the VP1 gene in Brazilian chicken flocks. Nevertheless, more studies are needed to determine the probability of a new genetic group of ChPV based on the analysis of the complete genome.

Detection and molecular characterization of chicken parvovirus and chicken megrivirus in layer breeders affected by intestinal dilatation syndrome.

RESEARCH HIGHLIGHTS: IDS presented pathognomonic dilatation of the jejunum up to Meckel's diverticulum.IDS caused weight loss, decreased egg production, and increased culling and mortality.Chicken parvovirus (ChPV) was consistently detected through PCR assays.Chicken megrivirus (ChMV) was consistently detected through viral metagenomics.

Lymphomas in seabirds: case reports in a black skimmer (Rynchops niger) and a brown booby (Sula leucogaster).

A black skimmer (Rynchops niger) and a brown booby (Sula leucogaster) were rescued and gross, histopathological, immunohistochemical and polymerase chain reaction evaluations were conducted to investigate the cause of death. There were neoplastic infiltrations of CD3+ PAX5- lymphocytes in the black skimmer and CD3- PAX5+ lymphocytes in the brown booby. Molecular assays for viral agents were negative in both cases. This is the first report of disseminated lymphoma as the cause of stranding and death in these species in Brazil.

Molecular characterization of the meq oncogene of Marek's disease virus in vaccinated Brazilian poultry farms reveals selective pressure on prevalent strains.

Marek's disease virus (MDV) has become an increasingly virulent pathogen in the poultry industry despite vaccination efforts to control it. Brazil has experienced a significant rise of Marek's disease (MD) outbreaks in recent years. Our study aimed to analyze the complete meq gene sequences to understand the molecular epidemiological basis of MD outbreaks in Brazilian vaccinated layer farms. We detected a high incidence rate of visceral MD (67.74%) and multiple circulating MDV strains. The most prevalent and geographically widespread genotype presented several clinical and molecular characteristics of a highly virulent strain and evolving under positive selective pressure. Phylogenetic and phylogeographic analysis revealed a closer relationship with strains from the USA and Japan. This study sheds light on the circulation of MDV strains capable of infecting vaccinated birds. We emphasize the urgency of adopting preventive measures to manage MDV outbreaks threatening the poultry farming industry.

Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease.

BACKGROUND: Enteric viruses are among the most prominent etiological agents of Runting-Stunting Syndrome (RSS). The Avian Nephritis Virus (ANV) is an astrovirus associated with enteric diseases in poultry, whose early diagnosis is essential for maintaining a good poultry breeding environment. ANV is an RNA virus that rapidly mutates, except for some conserved regions such as ORF1b. Therefore, the approach of a diagnostic method based on fast-RT-qPCR using SYBR® Green that focuses on the amplification of a fragment of ORF1b is presented as a feasible alternative for the diagnosis of this viral agent. In this study, the proposed assay showed a standard curve with an efficiency of 103.8% and a LoD and LoQ of 1 gene viral copies. The assay was specific to amplify the ORF 1b gene, and no amplification was shown from other viral genomes or in the negative controls. 200 enteric (feces) samples from chickens (broilers) and laying hens with signs of RSS from Ecuadorian poultry flocks were examined to validate the proposed method. RESULTS: Using our method, 164 positive results were obtained out of the total number of samples run, while the presence of viral RNA was detected in samples collected from one day to 44 weeks old in both avian lines. CONCLUSIONS: Our study presents a novel, rapid, robust, and sensitive molecular assay capable of detecting and quantifying even low copy numbers of the ANV in commercial birds, therefore introducing a handy tool in the early diagnosis of ANV in enteric disease outbreaks in poultry.

Antigenic and molecular characterization of isolates of the Brazilian genotype BR-I (GI-11) of infectious bronchitis virus supports its recognition as BR-I serotype.

