FACULTY OF VETERINARY MEDICINE
approved by the EAEVE
Department of Obstetrics, Reproduction and Herd Health
Datum: 9 februari 2016
Prof. DVM. A. de Kruif, Prof. DVM. A. Van Soom
Prof. DVM. G. Opsomer, Prof. DVM. D. Maes
Prof. DVM. J. Dewulf, Prof. DVM. S. De Vliegher
IWT: “PATHOGENESIS, DIAGNOSIS AND PREVENTION OF
DYSBACTERIOSIS IN BROILERS” – DISCUSSION MEETING
24/11/2014
Members of the Users Committee:
Company
Name
Present
Company
Name
Present
UGent
Filip Van Immerseel
Yes
Agrifirm
Albert van den Belt
No
UGent
Jeroen Dewulf
Yes
Agrifirm
Erik Bruininx
No
UGent
Richard Ducatelle
Yes
AMCRA
Evelyne De Graef
No
UGent
Venessa Eeckhaut
Yes
BEMEFA
Yvan Dejaegher
No
UGent
Iris Van Dosselaer
Yes
Bio-X Diagnostics
Anita Ginter
No
FAVV
Jozef Hooyberghs
No
IWT
Kirezi Kanobana
Yes
FOD Volksgezondheid
Luc Derolez
No
NBFB
Filip Ghekiere
No
AVEVE
Inge Peeters
Yes
Pharma.be
Davy Persoons
No
Boerenbond
Wouter Wytynck
Yes
PROVANT
Johan Zoons
No
Coris Bioconcept
Pascal Mertens
Yes
VEPEK
Erik Hoeven
No
DGZ
Herman Deschuytere
Yes
Elanco
Jos Jacobs
Yes
Frana VZW
Ludo Segers
Yes
Prodivet
Alexandre Pellegrims
Yes
Quartes
Joris Maes
Yes
Vanden Avenne - Ooigem
Jan Van Ginderachter
Yes
Vetworks
Maarten De Gussem
Yes
VIP
Nico Thooft
Yes
Vlaamse Bedrijfspluimvee en Konijnenhouders
Johan Venken
Yes
Faculteit Diergeneeskunde – Vakgroep Voortplanting, Verloskunde en Bedrijfsdiergeneeskunde
Salisburylaan 133, B-9820 Merelbeke
BTW: BE0248015142 - Partner F2K - Lid Researchgroep VVZ
Overview of the Presentations:
Introduction: Filip Van Immerseel
Agency for Innovation by Science and Technology: Kirezi Kanobana
IWT: “Pathogenesis, Diagnosis and Prevention of Dysbacteriosis in Broilers”
Introduction: Filip Van Immerseel
Work Package 1,2,5: Iris Van Dosselaer
Work Package 3,4: Venessa Eeckhaut
Introduction by Filip Van Immerseel:
Welcome Word
Short Introduction of the Members of Users Committee.
Short Overview of the Afternoon
Agency for Innovation by Science and Technology (IWT):
In the presentation given by Miss Kanobana, she has given us an insight about the organization itself and more
specifically how this organization sponsors question driven agricultural projects aimed at the primary sector.
Goal of these long-term projects is to come up with innovative solutions that can be applied on short-term and
result in evident changes with an economic added value for a broad stakeholder group.
Due to the integrated approach the research institute, in this case IWT and Ghent University, offer a result to the
primary sector based on knowledge generation, knowledge translation, innovative applications, change and
transitions.
The research institute benefits of the input of its stakeholders group, due to the fact that they have been chosen as
representatives who aim at given guidance and follow-up of the project and in the end also guidance on the
valorization of the project results.
General findings resulting out of the gathered data and results throughout the project will be distributed as broadly
as possible. Result however remain in the ownership of the IWT. Economical valuable results or user rights can
be transferred according to market price, to which ever company within the European Union and are nonexclusive
IWT: “Pathogenesis, Diagnosis and Prevention of Dysbacteriosis in broilers”
Work Package 1:
The aim of the 1st work package is to describe the risk factors that influence the prevalence of dysbacteriosis. To
be able to do this an analysis of the dynamics of possible predictive factors on the prevalence is necessary. In
order to have an idea about these possible predictive factors a quantitative description of the prevalence of
dysbacteriosis on herd and animal level is imperative.
This will be done throughout three specific field studies, starting off with a cross-sectional study on 50 broiler
farms to gain an idea about the prevalence on herd and animal level and to take as much samples as necessary to
give a correct description of dysbacteriosis. In a second study, a longitudinal or follow-up study, 20 farms
selected out of the first study, will be followed during an entire production round. This will give more
information about the dynamics the possible predictive factors. The last study is a case-control study on a 100
broiler farms that will allow us to predict more accurately the real prevalence of dysbacteriosis and the influence
of the risk factor on the prevalence.
Faculteit Diergeneeskunde – Vakgroep Voortplanting, Verloskunde
en Bedrijfsdiergeneeskunde, Salisburylaan 133, B-9820 Merelbeke
Partner F2K - Lid Researchgroep VVZ
www.ROHH.UGent.be
Work Package 2:
Work Package 2 aims to give a correct description between animals with and without dysbacteriosis, this based on
macroscopically visual lesion scoring, morph histological parameters and inflammatory parameters that we have
distinguished out of the samples taken in the 1st and 2 study of work package 1.
