ABSTRACT
Autogenous Escherichia coli vaccines to prevent the E. coli peritonitis syndrome (EPS) in laying hens are often used in the field, although their effectiveness has not been demonstrated yet. Therefore, in this study, which consisted of two experiments, their efficacy was assessed. In the first experiment, the EPS-inducing ability of three E. coli isolates originating from bone marrow of hens that died due to EPS and with different Pulsed-Field Gel Electrophoresis patterns, was examined by intravenous inoculation of the isolates in 17-week-old brown layers. Based on the results one isolate was chosen for the preparation of the vaccines and for homologous challenge and another one for heterologous challenge performed in the second experiment. In the named experiment, groups of laying hens which had been vaccinated intramuscularly at 14 and 18 weeks of age with inactivated vaccine either formulated as aqueous suspension or as water-in-oil emulsion were homologously or heterologously challenged per aerosol at 30 weeks of age. The vaccines contained ≥108.2 formaldehyde-inactivated colony-forming units (cfu) of E. coli per hen dose in 0.5 ml. The estimated E. coli challenge dose uptake ranged from 105.8 to 106.5 cfu per hen. Groups consisted of 18 hens each and were housed in separate isolators from 27 weeks of age. Control groups were included in this experiment, which was ended eight days after challenge. Vaccinations had no effect on body growth and both vaccine types induced (almost) complete protection against homologous challenge, while protection against heterologous challenge was inconclusive.
Introduction
In laying chickens two forms of colibacillosis prevail: salpingitis/peritonitis/salpingoperitonitis (SPS) and the Escherichia coli peritonitis syndrome (EPS). Coliform SPS (“egg peritonitis”), which has been observed since the beginning of poultry farming is a chronic condition and forms a substantial proportion of the “normal” mortality in flocks of productive hens (Bisgaard & Dam, Citation1981; Jordan et al., Citation2005; Stokholm et al., Citation2010). In contrast, EPS, which is of great economic importance for commercial poultry (Landman & Van Eck, Citation2015) is characterized by acute mortality, soaring up to 10% or more in combination with severe septicaemia and fibrinous polyserositis lesions, while salpingitis rarely occurs. A decrease in egg production and increase in downgraded eggs of a few percentages may be present. Flock and house associations are often observed with recurrent outbreaks within the same flock and in successive flocks in the same house. The disease has been observed in the USA and many European countries including the Netherlands from mid-1990s onwards (Dhillon & Jack, Citation1996; Zanella et al., Citation2000; Vandekerchove et al., Citation2004; Landman & Cornelissen, Citation2006; Raviv et al., Citation2007; Landman et al., Citation2013; Landman et al., Citation2014).
Although the pathogenesis of EPS is largely unknown, this condition was recently successfully induced in egg-producing commercial brown layers by exposure of the hens to E. coli (chicken/NL/Dev/SP01404Cou/05) isolated from the bone marrow of a dead brown layer with EPS, by both artificial (intravenous and intraperitoneal) and natural infection routes (intratracheal, aerosol and intravaginal). Air transmission of EPS-inducing E. coli strains seemed to be the most important route, although the intravaginal route could not be ruled out (Landman et al., Citation2013).
Attempts to control EPS outbreaks are often made using antimicrobials (Landman & Van Eck, Citation2015) and/or (autogenous) vaccines, while selection for reduced susceptibility to colibacillosis (Ask et al., Citation2006; Cavero et al., Citation2009) should be considered as a long-term solution. The use of antimicrobials is limited due to the possible occurrence of residues in meat and eggs, and the selection of multi-resistant strains. Moreover, the effect is temporary and therefore the treatment has to be repeated often (Landman & Van Eck, Citation2015). Although, vaccines are not hindered by the shortcomings of antimicrobial treatments, their efficacy has been questioned as no single trait or group of traits, which define avian pathogenic E. coli involved in EPS, have been identified. This makes selection of candidate vaccines difficult, while in general cross-protection is lacking (Ghunaim et al., Citation2014) likely due to the great diversity of serovars of E. coli (Ewers et al., Citation2007). Autogenous vaccines are therefore often used in the field and although they are expected to be highly effective against homologous challenge (Ghunaim et al., Citation2014), studies on the prevention of EPS are lacking.
