Jeanet A. van der Goot

Transmission of highly pathogenic avian influenza H5N1 virus in Pekin ducks is significantly reduced by a genetically distant H5N2 vaccine

Domestic ducks play an important role in the epidemiology of H5N1 avian influenza. Although it is known that vaccines that have a high homology with the challenge virus are able to prevent infection in ducks, little is yet known about the ability of genetically more distant vaccines in preventing infection, disease, and transmission. Here we study the effect of a widely used H5N2 vaccine (A/Chicken/Mexico/232/94/CPA) on the transmission of H5N1 virus (A/Chicken/China/1204/04) in ducks. The quantitative analyses show that despite the low level of homology between the virus and vaccine strain transmission was significantly reduced two weeks after a single or double vaccination. Mortality and disease rates were reduced markedly already one week after a single vaccination.

Presence of ESBL/AmpC -Producing Escherichia coli in the Broiler Production Pyramid: A Descriptive Study

Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the presence of these strains in broilers. As not much is known about the presence of these strains in the whole production pyramid, the epidemiology of ESBL/AmpC-producing E. coli in the Dutch broiler production pyramid was examined. Cloacal swabs of Grandparent stock (GPS) birds (one−/two-days (breed A and B), 18 and 31 weeks old (breed A)), one-day old Parent stock birds (breed A and B) and broiler chickens of increasing age (breed A) were selectively cultured to detect ESBL/AmpC-producing isolates. ESBL/AmpC-producing isolates were found at all levels in the broiler production pyramid in both broiler breeds examined. Prevalence was already relatively high at the top of the broiler production pyramid. At broiler farms ESBL/AmpC producing E. coli were still present in the environment of the poultry house after cleaning and disinfection. Feed samples taken in the poultry house also became contaminated with ESBL/AmpC producing E. coli after one or more production weeks. The prevalence of ESBL/AmpC-positive birds at broiler farms increased within the first week from 0–24% to 96–100% independent of the use of antibiotics and stayed 100% until slaughter. In GPS breed A, prevalence at 2 days, 18 weeks and 31 weeks stayed below 50% except when beta-lactam antibiotics were administered. In that case prevalence increased to 100%. Interventions minimizing ESBL/AmpC contamination in broilers should focus on preventing horizontal and vertical spread, especially in relation to broiler production farms.

Dynamics of CMY-2 producing E. coli in a broiler parent flock

Extended-spectrum β-lactamase and plasmid mediated AmpC β-lactamase (ESBL/pAmpC) producing bacteria are resistant to Extended Spectrum Cephalosporins (ESC), and are present in all levels of the broiler production chain. We determined the prevalence, concentration, and persistence of ESBL/pAmpC-Escherichia coli in a broiler parent flock during the rearing and laying period. One-day old chickens were housed in four separate pens. Until week 33 no antibiotics or coccidiostatics were used. During rearing 57 chickens in each pen (n=228), and in the laying period two groups of 33 chickens were individually sampled (n=66). Environmental samples were taken from week 16 onwards. ESBL/pAmpC-E. coli presence was determined by selective culturing. In the samples of week 16-19 the concentration of ESBL/pAmpC-E. coli was determined. All ESC-resistant isolates found were positive for pAmpC gene blaCMY-2 located on IncA/C plasmids, in several E. coli MLST types. CMY-2-E. coli prevalence decreased from 91% (95%CI 86-94%) at day 7 (week 1) to 0% (95%CI 0-5%) in week 21. However, CMY-2-E. coli remained present in the environmental samples during the whole study. CMY-2-E. coli concentration varied between detection limit (<10^3) and 2·10^4 cfu/g faeces. The sharp reduction of CMY-2-E. coli in this broiler parent flock in absence of antibiotics suggests a selective disadvantage of blaCMY-2 on IncA/C plasmids on animal level. The underlying mechanism should be studied further as this may provide new insights on how to reduce ESBL/pAmpC prevalence and transmission in the broiler production chain.

