Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: https://www.tandfonline.com/loi/cavp20

Development of an in-house ELISA for detection of

antibodies against Enterococcus cecorum in Pekin
ducks

Arne Jung & Silke Rautenschlein

To cite this article: Arne Jung & Silke Rautenschlein (2020): Development of an in-house ELISA
for detection of antibodies against Enterococcus�cecorum in Pekin ducks, Avian Pathology, DOI:
10.1080/03079457.2020.1753653

To link to this article: https://doi.org/10.1080/03079457.2020.1753653

Accepted author version posted online: 09

Apr 2020.

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Publisher: Taylor & Francis & Houghton Trust Ltd

Journal: Avian Pathology

DOI: 10.1080/03079457.2020.1753653

Short communication

Development of an in-house ELISA for detection of antibodies against

Enterococcus cecorum in Pekin ducks
Arne Jung* and Silke Rautenschlein

Clinic for Poultry, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover,
Germany

Running head: Enterococcus cecorum ELISA for Pekin ducks

*
Corresponding author: Arne Jung, Clinic for Poultry, University of Veterinary Medicine Hannover,
Bünteweg 17, 30559 Hannover, Germany. Tel: +49-511-953-8774, Fax: +49-511-953-8580. E-mail:
jung.arne@tiho-hannover.de
Abstract

Enterococcus cecorum (EC) is known to cause skeletal lesions in broiler chickens and also systemic

infections in Pekin ducks. Despite the importance of the pathogen, there is still a lack of serological

diagnostic tools for the detection of EC infections. Here we describe the development of an in-house

indirect enzyme-linked immunosorbent assay (ELISA) for the detection of EC-specific antibodies and

its application by examination of 67 sera from experimentally infected Pekin ducks, 710 field samples

from 4 Pekin duck breeder flocks previously vaccinated with inactivated vaccines and 80 samples from

commercial Pekin ducks coming from vaccinated parent flocks. All groups that had been experimentally

inoculated via air sac route were positive in the new ELISA, with significantly (P ≤ 0.05) increased

mean S/P ratios of 0.71 to 2.70 at days 7, 14 and 21 post infection, while orally inoculated ducks and

the EC-free control group remained negative with mean S/P ratios of 0.0 to 0.15. Antibodies were also

detected in each of 4 vaccinated Pekin duck breeder flocks, 67.8 % of the samples were antibody

positive. The highest S/P ratios were found between 16 and 26 weeks (median S/P ratios from 0.15 to

1.03), but antibodies were still detected in some serum samples in weeks 61 to 67 ph. No antibodies

were detected in the commercial Pekin ducks.

Antibody development in the ducks may be influenced by the composition of the inactivated vaccine.

The new ELISA assay provides a useful tool for investigations on response to EC infections and

vaccinations.

Keywords: Enterococcus cecorum, Pekin ducks, Pekin duck breeders, antibodies, ELISA, inactivated

vaccine
Introduction

Since 2002 it is known that Enterococcus cecorum (EC) can cause disease in broiler chickens

(Devriese et al., 2002; Wood et al., 2002) and today it is one of the most important bacterial pathogens

in broiler production (De Herdt et al., 2009; Szeleszczuk et al., 2013; Aitchison et al., 2014; Jung &

Rautenschlein, 2014; Zeshan et al., 2015; Talebi et al., 2016). Furthermore, EC infections were also

described in Pekin ducks (Metzner et al., 2010; Jung et al., 2013) and EC can be regularly isolated

from ducks (Dolka et al., 2017). In contrast to broilers, EC did not cause spondylitis and femoral head

necrosis in ducks. Instead, experimentally and naturally infected birds show a generalized infection

(Metzner et al., 2010; Jung et al., 2012; Jung et al., 2013), which is also seen in broilers in the early

septic phase of the infection (Jung & Rautenschlein, 2014; Borst et al., 2017). In Germany, some