In this manuscript two experiments are described. In Experiment 1, the EPS-inducing ability of three E. coli isolates with a distinct Pulsed-Field Gel Electrophoresis (PFGE) pattern and originating from the bone marrow of dead hens suffering from EPS was assessed in brown layer rearing hens by intravenous inoculation. In Experiment 2, a vaccination-challenge study was performed in laying hens using two E. coli isolates selected based on the results of Experiment 1. An inactivated vaccine was prepared using one of the isolates, which was also used for homologous challenge, while the other isolate was used for heterologous challenge. Two types of inactivated vaccines were applied; one as aqueous suspension and the other as water-in-oil emulsion. The challenge was performed by aerosol exposure.
Materials and methods
Experimental design
Experiment 1. The experimental design is outlined in . The EPS-inducing ability of three E. coli field isolates (chicken/NL/Dev/SP01404Cou/05, chicken/NL/Dev/SP01382/09 and chicken/NL/Dev/SP00419/10) was assessed. Isolates were obtained from the bone marrow of brown layers that died due to EPS at different commercial farms and had different PFGE banding patterns (types A, B and C) showing that they were not clonal.
Table 1. Intravenous inoculation of 17 week-old brown layer hens with E. coli PFGE-types to assess their virulence. E. coli bacteria had been isolated from bone marrow of laying hens with EPS. The experiment was ended one week after inoculation.
Three groups each of nine layers aged 17 weeks were made. The birds of these groups were inoculated intravenously (vena ulnaris) with either isolate.
Daily mortality was recorded and at the end of the experiment, one week after inoculation, the occurrence of EPS was assessed and bacteriology of bone marrow of all hens was performed. The relatedness of reisolates with the parent E. coli strains was assessed by PFGE.
Experiment 2. The experimental design is given in . The efficacy of two differently formulated (water or water-in-oil emulsion) inactivated E. coli vaccines was assessed. Six groups (groups 4–9) each of 18 layers which were vaccinated at the age of 14 and 18 weeks were made at 27 weeks of age. Each vaccine type was administrated to three groups. Another three groups (groups 1–3) with the same number of non-vaccinated layers were included in the study. This resulted in three categories of groups: non-vaccinated groups (groups 1–3), groups vaccinated with aqueous-based vaccine (groups 4–6) and groups vaccinated with oil-emulsion-based vaccine (groups 7–9). Groups of hens within each category were either not exposed to E. coli (groups 1, 4 and 7), exposed/challenged homologously (groups 2, 5 and 8) or heterologously (groups 3, 6 and 9) at the age of 30 weeks by aerosol.
Table 2. The protective effect of vaccination with inactivated E. coli vaccine against EPS in brown layers. Protection was assessed by aerosol challenge with homologous or heterologous E. coli PFGE-types. The experiment was ended eight days after challenge.
Daily mortality was recorded; moreover body weight had been assessed at 12, 14, 18, 22 and 27 weeks of age to determine whether the vaccines had an adverse effect on body weight.
At the end of the experiment, eight days after exposure/challenge, the occurrence of EPS was assessed and bacteriology of bone marrow of all hens was performed. The relatedness of reisolates with the parent E. coli strains was assessed by PFGE.
Chickens, housing and husbandry
Commercial brown layers were used. Non-E. coli-vaccinated hens free of Mycoplasma synoviae, which was assessed by rapid plate agglutination and PCR as described (Landman et al., Citation2004), were obtained from a commercial rearing farm. All birds were tagged with unique numbers.
The hens of Experiment 1 were obtained at 15 weeks of age and housed during the whole experimental period in three separated floor pens made within an experimental room, with wood shavings as bedding.
The layers (n = 174) used for Experiment 2 were obtained at 10 weeks of age and housed in an experimental room, with wood shavings as bedding. Fifty-seven birds were vaccinated with the aqueous vaccine (Group II), 60 with the water-in-oil vaccine (Group III) and 57 were left unvaccinated (Group I). At 27 weeks of age nine groups each consisting of 18 birds were formed and transferred to negative-pressure high-efficiency particulate air isolators with a volume of 1.34 m3 each (Beyer & Eggelaar, Utrecht, the Netherlands). Birds were kept in the isolators until the end of the experiment. Isolator temperature was maintained at 18–20°C; relative humidity was 60–80%.
During rearing, light was supplied for 8 h per day. On isolators, the hens were exposed to 16 h of light per day. Birds were fed a standard commercial layer diet ad libitum and had free access to drinking water.
