Bedeutung von elektrochemisch aktiviertem Wasser als Tränkwasserzusatz und als Interventionsmaßnahme gegen Campylobacter spp. in der
Hähnchenmast
INAUGURAL-DISSERTATION
Zur Erlangung des Grades einer Doktorin der Veterinärmedizin - Doctor medicinae veterinariae –
(Dr. med. vet)
Vorgelegt von Eva-Maria Bügener
Gronau-Epe
Hannover 2014
Institut für Lebensmittelqualität und -sicherheit
1. Gutachter: Univ.-Prof. Dr. Günter Klein
Institut für Lebensmittelqualität und -sicherheit
2. Gutachter: PD Dr. med. vet. Gerhard Glünder Klinik für Geflügel
Tag der mündlichen Prüfung: 28. Oktober 2014
Meiner Familie
„Man merkt nie, was schon getan wurde, man sieht immer nur, was noch zu tun bleibt.“
Marie Curie (1867-1934)
Inhaltsverzeichnis
1 Einleitung ... 1
2 Publikationen ... 4
2.1 Benefits of electrolyzed oxidizing water as drinking water additive for broiler chicken ... 5
2.2 Effect of electrolyzed oxidizing water on reducing Campylobacter spp. in broiler chicken at primary production ... 19
3 Zusammenfassung der Ergebnisse und übergreifende Diskussion ... 35
3.1 Einfluss von ECA-Wasser auf die Tränkwasserqualität im Hähnchenstall... 36
3.2 Einfluss von ECA-Wasser auf Leistungsparameter ... 38
3.3 Einfluss von ECA- Wasser auf die Tiergesundheit ... 39
3.4 Campylobacter ... 40
3.5 Einsatz in der Praxis ... 44
4 Zusammenfassung ... 46
5 Summary ... 48
6 Literaturverzeichnis... 50
7 Danksagung ... 60
Abkürzungsverzeichnis
In dieser Arbeit wurden folgende Kurzformen verwendet:
BMEL Bundesministerium für Ernährung und Landwirtschaft
CFU Colony forming units
E. coli Escherichia coli
ECA-Wasser Elektrochemisch aktiviertes Wasser EO water Electrolyzed oxidizing water
EPS Extrcelluläre polymere Substanzen
log Logarithmus
n Anzahl der Proben
ORP Oxidations-Reduktions-Potential
spp. Spezies
Tab Tabelle
VBNC Viable but non culturable
Teile dieser Arbeit sind bereits veröffentlicht:
BÜGENER, E., M. CASTEEL, A. WILMS-SCHULZE KUMP, G. KLEIN (2014) Effect of electrolyzed oxidizing water on reducing Campylobacter spp. in broiler chicken at primary production
Arch Lebensmittelhyg 65, 4-9
BÜGENER, E., A. WILMS-SCHULZE KUMP, M. CASTEEL, G. KLEIN (2014) Benefits of electrolyzed oxidizing water as drinking water additive for broiler chicken Poult Sci 93, 2320-2326
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eine herausragende Rolle. Die fast ausschließliche Nutzung von Tränkelinien als Transportweg für Impfstoffe, Arzneimittel und Ergänzungsfuttermittel stellt eine Besonderheit dar und verlangt einen hohen Anspruch an Tränkwasserqualität und Leitungshygiene im gesamten Stall. Neben einer guten Ausgangsqualität der Betriebsquelle, empfiehlt es sich somit die Wasserqualität möglichst auch bis zum Ende der Mast zu gewährleisten (KAMPHUES et al. 2007). Dies stellt in der Praxis eine große Herausforderung dar, denn hohe Staubbelastungen, Futter, Speichel, Exkremente und Einstreumaterial lassen die mikrobiologische Qualität des Tränkwassers stark variieren (KAMPHUES u. SCHULZ 2002). Ohne entsprechende Reinigungs- und Desinfektionsmaßnahmen kommt es zur Bildung von Biofilmen im Leitungssystem, die in der Lage sind, die Wirksamkeit von Arzneimitteln zu hemmen (ANWAR et al. 1992; DONLAN u. COSTERTON 2002). Durch die zahlreichen Einflussfaktoren besteht desweiteren die Möglichkeit, dass Tränkwasser zum Vektor für fäkal-oral aufgenommene pathogene Erreger sowie für Erreger mit zoonotischem Potenzial wie z.B. Campylobacter spp. (PEARSON et al. 1993) werden kann. Campylobacter spp.
