Protected Organic Acid and Essential Oils for Broilers Raised Under Field Conditions: Intestinal Health Biomarkers and Cecal Microbiota.

The objective of the present study was to evaluate the effect of protected organic acids (OA) and essential oils (EO) [P(OA + EO)] on the intestinal health of broiler chickens raised under field conditions. The study was conducted on four commercial farms. Each farm consisted of four barns, two barns under a control diet and two tested barns supplemented with P(OA + EO), totaling 16 barns [8 control and 8 under P(OA + EO)]. The control group was supplemented with antibiotic growth promoters [AGP; Bacitracin Methylene Disalicylate (50 g/ton) during starter, grower and finisher 1, and flavomycin (2 g/ton) during finisher 2]. The tested group was supplemented with 636, 636, 454, and 454 g/ton of P(OA + EO) during starter, grower, finisher 1 and 2, respectively. Eighty birds were necropsied (40/treatment; 20/farm; and 5/barn) to collect blood, jejunal tissue, and cecal contents. The data were submitted to analysis of variance (ANOVA) (P < 0.05) or Kruskal-Wallis' test and the frequency of antimicrobial resistant (AMR) genes was analyzed by Chi-Square test (P < 0.05). It was observed that the supplementation of P(OA + EO) reduced (P < 0.05) the histopathology scores, such as the infiltration of inflammatory cells in the epithelium and lamina propria and tended (P = 0.09) to reduce the serum concentration of calprotectin (CALP). The supplementation of P(OA + EO) reduced the serum concentration of IL-12 (P = 0.0001), IL-16 (P = 0.001), and Pentraxin-3 (P = 0.04). Additionally, P(OA + EO) maintained a cecal microbiota similar to birds receiving AGP. The substitution of AGP by P(OA + EO) reduced (P < 0.05) the frequency of four AMR genes, related to gentamicin (three genes), and aminoglycoside (one gene). Overall, the inclusion of P(OA + EO), and removal of AGP, in the diets of commercially raised broiler chickens beneficially changed the phenotype of the jejunum as shown by the lowered ISI scores which characterizes an improved intestinal health. Furthermore, P(OA + EO) significantly reduced the serum concentration of several inflammatory biomarkers, while maintaining the diversity and composition of the cecal microbiota similar to AGP fed chickens and reducing the prevalence of AMR genes.

mcr-1 Identified in Fecal Escherichia coli and Avian Pathogenic E. coli (APEC) From Brazil.

Colisitin-associated resistance in bacteria of food producing animals has gained significant attention with the mcr gene being linked with resistance. Recently, newer variants of mcr have emerged with more than nine variants currently recognized. Reports of mcr associated resistance in Escherichia coli of poultry appear to be relatively limited, but its prevalence requires assessment since poultry is one of the most important and cheapest sources of the world's protein and the emergence of resistance could limit our ability to treat disease outbreaks. Here, 107 E. coli isolates from production poultry were screened for the presence of mcr 1-9. The isolates were collected between April 2015 and June 2016 from broiler chickens and free-range layer hens in Rio de Janeiro, Brazil. All isolates were recovered from the trachea and cloaca of healthy birds and an additional two isolates were recovered from sick birds diagnosed with colibacillosis. All isolates were screened for the presence of mcr-1 to 9 using PCR and Sanger sequencing for confirmation of positive genes. Additionally, pulse field gel electrophoresis (PFGE) analysis, avian fecal E. coli (APEC) virulence associated gene screening, plasmid replicon typing and antimicrobial resistance phenotype and resistance gene screening, were also carried out to further characterize these isolates. The mcr-1 gene was detected in 62 (57.9%) isolates (61 healthy and 1 APEC) and the mcr-5 gene was detected in 3 (2.8%) isolates; mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, and mcr-9 were not detected in any isolate. In addition, mcr 1 and 5 positive isolates were phenotypically resistant to colistin using the agar dilution assay (> 8ug/ml). PFGE analysis found that most of the isolates screened had unique fingerprints suggesting that the emergence of colistin resistance was not the result of clonal dissemination. Plasmid replicon types IncI2, FIB, and B/O were found in 38, 36, and 34% of the mcr positive isolates and were the most prevalent replicon types detected; tetA and tetB (32 and 26%, respectively) were the most prevalent antimicrobial resistance genes detected and iutA, was the most prevalent APEC virulence associated gene, detected in 50% of the isolates. Approximately 32% of the isolates examined could be classified as APEC-like, based on the presence of 3 or more genes of APEC virulence associated path panel (iroN, ompT, hlyF, iss, iutA). This study has identified a high prevalence of mcr-1 in poultry isolates in Brazil, suggesting that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. Emergence of the mcr gene and associated colisitin resistance in production poultry warrants continued monitoring from the animal health and human health perspective.