Bacteriology
Transversally cut femur from a hen, which had died due to EPS, was sterilized with a hot scalpel blade after which a bone marrow sample was collected with a wire for the isolation of E. coli. A sheep blood agar plate (K004P090; Biotrading, Mijdrecht, the Netherlands) was then inoculated and subsequently incubated overnight at 37°C. Biochemical identification of colonies was performed using the indole and β-glucuronidase test. Isolates were stored at −70°C. Preparation of inocula was performed by rolling Frozen Protect Beads (TS70E; Biotrading) containing the E. coli isolate on a sheep blood agar plate (K004P090; Biotrading). After overnight incubation at 37°C, colonies were then scraped off and suspended in 3 ml physiological saline solution until a concentration of 0.5 McFarland (108 colony-forming units (cfu)/ml) was obtained. A volume of 0.1 ml of this suspension was mixed with 90 ml of 0.1% glucose broth (1000 ml purified water, 5 g Lab Lemco (Oxoid LP0029; Oxoid, Badhoevedorp, the Netherlands), 10 g bacteriological peptone (Oxoid LP0-037), 5 g NaCl (VWR 1.06404.1000; Merck Eurolab B.V., Amsterdam, the Netherlands) and 1 g glucose (VWR 1.08342.1000; Merck)), which was incubated for 17 h at 37°C. E. coli concentrations were assessed by means of bacterial counts according to international standards (ISO6887, Citation1983; ISO7402, Citation1985).
E. coli autogenous vaccines
Inactivated E. coli autogenous vaccines were prepared using E. coli strain chicken/NL/Dev/SP01404Cou/05 with PFGE-type A. E. coli was inactivated using 3.5 ml formaldehyde 37% (w/v) per litre physiological saline during 21 ± 3 h at 37 ± 1°C. Beforehand the E. coli colonies on sheep blood agar plates, which had been incubated 21 ± 3 h at 37 ± 1°C in an O2-enriched atmosphere, were scraped off and added to the saline until a suspension of ≥3.5 McFarland (≥109 cfu/ml) was obtained. The inactivated bacterial suspension was stored at 2–8°C after preparation. Sterility of the inactivated bacterial suspension was analysed using tryptone soya broth (casein soya bean digest medium) (OCM0129, Oxoid) and thioglycolate broth (CM0391, Oxoid). A volume of 1 ml of the inactivated bacterial suspension was added to 100 ml of each of the broths. Subsequently, the inoculated broths were incubated for 7 days at 37 ± 1°C in an O2-enriched atmosphere. At day 4 of the incubation, two sheep blood agar plates were each inoculated with a sample from either broth, which were incubated during 63 ± 9 h at 37 ± 1°C. The plate with the tryptone soya broth sample was incubated aerobically, while the other plate was incubated anaerobically. The inactivated bacterial suspension was considered sterile if after 63 ± 9 h of incubation no growth occurred on the sheep blood agar plates and the broths remained clear at visual inspection after 7 days of incubation.
Two vaccine formulations were prepared: one water-based and the other as water-in-oil emulsion. In order to obtain similar antigen concentrations in both formulations the same dilution was used to prepare both vaccine types. The aqueous vaccine suspension was prepared by adding 70 ml physiological saline to 30 ml inactivated bacterial suspension. The water-in-oil emulsion vaccine was prepared by adding 30 ml inactivated bacterial suspension to 70 ml Montanide ISA 70 VG, a natural metabolizable oil and a highly refined emulsifier from the mannide monooleate family (Seppic S.A., Puteaux, France), while applying high shear forces using a blender (IKA T18 basic Ultra-Turrax, IKA®-Werke GmbH & CO.KG, Staufen, Germany) equipped with a dispersing element (S18D-14G-KS, IKA®-Werke GmbH & CO.KG). The blender was set at 1200 rpm, while the inactivated E. coli suspension was added to Montanide at a maximum rate of 10 ml per minute using a syringe. The blender was set at full speed (25000 rpm) once the whole E. coli suspension was added. The temperature of the mixture was monitored continuously using an infrared thermometer (Thermo-Hunter PT-S80, Optex (Europe) Ltd., Maidenhead, Berkshire, UK) and the blending procedure was stopped once a temperature of 37°C was reached (approximately after 5–6 min of mixing). The prepared emulsified vaccine was thereafter subjected to microscopic analysis during which the sizes of the dispersed droplets were assessed. All droplets should be <10 µm and 90% below 1 µm.