Pekin duck breeder flocks are vaccinated with inactivated autogenous EC-vaccines to achieve

protection in the progeny through maternally derived antibodies. Autogenous vaccines can be used

when no licensed vaccine for the pathogen is available, which is the case for EC. Isolates from the

respective flock have to be used for production of the vaccine and the pathogen have to be safely

inactivated during production process. No data is available about the effectiveness of these vaccines so

far. However, no commercial ELISA kit for the detection of antibodies is available and there is also no

data published about the development of EC antibodies in ducks. Like chickens, ducks have three

immunoglobulin isotypes IgM, IgA and IgY (Lundqvist et al., 2006). Additionally, ducks express a

smaller form of IgY called IgY (ΔFc) with unknown function. IgY is the major systemic antibody,

involved in complement fixation and opsonization and is also transferred into the yolk sac of

developing embryo (Lundqvist et al., 2006). The aim of the present study was the development of an

in-house ELISA system for the detection of EC specific antibodies in Pekin ducks and evaluate the

usefulness of described ELISA for testing serum samples from experimentally infected Pekin ducks,

vaccinated breeders and their progeny.

Materials and Methods

Bacteria and growth conditions. The pathogenic Enterococcus cecorum (EC) strain D07-797-90/61

was used for coating of the 96-well plates. It was isolated from a disease outbreak in Pekin ducks

(Jung et al., 2013) and therefore categorized as a pathogenic isolate. The strain was grown on densely

inoculated Columbia sheep blood (CSB) agar plate (Oxoid, Wesel, Germany) and incubated for 24 h

at 37 °C in a CO2-enriched atmosphere using a desiccator and the candle method.

ELISA antigen preparation. Bacterial colonies from each CSB agar plate were washed off and

suspended in 3 ml sterile physiological saline and homogenized using 1 ml syringes and 20 G needles.

Afterwards, bacterial suspensions were collected in a 50 ml centrifuge tube and bacterial cells were

harvested by centrifugation at 3000 g for 30 min. The supernatant was discarded and the bacterial

pellet was resuspended in 40 ml carbonate/bicarbonate buffer (Na 2CO3: Merck, art. no. 6391;

NaHCO3: Riedel-de Haën, art. no. 31437; adjusted to pH 9.6) through vortexing and homogenized

using syringes and 20 G needles. The suspension was then adjusted with carbonate/bicarbonate buffer

to McFarland standard 4 and bacterial cells were disrupted using an ultrasonic disintegrator (MSE,

London, UK) with medium power, amplitude 4 and 15 cycles of 30 sec and 30 sec breaks. The

resulting suspension was used for coating of the ELISA plates and can be stored at -20 °C until further

use.

Antigen coating and blocking of ELISA plates. MaxiSorp 96-well plates (Nunc, Wiesbaden,

Germany) were washed once with carbonate/bicarbonate buffer and coated for 24 h at 4 °C with 100

µl of a 1:32 dilution with carbonate/bicarbonate buffer of the above described antigen suspension. The

protein concentration of the antigen suspension was determined through measurement of the

absorbance at 280 nm using a photometer (NanoDrop, Thermo Fisher, Waltham, USA) and was equal

to 40.31 µg/ml. After removal of the suspension, plates were washed 3 times. In each washing step,

every well was filled with 300 µl washing buffer (Synbiotics/Zoetis, cat. no. 02-3832-1103, Berlin,

Germany) and incubated for 3 min at 20 °C. Unspecific binding sites were blocked with 150 µl

phosphate-buffered saline buffer, pH 7.4, containing 1% bovine serum albumin (Carl Roth, Karlsruhe,
Germany), for 2 h at room temperature. After 3 times washing with washing buffer

(Synbiotics/Zoetis), plates were sealed with plastic film and stored at -20 °C.

ELISA procedure. Samples were diluted in ready to use dilution buffer (Synbiotics/Zoetis, cat. no.

