Application of different DNA extraction procedures, library preparation protocols and sequencing platforms: impact on sequencing results.

In this study three DNA extraction procedures, two library preparation protocols and two sequencing platforms were applied to analyse six bacterial cultures and their corresponding DNA obtained as part of a proficiency test. The impact of each variable on sequencing results was assessed using the following parameters: reads quality, assembly and alignment statistics; number of single nucleotide polymorphisms (SNPs), detected applying assembly- and alignment-based strategies; antimicrobial resistance genes (ARGs), identified on de novo assemblies of all sequenced genomes. The investigated nucleic acid extraction procedures, library preparation kits and sequencing platforms do not significantly affect de novo assembly statistics and number of SNPs and ARGs. The only exception was observed for two duplicates, which were associated to one PCR-based library preparation kit. Results from this comparative study can support researchers in the choice toward the available pre-sequencing and sequencing options, and might suggest further comparisons to be performed.

Multilocus sequence typing of Arcobacter butzleri isolates collected from dairy plants and their products, and comparison with their PFGE types.

AIMS: The present study aimed to determine, by multilocus sequence type (MLST), the heterogeneity level of Arcobacter butzleri isolates and to compare MLST and pulsed-field gel electrophoresis (PFGE) in terms of discriminatory power (DI) as well as unidirectional and bi-directional concordance. METHODS AND RESULTS: Arcobacter butzleri isolates (N = 133) from dairy products and environmental samples, collected from dairy plants, were characterized by MLST and PFGE with SacII and classified in 29 sequence types (STs), 47 PFGE and 62 type strains (TS). Among the 119 alleles, 19 were previously unreported and the same for all the STs but two. A significant linkage disequilibrium was detected when the complete ST data set was analysed The DIs of MLST, PFGE and their combination were 0·937, 0·953 and 0·965 respectively. The adjusted Wallace coefficients between MLST and PFGE as well as PFGE and MLST were 0·535 and 0·720 respectively; the adjusted Rand coefficient was 0·612. CONCLUSIONS: The A. butzleri studied population showed recombination to some degree. PFGE showed a DI higher than MLST. Both methods presented good concordance. The TS analysis seems to show persistence of the same strain on time and possible cross-contaminations between food and environmental sites. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights in the A. butzleri population found in raw milk, cheese, and dairy production plants. The data suggest that MLST and PFGE genotypes correlate reasonably well, although their combination results in optimal resolution.

Occurrence of ε-proteobacterial species in rabbits (Oryctolagus cuniculus) reared in intensive and rural farms.

In order to investigate the occurrence of Campylobacter, Helicobacter and Arcobacter species in caecal contents of rabbits reared in intensive and rural farms, a total of 87 samples from animals belonging to 29 farms were analysed by both cultural and PCR analyses. PCR analysis directly from faecal samples detected 100% positive samples for Campylobacter genus, 3.4% for Helicobacter genus and none for Arcobacter genus. 83 out of 87 animals (95.4%) and all the 29 farms were positive for Campylobacter cuniculorum as also determined by cultural examination. Campylobacter coli and Campylobacter jejuni were isolated only from three animals reared in two rural farms. No Helicobacter and Arcobacter species were isolated. To evaluate a possible genetic variability, one strain of C. cuniculorum from each farm was analysed by Pulsed Field Gel Electrophoresis (PFGE) and Amplified Fragment Length Polymorphism (AFLP). Genotyping revealed that C. cuniculorum population is heterogeneous among the different sources and no dominant clone has spread in the investigated farms. This survey highlighted a high presence of C. cuniculorum with a high rate of intestinal colonization, low presence of C. jejuni-coli, Helicobacter spp. and any Arcobacter spp. in farmed rabbits.

Modified-atmosphere packaging of hen table eggs: effects on pathogen and spoilage bacteria.