Comparing PFGE, MLST, and WGS in monitoring the spread of macrolide and rifampin resistant Rhodococcus equi in horse production.

BACKGROUND: Rhodococcus equi (R. equi) infections are endemic in many horse facilities in the United States resulting significant economic loses annually. Currently, there is no commercial vaccine available and the emergence of isolates that are resistant to the current treatment and prophylaxis using antibiotics prompts closer surveillance of this pathogen. OBJECTIVE: This study compares three different genotyping techniques, Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and whole genome SNP-based phylogeny to determine the most accurate method to monitor the spread of macrolide-and-rifampin-resistant R. equi. METHODS: 16 macrolide and rifampin-resistant and 6 susceptible R. equi and their Illumina Miseq whole genome sequences were used in this study. The isolates were sub-typed by PFGE with VspI and a dendrogram based on their similarities generated. Additionally, three phylogenetic trees were constructed using CSI phylogeny on (i) whole genome sequences (WGS), (ii) in silico MLST sequences and (iii) MLST sequences obtained after PCR-amplification and Sanger sequencing. RESULTS: PFGE identified 18 different genetic profiles and grouped the 22 isolates into 3 clusters independently of their susceptibilities. The phylogenetic trees built from WGS and MLST data showed similar topology, separating the isolates into 2 major clades in accordance with their susceptibility profiles (susceptible and resistant). However, only the trees generated with next generation sequencing data could detect the clonality of the resistant isolates.

Prevalence of ColV Plasmid-Linked Genes and In Vivo Pathogenicity of Avian Strains of Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in birds, leading to an increase in the cost of poultry production. The ColV plasmid-linked genes iroN, ompT, hlyF, iss, and iutA have previously been suggested to be predictors of the virulence of APEC. In this research, we analyzed the frequencies of these genes in a Brazilian collection of E. coli isolated from birds with colibacillosis (APEC) and from apparently healthy birds (avian fecal [A(fecal)]), as well as from the litter of poultry houses of apparently healthy flocks (avian litter [A(litter)]). All the isolates that harbored ompT also harbored hlyF, so they were considered as one trait for statistical analysis. The relationship between in vivo virulence in 1-day-old chicks, expressed as a pathogenicity score, and the number of genes in each isolate showed that isolates with less than two of the four genes were rarely pathogenic, while most pathogenic isolates contained two or more genes. Nevertheless, about half of the nonpathogenic isolates also harbored two or more genes, in agreement with previous observations that commensal E. coli isolates from the birds' microbiota can serve as a reservoir of virulence genes. Thus, the pentaplex polymerase chain reaction can be used to indicate that a strain carrying none or only one gene would be nonpathogenic, but it cannot be used to indicate that a strain with two to four genes would be an APEC. Isolates allocated to phylogenetic group B2, which is frequently associated with extraintestinal infections, had the highest pathogenicity scores, while isolates allocated to group B1 had the lowest.

Molecular characterization and clonal relationships among Escherichia coli strains isolated from broiler chickens with colisepticemia.

This study characterized 52 Escherichia coli isolates from distinct diseased organs of 29 broiler chickens with clinical symptoms of colibacillosis in the Southern Brazilian state of Rio Grande do Sul. Thirty-eight isolates were highly virulent and 14 were virtually avirulent in 1-day-old chicks, yet all isolates harbored virulence factors characteristic of avian pathogenic E. coli (APEC), including those related to adhesion, iron acquisition, and serum resistance. E. coli reference collection phylogenetic typing showed that isolates belonged mostly to group D (39%), followed by group A (29%), group B1 (17%), and group B2 (15%). Phylogenetic analyses using the Amplified Ribosomal DNA Restriction Analysis and pulse-field gel electrophoresis methods were used to discriminate among isolates displaying the same serotype, revealing that five birds were infected with two distinct APEC strains. Among the 52 avian isolates, 2 were members of the pandemic E. coli O25:H4-B2-ST131 clone.