By combining the outcome of the macroscopically visual lesions with morph histological parameters based on
villus length, villus clomping, thickness of the intestinal wall, the amount of goblet cells and the infiltration of
immune cells, we hope to be able to clearly distinguish a healthy intestine from one damaged by dysbacteriosis.
In order to provide even more markers to distinguish we try to increase these parameters by adding an extra value
in the form of micro biota differentiation between healthy and injured intestines. This will be done by 16S
sequencing to give a correct description of the phyla that are present. Once the phyla are known we determine the
proportion between each phyla in order to detect differences between healthy intestines and injured intestines.
In the last part of the study we also try to quantify the transcription level of cytokines en inflammatory mediators.
Combination of all above mentioned parameters will hopefully give us more detailed information whether certain
parameters are correlated or not with each other.
Work Package 3:
The aim of the 3rd work package is to identify a number of non-invasive biomarkers to diagnose chickens with
dysbacteriosis. We hypothesize that in chickens with dysbacteriosis an impairment of the integrity of the intestinal
wall results in paracellular leakage of bacterial products and endogenous cytosolic enterocyte proteins which may
be quantified in plasma.
One of the bacterial products that will be analyzed is LPS which is a major constituent of the outer membrane of
gram-negative bacteria and is released during replication and dying of the cells.
The second parameter is D-lactate, an end product of bacterial fermentation. Unlike the L-from, D-lactate is not
metabolized by the host. Increased levels are therefore a reflection of bacterial overgrowth in the gut due to the
impaired immunological barrier.
The first product of cellular origin that will be quantified is the intestinal-fatty acid binding protein (I-FABP).
This protein is found in high concentrations in tissues, like the intestine, involved in uptake and consumption of
fatty acids. I-FABP may be used as a marker for early enterocyte death as levels rise rapidly after episodes of
acute inflammation.
Another marker for reduced small bowel epithelial cell mass is citrulline, an amino acid not incorporated into
proteins and synthesized exclusively in small intestinal enterocytes from glutamine.
Claudin-3, an important tight junction (TJ) protein establishing the paracellular barrier is present in high quantity
in the intestine but disappears rapidly from TJ.
Another marker is alpha1-antitrypsin (A1AT), a serine protease inhibiting pro-inflammatory mediators which
increase in case of inflammation. Therefore A1AT is used to diagnose protein losing enteropathies.
We will also search for a microbial biomarker by performing qPCR on the content of different intestinal segments.
We will take into consideration different ratio’s after quantifying the number of bacteria within the phyla
Firmicutes, Bacteroidetes and Proteobacteria, the family Ruminococcaceae (= Clostridium cluster IV) and
Lachnospiraceae (Clostridium cluster XIVa) and the number of the highly abundant butyrate producing species,
Faecalibacterium prausnitzii.
Faculteit Diergeneeskunde – Vakgroep Voortplanting, Verloskunde
en Bedrijfsdiergeneeskunde, Salisburylaan 133, B-9820 Merelbeke
Partner F2K - Lid Researchgroep VVZ
www.ROHH.UGent.be
Work Package 4:
In the 4th work package we want to optimize an in vivo model for dysbacteriosis in broilers based on the risk
factors that come out of work package 1. If possible we would like to start earlier by simulating different
conditions based on input from the members of the users committee. For instance we already know that use of less
digestible feed compounds and compounds that are rich in NSPs are predisposing for dysbiosis. Once the model is
optimized it should be used to validate different prevention protocols.
Work Package 5:
Work Package 5 describes in detail the way the gathered information will be spread to the different members of
interest. This will not only be done by writing articles in national and (peer-reviewed) international journals, but
will also be done through direct contact with the primary sector on so called demonstration days, newsletters by
mail or post and if wanted local meetings with the professional organization.
A symposium that will allow members of not only the scientific community but also the organization directly
involved with the primary sector is scheduled.
Question, Remarks and Advises about Work Package 1:
1) Question Richard Ducatelle: How are you going to measure the moisture in the stable / litter.
Answer Iris Van Dosselaer: At this moment multiple methods are under study. Depending on the outcome we
will use one or more of them in our project to estimate the humidity.
Advice Herman Deschuytere: Take the ambient temperature outside also in to account.
2) Question Richard Ducatelle: What do you do with coccidiosis, do you take it in to account?
Answer Jos Jacobs: One should take it in to account, since the different coccidiostats can have an effect on the
litter quality.
Answer Maarten De Gussem: Not only the coccidiostats program in general should be taken in to account, but
each specific program, the time it is present in the feed, the changes in programs and even the fact of vaccination
or not.
Answer Iris Van Dosselaer: The mentioned items will be taken into account during the gathering of the data
about farm and stable specific elements. We also will collect information from the nutrition companies on top of
what we can find on the label. Moreover we will collect detailed info on all vaccinations (including coccidiosis) as
well as all antimicrobial and other treatments.