gehören sowohl in Deutschland als auch in der Europäischen Union zu den am häufigsten vorkommenden Verursachern menschlicher Enteritiden. Frisches Geflügelfleisch ist häufig mit Campylobacter spp. kontaminiert und gilt als Hauptansteckungsquelle für die menschliche Campylobakteriose (EFSA 2012). Die Senkung der Kontamination im Geflügelfleischbereich ist somit dringend erforderlich. Bestrebungen zur Kontrolle des Erregers in der Lebensmittelkette erweisen sich jedoch, vor allem aufgrund von Kreuzkontaminationen während des Schlachtprozesses, bis dato weitestgehend als uneffektiv (HERMANS et al. 2011). Zunehmend gibt es daher Überlegungen, Maßnahmen zur Senkung der Prävalenz von Campylobacter spp. in der Primärproduktion zu etablieren, um das Risiko für den Verbraucher direkt zu Beginn der Produktion zu senken. Somit gilt es neue Interventionsmaßnahmen zu untersuchen, um den Eintrag von Campylobacter spp. in die weiteren Produktionsstufen der Lebensmittelkette in Zukunft zu vermeiden. Bislang sind nur wenige Daten über Interventionsmaßnahmen auf Betriebsebene verfügbar. Die Verwendung von Probiotika (GHAREEB et al. 2012), sowie von Gemischen aus organischen Säuren (JANSEN et al. 2014) oder von Bakteriophagen (KITTLER et al. 2013) sind jedoch
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vielversprechend. Eine weitere Option stellt der Einsatz von elektrochemisch aktiviertem (ECA) Wasser dar. Die desinfizierende Wirkung von ECA-Wasser ist bekannt und konnte sowohl bei klinisch relevanten Bakterien und Pilzen (FENNER et al. 2006) als auch bei lebensmittelhygienisch bedeutenden Erregern (HUANG et al. 2008) schon belegt werden.
Untersuchungen von MORITA et al. (2011) an Mäusen bestätigen desweiteren, dass keine systemischen Auswirkungen auf den Magendarmtrakt zu erwarten sind. ECA-Wasser entsteht durch einen Elektrolysevorgang. Bevor dieser jedoch stattfinden kann, werden dem Wasser der Betriebsquelle im Rahmen einer Umkehrosmose frei gelöste Stoffe (z.B. Salze, Nitrat, Calcit) entzogen. Anschließend beginnt die Elektrolyse einer gesättigten Natriumchloridlösung, welche in ein Zweikammersystem geleitet wird. In der einen Kammer befindet sich eine Anode und in der anderen Kammer eine Kathode. Beide Kammern sind durch eine semipermeable Membran voneinander getrennt. Durch Anlegen einer Gleichstromspannung entstehen zwei unterschiedliche Lösungen in den Kammern (SCHULZ SYSTEMTECHNIK 2010). Auf der Seite der Anode entsteht alkalisches elektrochemisch aktiviertes Wasser mit einem pH-Wert > 11 und einem Oxidations-Reduktions-Potential (ORP) < - 800mV. In der anderen Kammer, welche die Kathode enthält, entsteht saures elektrochemisch aktiviertes Wasser mit einem pH-Wert < 2,7 und einem ORP-Wert von >
1100 mV.
Der Mechanismus der Desinfektion von ECA-Wasser ist abhängig von einem möglichst hohen ORP, denn je höher der Wert, desto höher die Oxidationskraft und die bakterizide Wirkung der Lösung (LIAO et al. 2007). Desweiteren sind die Anwesenheit von hypochloriger Säure, sowie die Konzentration von freiem Chlor ausschlaggebend für den antimikrobiellen Effekt von ECA-Wasser (LEN et al. 2002; PARK et al. 2004). Aufgrund des geringeren Verlustes von freiem Chlor und fehlender Korrosivwirkung auf das Leitungsmaterial gilt neutrales elektrochemisch aktiviertes Wasser als die praktikabelste Variante dieses Verfahrens (LEN et al. 2002; AYEBAH u. HUNG 2005). Zur Herstellung von so genanntem neutralem ECA-Wasser werden je 5-10 % der hergestellten alkalischen Lösung mit der sauren Lösung durch den Generator in einem zusätzlichen Schritt vermischt.
