A cross-sectional study was conducted to evaluate the prevalence of extended-spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC-producing Escherichia coli and associated risk factors in dairy herds. One hundred dairy herds were randomly selected and sampled to study the presence of ESBL- and AmpC-producing E. coli in slurry samples. The sensitivity of testing slurry samples for ESBL/AmpC herd status is less than 100%, especially for detecting herds with a low ESBL/AmpC prevalence. Therefore, whereas herds that tested positive for ESBL/AmpC-producing E. coli in slurry were defined as positive herds, herds with negative slurry samples were defined as unsuspected. Isolates of ESBL- and AmpC-producing E. coli were further characterized by detection and typing of their ESBL/AmpC gene. At the initial sampling, a comprehensive questionnaire was conducted at the participating farms. The farmers were asked questions about management practices potentially associated with the ESBL/AMPC herd status. Also, data on antimicrobial purchases during 2011 were acquired to evaluate whether the animal-defined daily dose of antimicrobials per year at farm level was associated with the ESBL/AmpC herd status. Multivariable logistic regression models were used to determine the association between management practices and the ESBL/AmpC herd status. Six months after the initial slurry sampling, 10 positive herds and 10 herds that had an unsuspected ESBL/AmpC herd status during the first visit were resampled. At each farm, slurry samples and feces from 24 individual cows were collected to evaluate within herd dynamics. During the first sampling, ESBL/AmpC-producing E. coli were isolated from the slurry samples collected at 41% of the herds. In total, 37 isolates were further characterized, revealing 7 different ESBL genes (bla and bla), 1 plasmid-encoded AmpC gene (bla), and 1 chromosomally encoded ampC gene (ampC type 3). The total animal-defined daily dose of antimicrobials per year at farm level was not significantly different between ESBL/AmpC-positive and unsuspected dairy herds. The use of third- and fourth-generation cephalosporins, however, was found to be associated with ESBL/AmpC status, with higher use of these antimicrobials resulting in a significant higher odds to be ESBL/AmpC-positive. Management factors that were associated with a higher odds of being ESBL/AmpC-positive were treatment of all cases of clinical mastitis with antimicrobials, a higher proportion of calves treated with antimicrobials, not applying teat sealants in all cows at dry off, and the use of a floor scraper. This last association, however, was considered a methodological effect rather than a true risk factor. On 5 of the 10 initially positive farms, no ESBL/AmpC-producing E. coli were cultured from the slurry or any of the individual cow samples collected during the second sampling. In 4 of the initially unsuspected farms, slurry or individual cow samples tested positive during the second sampling. In conclusion, ESBL/AmpC could frequently be cultured...

Porcine epidemic diarrhea virus (PEDV) is the highly contagious, causative agent of an economically important acute enteric disease in pigs of all ages. The disease is characterized by diarrhea and dehydration causing mortality and growth retardation. In the last few decades, only classical PEDV was reported sporadically in Europe, but in 2014 outbreaks of PEDV were described in Germany. Phylogenetic analysis showed a very high nucleotide similarity with a variant of PEDV that was isolated in the US in January 2014. The epidemiological situation of PEDV infections in the Netherlands in 2014 was unknown and a seroprevalence study in swine was performed. In total, 838 blood samples from sows from 267 farms and 101 samples from wild boars were collected from May till November 2014 and tested for antibodies against PEDV by ELISA. The apparent herd prevalence of 0.75% suggests that PEDV was not circulating on a large scale in the Netherlands at this time. However, in November 2014 a clinical outbreak of PEDV was diagnosed in a fattener farm by PCR testing. This was the first confirmed PEDV outbreak since the early nineties. Sequence analyses showed that the viruses isolated in 2014 and 2015 in the Netherlands cluster with recently found European G1b strains. This suggests a one event introduction of PEDV G1b strains in Europe in 2014, which made the Netherlands and other European countries endemic for this type of strains since then.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a devastating pig disease present all over the world. The remarkable genetic variation of PRRSV, makes epidemiological and molecular analysis of circulating viruses highly important to review current diagnostic tools and vaccine efficacy. Monitoring PRRS viruses supports modern herd management by explaining the source of found viruses, either internally or externally from the herd. No epidemiological or molecular study has been published on circulating PRRS-viruses in the Netherlands, since the early nineties. Therefore, the objective of this study is to investigate circulating PRRS-viruses in the Netherlands in 2014, 2015 and 2016 on a molecular level by sequencing ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7. The results demonstrate that the 74 PRRSV strains belong to PRRSV-1, but the diversity among strains is high, based on nucleotide identity, individual ORF length and phylogenetic trees of individual ORFs. Furthermore, the data presented here show that the phylogenetic topology of some viruses is ORF dependent and suggests recombination. The identity of the strain of interest might be misinterpreted and wrong conclusions may be drawn in a diagnostic and epidemiological perspective, when only ORF5 is analyzed, as performed in many routine sequencing procedures.

Escherichia coli colonies isolated from the bone marrow of fresh dead hens of laying flocks with the E. coli peritonitis syndrome (EPS) were genotyped using pulsed-field gel electrophoresis (PFGE). Typing is important from an epidemiological point of view and also if the use of autogenous (auto)vaccines is considered. Birds with EPS originated from one house of each of three layer farms and one broiler breeder farm. Farms were considered as separate epidemiological units. In total, six flocks were examined including two successive flocks of one layer farm and the broiler breeder farm. E. coli colonies (one per bird) from nine to 16 hens of each flock were genotyped. The clonality of E. coli within birds was studied using five colonies of each of nine to 14 birds per flock. E. coli genotypes, which totalled 15, differed between farms and flocks except for two successive layer flocks that shared three genotypes. One to five genotypes were found per flock with one or two genotypes dominating each outbreak. Within hens, E. coli bacteria were always clonal. Colonies of the same PFGE type always had the same multilocus sequence type. However, four PFGE types shared sequence type 95. Neither PFGE types nor multilocus sequence types were unambiguously related to avian pathogenic E. coli from EPS. In cases where persistence of E. coli strains associated with EPS is found to occur frequently, routine genotyping to select strains for autovaccines should be considered.

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