Each hen was inoculated in the musculus pectoralis with 0.5 ml of either vaccine containing ≥108.2 inactivated cfu of E. coli.
Challenge: aerosol fluids, application, assessment of aerosol concentration and dose uptake
A volume of 50 ml 0.1% glucose broth containing 108.3–8.4 cfu E. coli per ml was nebulized using a spray head with an orifice diameter of 0.5 mm (Walther Pilot I spray-head; Walther Spritz- and Lackiersysteme, Wuppertal, Germany) coupled to an air compressor (Mecha Concorde, type 7SAX, 1001, 10 bar/max; SACIM, Verona, Italy) at a pressure of two bar and an air yield of 30 l/min, resulting in a flow of 30 ml/min (aerosol generation time = 100 s). The droplet spectrum of the aerosol (assessed as described (Landman et al., Citation2004)) was: Dv(0.1) = 2.7 µm, Dv(0.5) = 9.9 µm and Dv(0.9) = 30.7 µm. From the start of the aerosol generation until 30 min after ending, the isolator ventilation was switched off, resulting in a temperature increase up to 25–27°C. During aerosolization the relative humidity of the isolator air raised to 100% within a few minutes. Immediately and 20 min after ending the aerosol generation, air sampling (67 l air per sampling) was performed using gelatine filters (3 µm pore size and 80 mm diameter, type 17528-ACD, Sartorius B.V., Nieuwegein, the Netherlands) mounted to a MD8 airscan (Sartorius B.V.) as described (Landman et al., Citation2004) in order to calculate the bacterial concentrations per m3 of isolator air and subsequently the dose uptake of the birds by inhalation. Bacteriology of gelatine filters was performed after dissolving them in 50 ml buffered peptone water kept at 37°C. Subsequently, tenfold dilutions (10−1–10−6) were prepared using peptone physiological saline. A volume of 0.1 ml of the undiluted gelatin solution and of each dilution was plated on sheep blood agar (K004P090; Biotrading) and incubated at 37°C for 24 h. Only plates containing between 30 and 300 colonies were counted. The detection limit was 103.9 cfu/m3 isolator air. Validation of bacteriology of filters was performed earlier (Landman et al., Citation2004). Directly after the aerosol generation and 20 min later, E. coli concentrations in isolator air ranged from 108.0 to 108.8 cfu/m3 and from <103.9 to 106.1 cfu/m3, respectively.
The uptake of aerosolized E. coli was calculated as described (Landman et al., Citation2013) using a ventilation volume of 30 l/hen/hour, which was estimated based on the minute ventilation rate of White Leghorn hens (261 ml/min/kg) (Gleeson et al., Citation1985) and ranged from 105.8 to 106.5 cfu/hen ().
Pulsed-field gel electrophoresis
The clonal relationship between the inoculation strains used in experiments 1 and 2 and the reisolated E. coli bacteria (one colony per positive bird) was assessed by PFGE.
The PFGE-technique of contour-clamped homogeneous electric fields as described earlier (Landman et al., Citation2015) was used.
Statistics
Normality of the body weights was assessed by visual inspection of the normal probability plot. Body weight of vaccinated layers was compared to that of their non-vaccinated counterparts using ANOVA (StataCorp., Citation2015), with group and age as independent variables. As post hoc test Bonferroni correction was applied.
Differences in the number of hens with EPS between groups were analysed using Fisher’s exact test (Analytical Software, Citation2010).
Differences were considered significant if P < 0.05. Statistically significant is further referred to as significant.
Ethics
The study was approved by the Institutional Animal Experimental Committee, DEC-Consult Foundation, according to Dutch law on experimental animals (Wet op de dierproeven).
Results
Experiment 1
Results are presented in . Although the mortality was highest in the group inoculated with E. coli PFGE-type A (6/9) (Group 1), there was no statistical difference with the mortality in the other groups (groups 2 and 3). Also, the mean death time was the shortest in Group 1. All mortality was due to EPS as E. coli was isolated from the bone marrow of the dead hens. Some surviving hens had E. coli arthritis and overfilled joints.
E. coli PFGE-type A was chosen for vaccine preparation and homologous challenge and E. coli PFGE-type B for heterologous challenge in Experiment 2.