Die entstandene Lösung besitzt einen pH-Wert zwischen 6,2 und 7,5 und ist daher annähernd neutral (SCHULZ SYSTEMTECHNIK 2010).
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PARK et al. (2002) gelang es mit Hilfe von neutralem ECA-Wasser in vitro die Anzahl von Campylobacter spp. auf der Oberfläche von Hähnchenflügeln durch den Kontakt mit ECA- Wasser deutlich zu senken. BODAS et al. (2013) zeigten eine Verbesserung der Wasserqualität nach Einsatz von ECA-Wasser im Tränkwasser von Milchschafen. Ziel dieser Studie war es daher, in einem Feldversuch die Wirkung von neutralem ECA-Wasser als Tränkwasserzusatz auf die Wasserqualität, die Tiergesundheit und die Mastleistung im Hähnchenstall zu ermitteln. Desweiteren wurde der Einfluss auf die Verbreitung von Campylobacter spp. in natürlich kolonisierten Herden, sowie den jeweils zugehörigen Karkassen untersucht.
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Publikation 1: BÜGENER, E., A. WILMS-SCHULZE KUMP, M. CASTEEL, G. KLEIN (2014) Benefits of electrolyzed oxidizing water as drinking water additive for broiler chicken
Poult Sci 93, 2320-2326
Die erste Publikation beinhaltet die Untersuchungen zum Einfluss von elektrochemisch aktiviertem Wasser auf die Tiergesundheit und die Leistungsparameter beim Masthähnchen.
Idee und Konzeption: E.Bügener, Prof. Dr. G. Klein, Dr. A. Wilms-Schulze Kump,
Experimentelle Umsetzung: E. Bügener, Dr. M. Casteel
Auswertung der Ergebnisse: E. Bügener
Verfassen des Manuskriptes: E. Bügener, Dr. M. Casteel, Dr. A. Wilms-Schulze Kump, Prof. Dr. G. Klein
Publikation 2: BÜGENER, E., M. CASTEEL, A. WILMS-SCHULZE KUMP, G. KLEIN (2014) Effect of electrolyzed oxidizing water on reducing Campylobacter spp.
in broiler chicken at primary production Arch Lebensmittelhyg 65, 4-9
Die zweite Publikation behandelt den Einfluss von elektrochemisch aktiviertem Wasser auf die Senkung von Campylobacter spp. von natürlich kolonisierten Masthähnchenherden im Bereich der Primärproduktion.
Idee und Konzeption: E.Bügener, Prof. Dr. G. Klein, Dr. A. Wilms-Schulze Kump,
Experimentelle Umsetzung: E. Bügener, Dr. M. Casteel
Auswertung der Ergebnisse: E. Bügener
Verfassen des Manuskriptes: E. Bügener, Dr. M. Casteel, Dr. A. Wilms-Schulze Kump, Prof. Dr. G. Klein
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2.1 Benefits of electrolyzed oxidizing water as drinking water additive for broiler chicken
Eva-Maria Bügener*†, Andreas Wilms-Schulze Kump†, Maximilian Casteel‡, Günter Klein*
*Institute of Food Quality and Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover; † WEK Veterinary practice, Lohe 13, 49429 Visbek; ‡ WEK Laboratory, Lohe 13, 49429 Visbek
ABSTRACT
In the wake of discussion about the use of drugs in food producing farms it seems to be more and more important to search for alternatives and supportive measures to improve health. In this field trial the influence of electrolyzed oxidizing (EO) water on water quality, drug consumption, mortality and performance parameters like body weight and feed conversion rate, was investigated on 2 broiler farms. At every farm, 3 rearing periods were included in the study. With EO water as water additive, the total viable cell count and the number of Escherichia coli in drinking water samples were reduced compared to the respective control group. The frequency of treatment days was represented by the number of used daily doses per population and showed lower values in the EO-water-treated groups at both farms. Furthermore, the addition of EO water resulted in a lower mortality rate. In terms of analyzed performance parameters, no significant difference could be determined. In this study, the use of EO water improved drinking water quality and seemed to reduce the drug use without showing negative effects on performance parameters and mortality rates.
