Prevalence of extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in young calves on Dutch dairy farms.

In young calves on dairy farms the animal prevalence of extended-spectrum and AmpC ß-lactamase-producing Escherichia coli (ESBL/AmpC-EC) is significantly higher compared with the animal prevalence in young stock and dairy cows. Hitherto it was unknown at what age antimicrobial resistant bacteria appear for the first time in the gut of calves on dairy farms, and how long these infections persist. The aim of this study was to examine the prevalence of ESBL/AmpC-EC, the number of excreted ESBL/AmpC-EC (in cfu/g of feces), as well as the ESBL/AmpC genotypes in young dairy calves (0-21 d of age) and the variation of these parameters between calves of different ages. Next to this, the course of shedding ESBL/AmpC-EC during the first year in dairy calves was studied. In a cross-sectional study, fecal samples from 748 calves, from 0 to 88 d of age, on 188 Dutch dairy farms were collected. The prevalence of calves testing positive for ESBL/AmpC-EC in a phenotypic assay was determined for different age categories (per 2 d of age). Positive samples were subjected to a semiquantitative test to determine the numbers of ESBL/AmpC-EC per gram of feces and for a selection of ESBL/AmpC-EC isolates the ESBL/AmpC genotype was determined. Ten of the 188 farms were selected for a longitudinal study based on the presence of at least 1 female calf with ESBL/Amp-EC in the cross-sectional study. These farms were additionally visited 3 times with a 4-mo interval. All calves that were sampled in the cross-sectional study were, if still present, resampled during the follow-up visits. Results show that from the day of birth ESBL/AmpC-EC can be present in the gut of calves. The phenotypic prevalence of ESBL/AmpC-EC was 33.3% in 0- to 21-d-old calves and 28.4% in 22- to 88-d-old calves. The prevalence of ESBL/AmpC-EC positive calves varied per age category among calves up to 21 d of age: significant increases and decreases at an early age were shown. Results of the longitudinal study show that after 4, 8, and 12 mo the prevalence of ESBL/AmpC-EC positive calves dropped to 3.8% (2/53), 5.8% (3/52), and 2.0% (1/49), respectively. This indicates that early gut colonization in young calves with ESBL/AmpC-EC is transient and does not lead to long-term shedding of these bacteria.

Assessment of the best inoculation route for virulotyping Enterococcus cecorum strains in a chicken embryo lethality assay.

The study aim was to determine the best inoculation route for virulotyping Enterococcus cecorum in a chicken embryo lethality assay (ELA). Twenty-eight genetically different strains were used. Fourteen strains were isolated from cloaca swabs of broiler reproduction chickens (cloaca strains) and 14 strains from broilers with E. cecorum lesions (lesion strains). In all ELAs, 12-day incubated embryonated broiler eggs were inoculated with approximately 100 colony-forming units of E. cecorum/egg. Twenty embryos per inoculation route and strain were used in each of three experiments. In Experiment 1, four cloaca and four lesion strains were inoculated via various routes, i.e. albumen, amniotic cavity, allantoic cavity, chorioallantoic membrane, intravenous or air chamber. The albumen inoculation route showed low mortality with cloaca strains, high mortality with lesion strains and the largest difference in mortality between these groups of strains (≥60%). This route was therefore used in subsequent experiments. In Experiment 2, the same strains were used to test reproducibility, which proved to be generally good. All 28 strains were thereafter used in Experiment 3. In the three experiments, mortality caused by cloaca and lesion strains ranged from 0-25% and from 15-100%, respectively. Recovery rates, assessed in all experiments after albumen inoculation, were significantly lower from eggs inoculated with cloaca strains, compared to lesion strain-inoculated eggs (P < 0.05). However, the bacterial load of eggs with positive recovery was similar in both groups. In conclusion, the albumen inoculation route appeared to be the best to virulotype E. cecorum strains.RESEARCH HIGHLIGHTS The albumen route is the best to differentiate between E. cecorum strains.Egg albumen likely affects cloaca E. cecorum strains more than lesion strains.Based on SNPs, E. cecorum cloaca strains are clustered as well as lesions strains.

In vivo typing of Escherichia coli obtained from laying chickens with the E. coli peritonitis syndrome.