02-3830-0103) as follows: field samples 1:500, negative control serum 1:500 and positive control

serum 1:800. The dilutions were determined after checkerboard titrations with positive/negative

control sera and the conjugate. Therefore, twofold dilutions of negative and positive sera were added

horizontally and twofold dilutions of conjugate were added vertically to a 96-well plate. Dilutions with

highest signal to background ratios were selected. Afterwards, 100 µl diluted serum was added to each

well of the ELISA plates. Controls were added to each plate in triplicates and samples were incubated

for 30 min at room temperature. After removal of the serum samples, plates were washed 3 times (see

above). Horseradish peroxidase conjugated rabbit anti-duck IgG (antibodies-online GmbH, cat. no.

ABIN457698, Aachen, Germany) was used as the secondary antibody at a dilution of 1:1750 (3.6

µg/ml) in dilution buffer. A volume of 100 µl conjugate solution was added per well and plates were

incubated for 30 min at room temperature. Subsequently, plates were washed 3 times, 100 µl of the

substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS; Synbiotics/Zoetis, cat. no.

02-3850-0103) was added to each well. After 6 min incubation in darkness and at room temperature,

100 µl stop solution (Synbiotics/Zoetis, cat. no. 02-3750-0103) was added to each well and bubbles

were removed, if necessary. OD (optical density) values were measured at 405 nm wavelength using a

Tecan Sunrise ELISA reader (Tecan, Crailsheim, Germany). For correction of OD values, the mean

OD value of negative controls was subtracted from the OD values of samples and positive controls.

ELISA results are presented as ratios of the corrected sample OD and the corrected mean OD of

positive controls (S/P ratio).

Negative and positive control sera. Sera from a previous study were used as negative and positive

controls (Jung et al., 2013). Pekin ducks in this study were obtained from a parent flock that was not

vaccinated against EC (Jung et al., 2013). Pekin ducks were confirmed to be free from EC via
euthanasation of five 1-day-old ducklings. The yolk sac and caeca were removed for EC isolation.

Furthermore, swabs from the oral cavity and the cloaca from five birds per group were collected at day

5 post hatch. Samples were tested for EC by isolation on agar plates. Negative control sera were

collected from the negative control group at 33 days of age. Positive control sera were obtained from

Pekin ducks which received 8.5 × 107 colony forming units (CFU) of EC strain D07-797-90/61 per

bird in the left caudal thoracic air sac at 12 days post hatch (Jung et al., 2013). Sera were collected at

33 days of age (21 days post infection).

Sera from experimentally infected Pekin ducks. Sera from a previous study (Jung et al., 2013) were

examined with the newly developed ELISA. The study design was as follows: at 12 days of age, one

group of ducks was inoculated orally with 0.5 ml EC suspension containing 1.5 × 109 colony-forming

units (CFU) per bird. Three more groups received 0.5 ml EC suspension into the air sac containing 8.5

× 105, 8.5 × 107 and 1.5 × 109 CFU per bird respectively. Another group was infected intravenously

with 0.5 ml EC suspension containing 1.5 × 109 CFU per bird. Birds of the control group were

inoculated with sterile physiological saline solution. At days 7, 14 and 21 post infection (pi), serum

samples of five birds of each group were collected and stored at -20 °C (Jung et al., 2013).

Field sera. Four Pekin duck breeder flocks (Cherry Valley) of one breeding farm were vaccinated

with 0.5 ml/animal inactivated autogenous vaccines (RIPAC-Labor GmbH, Potsdam, Germany) at

weeks 10, 15, 19, 21 and 40 ph subcutaneously in the neck. The vaccine included EC and Escherichia coli

with aluminium hydroxide as an adjuvant. Each vaccine contained 5 to 8 different EC isolates in a

concentration of about 7 x 108 CFU/vaccine dose and Escherichia coli with about 3 x 109

CFU/vaccine dose. Serum samples were collected after vaccination at weeks 16, 19, 22, 25, 28, 31, 34,

37, 40, 43, 46, 49, 52, 55, 58, 61, 64 and 67.

