Detection of Campylobacter spp. in chilled and frozen broiler carcasses comparing immunoassay, PCR and real time PCR methods / Detecção de Campylobacter spp. em carcaças de frango de corte resfriadas e congeladas pelos métodos imunoenzimático, PCR e PCR em tempo real

ABSTRACT: In order to detect and identify Campylobacter spp. in broiler chicken carcasses, and to compare detection methods, 43 chilled and 43 frozen carcasses were collected and analyzed. Three methodologies were evaluated: an automated Enzyme Linked Fluorescent Assay (ELFA) VIDAS®30, Polymerase Chain Reaction (PCR) and real-time PCR. Only four chilled carcasses (4.6%) were considered positive for Campylobacter spp. by VIDAS®30 and no sample was positive when the conventional PCR technique was used. However, real-time PCR showed a higher incidence of contamination by Campylobacter spp. in broiler carcasses, with 45 (52.3%) positive samples. C. jejuni was the species most frequently reported in the samples (88.8%). No differences in the frequencies of Campylobacter spp. were observed between the chilled and frozen broiler carcasses. In conclusion, real-time PCR was the most sensitive method for the detection of Campylobacter spp. in chilled or frozen broiler carcasses, which were mainly contaminated by C. jejuni.

RESUMO: Com o objetivo de detectar e identificar Campylobacter spp. em carcaças de frango de corte utilizando três metodologias distintas - ensaio imunoenzimático VIDAS®30, Reação em Cadeia da Polimerase (PCR) e PCR em tempo real - foram coletadas e analisadas 43 carcaças de frango resfriadas e 43 congeladas. Quatro carcaças refrigeradas (4,6%) foram consideradas positivas para Campylobacter spp. pelo VIDAS®30 e nenhuma amostra positiva foi identificada quando utilizada a técnica de PCR. Porém, ao analisar as carcaças pela metodologia da PCR em tempo real, foi observada uma maior incidência de Campylobacter spp., com 45 amostras (52,3%) positivas, sendo que Campylobacter jejuni foi a espécie mais frequentemente encontrada nas amostras (88,8%). Não foi observada diferença na frequência do micro-organismo entre carcaças de frangos resfriadas e congeladas. Concluiu-se que a técnica de PCR em tempo real apresentou maior sensibilidade na detecção de Campylobacter spp. em carcaças de frangos de corte e que foi encontrada elevada presença de carcaças contaminadas, especialmente por C. jejuni.

Detection of fluoroquinolone resistance by mutation ingyrA gene of Campylobacterspp. isolates from broiler and laying (Gallus gallus domesticus) hens,from Rio de Janeiro State, Brazil / Detecção de resistência às fluoroquinolonas através da mutação no gene gyrA em Campylobacter spp. isolados de frangos de corte e galinhas (Gallus gallus domesticus) poedeiras, no Estado do Rio de Janeiro, Brasil

Poultry are considered to be the main reservoir of Campylobacterspp. bacteria, an important pathogen for humans. Many studies have reported a rapid selection of fluoroquinolone-resistant strains following the widespread use of these antimicrobials in poultry production and human medicine. The main mechanism of fluoroquinolone resistance in Campylobacteris a mutation in the Quinolone Resistance Determinant Region (QRDR) in the gyrA gene, which codes for the subunit of the enzyme DNA gyrase, the target for fluoroquinolone. The aim of this study was to investigate the mutation in QRDR in the gyrA gene of Campylobacterstrains previously isolated from broiler carcasses and feces of laying hens. Thirty-eight strains of C. jejuniand 19 C. colistrains (n=57), previously characterized as resistant to ciprofloxacin and enrofloxacin by the disk diffusion method and minimum inhibitory concentration (MIC), were selected. For detection of the mutation, a fragment of 454pb QRDR in the gyrA gene was used for direct sequencing. All strains presented the QRDR mutation in the gyrA gene at codon 86 (Thr-86-Ile), which confers resistance to fluoroquinolones. Other known silent mutations were observed. This genotypic characterization of fluoroquinolone resistance inCampylobacterstrains has confirmed the prior phenotypic detection of the resistance. The Thr-86-Ile mutation was observed in all samples confirming that this is the predominant mutation in enrofloxacin and ciprofloxacin resistant strains of C. jejuniand C. coli.

As aves são consideradas o principal reservatório deCampylobacterspp., um importante patógeno para humanos e muitos estudos têm relatado uma rápida seleção de cepas resistentes às fluoroquinolonas após o uso destes antimicrobianos na produção avícola e na medicina humana. O principal mecanismo de resistência às fluoroquinolonas em Campylobacterconsiste na mutação na Região Determinantes de Resistência às Quinolonas (RDRQ) do gene gyrA, que codifica para a subunidade A da enzima DNA girase, alvo das fluoroquinolonas. O objetivo deste estudo foi investigar a mutação na RDRQ do gene gyrA em cepas de Campylobacterpreviamente isolados de carcaças de frangos de corte e fezes de galinhas poedeiras. Foram selecionadas 38 cepas de C. jejunie 19 cepas de C. coli(n=57), previamente caracterizadas como resistentes à ciprofloxacina e enrofloxacina, pelo método da difusão em disco e pela determinação da concentração inibitória mínima. Para detecção da mutação, foi utilizado sequenciamento direto de um fragmento de 454pb da RDRQ do gene gyrA gerado por PCR. Todas as cepas apresentaram a mutação na RDRQ do gene gyrA no códon 86 (Tre-86-Ile), que confere resistência às fluoroquinolonas e outras mutações silenciosas foram observadas. A caracterização genotípica da resistência às fluoroquinolonas em Campylobacterconfirmou a prévia detecção fenotípica dessa resistência e a mutação Tre-86-Ile foi observada na totalidade das amostras, comprovando ser esta a mutação predominante em cepas de C. jejunie C. coliresistentes à enrofloxacina e ciprofloxacina.