Genotypes and pathogenicity of cellulitis isolates reveal traits that modulate APEC virulence.

We characterized 144 Escherichia coli isolates from severe cellulitis lesions in broiler chickens from South Brazil. Analysis of susceptibility to 15 antimicrobials revealed frequencies of resistance of less than 30% for most antimicrobials except tetracycline (70%) and sulphonamides (60%). The genotyping of 34 virulence-associated genes revealed that all the isolates harbored virulence factors related to adhesion, iron acquisition and serum resistance, which are characteristic of the avian pathogenic E. coli (APEC) pathotype. ColV plasmid-associated genes (cvi/cva, iroN, iss, iucD, sitD, traT, tsh) were especially frequent among the isolates (from 66.6% to 89.6%). According to the Clermont method of ECOR phylogenetic typing, isolates belonged to group D (47.2%), to group A (27.8%), to group B2 (17.4%) and to group B1 (7.6%); the group B2 isolates contained the highest number of virulence-associated genes. Clonal relationship analysis using the ARDRA method revealed a similarity level of 57% or higher among isolates, but no endemic clone. The virulence of the isolates was confirmed in vivo in one-day-old chicks. Most isolates (72.9%) killed all infected chicks within 7 days, and 65 isolates (38.1%) killed most of them within 24 hours. In order to analyze differences in virulence among the APEC isolates, we created a pathogenicity score by combining the times of death with the clinical symptoms noted. By looking for significant associations between the presence of virulence-associated genes and the pathogenicity score, we found that the presence of genes for invasins ibeA and gimB and for group II capsule KpsMTII increased virulence, while the presence of pic decreased virulence. The fact that ibeA, gimB and KpsMTII are characteristic of neonatal meningitis E. coli (NMEC) suggests that genes of NMEC in APEC increase virulence of strains.

Characterization of extraintestinal Escherichia coli isolated from a peacock (Pavo cristatus) with colisepticemia.

Extraintestinal infections by avian pathogenic strains of Escherichia coli (APEC) are commonly reported in poultry, but there is little information on infections by APEC in other bird species. Here we report on the characterization of extraintestinal E. coli isolated from a domesticated peacock, from the south of Brazil, that died of colisepticemia. Necropsy examination revealed congested liver, hypertrophied kidneys, peritonitis, severe typhlitis suggestive of coligranuloma, pneumonia, and airsacculitis--typical signs of colisepticemia. The isolates from lungs, kidney, heart, intestine, liver, and bone marrow all harbored the same virulence-associated factors (iucD, colV, iss, mat, fimC, ompA, traT crl, csgA vgrG, and hcp), yielded the same band pattern in amplified ribosomal DNA restriction analysis, and were allocated to the Escherichia coli Reference Collection group B1. The isolates were resistant to bacitracin, trimethoprim, and tetracycline, but displayed slight differences in their resistance to other antimicrobials. The isolates also differed in their virulence in 1-day-old chickens, but none displayed high virulence in vivo. We conclude that the peacock died of colisepticemia after it was infected with an extraintestinal E. coli strain of low virulence that nevertheless harbored virulence factors generally associated with APEC. This study represents the first characterization of an APEC isolated from a nonpoultry bird species.

Infections with avian pathogenic and fecal Escherichia coli strains display similar lung histopathology and macrophage apoptosis.