The antigenic and molecular characteristics of BR-I infectious bronchitis viruses (IBVs) isolated from Brazil are reported. IBVs isolated from commercial flocks with different clinical manifestations between 2003 and 2019 were submitted to antigenic and molecular characterization. The complete S1 glycoprotein gene of 11 field isolates was amplified and sequenced. The virus neutralization (VN) test showed 94.75% neutralization with a BR-I isolate and 30% or less against other worldwide reference strains. The nucleotide and amino acid sequence analyses revealed 84.3-100% and 83.5-100% identity among them, respectively. The identity values ranged from 57.1 to 82.6% for nucleotides and from 46.6-84.4% for amino acids compared with those of other genotypes. By phylogenetic tree analysis, the Brazilian isolates were branched into the BR-I genotype (lineage GI-11), which was differentiated from foreign reference strains. Selective pressure analyses of BR-I IBVs revealed evolution under purifying selection (negative pressure) for the complete S1 gene but four specific sites (87, 121, 279, and 542) under diversifying selection (positive pressure). Profiles of cleavage sites and potential N-glycosylation sites differed from those of other genotypes. The low molecular relationship among the Brazilian viruses and foreign serotypes was concordant with the VN test results. The low antigenic relatedness (ranging from 5.3-30% between Brazilian genotype BR-I and reference IBV serotypes of North America, Europe, and Asia) indicates that the BR-I genotype is a different serotype, referred to for the first time and hereafter as serotype BR-I. RESEARCH HIGHLIGHTSStrains of the BR-I genotype presented robust antigenic and molecular similarity.BR-I strains evolved under purifying selection mode (negative pressure).The BR-I genotype originated in Brazil and dispersed to other countries.BR-I genotype viruses can be referred to as the BR-I serotype.

Genomic Characterization and Genetic Profiles of Salmonella Gallinarum Strains Isolated from Layers with Fowl Typhoid in Colombia.

Salmonella Gallinarum (SG) is the causative agent of fowl typhoid (FT), a disease that is harmful to the poultry industry. Despite sanitation and prophylactic measures, this pathogen is associated with frequent disease outbreaks in developing countries, causing high morbidity and mortality. We characterized the complete genome sequence of Colombian SG strains and then performed a comparative genome analysis with other SG strains found in different regions worldwide. Eight field strains of SG plus a 9R-derived vaccine were subjected to whole-genome sequencing (WGS) and bioinformatics analysis, and the results were used for subsequent molecular typing; virulome, resistome, and mobilome characterization; and a comparative genome study. We identified 26 chromosome-located resistance genes that mostly encode efflux pumps, and point mutations were found in gyrase genes (gyrA and gyrB), with the gyrB mutation S464T frequently found in the Colombian strains. Moreover, we detected 135 virulence genes, mainly in 15 different Salmonella pathogenicity islands (SPIs). We generated an SPI profile for SG, including C63PI, CS54, ssaD, SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-6, SPI-9, SPI-10, SPI-11, SPI-12, SPI-13, and SPI-14. Regarding mobile genetic elements, we found the plasmids Col(pHAD28) and IncFII(S) in most of the strains and 13 different prophage sequences, indicating a frequently obtained profile that included the complete phage Gifsy_2 and incomplete phage sequences resembling Escher_500465_2, Shigel_SfIV, Entero_mEp237, and Salmon_SJ46. This study presents, for the first time, the genomic content of Colombian SG strains and a profile of the genetic elements frequently found in SG, which can be further studied to clarify the pathogenicity and evolutionary characteristics of this serotype.

Complete genome sequence data of two Salmonella enterica subsp. enterica serovar Gallinarum: A 9R vaccine strain and a virulent Brazilian field strain.

Salmonella Gallinarum (SG) is a host-restricted enterobacteria and the causative agent of fowl typhoid in poultry. Here, we report the complete genomes of two strains belonging to this serotype. SA68 is a field strain isolated from the livers of dead hen carcasses of a commercial layer farm presenting high mortality located in São Paulo city, Brazil, in 1990. Strain 9R corresponds to a live attenuated SG commercial vaccine. DNA was extracted from pure cultures and subjected to whole genome sequencing (WGS) using the Ion Torrent PGM System. The assemblies reached lengths of 4,657,435 (SA68) and 4,657,471 (9R) base pairs. Complete genomes were deposited in GenBank under the accession numbers CP110192 (SA68) and CP110508 (9R). Both genomes were analyzed and compared in terms of molecular typing, antibiotic resistance genes, virulence genes, Salmonella pathogenic islands (SPIs), insertion sequences and prophages. The data obtained show many similarities in the genetic content, with the exception of the SPI-12 and CS54 pathogenic islands, which are exclusive to the field strain. The information generated will help to understand the virulence differences of field and vaccinal SG strains and can be used to perform evolutionary and epidemiologic studies.