In vitro susceptibility of Campylobacter jejuni from Kuwait to tigecycline & other antimicrobial agents.

Background & objectives: There is an increasing frequency of resistance of Campylobacter jejuni to antimicrobial agents making treatment difficult. In this study, the in vitro susceptibility of C. jejuni isolates collected over an eight year period was tested against tigecycline, a glycylcycline, the previously tested antimicrobial agents in Kuwait, ciprofloxacin, erythromycin and tetracycline, and other antimicrobial agents not previously tested in Kuwait, amoxicillin-clavulanic acid, gentamicin, imipenem and meropenem. Methods: A total of 97 C. jejuni isolates from diarrhoeal stools of Kuwaiti patients during 2002-2010 were studied for susceptibility to the above antimicrobial agents by E test. Results: Erythromycin resistance increased from 5.0 per cent in 2002-2003 to 13.8 per cent in 2007-2010. The figures for ciprofloxacin resistance for the same periods were 53 and 65.5 per cent, respectively. Tetracycline resistance increased from 40.0 per cent in 2003-2006 to 62.1 per cent in 2007-2010 (P=0.05). However, all isolates were uniformly susceptible to tigecycline and other antimicrobial agents. Interpretation & conclusions: There was a progressive increase in the prevalence of resistance to ciprofloxacin, erythromycin and tetracycline. As all isolates were uniformly susceptible to tigecycline, this antimicrobial agent can be considered as a potential candidate for treatment in clinical studies.

Influences of both genetic background and sex on the production of autoantibodies induced by vaccine of campylobacter jejuni / 中国免疫学杂志

Five strains of mice (C57BL/6, BALB/c, ICR, KM and SMMC/B) were respectively orally immunized with CJ-S131 vaccine for 16 weeks, Autoantibodies against ss-DNA, ds-DNA, histones, extractable nuclear antigens (ENA)and thymocytes were detected by ELISA or CELIS(?)One or more kind (s) of autoantibody significantly raised in the serum of SMMC/B, KM, BALB/c and ICR mice, but not in C57BL/6 mice. The mean enzyme index (EI) of the autoantibodies of SMMC/B and KM mice was higher than that of ICR and BALB/c. The influence of the sex on the production of autoantibody induced by CJ-S131 vaccine seemed to be demonstrated on the basis of genetic background.It was characterized by (1) autoantibodies raising mainly in the female mice,e.g. in KM mice; (2) one kind of autoantibody or more raising in both male and female mice, as seen in SMMC/B and BALB/c mice; and (3) autoantibody no in male and female of C57BL/6 mice. The results revealed that the profile of autoimmune response induced by CJ-S131 vaccine was different in strains and sex of mice.

Kinetics of autoantibody induced by oral immunization with formalized campylobacter jejuni in mice / 中国免疫学杂志

KM mice were orally immunized with formalized C. jejuni (CJ-S131 vaccine) for 16 weeks. The production kinetics of autoanti bodies against ss-DNA, ds-DNA, histories and extractable nuclear antigens (ENA) was investigated and two peaks of autoantibody production were observed within 16 weeks after immunizalion. The first peak appeared at 6th week after immunization when the positive percentage of autoantibody against histones (83%) and ENA (67%) was higher than that against ds-DNA (33%) and ss-DNA ( 0 %), and the second peak at 16th week when the positive percentage of anti-ds-DNA (50%) and anti-ss-DNA (83%) antibody higher than that of anti-histones (33%) and anti-ENA (33%) anti body. Natural autoanti bodies against nuclear acid (ds-DNA and ss-DNA) in the controlled mice could be detected, too, by ELISA, which raised with aging but obviously demonstrating individual difference; however, autoanti bodies to nucleoproteins (histones and ENA) could not be detected at the matched times. The results suggested that autoantibody against histones or ENA was more specific for autoimmune diseases than that against DNA, and might be of significance for earlier diagnosis of the systemic autoimmune diseases, e. g. SLE, in clinic.

Autoimmune syndrome induced by chronic mucosal immune response / 中国免疫学杂志

To explore me chanisms of successful induction of autoimmunity by chronic Campylobacter jejuni (CJ-S131 ) infection [3, 4, 5, 6], a chronic mucosal immune response mooe(?) was established by oral immunization of BALB /C mice with formalized CJ-S131 bacteria.in a dosage of 4 xlO8 bacterial cells per mouse, twice a week for 14 weeks, which mimicked the released antigens persistently stimulating the mucosal immune system when mice were chronically infected by C. jejuni. It was also found that the immunized mice demonstrated (?)upus-like autoimmune syndro me, sim ilar, but more severe.to those seen in the mice chronically infected by C. jejuni. It was characterized by (1) significant lymphoproliferation of both mucosal and systemic immune systems; (2) polyclonal activation of B lymphocytes; (3) significantly elevated level of multiple autoanti bodies against ss- DNA, ds- DNA and histones; (4) immunocom plex glomerulonephritisi; (5) chronic inflammation of multiple organs or tissues including the intestine, liver and blood vessels. In the polyclonal activation testin vitro, the levels of total immunoglobulins and autoantibody against DNA in the supernatan ts of the splenic culture cells from the immunized mice were significantly higher than that from the controll mice. The results verified that chronic C. jejuni infection in the gut could induce abnormal. chronic mucosal immune response which led to perturbation of the systemic immune system, resulting autoimmunity or autoimmune syndrom.