As part of a more comprehensive research activity on the use of modified-atmosphere packaging for the improvement of quality and functional properties of table eggs, the effects of air, 100% CO(2), and 100% O(2) packaging were also evaluated on the survival of experimentally inoculated pathogen bacteria (Salmonella Enteritidis, Escherichia coli, and Listeria monocytogenes) as well as on spoilage bacteria (total aerobic mesophilic bacteria) on table eggs during 30 d of storage at 4, 25, and 37°C by colony count method. In general, temperatures played a major role, rather than gasses, in influencing the bacterial survival. In particular, the lowest microbial loads were registered at 4°C on E. coli and spoilage bacteria, whereas 37°C was the best storage temperature to avoid the psychrotropic microorganism L. monocytogenes development regardless of the gas used. One hundred percent CO(2) packaging, in association with a low storage temperature (4°C), had a significant positive effect in reducing Salmonella loads. On eggs inoculated with L. monocytogenes and stored at 4°C as well as on eggs containing only spoilage bacteria and stored at 25°C, 100% CO(2) resulted the best gas in comparison with air and O(2). One hundred percent CO(2) packaging showed no negative effect on pathogen survival compared with air. Although further improvements are required to control RH within packaging to limit bacteria growth/survival, in view of the positive effects of CO(2) packaging on quality traits of table eggs, 100% CO(2) packaging might represent a promising innovative technique for the maintenance of egg characteristics during transport, retail, and domestic storage.

Hot air treatment for surface decontamination of table eggs experimentally infected with Salmonella, Listeria, and Escherichia coli.

Hot-air pasteurization was investigated in the EU-funded project "Reducing Egg Susceptibility to Contaminations in Avian Production in Europe (RESCAPE)" as an innovative treatment for surface bacterial decontamination of table eggs. Possible side effects of the treatment on egg quality traits were also studied. The decontamination power of hot air was evaluated over 1 month on shell eggs that were experimentally inoculated with Salmonella enteritidis, Escherichia coli, or Listeria monocytogenes. The S. Enteritidis and L. monocytogenes populations on the surfaces of treated eggs showed a significant reduction compared with untreated eggs. No statistically significant results were obtained comparing E. coli loads on treated and untreated eggs. No detrimental effects on quality traits either immediately after treatment or after 28 days of storage at 20 degrees C were recorded.

Clostridium perfringens occurrence and ribotypes in healthy broilers reared in different European countries.

The main aim of this study was to investigate the occurrence and ribotypes of Clostridium perfringens in broiler flocks reared in 2 European countries that apply European Union Regulation 1831/2003. A total of 1,532 cecum contents were collected between June 2005 and November 2006 from birds belonging to 51 intensively reared flocks produced in the Czech Republic and 41 intensive production, organic, and free-range flocks reared in Italy. Clostridium perfringens was detected in 64.7 and 82.9% of the Czech Republic and Italian flocks, respectively, at mean loads ranging between 3.65 and 4.77 log10 cfu per gram of cecum content. More than 1 ribotype was identified among isolates belonging to the same flock in 57.1 and 76.5% of the Czech Republic and Italian flocks, respectively. Moreover, common ribotypes were identified between strains belonging to 2 up to 8 different flocks. In particular, 4 ribotypes were shared between strains isolated in the 2 European countries. The results of this study report on C. perfringens occurrence and mean populations in broilers reared on diets devoid of antibiotic growth promoters. Moreover, these findings show for the first time the presence of common ribotyping profiles among isolates collected from birds reared more than 1,000 km apart.

Genotypic and phenotypic diversity within three Campylobacter populations isolated from broiler ceca and carcasses.

The main aim of this study was to trace Campylobacter subtypes colonizing Italian broilers and carcasses in and between flocks. Overall, 209 Campylobacter isolates were collected from ceca (n = 94) and carcasses (n = 115) of broilers belonging to 3 different flocks reared in the same farm during subsequent rotations and processed in the same slaughterhouse. All isolates were identified by multiplex polymerase chain reaction and genotyped by amplified fragment length polymorphism. Furthermore, 166 out of 209 strains were phenotyped by antimicrobial resistance profile (R-type). The results of genetic and phenotypic characterization showed that (1) multiple Campylobacter species and subtypes can colonize the same broiler and carcass; (2) common Campylobacter subtypes in ceca and carcasses seem to be rare; and (3) carryover of Campylobacter subtypes between broiler flocks in the same house rarely occurs. The outcomes of this study should be taken into account for setting of isolate collection during epidemiological investigations to check sources and transmission routs of Campylobacter in broilers and poultry products.

Application of automated ribotyping to support the evaluation of Listeria monocytogenes sources in a Taleggio cheese producing plant.