The purpose of this study was to compare histopathological changes in the lungs of chickens infected with avian pathogenic (APEC) and avian fecal (A(fecal)) Escherichia coli strains, and to analyze how the interaction of the bacteria with avian macrophages relates to the outcome of the infection. Chickens were infected intratracheally with three APEC strains, MT78, IMT5155, and UEL17, and one non-pathogenic A(fecal) strain, IMT5104. The pathogenicity of the strains was assessed by isolating bacteria from lungs, kidneys, and spleens at 24 h post-infection (p.i.). Lungs were examined for histopathological changes at 12, 18, and 24 h p.i. Serial lung sections were stained with hematoxylin and eosin (HE), terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) for detection of apoptotic cells, and an anti-O2 antibody for detection of MT78 and IMT5155. UEL17 and IMT5104 did not cause systemic infections and the extents of lung colonization were two orders of magnitude lower than for the septicemic strains MT78 and IMT5155, yet all four strains caused the same extent of inflammation in the lungs. The inflammation was localized; there were some congested areas next to unaffected areas. Only the inflamed regions became labeled with anti-O2 antibody. TUNEL labeling revealed the presence of apoptotic cells at 12 h p.i in the inflamed regions only, and before any necrotic foci could be seen. The TUNEL-positive cells were very likely dying heterophils, as evidenced by the purulent inflammation. Some of the dying cells observed in avian lungs in situ may also be macrophages, since all four avian E. coli induced caspase 3/7 activation in monolayers of HD11 avian macrophages. In summary, both pathogenic and non-pathogenic fecal strains of avian E. coli produce focal infections in the avian lung, and these are accompanied by inflammation and cell death in the infected areas.

Avian pathogenic Escherichia coli MT78 invades chicken fibroblasts.

Avian pathogenic Escherichia coli (APEC) are responsible for extraintestinal diseases, called colibacillosis, in avian species. The most severe manifestation of the disease is colisepticemia that usually starts at the respiratory tract and may result in bird death. However, it is not yet clear how APEC cross the respiratory epithelium and get into the bloodstream. In this work, we studied the interaction between 8 APEC strains (UEL31, UEL17, UEL13, UEL29, MT78, IMT5155, IMT2470, A2363) and a chicken non-phagocytic cell, the fibroblast CEC-32 cell line. We investigated the association profile, the invasion capability, the cytotoxicity effect and the induction of caspase-3/7 activation in an attempt to understand the way the pathogen gains access to the host bloodstream. Association to cells was determined after 1 h of infection, while cell invasion was determined after 4 and 24 h of infection. The cytotoxic effect of bacterial infection was measured by lactate dehydrogenase (LDH) release and the activation of the apoptotic program was verified by caspase-3/7 activation. Also, the presence of genes for adhesins, invasins and other related virulence-associated factors was verified by PCR. All bacterial strains showed similarity in relation to adhesion, LDH release and caspase-3/7 activation. However, one APEC strain, MT78, showed high invasion capability, comparable to the invasive Salmonella typhimurium strain SL1344. Since an APEC strain was capable of invading non-phagocytic cells in vitro, the same may be happening with the epithelial cells of the avian respiratory tract in vivo. CEC-32 monolayers can also provide a useful experimental model to study the molecular mechanisms used by APEC to invade non-phagocytic cells.

Enteroparasitoses em escolares residentes na periferia de Porto Alegre, RS, Brasil / Enteroparasites in schoolchildren living in a suburban community of Porto Alegre, RS, Brazil

Análises coproparasitológicas foram realizadas em 222 crianças de um loteamento da periferia de Porto Alegre-RS, entre agosto de 2004 e setembro de 2005, pelo método de Hoffman, Pons e Janner. A positividade para enteroparasitos foi de 102 (46por cento) amostras. A faixa etária de 12 a 14 anos foi a que apresentou maior percentual de positividade (58,5por cento). O parasito mais freqüente foi Trichuris trichiura (18,9por cento), seguido por Ascaris lumbricoides e pelo comensal Entamoeba coli (ambos com ocorrência de 16,7por cento), Giardia lamblia (2,7por cento), Strongyloides stercoralis (4,5por cento), Hymenolepis nana (2,7por cento) e Enterobius vermicularis (2,2por cento). O monoparasitismo foi observado em 63,7por cento dos casos positivos. A associação entre A. lumbricoides e T. trichiura ocorreu 16 vezes no estudo. Os resultados obtidos apontam para a necessidade contínua de investimentos dos setores públicos em saúde e infra-estrutura somados a investimentos em educação e treinamento de educadores visando a uma melhor aplicação dos conhecimentos sobre a prevenção das parasitoses e à conseqüente melhora na qualidade de vida da população.