Experiment 2
An overview of the results is presented in . In the negative control group (Group 1) and the vaccine control groups (groups 4 and 7) mortality and EPS did not occur. In the positive control group exposed to an aerosol of E. coli PFGE-type A (Group 2), mortality and number of birds with EPS were 5/18 and 7/18, respectively. These parameters were 2/18 and 4/18, respectively, in the group exposed to an aerosol of E. coli PFGE-type B (Group 3). Mortality due to EPS occurred between 2 and 5 days post exposure. E. coli was isolated from the bone marrow of the dead hens. Some of the surviving hens showed chronic peritonitis, degenerated ovaries or shell-less abdominal eggs.
Both the aqueous suspension (Group 5) and the water-in-oil emulsion (Group 8) vaccines (almost) fully protected against homologous challenge: the number of birds with EPS was significantly reduced in vaccinated groups compared to the non-vaccinated E. coli aerosol-exposed positive control group (Group 2).
Protection induced by both vaccines against heterologous challenge was not assessed for significance as the positive control group exposed to E. coli PFGE-type B (Group 3) did not differ significantly from the negative control group (Group 1).
Effect of vaccination on growth
Mean body weight (± SD) of the three groups of hens (groups I–III) are given in and did not differ significantly between groups at any age.
Table 3. Mean body weight ± SD (g) of brown layer hens intramuscularly vaccinated against EPS with inactivated E. coli autogenous vaccine at 14 and 18 weeks of age.
Pulsed-field gel electrophoresis
Re-isolates showed the same banding pattern as the parent strain and were therefore considered genetically indistinguishable.
Discussion
Deb and Harry were the first to examine the efficacy of inactivated E. coli vaccines based on serotypes O78 K80 (Deb & Harry, Citation1976) or O2 K1 (Deb & Harry, Citation1978) and containing approximately 108.6–9.2 inactivated E. coli bacteria per dose in 0.5 ml. Vaccination of Light Sussex chickens at 2–3 weeks of age resulted in protection against homologous challenge 3–4 weeks later. Heterologous protection was not found. Several publications on E. coli vaccination have been issued thereafter; an overview can be found in the review of Ghunaim and others (Citation2014) in which the various E. coli vaccine types are described, i.e. live attenuated, inactivated and subunit vaccines. All these vaccines were studied experimentally and administered to chickens and turkeys. Vaccinations were performed once or twice in the first seven weeks of life and subsequently the birds were challenged homologously or heterologously 10 days to 5 weeks after the last vaccination. As a rule vaccines only conferred protection against homologous challenge (Ghunaim et al., Citation2014).
In young birds the most relevant form of colibacillosis is fibrinous polyserositis, which commonly occurs in broilers during the second half of the grow-out period. It is mainly caused by opportunistic E. coli bacteria as this disease is favoured by a number of triggers such as infections with both, field and vaccine respiratory viruses (infectious bronchitis virus (Matthijs et al., Citation2003; Matthijs et al., Citation2005), Newcastle disease virus (Van Eck & Goren, Citation1991), avian pneumovirus (Al-Ankari et al., Citation2001)) and mycoplasma infections (Gross, Citation1961; Fabricant & Levine, Citation1962).
Passive immunity by maternal derived antibodies protected the offspring of vaccinated breeders during the first two weeks of life (Rosenberger et al., Citation1985; Heller et al., Citation1990). In contrast, Li et al. (Citation2017) did not find transfer of E. coli antibodies from vaccinated parent stock to their progeny. In any case, maternal antibodies do not protect broilers during the second half of the grow-out period.
Rather than relying only on partially effective E. coli vaccinations (heterologous protection is not satisfactorily obtained) a solution for the colibacillosis problem in broilers should be sought to prevent the occurrence of the triggers for this disease.
In laying hens EPS is the most relevant colibacillosis form, which is likely caused by non-opportunistic virulent E. coli strains (Landman et al., Citation2013). As specific triggers do not seem to be of importance, vaccination is the only option to be considered.