Fourteen genetically different chicken Escherichia coli strains were biotyped in hens to examine if any E. coli strain at high dose can induce the E. coli peritonitis syndrome (EPS). Moreover, biotyping was performed in embryos and the median lethal dose (LD50) of three strains was determined in hens. Nine strains were obtained from femur marrow and one strain from caecum of hens that had died from EPS. One strain originated from the inflamed pericardium of a broiler and three strains from the cloaca of specified pathogen-free (SPF) broiler breeders. Strains were inoculated intratracheally into separate groups of 32 productive SPF White Leghorn (WL) hens at a dose of 7.8-9.2 log10 colony forming units (CFU) per hen and into the allantoic cavity of separate groups of 20 SPF WL embryos incubated during 14 days in a dose of 4.2-4.6 log10 CFU per embryo. The embryo test was replicated. Bone marrow and pericardium strains induced EPS, the other strains did not. Based on mortality in hens, EPS-inducing strains could be classified as very virulent (59-100% mortality), moderately virulent (38% mortality) and low virulent (6% mortality). In productive SPF WL hens, the LD50 of three very virulent strains ranged from <2.7 to 5.3 log10 CFU. Virulent and avirulent strains killed 60-95% and 0-30% of embryos, respectively. The embryo lethality test, which showed good reproducibility, did not discriminate within virulent strains, but can nevertheless be considered as a useful alternative for biotyping E. coli in productive hens.RESEARCH HIGHLIGHTSEven at high doses, no E. coli strain could induce EPS.Substantial differences in virulence exist within very virulent E. coli strains.The embryo lethality test is a useful alternative for biotyping E. coli in laying hens.Broiler colibacillosis may represent a source of EPS strains for layers and vice versa.

Molecular characterization of type 1 porcine reproductive and respiratory syndrome viruses (PRRSV) isolated in the Netherlands from 2014 to 2016.

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.

Porcine epidemic diarrhea virus (PEDV) introduction into a naive Dutch pig population in 2014.

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.

Prevalence and risk factors for extended-spectrum ß-lactamase- and AmpC-producing Escherichia coli in dairy farms.

A cross-sectional study was conducted to evaluate the prevalence of extended-spectrum ß-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 (blaCTX-M-1, -2, -14, -15, -32, -55 and blaTEM-52), 1 plasmid-encoded AmpC gene (blaCMY-2), 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 from slurry samples collected from Dutch dairy farms and the ESBL/AmpC genes carried by the isolates were consistent with those reported earlier. The use of third- and fourth-generation cephalosporins appeared to be associated the ESBL/AmpC herd status.

Molecular typing of avian pathogenic Escherichia coli colonies originating from outbreaks of E. coli peritonitis syndrome in chicken flocks.

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.

The seroprevalence of Lawsonia intracellularis in horses in The Netherlands.

Equine proliferative enteropathy caused by Lawsonia intracellularis is an emerging disease of weanling foals and affects their growth and development. The prevalence of Lawsonia intracellularis in The Netherlands is not known. The aim of the study was to investigate the seroprevalence of Lawsonia intracellularis in horses in The Netherlands. Blood samples were taken from healthy foals before and after weaning and from healthy yearlings and mature horses on farms throughout The Netherlands. These samples were analysed for the presence of Lawsonia intracellularis-specific antibodies with a blocking ELISA. White blood cell count, packed cell volume, and total protein concentration were also measured in all foals. Information regarding housing, pasture access, and contact with pig manure on the premises was obtained for all animals. The prevalence of Lawsonia intracellularis antibodies in foals increased significantly from 15% before weaning to 23% after weaning (p = 0.019); it was 89% in yearlings and 99% in horses older than 2 years. There was no significant difference in seroprevalence between the pasture-kept and stable-confined adult horses (97% and 100%, respectively), and there was no significant influence of contact with pig manure. None of the sampled animals showed clinical disease. In conclusion, the results suggest that Lawsonia intracellularis is widespread in The Netherlands and that seropositivity is not necessarily associated with clinical problems. The high seroprevalence in adult horses suggests long-term persistence of antibodies against Lawsonia intracellularis or constant exposure to the bacterium.