Susceptibilidade de duas linhagens comerciais de frango de corte no desenvolvimento de dermatite necrótica e possível relação dos genes iss e iutA de Escherichia coli com a reprodução experimental da doença / Susceptibility of two commercial lineages of broilers in the development of necrotic dermatitis and relationship of iss and iutA genes from Escherichia coli with the experimental reproduction of the disease

Celulite aviária é uma enfermidade de grande importância para a avicultura mundial, sendo relacionada principalmente à Escherichia coli (E. coli). Neste estudo foi comparada a susceptibilidade de duas linhagens de aves no desenvolvimento da celulite diante do desafio com diferentes concentrações de E. coli. Além disso, foi avaliada a relação dos genes iss e iutA com a patogenicidade de amostras de E. coli de diferentes origens (fecal/casos clínicos) em pintinhos e com a reprodução experimental da doença em aves de 35 dias de idade. Através da inoculação de frangos de corte (Cobb/Ross) com diferentes níveis de desafio (105 a 108 UFC/mL) de E. coli, não foram observadas diferenças significativas entre as linhagens quanto à sensibilidade à dermatite necrótica para a mesma dosagem (p≤0,05). A detecção dos genes iss e iutA demonstrou que estes estiveram presentes somente nas amostras provenientes de casos clínicos. Da mesma forma, estes isolados foram considerados de alta patogenicidade para pintinhos (>80% letalidade), levando a formação de áreas de lesão mais extensas (≥3cm2) em aves de 35 dias, quando comparado às amostras de origem fecal (p≤0,05). Ainda, as diferenças com relação ao tamanho de lesão foram constatadas também entre os isolados de mesma origem (p≤0,05). Desta forma, a linhagem não pode ser considerada um fator primordial para o desenvolvimento de dermatite necrótica em frangos. Ainda, sugere-se que os genes iss e iutA, quando presentes em conjunto ou isoladamente, poderiam ser considerados marcadores de virulência em cepas de E. coli causadoras de celulite aviária.(AU)

Avian cellulitis is a disease of great importance for the global poultry industry, being mainly related to Escherichia coli. In this study the susceptibility of two lineages of broilers in the development of cellulite was compared to the challenge with different concentrations of E. coli. In addition, it evaluated the relationship of the iss and iutA genes with pathogenicity of E. coli samples from different origins (fecal/clinical cases) in chicks and with the experimental reproduction of disease in 35-day-old broilers. By inoculating broilers (Cobb/Ross) with different levels of challenge (105-108 CFU/mL) of E. coli, no significant differences had been observed between strains for sensitivity to necrotic dermatitis for the same dosage (p≤0.05). Detection of the iss and iutA genes showed that they were only present in samples from clinical cases. Likewise, these strains were considered high pathogenicity for chickens (>80% lethality), leading to the formation of more extensive lesion areas (≥3cm2) at 35 days of birds compared to the samples from fecal origin (p≤0.05). Still, the differences with respect to lesion size were also found among isolates of the same origin (p≤0,05). Thus, the lineage can not be considered a primary factor in the development of necrotic dermatitis in broilers. Furthermore, it is suggested that iss and iutA genes, when present together or separately, could be considered as virulence markers for E. coli strains that cause avian cellulite.(AU)

Genomic analysis and clinical importance of Escherichia coli isolate from patients with sepsis.

Context: Escherichia coli is a major cause of bloodstream infections and death due to sepsis. Bacteremic isolates harbor a signifi cantly greater repertoire of virulence factors (VFs) in contrast with commensal E. coli isolates. Aims: The aim was to determine the relationships between E. coli VFs, phylogenetic groups, and their clinical importance. Settings and Design: This descriptive study was carried out in a multi-specialty tertiary care hospital. Materials and Methods: Escherichia coli isolates from consecutive episodes of bacteremia in 100 patients were screened for their VFs, phylogenetic group, and their effect on patient’s clinical outcome. Virulence genes of all isolates were determined by multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Estimation of risk of death was calculated using APACHE score II calculator. Results: Of the 100 patients, the most common predisposing factors were diabetes (42%), followed by carcinoma (23%). On analysis of the VF genes of the isolates, a majority of strains (88%) were possessing the fi mH gene followed by iutA (76%), papC (44%), cnf1 (16%), hlyA (16%) and neuC (5%) respectively. Phylogenetic analysis revealed that 25 (25%) isolates belonged to phylogroup A, 8(8%) strains to group B1, 30 (30%) were from group B2 and 37 (37%) were from group D. The incidence of iutA gene was signifi cant in higher APACHE II score group. Conclusions: Our fi ndings indicate that virulent as well as commensal strains are capable of causing sepsis. Host related predisposing factors, adherence factors, and iron uptake are essential for the survival of the sepsis inducing strains.