3) Question Richard Ducatelle: What do you do with farms that have used antibiotics?
Answer Iris Van Dosselaer: It is likely that at least some of the selected farms in the random sample will be
treating or have been treating against dysbacteriosis. This means that we will only be able to determine the
prevalence under a situation of treatment as it occurs in the field.
Answer Jeroen Dewulf: In the longitudinal study we will ask the farmers to delay a treatment as long as possible,
to allow a more accurate evaluation of the incidence and within herd prevalence at different periods in time.
4) Question Johan Venken: How are you going to find so many broiler farms? Because there are already too
many researchers that need field samples. Farmers are fed up.
Instant Remark Herman Deschuytere: Is collaboration of research groups to lower the pressure on farmers not
possible?
Faculteit Diergeneeskunde – Vakgroep Voortplanting, Verloskunde
en Bedrijfsdiergeneeskunde, Salisburylaan 133, B-9820 Merelbeke
Partner F2K - Lid Researchgroep VVZ
www.ROHH.UGent.be
Answer Iris Van Dosselaer: A request to the privacy commission will be send out to obtain the authorization to
use the Sanitel I&R database from the food agency. Based upon this dataset and taken into account the selection
criterion of >10.000 animals per farm, an ad random selection of 50 broiler farmers will be made. These farmers
will receive an email or letter by post and will be contacted one week after sending by phone, to see whether they
are interested to participate. Since the first study is planned during the whole year of 2015 not all farmers can be
contacted immediately. If we notice that the response rate is to low, we can ask veterinary practitioners or
nutrition companies to join us in our quest for motivated farmers. But this will increase the risk of bias which
would result in an inaccurate estimation of the prevalence.
Remark Wouter Wytynck: We can always put an advertisement in the Agricultural Journal to inform
stakeholders about the project.
Answer Jeroen Dewulf: We have ample experience in performing this type of field studies and have always been
able to select sufficient number of herds. At this moment we see that it is easier to get the input from broiler
farmers then from pig farmers. A lot of projects are running in our department either on poultry or pigs and we do
try to collaborate as much as possible. Yet sometimes the selection criteria or the day of sampling are different
and therefore combinations are impossible.
5) Question Jan Van Ginderachter: Do you take the water consumption also in to account?
Answer Iris Van Dosselaer: Not only the consumption but also the way the systems has been build up will be
taken into account.
6) Question Wouter Wytynck: Where do you take the water samples?
Answer Iris Van Dosselaer: The samples will be taken at the source, at the entrance of the stable and at the end
of the line. Preferably the one that is located as far away from the source as possible.
7) Question Jan Van Ginderachter: Do you follow up the end result of the herds that you visit?
Answer Iris Van Dosselaer: In the Cross-Sectional study we will not only focus on the visited flock, but we will
also ask data from 6 to 7 production rounds before. This from the time they have entered the stable until the
slaughter results have come back. During the Follow Up study we can actually monitor much closer since we
follow the herd from the beginning ourselves.
Question, Remarks and Advises about Work Package 3:
Question Kirezi Kanobana: Do you only use the samples that you have gathered in your Cross-Sectional study
to develop the markers.
Answer Filip Van Immerseel, Venessa Eeckhaut, Iris Van Dosselaer: No we will also use data from the
longitudinal study. Ideally we need a marker that is stable and that tells you when it changes that you go towards
a state of dysbacteriosis. This marker should be able to be detected before the dysbacteriosis is present.
Answer Filip Van Immerseel: Suppose that detecting a lot of death cells in fecal material would be a way to
indicate that dysbacteriosis will become present then a good positive control for this would be to test this on
animals suffering from Necrotic Enteritis.
Question, Remarks and Advises about Work Package 4:
Question Jan Van Ginderachter: Will you try out different types of feed, meaning pelleted feed, long or short
pellets, etc… .
Faculteit Diergeneeskunde – Vakgroep Voortplanting, Verloskunde
en Bedrijfsdiergeneeskunde, Salisburylaan 133, B-9820 Merelbeke
Partner F2K - Lid Researchgroep VVZ
www.ROHH.UGent.be
Remark Richard Ducatelle: Would you suggest to establish different types of model?
Answer Filip Van Immerseel, Venessa Eeckhaut, Iris Van Dosselaer: We will evaluate different model options
and search until we find the best model. Possibly this will result in more than one model.
Question, Remarks and Advises about Work Package 5:
Question Kirezi Kanobana: How do plan on informing the primary sector about the project?
Answer Iris Van Dosselaer: Multiple communication routes will be used. We will inform the farmers by
sending out the news letters that we have planned. Also articles in specialized journals will be written. At the end
of the project a demonstration day is planned.
Venessa Eeckhaut
Prof. Ir. Filip Van Immerseel
Iris Van Dosselaer
Prof. Jeroen Dewulf
Faculteit Diergeneeskunde – Vakgroep Voortplanting, Verloskunde
en Bedrijfsdiergeneeskunde, Salisburylaan 133, B-9820 Merelbeke
Partner F2K - Lid Researchgroep VVZ
www.ROHH.UGent.be