Metagenomics analysis of the morphological aspects and bacterial composition of broiler feces.

In this descriptive study, we used metagenomics to analyze the relationship between the morphological aspects of chicken feces and its respective bacterial compositions. The microbiota composition was determined by sequencing the V4 region of the 16S rRNA genes collected from fresh broiler feces at 19 d old. In total, 48 samples were collected and divided into 8 groups of 6 samples each. The morphological changes studied were feed passage (FP) and reddish mucus (RM). Each was classified into 3 levels of intensity: 1 (slight), 2 (moderate), or 3 (intense). Thus, the 8 groups studied were feed passage (FP-1; FP-2; FP-3), reddish mucus (RM-1; RM-2; RM-3), normal ileal feces (NIF), and cecal discharge (CD). The alpha diversity (Shannon's index) revealed that the CD group showed greater diversity, and was significantly different from FP-2, FP-3, and RM-1. The beta diversity showed that the CD group samples were more homogeneous than the ileal feces groups. The relative abundance analysis revealed that Firmicutes and Proteobacteria were the most abundant phyla in the ileal feces groups. In CD, Firmicutes and Bacteroidetes were the most abundant. The relative abundance at the genus level revealed 136 different bacterial genera. In the ileal feces groups, the two most abundant genera were Lactobacillus and Escherichia/Shigella, except in the FP-1 and RM-2 groups, which had the opposite order. Unlike the others, the CD group had a higher abundance of Bacteroides and Faecalibacterium. When comparing the NIF group with the others, significant changes were found in the fecal microbiota, with nine genera for the FP groups, 19 for the RM groups, and 61 when compared to CD. The results of the present study suggest that evaluation of fecal morphology is a fundamental task that makes it possible to act quickly and assertively, as the morphological aspects of the feces may be related to the composition and structure of fecal microbiota.

Predictive models for starting antiseizure medication withdrawal following epilepsy surgery in adults.

More than half of adults with epilepsy undergoing resective epilepsy surgery achieve long-term seizure freedom and might consider withdrawing antiseizure medications. We aimed to identify predictors of seizure recurrence after starting postoperative antiseizure medication withdrawal and develop and validate predictive models. We performed an international multicentre observational cohort study in nine tertiary epilepsy referral centres. We included 850 adults who started antiseizure medication withdrawal following resective epilepsy surgery and were free of seizures other than focal non-motor aware seizures before starting antiseizure medication withdrawal. We developed a model predicting recurrent seizures, other than focal non-motor aware seizures, using Cox proportional hazards regression in a derivation cohort (n = 231). Independent predictors of seizure recurrence, other than focal non-motor aware seizures, following the start of antiseizure medication withdrawal were focal non-motor aware seizures after surgery and before withdrawal [adjusted hazard ratio (aHR) 5.5, 95% confidence interval (CI) 2.7-11.1], history of focal to bilateral tonic-clonic seizures before surgery (aHR 1.6, 95% CI 0.9-2.8), time from surgery to the start of antiseizure medication withdrawal (aHR 0.9, 95% CI 0.8-0.9) and number of antiseizure medications at time of surgery (aHR 1.2, 95% CI 0.9-1.6). Model discrimination showed a concordance statistic of 0.67 (95% CI 0.63-0.71) in the external validation cohorts (n = 500). A secondary model predicting recurrence of any seizures (including focal non-motor aware seizures) was developed and validated in a subgroup that did not have focal non-motor aware seizures before withdrawal (n = 639), showing a concordance statistic of 0.68 (95% CI 0.64-0.72). Calibration plots indicated high agreement of predicted and observed outcomes for both models. We show that simple algorithms, available as graphical nomograms and online tools (predictepilepsy.github.io), can provide probabilities of seizure outcomes after starting postoperative antiseizure medication withdrawal. These multicentre-validated models may assist clinicians when discussing antiseizure medication withdrawal after surgery with their patients.

Evolutionary Analysis of a Parrot Bornavirus 2 Detected in a Sulphur-Crested Cockatoo (Cacatua galerita) Suggests a South American Ancestor.