Detecção de fatores de virulência de Escherichia coli e análise de Salmonella spp. em psitacídeos / Detection of virulence factors in Escherichia coli and analysis of Salmonella spp. in psittacines

The enteric flora of psittacines is mainly composed of Gram positive bacteria. Gram negative bacteria, like Escherichia coli and Salmonella spp., have a high pathogenic potential and can be considerate as an indicative of management problems that may culminate in disease manifestation due to stress factors, poor diets and overcrowding, in combination with a high bacterial load on the environment. The objective of this study was evaluated the presence of Salmonella spp., Escherichia coli and the virulence genes iss and iutA from E. coli isolates. Forty-four samples were analyzed from psittacines living in captivity, which fifteen samples were from organs fragments of necropsied birds, and twenty-nine were from cloacal and crop swabs of red-spectacled parrots (Amazona pretrei) keeping in captivity. No samples were positive for Salmonella spp. In the samples in which E. coli was detected, both virulence factors (genes iss and iutA) were present.

A flora entérica dos psitacídeos é composta principalmente por bactérias Gram positivas. Bactérias Gram negativas, como Escherichia coli e Salmonella spp., apresentam elevado potencial patogênico, sendo consideradas indicativo de problemas de manejo, que poderão culminar em manifestação de doenças em decorrência de fatores estressantes, dietas deficientes e superlotação, combinados com alta carga bacteriana no ambiente. O objetivo deste trabalho foi avaliar a presença de Salmonella spp., Escherichia coli e os fatores de virulência dos genes iss e iutA dos isolados de E. coli. Analisou-se um total de 44 amostras provenientes de psitacídeos criados em cativeiro, sendo estas 15 fragmentos de órgãos de aves submetidas a exame de necropsia e também 29 amostras de swabs de cloaca e inglúvio de papagaios-charão (Amazona pretrei) criados em cativeiro. Nenhuma amostra foi positiva para Salmonella spp. Nas amostras de E. coli detectou-se ambos os fatores de virulência pesquisados.

Evaluation of systemic and mucosal immune responses in mice administered with recombinant Salmonella Typhimurium expressing IutA protein / 대한수의학회지

Avian pathogenic Escherichia coli (APEC) are known to cause extraintestinal disease in poultry, leading to substantial losses in the industry. IutA, iron-regulated aerobactin receptor is firmly associated with APEC. To assess the potential of IutA to induce protective immune responses, attenuated Salmonella Typhimurium strain expressing IutA was constructed and administered orally to BALB/c mice. The IutA-specific immune responses were measured with sera, vaginal and fecal samples by an enzyme-linked immunosorbent assay. We found that the Salmonella-IutA vaccine induced significantly higher immune responses as compared to the control inoculated with the attenuated S. Typhimurium containing the plasmid only. The IutA-specific immune responses were increased by second immunization at third week after initial immunization, whereas triple immunization induced lower immune responses than those induced by the double immunization. The Salmonella-IutA vaccine induced a nature of immunity biased to the Th1-type, as judged by the ratio of IutA-specific IgG isotypes (IgG2a/IgG1). Overall, these results suggest that the Salmonella-IutA vaccine appear to be suitable candidate for a vaccine against APEC.

Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.

A proteína de membrane externa IutA (iron uptake transport) é o receptor para aerobactina férrica, um fator de virulência encontrado mais frequentemente entre as amostras de E. coli pathogênicas para aves (APEC) do que entre os isolados fecais de aves saudáveis. O gene iutA da amostra APEC 9, sorotipo O2:H9, foi amplificado e clonado no vetor pET101/D-TOPO. O gene iutA 2.2 Kb foi sequenciado (AY602767) e mostrou alta similaridade para gene iutA de três plasmidios, dois da APEC, pAPEC-02-ColV (AY545598.4) e pTJ100 (AY553855.1), e um da amostra E. coli invasiva humana, pColV K30. A proteína IutA recombinante (r-IutA) foi produzida em Escherichia coli BL21(DE-3), solubilizada com uréia e purificada em coluna de níquel Ni-NTA. A r-IutA tem aproximadamente 74kDa e foi utilizada para produzir anticorpos anti-IutA. Este anticorpo reagiu com a r- IutA e com IutA da APEC13, como demonstrado por Western blot, mostrando que a r-IutA tem epitopos conservados e sua antigenicidade foi preservada. O anticorpo anti-IutA foi capaz de inibir a atividade biológica da IutA, inibindo o teste positivo de sensibilidade à cloacina DF13 apresentada pela APEC 9, contudo não inibiu o crescimento da APEC9 crescida em M9 e não protegeu os pintinhos inoculados com APEC 9, sugerindo que a APEC possui outro mecanismo de captação de íons ferro distinto da aerobactina.