In March 2005, Listeria monocytogenes was detected on the rinds of Taleggio cheeses produced in an Italian plant. To identify the pathogen source, 154 rinds of cheeses that had been manually and automatically salinated and 52 environmental swabs collected from salting equipment, ripening cloths, and ripening boxes were tested for L. monocytogenes. Twenty-seven strains isolated from cheese samples and 16 strains isolated from environmental samples were genotyped by EcoRI and PvuII automated ribotyping. The microbiological results revealed a significant incidence of contamination of cheeses that were automatically salinated and contamination on the salting equipment, ripening cloths, and boxes. All cheese and environmental strains had the same EcoRI and PvuII ribotyping profiles, designated 153-204-S5 and 153-210-S-2, respectively. The only exception were three Taleggio strains, isolated from the same lot of product, that had EcoRI and PvuII ribotyping profiles designated 153-289-S6 and 153-214-S-5, respectively. Strains with EcoRI profile 153-204-S5 were classified as DUP-ID 1045 and serotype 1/2a, whereas strains with EcoRI profile 153-289-S6 were classified as DUP-ID 1034 and serotype 1/2b. The microbiological and molecular typing data collected in this study suggest that the source of the L. monocytogenes contamination in the Taleggio plant under study was the automated salting equipment. The isolate DUP-IDs were used to trace the introduction of potentially dangerous strains, such as those characterized as DUP-ID 1034, in the processing plant.

Occurrence and antibiotic susceptibility of Helicobacter pullorum from broiler chickens and commercial laying hens in Italy.

In 2005, in order to investigate the occurrence of Helicobacter pullorum in poultry, the caecal contents collected from a total of 60 animals intensively reared in Italy on 15 different farms (9 farms of broiler chicken and 6 of laying hens) were examined at the slaughterhouse. A modified Steele-McDermott membrane filter method was used. Small, greyish-white colonies of Gram-negative, gently curved, slender rod bacteria were preliminarily identified as H. pullorum by a Polymerase Chain Reaction (PCR) assay based on 16S rRNA and were then subjected to an ApaLI digestion assay to distinguish H. pullorum from Helicobacter canadensis. One isolate from each farm was phenotypically characterized by biochemical methods and 1D SDS-PAGE analysis of whole cell proteins; antibiotic susceptibility was also tested. According to the PCR and PCR-RFLP results, all the animals examined were positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 15 isolates tested. A monomodal distribution for the Minimum Inhibitory Concentrations (MICs) was found for ampicillin, chloramphenicol, gentamicin and tetracycline. For erythromycin and ciprofloxacin, a bimodal trend having a second peak at >128 micro(-1) and 32 micro(-1) was found. The isolation method used in this study seems to be highly suitable for isolating H. pullorum from chicken caecal contents. Moreover, the detection of a high number of colonies phenotypically similar to H. pullorum suggests that this microorganism, when present, colonizes the caecum at high concentration.

Enumeration and identity of Campylobacter spp. in Italian broilers.

Transmission of Campylobacter to humans has been prominently associated with mishandling or improperly preparing contaminated poultry carcasses. The number of organisms per carcass represents an important measure of human exposure to the agent. Therefore, we wished to estimate this public exposure over 1 yr among Italian broiler carcasses. We sampled 213 broiler carcasses from rinse water samples collected from a single slaughterhouse. Groups of carcasses had mean processed weights ranging from 1.2 to 2.7 kg. These were produced from 22 commercial broiler chicken flocks collected from 12 different farms, 3 of which were seasonally tested. Carcasses were rinsed with sterile water, and the rinse suspension was then serially diluted and spread-plated directly onto Campy-Cefex agar plates. One to 5 typical Campylobacter colonies per plate were identified by polymerase chain reaction as Campylobacter thermo-tolerant species. The overall estimated mean count per carcass in our study was 5.16 +/- 0.80 log10 cfu. This value increased in summer and autumn, as well as on carcasses collected from farms located > 100 km far from the slaughterhouse. A total of 678 Campylobacter colonies were identified by polymerase chain reaction. The majority of isolates were classified as Campylobacter jejuni (49.2%) or Campylobacter coli (47.5%). The overall number of C. jejuni was significantly higher on 1) carcasses weighing > 2 kg, 2) carcasses belonging to flocks with > 10,000 birds, and 3) carcasses collected from farms located > 100 km from the slaughterhouse. Moreover, among farms tested seasonally, C. jejuni was significantly greater than C. coli in winter. These data provide the first results of a continuing survey on Campylobacter loads and species identification from Italian broiler carcasses and represents an important baseline to estimate the human exposure to Campylobacter in Italy.