Parrot bornavirus (PaBV) is an RNA virus that causes Proventricular Dilatation Disease (PDD), neurological disorders, and death in Psittaciformes. Its diversity in South America is poorly known. We examined a Cacatua galerita presenting neuropathies, PDD, and oculopathies as the main signs. We detected PaBV through reverse transcription polymerase chain reaction (RT-PCR) and partial sequencing of the nucleoprotein (N) and matrix (M) genes. Maximum likelihood and Bayesian phylogenetic inferences classified it as PaBV-2. The nucleotide identity of the sequenced strain ranged from 88.3% to 90.3% against genotype PaBV-2 and from 80.2% to 84.4% against other genotypes. Selective pressure analysis detected signs of episodic diversifying selection in both the N and M genes. No recombination events were detected. Phylodynamic analysis estimated the time to the most recent common ancestor (TMRCA) as the year 1758 for genotype PaBV-2 and the year 1049 for the Orthobornavirus alphapsittaciforme species. Substitution rates were estimated at 2.73 × 10-4 and 4.08 × 10-4 substitutions per year per site for N and M, respectively. The analysis of population dynamics showed a progressive decline in the effective population size during the last century. Timescale phylogeographic analysis revealed a potential South American ancestor as the origin of genotypes 1, 2, and 8. These results contribute to our knowledge of the evolutionary origin, diversity, and dynamics of PaBVs in South America and the world. Additionally, it highlights the importance of further studies in captive Psittaciformes and the potential impact on endangered wild birds.

Detection and molecular characterization of infectious laryngotracheitis virus (ILTV) in chicken with respiratory signs in Brazil during 2015 and 2016.

Avian infectious laryngotracheitis (ILT) is a respiratory disease that causes severe economic losses in the poultry industry, mainly due to high morbidity and mortality and reduced egg production. Molecular characterization was performed on samples collected from flocks in the Brazilian States of São Paulo, Pernambuco, and Minas Gerais during 2015 and 2016 that presented clinical signs of respiratory disease. End-point PCR was used for viral detection, and DNA sequencing was used for differentiation of vaccine and field strains. Molecular analysis based on the infected cell protein (ICP4) gene separated four of the nine samples together with previous Brazilian isolates (São Paulo and Minas Gerais), one sample was grouped on the same branch as Minas Gerais strains (along with another related sample), one sample was separately branched but still related to the tissue culture origin (TCO) vaccine strain, and two samples were grouped on the same branch as the TCO vaccine strain. Molecular analysis of the thymidine kinase (TK) gene showed the existence of strains of both high and low virulence. The characterization of two fragments of the ICP4 gene and a fragment of the TK gene in this study suggested that the virus circulating in Guatapará, as well as those in Barretos and Itanhandu, that is causing respiratory problems in birds is a highly virulent field strain. The clinical signs point to a TCO vaccine strain that most likely underwent some reversal event and is a latent reactivated infection.

Molecular characterization of chicken astrovirus from high embryonic mortality, based on the capsid protein (ORF2) gene, in Brazil.

CAstV infections were found in farms and incubators with increased embryo mortality.Brazilian CAstV Biv strains were associated with white chick syndrome.Antigenic peptides were predicted on the surface of the capsid protein.

Detection of aves polyomavirus 1 (APyV) and beak and feather disease virus (BFDV) in exotic and native Brazilian Psittaciformes.

There are several viral diseases in captive birds. Aves polyomavirus 1 (APyV) and beak and feather disease virus (BFDV) are among the most important in Psittaciformes. The occurrence of these agents has been widely described in various parts of the world; however, little is known about these viruses in South America. APyV and BFDV can cause high morbidity with feather alterations and even mortality. Other variable symptoms could appear depending on the host's age and taxonomic group. The aim of this study was to detect APyV and BFDV in samples of captive exotic and native Psittaciformes in Brazil. Samples from 120 birds with clinical signs compatible with APyV and/or BFDV were examined. In total, 57 (47.5%) positive birds were found, of which 21 (17.5%) had APyV and 41 (34.17%) had BFDV. Five animals (4.17%) presented concurrent infection. Phylogenetic analysis showed a divergent APyV strain and a diversity of Brazilian BFDV strains. Our study shows that these viruses are present at a significant frequency in captive exotic and native Psittaciformes in Brazil. This study also highlights the need for constant epidemiologic surveillance to preserve bird biodiversity with a focus on endangered Psittaciformes species.