Literature regarding vaccination for the protection of laying hens against colibacillosis is very scare; to date only two manuscripts on this subject have been published (Gregersen et al., Citation2010; Li et al., Citation2017). Gregersen and others performed a field study in broiler breeders using a subunit vaccine (E. coli fimbrial antigen F11 and E. coli flagellar toxin in a water-in-oil emulsion). Although the E. coli-associated mortality was lower in the vaccinated flocks, the total mortality did not differ between vaccinated and non-vaccinated birds. Therefore, drawing unambiguous conclusions is difficult. In the study by Li and others, broiler breeders were vaccinated at day old and 12 weeks of age with a live attenuated E. coli vaccine by coarse spray. Additionally, birds were given once or twice subcutaneous injections with a formalin-inactivated mineral-oil-adjuvanted E. coli vaccine containing three E. coli isolates (two liver isolates and one from a cellulitis lesion in a broiler breeder). The inactivated vaccine was administered at 14 and 18 or only at 18 weeks of age. Challenge was performed at 28 weeks of age by injecting 105.7 cfu per bird into the salpinx following laparotomy under general anaesthesia. Birds were either challenged with one of the E. coli liver strains of the vaccine or with an E. coli isolate obtained from periorbital skin from a chicken with swollen head syndrome (heterologous challenge). The experiment was ended at 29 weeks of age. The main conclusion of this work is that the autogenous vaccine protected homologously against acute E. coli mortality, but not against the occurrence of colibacillosis lesions after homologous or heterologous challenge. The effect of this vaccine on E. coli mortality following heterologous challenge was inconclusive as mortality did not occur in non-vaccinated heterologously challenged birds. Apparently, Li and others studied the effect of vaccination on SPS, but surprisingly they did not use E. coli strains originating from SPS lesions to produce their autogenous vaccine, neither were such strains used for homologous and heterologous challenge. Moreover, two of the three E. coli strains included in the vaccine originated from broilers and not from laying hens.
In the present manuscript the protective effect of autogenous vaccine against EPS is outlined. Both, the vaccine and challenge strains originated from the bone marrow of laying hens that died due to EPS. The two vaccine formulations used (aqueous and water-in-oil) administered at 14 and 18 weeks of age conferred (almost) complete protection against homologous challenge. The results of the heterologous challenge were inconclusive as no difference in EPS prevalence was found between the negative and positive control group (groups 1 and 3, respectively; ). Although, no significant difference in protective effect was found between the two vaccine formulations, a long-term difference in favour of the water-in-oil emulsion vaccine might be present.
In the study of Li and others and in Experiment 2 of our study similar challenge doses were used (105.7 cfu per bird versus 105.8–6.5 cfu per bird). Also differences between the mentioned two studies exist, which might explain the different results, amongst others, difference in breed of experimental birds, in challenge route and possibly also in antigen content of the inactivated vaccine. The latter was not presented by Li and others, making comparison with the vaccine used in the present study impossible. However, most likely the difference in outcome of the two studies, i.e. almost complete protection against EPS in our study versus protection against acute mortality but not against E. coli lesions in the study of Li and others, might be explained by the difference in challenge route.
In the present work a natural challenge route and the most plausible infection route for EPS in the field was used, i.e. aerosol exposure (Landman et al., Citation2013). In contrast, Li and others used a rather cumbersome non-natural challenge route, by performing laparotomy under general anaesthesia and inoculating the challenge strain from a swollen head directly into the salpinx. Also, a semisynthetic opioid (buprenorphine) was administered to diminish post-surgical pain and discomfort. Possibly, bypassing the natural host defences using a non-natural challenge route and/or the administration of buprenorphine have enabled the persistence of E. coli lesions in vaccinated birds.
As a rule, inactivated vaccines administered via injection elicit high systemic antibody titres, even more so if they are given as booster. Chickens with a cyclophosphamide-induced B cell depletion did not produce avian pathogenic E. coli-specific IgY and were as susceptible to challenge as age-matched unvaccinated controls, showing that protection against colibacillosis requires a cyclophosphamide-sensitive cell population including B cells (Sadeyen et al., Citation2015). The protective effect observed in our study and by Li and others is therefore explained by the fact that vaccine-induced systemic antibodies likely hinder the access of E. coli to the bloodstream and/or favour its rapid clearance.
In conclusion, for the first time (almost) full homologous protection by an autogenous E. coli vaccine against EPS, which is the most important cause of death in laying hens, was shown.
Acknowledgements
We thank Wilko Vije and Gert Jan Boelm for their skilful technical assistance. We also thank Rianne Buter for performing the PFGE analysis and Anouk Veldhuis for performing the statistical analysis.
Disclosure statement
No potential conflict of interest was reported by the authors.
Additional information
Funding
References
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