Phage types, ribotypes and tetracycline resistance genes of Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from different origins in Italy.

The tetracycline resistance (tet) gene patterns of 52 tetracycline resistant Salmonella enterica subsp. enterica (S.) serovar Typhimurium isolates collected from animals, food of animal origin, and humans in Italy, were investigated to evaluate whether the tet gene patterns could be used for strain differentiation in addition to phage typing and ribotyping. The detection of tet genes was performed by specific PCR assays. Ribotyping was performed automatically using PvuII as restriction enzyme. Ten different ribotyping patterns were detected. All isolates were positive for at least one of the tet genes studied and six different tet gene patterns were observed. Ribotyping and tet gene patterns showed discriminatory indices of 0.741 and 0.812, respectively. Multiple tet genes were commonly found among tetracycline resistant S. typhimurium isolates from various sources. The resulting tet gene patterns allowed further discrimination of strains which were otherwise indistinguishable by their phage type, ribotype and origin. Thus, the analysis of tet gene patterns might represent an additional tool for the differentiation of S. typhimurium isolates.

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy.

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Effects of dietary vitamin E on the quality of table eggs enriched with n-3 long-chain fatty acids.

Because of the proposed cardioprotective benefits of n-3 fatty acids and vitamin E, a trial was carried out to investigate the possibility of enriching eggs with n-3 fatty acid and vitamin E added to the hen's diet. One hundred ninety-two Hy-Line Brown hens, 39-wk-old, were divided into eight groups: four groups received the basal diet supplemented with 3% lard and four doses of dl-alpha-tocopheryl acetate (0, 50, 100, and 200 ppm), whereas the diets of the other groups were supplemented with 3% of fish oil and the same doses of vitamin E. The performances of the hens and egg weights were not affected either by the type of lipid supplement or by the vitamin level. The treatment with fish oil caused a dramatic increase (P < 0.01) of all n-3 fatty acids of the yolk, particularly EPA (19.53 vs. 0.74 mg/egg) and DHA (143.70 vs. 43.66 mg/egg), and an appreciable decrease of arachidonic acid (25.54 vs. 67.72 mg/egg). The different levels of dietary vitamin E slightly affected the fatty acid composition of the yolk. Yolk alpha-tocopherol increased linearly as dietary dl-alpha-tocopheryl acetate increased (P < 0.01) from the control level of 90.93 microg/g of yolk to 313.84 microg/g of yolk when 200 ppm were added to the hen diets. Twenty-eight days of storage at room temperature (20 to 25 C) did not alter the yolk fatty acid profile, and, moreover, the levels of vitamin E remained still very close to those observed in fresh egg.

Vitamin E in viral inactivated vaccines.

This research aimed at verifying whether vitamin E added to inactivated and emulsified vaccines enhances the immune response to viral antigens in chicken. Three hundred and twenty broilers (males and females) and 16 types of vaccines, varying in viral antigen [Newcastle disease virus, egg drop syndrome 1976 virus (EDS76V), and infectious bursal disease virus] and vitamin E amount (replacing 10, 20, and 30% of mineral oil) were used. Results show that vaccines with vitamin E, especially when it replaces 20 or 30% of mineral oil, induces a more rapid and higher antibody response than control vaccines. An adjuvant effect of vitamin E was also present in viral vaccine lacking bacterial antigens. Apart from vitamin E content, the Newcastle disease virus and infectious bursal disease virus monovalent vaccines induced higher titers of specific circulating antibodies in birds than did trivalent vaccines.

Vitamin E as adjuvant in emulsified vaccine for chicks.

Mineral oil was partially replaced with D, L-alpha-tocopheryl acetate (vitamin E) in bacterial and viral inactivated emulsified vaccines. Vitamin E increased the immune response to the viral antigen (Newcastle disease virus) used but not to the bacterial antigen (Escherichia coli) when its presence in the oil phase did not exceed 30%. Inoculated vitamin E may have enhanced the immune response by interacting with the immune-competent cells involved in the inflammatory reaction that followed inoculation of emulsified vaccines.