Outbreaks of Avipoxvirus Clade E in Vaccinated Broiler Breeders with Exacerbated Beak Injuries and Sex Differences in Severity.

Avipoxvirus affects chickens and wild birds, and it is characterized by lesions on the nonfeathered parts of the body (the cutaneous form), or necrotic lesions in the upper respiratory tract (the diphtheritic form). In poultry farming, avian pox is usually controlled by live attenuated vaccines. However, there have been many reports of outbreaks, even in flocks of vaccinated birds. In the present study, different outbreaks of the emerging clade E avipoxvirus were detected in commercial breeder flocks of chickens vaccinated against fowlpox virus in Southeast Brazil. Clinical manifestations of these outbreaks included a marked prevalence of moderate to severe progressive lesions in the beaks of affected birds, especially in roosters with increased mortality (up to 8.48%). Also, a reduced hatchability (up to 20.77% fewer hatching eggs) was observed in these flocks. Analysis of clinical samples through light and transmission electron microscopy revealed the presence of Bollinger bodies and poxvirus particles in epithelial cells and affecting chondrocytes. PCR, sequencing, and phylogenetic analysis of major core protein (P4b) and DNA polymerase (pol) genes identified this virus as clade E avipoxvirus. We also developed qPCR assays for open reading frames (ORFs) 49, 114, and 159 to detect and quantify this emergent virus. These results show the arrival and initial spread of this pathogen in the poultry industry, which was associated with harmful outbreaks and exacerbated clinical manifestations in vaccinated commercial breeder flocks. This study also highlights the relevance of permanent vigilance and the need to improve sanitary and vaccination programs.

Complete Genome Characterization of Reticuloendotheliosis Virus Detected in Chickens with Multiple Viral Coinfections.

Reticuloendotheliosis virus (REV) is a retroviral pathogen capable of infecting several avian hosts and is associated with immunosuppression, anemia, proventriculitis, neoplasia, and runting-stunting syndrome. Its genome contains the three major genes, gag, pol, and env, and two flanking long terminal repeat (LTR) regions. Complete genome sequences of REV are limited in terms of geographical origin. The aim of this study was to characterize the complete genome of REV detected in Brazilian chickens with multiple viral coinfections and analyze the polymorphisms in the deduced amino acids sequences corresponding to its encoded proteins. We tested the presence and completeness of REV as well as other viral pathogens in samples from Brazilian poultry farms by qPCR. The complete genomes of two REV strains were sequenced by overlapping fragments through the dideoxy method. Phylogenetic analysis, pairwise identity matrix, polymorphism identification and protein modeling were performed along the entire genome. We detected REV in 65% (26/40) of the tested samples. Concomitant viral infections were detected in 82.5% (33/40) of the samples and in 90% (9/10) of the farms. Multiple infections included up to seven viruses. Phylogenetic analysis classified both Brazilian strains into REV subtype 3, and the pairwise comparison indicated that strains from the USA and fowlpox virus (FWPV)-related strains were the most identical. The subdomain p18 in gag, the reverse transcriptase/ribonuclease H in pol, and the surface (SU) in the env protein were the most polymorphic in genomic comparisons. The relevant motifs for each protein were highly conserved, with fewer polymorphisms in the fusion peptide, immunosuppression domain, and disulfide bonds on the surface (SU) and transmembrane (TM) of env. This is the first study to include complete genomes of REV in Brazil and South America detected in farms with multiple viral coinfections. Our findings suggest an involvement of REV as an immunosuppressor and active agent in the emergence and progression of multiple infectious diseases. We also found a possible etiological relationship between Brazilian strains and the USA and FWPV recombinant strains. This information highlights the need for epidemiological vigilance regarding REV in association with another pathogens.

In vitro and in vivo persistence of IncN plasmids carrying qnr genes in uropathogenic Escherichia coli isolates.