Risk-based testing of imported animals: A case study for bovine tuberculosis in The Netherlands

In intra-EU trade, the health status of animals is warranted by issuing a health certificate after clinical inspection in the exporting country. This certificate cannot provide guarantee of absence of infection, especially not for diseases with a long incubation period and no overt clinical signs such as bovine tuberculosis (bTB). The Netherlands are officially free from bTB since 1999. However, frequent reintroductions occurred in the past 15 years through importation of infected cattle. Additional testing (AT) of imported cattle could enhance the probability of detecting an imported bTB infection in an early stage. The goal of this study was to evaluate the effectiveness of risk-based AT for bTB in cattle imported into The Netherlands. A generic stochastic import risk model was developed that simulates introduction of infection into an importing country through importation of live animals. Main output parameters are the number of infected animals that is imported (Ninf), the number of infected animals that is detected by testing (Ndet), and the economic losses incurred by importing infected animals (loss). The model was parameterized for bTB. Model calculations were optimized to either maximize Ndet or to minimize loss. Model results indicate that the risk of bTB introduction into The Netherlands is very high. For the current situation in which Dutch health checks on imported cattle are limited to a clinical inspection of a random sample of 5-10% of imported animals, the calculated annual Ninf=99 (median value). Random AT of 8% of all imported cattle results in Ndet=7 (median value), while the median Ndet=75 if the sampling strategy for AT is optimized to maximize Ndet. However, in the latter scenario, loss is more than twice as large as in the current situation, because only calves are tested for which cost of detection is higher than the expected gain of preventing a possible outbreak. When optimizing the sampling strategy for AT to minimize loss, only breeding and production cattle are selected for AT resulting in Ndet=1 (median value). Loss is; however, reduced by 75% if compared to the current situation. We conclude that the effectiveness of AT can greatly be improved by risk-based sampling. The optimal sampling strategy for risk-based AT for bTB is highly dependent on the objective of AT. If economic losses are to be contained, AT should focus on breeding and production cattle originating from high-risk countries.

A cross sectional study on Dutch layer farms to investigate the prevalence and potential risk factors for different Chlamydia species.

In poultry several Chlamydia species have been detected, but Chlamydia psittaci and Chlamydia gallinacea appear to be most prevalent and important. Chlamydia psittaci is a well-known zoonosis and is considered to be a pathogen of poultry. Chlamydia gallinacea has been described more recently. Its avian pathogenicity and zoonotic potential have to be further elucidated. Within the Netherlands no data were available on the presence of Chlamydia on poultry farms. As part of a surveillance programme for zoonotic pathogens in farm animals, we investigated pooled faecal samples from 151 randomly selected layer farms. On a voluntary base, 69 farmers, family members or farm workers from these 151 farms submitted a throat swab. All samples were tested with a generic 23S Chlamydiaceae PCR followed by a species specific PCR for C. avium, C. gallinacea and C. psittaci. C. avium and psittaci DNA was not detected at any of the farms. At 71 farms the positive result could be confirmed as C. gallinacea. Variables significantly associated with the presence of C. gallinacea in a final multivariable model were ‘age of hens,’ ‘use of bedding material’ and ‘the presence of horses.’ The presence of C. gallinacea was associated with neither clinical signs, varying from respiratory symptoms, nasal and ocular discharges to diarrhoea, nor with a higher mortality rate the day before the visit. All throat swabs from farmers, family members or farm workers tested negative for Chlamydia DNA, giving no further indication for possible bird-to-human (or human-to-bird) transmission.

Competitive exclusion reduces transmission and excretion of extended-spectrum β-lactamase producing Escherichia coli in broilers

ABSTRACT Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpC) are enzymes able to hydrolyze a large variety of β-lactam antibiotics, including third-generation cephalosporins and monobactams. Broilers and broiler meat products can be highly contaminated with ESBL- and pAmpC-producing Escherichia coli strains, also known as extended-spectrum cephalosporin (ESC)-resistant E. coli strains, and can be a source for human infections. As few data on interventions to reduce the presence of ESC-resistant E. coli in broilers are available, we used transmission experiments to examine the role of competitive exclusion (CE) on reducing transmission and excretion in broilers. A broiler model to study the transmission of ESC-resistant E. coli was set up. Day-old chickens were challenged with an ESBL-producing E. coli strain isolated from healthy broilers in the Netherlands. Challenged and not challenged chicks were housed together in pairs or in groups, and ESBL-producing E. coli transmission was monitored via selective culturing of cloacal swab specimens. We observed a statistically significant reduction in both the transmission and excretion of ESBL-producing E. coli in chicks treated with the probiotic flora before E. coli challenge compared to the transmission and excretion in untreated controls. In conclusion, our results support the use of competitive exclusion as an intervention strategy to control ESC-resistant E. coli in the field. IMPORTANCE Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases are a primary cause of resistance to β-lactam antibiotics among members of the family Enterobacteriaceae in humans, animals, and the environment. Food-producing animals are not exempt from this, with a high prevalence being seen in broilers, and there is evidence pointing to a possible foodborne source for human contamination. We investigated the effect of administration of a commercial probiotic product as an intervention to reduce the amount of ESBL-producing Escherichia coli in broilers. Our results showed a substantial reduction in the level of colonization of broiler intestines by ESBL-producing E. coli after administration of commercial probiotic product. The protective effect provided by these probiotics could be implemented on a larger scale in poultry production. Reductions in the levels of ESBL-producing Enterobacteriaceae in the food chain would considerably benefit public health.

