Isolation and molecular characterization of Salmonella enterica serovar Enteritidis from poultry house and clinical samples during 2010
ABSTRACT A total of 60 Salmonella enterica serovar (ser.) Enteritidis isolates, 28 from poultry houses and 32 from clinical samples, were isolated during 2010. These isolates were subjected to testing and analyzed for antibiotic resistance, virulence genes, plasmids and plasmid replicon types. To assess genetic diversity, pulsed-field gel electrophoresis (PFGE) fingerprinting, using the XbaI restriction enzyme, Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA) and plasmid profiles were performed. All isolates from poultry, and 10 out of 32 clinical isolates were sensitive to ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, sulfisoxazole, streptomycin, and tetracycline. Twenty-one of thirty-two clinical isolates were resistant to ampicillin and tetracycline, and one isolate was resistant to nalidixic acid. PFGE typing of sixty ser. Enteritidis isolates by XbaI resulted in 10-12 bands and grouped into six clusters each with similarity from 95% to 81%. The MLVA analysis of sixty isolates gave 18 allele profiles with the majority of isolates displayed in three groups, and two clinical isolates found to be new in the PulseNet national MLVA database. All isolates were positive for 12 or more of the 17 virulence genes mostly found in S. enterica (spvB, spiA, pagC, msgA, invA, sipB, prgH, spaN, orgA, tolC, iroN, sitC, IpfC, sifA, sopB, and pefA) and negative for one gene (cdtB). All isolates carried a typical 58 kb plasmid, type Inc/FIIA. Three poultry isolates and one clinical isolate carried small plasmids with 3.8, 6, 7.6 and 11.5 kb. Ten of the clinical isolates carried plasmids, with sizes 36 and 38 kb, types IncL/M and IncN, and one isolate carried an 81 kb plasmid, type IncI. Southern hybridization of a plasmid with an Inc/FIIA gene probe hybridized one large 58 kb plasmid in all isolates. Several large and small plasmids from poultry isolates were not typed by our PCR-based method. These results confirmed that PFGE fingerprinting has limited discriminatory power for ser. Enteritidis in both poultry and clinical sources. However, the plasmid and MLVA allele profiles were a useful and important epidemiology tool to discriminate outbreak strains of ser. Enteritidis from poultry and clinical samples.
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ABSTRACT: Salmonelosis is a major zoonotic disease transmitted by food products worldwide. Although Salmonella spp., are usually isolated from intensive livestock production systems, such as the bovine and swine, chicken meat, eggs and poultry byproducts are considered the main source of transmission of S. enteritidis to humans, and this is partially due to the feasibility of cross-contamination in poultry processing plants. In addition, Salmonella-infected chickens and other birds can be asymptomatic or may not develop the disease and often are natural carriers of this pathogen. Epidemiological studies on Salmonella spp., present on poultry farms in Tolima district are very limited, and the prevalence, impact on bird’s health status and public health is currently unknown. This study was designed to estimate the prevalence of Salmonella spp., in laying hen farms, by attempting microbiological culture and biochemical characterization, followed by serotyping and a preliminary molecular characterization using pulsed field gel electrophoresis. The study also looks for potential risk factors associated with the presence of this microorganism that could not be covered by routine farm biosecurity measures. Samples from cloacal swabs (155 pools), food (31), drinking water (31), eggs (310), drag swabs (31), and operator´s stool sample (31), were collected and cultured for Salmonella isolation following standard protocols. The results indicate that Salmonella spp., could be recovered from various sources, however, attention should be placed on the feed. Molecular characterization is in progress to establish the relationships between the Salmonella isolates.International Poultry Scientific Forum, Atlanta, Georgia; 01/2014
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ABSTRACT: Salmonella infection is one of the major food-borne illnesses in the United States. Several Gram-negative bacterial pathogens, including Salmonella Typhi, produce cytolethal distending toxin (CDT), which arrests growth, induces apoptosis of infected host cells and extends persistence of pathogenic bacteria in the host. The aim of this study was to characterize the functionality of CDT (cdtB, pltA and pltB) from non-typhoidal Salmonella isolates. Fifty Salmonella enterica serovar Javiana isolates from food, environmental and clinical samples were screened for cdtB, pltA and pltB genes by PCR and all were positive for all three genes. Nucleotide sequence analysis of all amplified PCR products showed 100% identity to S. Typhi cdtB. To understand the roles of CdtB, PltA and PltB in S. Javiana, cdtB, pltA and pltB deletion mutants were constructed by using a lambda Red-based recombination system. In vitro cultured HeLa cell lines were infected with a wild type strain and its isogenic ∆cdtB, ∆pltA and ∆pltB to determine whether the strains of S. Javiana are responsible for invasion and cytolethal distending intoxication, including cell cycle arrest, cytoplasmic distension, and nuclear enlargement of host target cells. The results showed that HeLa cells infected with S. Javiana wild type were arrested in G2 /M and had distended cytoplasm and nuclei that were larger than those infected with S. Javiana ∆cdtB and ∆pltA strains. The S. Javiana ∆pltB strain retained the ability to induce cytoplasmic distension and cell cycle arrest, whereas the complemented ∆cdtB and ∆pltA S. Javiana strains showed activity like the wild type strains. CdtB and pltA from S. Javiana had apparent effects on the distension of both cytoplasm and nucleus as well as cell cycle arrest of HeLa cell lines after 72 h of infection. Our data show a significant difference between the wild type cdtB strain and its isogenic ∆cdtB for invasion of the cell lines. Therefore, CdtB produced from S. Javiana strains may play an important role in pathogenesis in host cells. This article is protected by copyright. All rights reserved.Foodborne Pathogens and Disease 06/2014; 72(2). DOI:10.1111/2049-632X.12191 · 2.09 Impact Factor
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ABSTRACT: Salmonellosis affects humans more frequently than any other foodborne disease, and it causes severe economic losses in the poultry industry. A cross-sectional study was carried out to estimate the prevalence of Salmonella spp. in laying hen farms in the Tolima region of Colombia. Fifteen egg-laying hen farms were sampled, and a total of 589 samples were cultured to isolate Salmonella spp. A total of 14 isolates of Salmonella spp. were recovered from five farms, resulting in a prevalence of 33.33% (95%, confidence interval = 14%–53%) at the farm level. Salmonella spp. were recovered from eggshells (57.15%, n = 8), feed (28.57%, n = 4), and environmental samples (14.29%, n = 2). Farm practices, such as the milling of feed (odds ratio [OR] = 24) and the storage of eggs in the henhouses (OR = 11.25), in addition to the feed type (OR = 7.64) and the use of bamboo for construction of the facility (OR = 5.24), were identified as risk factors for Salmonella spp. The 14 isolates were identified as Salmonella Enteritidis (n = 6) and Salmonella Shannon (n = 8), and both serovars were resistant to a number of antibiotics. Pulsed-field gel electrophoresis presented three different XbaI macrorestriction patterns. The Salmonella Enteritidis isolates all presented a single pattern, whereas the Salmonella Shannon isolates were grouped into two distinct patterns. The results indicate that Salmonella spp. could be recovered from various sources at laying hen farms, and eggshell contamination is a particular concern.Avian Diseases 03/2015; 59:57-63. DOI:10.1637/10873-052714-Reg · 1.11 Impact Factor