OBJECTIVES: This study aimed to evaluate the persistence of the plasmid-mediated quinolone resistance (PMQR) among uropathogenic Escherichia coli strains grown under or without exposure to subinhibitory concentrations of ciprofloxacin. Based on that, we evaluated the possible spontaneous loss or maintenance of PMQR and the possible appearance of compensatory mutations in gyrA and parC genes. METHODS: Three uropathogenic E. coli strains harbouring chromosomal mutations in the gyrA and/or parC genes coupled with qnrS1 or qnrB2 determinants carried by distinct plasmid sizes and incompatibility N groups (IncN/ST1, IncN/ST5) were evaluated using in vitro and in vivo assays. RESULTS: PMQRs remained stable in all strains throughout the generations evaluated, independently of exposure to ciprofloxacin in both in vivo and in vitro assays. Analysis of gyrA and parC genes after in vivo and in vitro assays revealed that no changes occurred in quinolone-resistance determining regions (QRDR). CONCLUSION: We demonstrated that IncN plasmids were persistent over 14 days in E. coli clinical strains independently of exposure to ciprofloxacin, as well as previous mutations in QRDR.

Molecular Characterization and Determination of Relative Cytokine Expression in Naturally Infected Day-Old Chicks with Chicken Astrovirus Associated to White Chick Syndrome.

White chick syndrome (WCS) is an emergent disease that affects hatchability and hatched chicks, resulting in high mortality and economic losses, and is related to chicken astrovirus (CAstV). This syndrome has been reported in several countries worldwide, and groups A iii and B vi of CAstV have been determined; however, in Brazil, the virus has not been genotyped. The innate immunity of chicks affected by WCS or any CAstV is poorly understood and studied, and it is important to determine whether relative cytokine expression occurs during the early stages of the life of chicks. The aim of the present investigation is to detect and molecularly characterize CAstV associated with WCS, examine the macroscopic and microscopic lesions in the jejunum and spleen, and determine cytokine expression in the jejunum, liver, spleen and thymus of chicks naturally infected with WCS. To do so, we applied a pathological and molecular approach for CAstV detection and characterization, as well as the quantification of the relative mRNA expression of several cytokine genes. The phylogenetic analyses of the sequences obtained herein classified CAstV as uniquely belonging to group B iv, showing a high similarity of nucleotides (NT) (75.7-80.6%) and amino acids (AA) (84.2-89.9%) with the members of group B and a low similarity of NT (46.7-47.9%) and AA (37.8-38.9%) with the virus belonging in group A. CAstV was also detected and quantified in the serum, spleen, thymus and jejunum, the latter being the organ where CAstV had the highest viral concentration. However, this organ did not present any microscopical alterations. In contrast, we observed necrotic hepatitis in the liver of the affected subjects. On the other hand, we observed the activation of several T helper 1 (Th1)- and T helper 2 (Th2)-cytokines (IFN-γ, IL-2, IL-8, IL-12p40, IL-15, TGF-ß4, TNF-SF-15 and t-BET), without being able to control the viral replication due to the high concentration of viral particles in some organs, principally in the gut. One possible role of these cytokines is contributing to the control of inflammation and cell protection of intestinal cells, principally during the early activation of immune responses. However, the fact that these responses are not mature enough to control the viral infection means that more studies need to be carried out to elucidate this topic.

Molecular Characterization and Pathogenicity of Chicken Parvovirus (ChPV) in Specific Pathogen-Free Chicks Infected Experimentally.

Chicken parvovirus (ChPV) is an agent frequently associated with runting stunting syndrome (RSS). This syndrome has been reported in association with ChPV in many countries, including Brazil; however, studies characterizing the virus on a molecular level are scarce, and ChPV pathogenicity in day-old chicks remains unclear. The aim of the present work was to establish the molecular characteristics of ChPV, determine the pathogenicity of ChPV in SPF chicks and detect and quantify ChPV by qPCR in several tissues and chicks of different ages. The experimental challenge was performed at one day of age, and daily and weekly observations were performed and five birds from each experimental group (mock and infected birds) were euthanized to perform the different analysis. ChPV genome copies were detected and quantified by qPCR in gut, spleen, thymus, kidney, pancreas, proventriculus and bursa. Clinically, the infected group presented with diarrhea 24 h post-infection, which persisted until 42 days of age. The small intestine was distended, and its contents were aqueous and foamy. Enteritis and dilated crypts with cyst shapes were observed in intestinal segments. Acute pancreatitis associated with lymphocytic nodules, infiltrating lymphocytes and plasma cells between the pancreatic acinus was observed. Koch's postulate was demonstrated and the genetic characterization of the VP1 gene showed that the Brazilian ChPV isolate belongs to the ChPV II group.