Serum samples (n=20) from commercial Pekin ducks (progeny) coming from vaccinated parent flocks

were collected at days 5, 10, 15 and 22 post hatch. The Pekin duck parents were vaccinated
subcutaneously in the neck at weeks 2, 4, 6, 10, 13, 15 and 17 with 0.5 ml/animal of an inactivated

autogenous vaccines against EC. Above mentioned progeny hatched from eggs laid in week 31.

In total, 710 field serum samples from 4 Pekin duck breeder flocks (flock A: 180, B: 180, C: 180, D:

170 samples) and 80 field serum samples from one commercial Pekin duck flock were examined for

EC antibodies with the new developed in house ELISA.

Determination of the negative-positive cut-off value. The mean S/P ratio (x) and standard deviation

(SD) were calculated from serum samples of 11 EC-negative control birds from the infection

experiment. The cut-off value was calculated as x + (3 × SD).

Reproducibility. Intraassay variation was determined with 12 replicates of three different dilutions

(1:400; 1:800; 1:1600) of the positive control respectively. Interassay variation was determined using

3 different duck sera from the experimentally infected ducks (high, medium and low antibody titers) in

duplicates on 4 different plates at different days. Arithmetic mean, standard deviation and coefficient

of variation (CV) were calculated from the raw OD values.

Statistics. Comparison of S/P ratios from sera of experimentally infected ducks was done using

analysis of variance with Fisher’s least-significant difference test as post hoc-test. For all calculations,

Statistix 10 (Analytical Software, Tallahassee, Florida, USA) was used. Differences in all statistical

tests were considered significant at P ≤ 0.05.

Results

Determination of the negative-positive cut-off value. The cut-off value defines the threshold

between negative and positive samples and was equal to 0.16.

Reproducibility. Concerning the intraassay variation, the CV values for the 1:400, 1:800 and 1:1600

dilutions of the positive control were 5.15%, 4.93% and 4.24% respectively. Concerning the interassay

variation, the CV values for the three different sera were 14.41%, 13.14% and 13,18% respectively.

Examination of sera from experimentally infected ducks. In total, 67 sera from experimentally

infected Pekin ducks were examined with the EC ELISA. Significantly (P ≤ 0.05) increased S/P ratios

were detected in all air sac inoculated groups from day 7 to day 21 pi with a peak at 7 dpi (mean S/P

ratios of 0.71 to 2.70; Figure 1). In total, 76.3 % of the air sac inoculated ducks were seropositive (CV

values from 0.39 to 1.09). Ducks of the orally inoculated group were negative, except for one animal

at day 7 pi, which was clearly positive for EC specific antibodies (mean S/P ratios of 0.0 to 0.15). In

general, there was a tendency of declining antibody titers from day 7 to day 21 pi.

Examination of field sera from vaccinated Pekin duck breeder and Pekin duck flocks. EC

antibodies were detected in all 4 Pekin duck breeder flocks and the highest titers were found from 16

to week 26 (Figure 2). In all 4 flocks there is tendency of declining antibody titers during the sampling

period, although at week 61 for flock D and 64 for flock A the median antibody titers were still clearly

above the negative-positive cut off. Additionally, there were some differences at the beginning of the

sample period. While the median S/P ratio of flock A and B at week 16 was around the negative-

positive cut off of 0.16, the median S/P ratio at this week of flock D was 1.03. In total, 482 of 710

(67.8 %) tested serum samples were positive for EC antibodies.

All serum samples from the Pekin duck flock were negative for EC specific antibodies (data not

shown).
Discussion

In this study, a new in-house ELISA for the detection of EC antibodies in ducks was successfully

developed. It is generally accepted that CV values of ELISA systems should be below 10% for

intraassay variation and below 15% for interassay variation (Anonymous), which was achieved for

this ELISA. In experimentally infected ducks, EC specific serum antibodies were detected with the

new ELISA with a peak at 7 days post infection (dpi) and a decline until 21 dpi, which was the end of

the experiment. The control birds remained negative through the whole trial. These results are in

concordance with the development of serum antibodies in poultry infected with other bacterial

pathogens (Knab et al., 2018), where the highest antibody titers were also found from 7 to 11 days

post experimental infection.

