No abstract

This study evaluates the multiple-locus variable-number tandem-repeat assay (MLVA) and pulsed-field gel electrophoresis (PFGE) when using restriction enzymes BstZI, SacII, and ApaI to fingerprint a diverse collection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) isolates. These isolates had been characterized previously by multilocus sequence typing, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed, and the concordance between the various methods was determined. All MRSA ST398 isolates were typeable by the MLVA and PFGE using BstZI, SacII, and ApaI. With each method, the MRSA ST398 isolates formed a separate group from the two non-ST398 MRSA strains. PFGE, performed with any of the three restriction enzymes, had the most discriminatory power, followed by MLVA, spa typing, and SCCmec typing. The MLVA showed the highest concordance with PFGE using ApaI and spa typing. As further expressed by the Wallace coefficient, the MLVA type was poorly predicted by spa typing, whereas the spa type was well predicted by MLVA. PFGE, using a combination of all three restriction enzymes, had the highest concordance with the MLVA but had a low probability of being predicted by MLVA. PFGE, using a combination of all three restriction enzymes, was able to predict SCCmec type and MLVA type completely and had a high probability of predicting spa type. Both the MLVA and PFGE could be used to discriminate among the MRSA ST398 isolates. Although the MLVA is a faster technique, PFGE had more discriminatory power than the MLVA, especially when a combination of restriction enzymes was used.

Salmonella Enteritidis (SE) is a genetically homogenous serovar, which makes optimal subtype discrimination crucial for epidemiological research. This study describes the development and evaluation of an optimized multiple-locus variable number tandem-repeat assay (MLVA) for characterization of SE. The typeability and discriminatory power of this MLVA was determined on a selected collection of 60 SE isolates and compared with pulsed-field gel electrophoresis (PFGE) using restriction enzymes XbaI, NotI, or SfiI. In addition, the estimated Wallace coefficient (W) was calculated to assess the congruence of the typing methods. Selection of epidemiologically unrelated isolates and more related isolates (originating from layer farms) was also based on the given phage type (PT). When targeting six loci, MLVA generated 16 profiles, while PFGE produced 10, 9, and 16 pulsotypes using XbaI, NotI, and SfiI, respectively, for the entire strain collection. For the epidemiologically unrelated isolates, MLVA had the highest discriminatory power and showed good discrimination between isolates from different layer farms and among isolates from the same layer farm. MLVA performed together with PT showed higher discriminatory power compared to PFGE using one restriction enzyme together with PT. Results showed that combining PT with the optimized MLVA presented here provides a rapid typing tool with good discriminatory power for characterizing SE isolates of various origins and isolates originating from the same layer farm.

Since the ban of antimicrobial growth promotors, the importance of necrotic enteritis in broilers increases. Reliable and reproducible infection models are required for pathogenesis studies and product screening. Two major predisposing factors in necrotic enteritis models are fishmeal supplementation to feed and Eimeria infection. However, many unsolved issues regarding these predisposing factors still exist. Therefore, the influence of timepoint of fishmeal administration (onset on day 8 or day 18), timing of coccidiosis challenge (day 15 or day 19) and strain of coccidiosis challenge (field strain vs. commercial vaccine) on the induction of necrotic enteritis lesions was investigated. The birds were inoculated with Clostridium perfringens three times per day for four consecutive days (day 17 until day 20) and were scored for the presence of necrotic enteritis on days 22, 23, 24, 25 and 26. Supplementation of the diet with fishmeal from day 8 onwards increased the likelihood of necrotic enteritis compared to supplementation from day 18 onwards. Birds challenged on day 19 with coccidiosis were more likely to have necrotic enteritis on scoring days 23 and 24 compared to birds challenged on day 15. Differences on other scoring days were less pronounced. Finally, the strain of coccidiosis challenge had little influence on the induction of necrotic enteritis. Findings of this study can help researchers to set up successful necrotic enteritis infection models.

The aim of this study was to closely examine the Salmonella enterica serovar Enteritidis environmental contamination on persistently positive layer farms in Belgium during successive laying cycles. All of the farms were required to vaccinate their layers under the national control program for Salmonella. Seven farms with previous or current Salmonella Enteritidis contamination were monitored during different stages of the laying period and after cleaning and disinfection (CD). Environmental samples, including from the equipment and vermin, were taken in the henhouse and egg-collecting area. Dilutions were performed to define the degree of Salmonella Enteritidis contamination. Eggshells, egg contents, and ceca were also tested for Salmonella. At the end of the first sampled laying period, 41.6% of the environmental samples were contaminated with Salmonella Enteritidis. After CD, the prevalence dropped to 11.4%. On average, the prevalence in the second laying period increased again: 17.8, 18.4, and 22.3% at the onset, middle, and end of the lay period, respectively. After CD before the third laying period, the prevalence decreased to 6.6% and stabilized at the onset of lay (6.3%). During lay, as well as after CD, a wide variety of contaminated environmental samples were found; for example, in the henhouse, in the egg-collecting area, on mobile equipment and in or on vermin. In the henhouse during laying, the most recurrent and highly contaminated sites were the overshoes, floor, manure belt, and hen feces. The egg-collecting area had a significantly higher number of contaminated samples compared with that of the henhouse. For both sites, the floor appeared to be the most suitable sampling site to estimate the Salmonella Enteritidis status of the farms. Eggshell and egg content contamination varied between 0.18 and 1.8% and between 0.04 and 0.4%, respectively. In total, 2.2% of the analyzed ceca contained Salmonella Enteritidis. This study revealed that Salmonella Enteritidis is present in the environment of persistently Salmonella Enteritidis-contaminated layer farms, demonstrated that in many cases Salmonella Enteritidis contamination was not eliminated after CD, and identified the egg-collecting area as a critical point on most farms.