Dynamics of cefotaxime resistant Escherichia coli in broilers in the first week of life

Extended-spectrum beta-lactamase producing E. coli (ESBL-E) are wide spread among broilers, with the highest prevalence among individual birds at broiler production farms. Previous research describes low prevalences among individual birds at arrival at the farm (below 30%), and a rapid increase up to 100% within the first week. Our goal was to investigate whether this rapid increase was due to latent contamination of ESBL-E or to contamination at the broiler farm. Two broiler groups, one hatched at a conventional hatchery and the other individually hatched in an ESBL-free environment, were housed individually in an experimental ESBL-free environment. A third group was hatched at a conventional hatchery and kept at a conventional broiler farm. The birds were sampled daily during the first week after hatch and tested for the presence of ESBL-E. In addition ESBL-E presence in eggs that were not incubated was investigated. All birds and eggs came from one ESBL-E positive parent flock. ESBL/AmpC genes, plasmids and E. coli sequence types were determined for a selection of isolates. ESBL-E was never found in the two groups kept in the ESBL-free experimental environment or in the sampled eggs, whereas all broilers sampled at the conventional farm became positive for ESBL-E within three days. One dominant E. coli strain (ST88) carrying blaCTX-M-1 gene on an IncI1/pST3 plasmid was found in parent and broiler samples. We conclude that the rapid increase in ESBL-E prevalence in the first week of life is not caused by a latent contamination of the majority of birds at arrival, but that this increase must be caused by other factors.

Similar transmissibility of the Italian H7N1 highly pathogenic avian influenza virus and its low pathogenic avian influenza virus predecessor

The transmissibility of the H7N1 highly pathogenic avian influenza virus (HPAIV), which caused a large epidemic in commercial poultry in Italy in 1999-2000, was studied in chickens and compared with that of the low pathogenic precursor virus (LPAIV). Group transmission experiments using the HPAIV were executed to estimate the infectious period (IP), the transmission parameter (β) and the basic reproduction number (R0). These estimates were then compared with those reported for the LPAIV. The estimated β and R0 were similar for both viruses, whilst the IP of the LPAIV was longer than that of the HPAIV. These findings indicate that transmissibility from chicken-to-chicken alone does not appear to confer an advantage for this LPAIV to evolve to a HPAIV.

Genomic and functional characterisation of IncX3 plasmids encoding bla SHV-12 in Escherichia coli from human and animal origin

The blaSHV-12 β-lactamase gene is one of the most prevalent genes conferring resistance to extended-spectrum β-lactams in Enterobacteriaceae disseminating within and between reservoirs, mostly via plasmid-mediated horizontal gene transfer. Yet, studies regarding the biology of plasmids encoding blaSHV-12 are very limited. In this study, we revealed the emergence of IncX3 plasmids alongside IncI1α/γ in blaSHV-12 in animal-related Escherichia coli isolates. Four representative blaSHV-12-encoding IncX3 plasmids were selected for genome sequencing and further genetic and functional characterization. We report here the first complete sequences of IncX3 plasmids of animal origin and show that IncX3 plasmids exhibit remarkable synteny in their backbone, while the major differences lie in their blaSHV-12-flanking region. Our findings indicate that plasmids of this subgroup are conjugative and highly stable, while they exert no fitness cost on their bacterial host. These favourable features might have contributed to the emergence of IncX3 amongst SHV-12-producing E. coli in the Netherlands, highlighting the epidemic potential of these plasmids.