Analyzing field samples from vaccinated animals, antibodies against EC were detected in all 4

investigated Pekin duck parent flocks. This shows that the administration of inactivated vaccines can

induce detectable EC antibody titers in the vaccinated Pekin ducks. Compared to the experimental

infection, the S/P ratios in the vaccinated parent ducks were lower. One reason may be antigenic

differences between the vaccine strains and the EC strain which was used for coating of the ELISA

plates. Different serotypes of EC were recently described (Jung et al., 2017). Therefore, for a broad

application of the ELISA, coating of the 96-well-plates with a mixture of different EC serotypes might

be reasonable to improve the validity of the ELISA results. Another possible reason for the low S/P

ratios might be the composition of the inactivated vaccine with EC at a lower antigen concentration

than E. coli. A vaccine incorporating only EC strains in a higher concentration might induce higher

antibody titers. However, in this study, Pekin duck parent flocks were vaccinated using autogenous

vaccines with different antigen compositions. Therefore, anti-EC antibody development may have

been influenced by antigen compositions and direct comparison of EC antibody levels should be

conducted with caution. Additionally, no statement about the efficacy of the vaccine is possible,

because challenge studies under controlled laboratory conditions with vaccinated Pekin ducks or Pekin

duck parents are still missing.

No antibodies were detected in Peking ducklings whose parents were vaccinated against EC. The

underlying reason remains speculative, because the antibody titers of this parent flock were not

examined. As described above, the composition of the inactivated vaccine with different antigens may

have negatively affected the antibody production against EC.

Vaccination of broiler parents led to detectable maternally derived antibodies against EC in broiler

birds at day 0 but no longer at day 14 post hatch (Borst et al., 2019). However, these birds were not

protected against homologous or heterologous challenge with EC. Dual vaccination of Pekin duck

parents against Riemerella anatipestifer led to detectable maternally derived antibodies in the progeny

up to day 10 post hatch (Lobbedey et al., 2003). However, these data were collected in an

experimental setting, which is in contrast to our study, where field sera were examined.

In-house ELISAs were already developed for Riemerella anatipestifer and Pasteurella multocida

(Huang et al., 2002; Lobbedey et al., 2003; Liu et al., 2017; Poolperm et al., 2017), which are

important bacterial pathogens of ducks. This is the first description of an ELISA for the detection of

EC antibodies in ducks. It may help to answer open questions concerning EC epidemiology and

immune response to vaccinations in Pekin ducks. The development of anti-EC antibody titers in Pekin

ducks may be influenced by the composition of used inactivated vaccine.

Disclosure Statement

The authors declare that they have no competing interests.

Acknowledgements
The authors would like to thank Katja Hartmann for the excellent technical support.
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Figure 1. Development of serum antibodies after inoculation with Enterococcus cecorum (EC) via
different routes. Air sac low dose: 8.5 × 105 CFU EC/bird. Oral: 1.5 × 109 CFU EC/ bird. Air sac
middle dose: 8.5 × 107 CFU EC/bird. Air sac high dose: 1.5 × 109 CFU EC/ bird. In the air sac high-
dose group no birds were left at days 14 and 21 p.i. because of death or euthanasia due to welfare
reasons. n = 4 to 5. Data are presented as mean values of S/P-Ratios with positive standard deviations.
Different uppercase superscript letters indicate significant differences between groups (P ≤ 0.05;
analysis of variance, post-hoc test: Fisher’s least-significant difference).

Figure 2. Development of serum antibodies in 4 Pekin duck breeder flocks (A to D) after vaccination
with an Enterococcus cecorum (EC) inactivated vaccine at weeks 10, 15, 19, 21 and 40 ph, n = 10 to 15.
Data is presented as boxplots. The boxes indicate the upper and lower quartiles, the horizontal bars in
the boxes indicate the median, while the whiskers indicate the minimum and maximum. The
horizontal lines through the whole diagram indicate the negative-positive cut off. ND: not done.