Keywords: electrolyzed oxidizing water, broiler chicken, water additive
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INTRODUCTION
The use of drinking water in sufficient amounts and in adequate quality plays an important role in maintaining health and performance for food producing animals (KAMPHUES u. SCHULZ 2002). In broiler chicken production, many chicken houses are equipped with closed drinking systems, which in comparison to open systems, provide a better water quality (QUICHIMBO et al. 2013). Nonetheless, even in this kind of drinking system, water is often contaminated with pathogens introduced between the water source and the drinkers (AMARAL 2004). However, water quality should be guaranteed not only before entering the chicken house but also in the whole drinking line system until the last day of grow-out (KAMPHUES et al. 2007). Therefore, water disinfection with different chemical solutions is a usual method to eliminate water as a transmission route for pathogens during the rearing period.
Furthermore, disinfection plays an important role in maintaining the drinking line system. In modern chicken houses vaccination, medical treatment and most special treatments (for example, probiotics or organic acids) are administered as water additives. Safety and efficiency of these applications depend on clean drinking lines which are free from nonpathogen contaminants (dust, feeding residues, and so on) and biofilms.
In recent years, increasing attention has been dedicated to electrolyzed oxidizing (EO) water and its antimicrobial effects (FENNER et al. 2006). In poultry production, studies have been carried out in different parts of the production system. BIALKA et al. (2004) showed the sanitizing effect of EO water when sprayed on eggshells. Furthermore, EO water could be used for sensitive disinfecting measurement of air and surfaces in layer breeding houses (HAO et al. 2013). By using EO water while rearing broiler chicken, drinking lines could be excluded as a vector for campylobacter colonization (MEEMKEN et al. 2014). Even at the end of the production chain, EO water showed its effect on disinfecting carcasses after spraying at the slaughterhouse (NORTHCUTT et al. 2007;
RASSCHAERT et al. 2013). Electrolyzed oxidizing water is generated by electrolysis. Before starting the EO water generator, a reverse osmosis runs. The next step is an electrolysis of a saturated solution of sodium chloride, which is conducted in a chamber containing an anode and a cathode separated by a ceramic membrane. By applying a direct current voltage 2 different solutions are created (SCHULZ SYSTEMTECHNIK 2010). On the negative charged side of the electrolysis chamber, alkaline EO water with pH > 11 and oxidation-reduction potential (ORP) < -800 mV is produced. On the other side, acidic water with pH < 2.7 and ORP > 1,100 mV is generated (HSU 2005). The disinfection mechanism is based on a high ORP, hypochlorous acid and available chlorine concentration (LEN et al. 2000; PARK et al. 2004; LIAO et al. 2007). Due to its better stability, lower loss of free chlorine and less corrosion effects, neutral EO water appears to be the most appropriate variant for the use of an EO water generator (LEN et al. 2002; EZEIKE u. HUNG 2004; AYEBAH u. HUNG 2005). To
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produce neutral EO water, 5 to 10 % of the alkaline EO water has to be mixed with the acidic EO water by a computer-controlled generator. In this way, the neutral EO water solution had a pH of 6.2 to 7.5 and an ORP of 800–1,100 mV (SCHULZ SYSTEMTECHNIK 2010). In light of this background, the primary objective of this study was to evaluate the effect of neutral EO water as a permanently administered water additive on water quality and performance of broiler chickens.
Furthermore, the aim of this study was to investigate its benefit for the production of a poultry meat product obtained with minimal use of medical treatment.
MATERIAL AND METHODS
Rearing farms
As part of a field trial, 2 broiler farms were examined for 3 rearing periods under conventional production conditions. The selection of farms was carried out according to criteria regarding type of chicken house, birds and biosecurity. Chicken houses on each farm were built in the same year and were identical in size. Equipment for water and feed was the same and could be individually operated.
On each farm, 2 chicken houses were included in the study. One of these houses served as a control group, whereas the other one was the test group. Two chicken flocks were examined from the same breeder flock. In both houses, chicken consistently received the same feed. Farm A was rearing for integration A. Chickens were slaughtered at slaughterhouses A and B after batch depletion and at slaughterhouse C after main catching. The farm had 2 identical chicken houses, which were connected via a shared entrance hall. In every chicken house, 40, 000 chickens were kept. There was no other broiler farm within a radius of 3 km. The access road was paved. To gain access to the entrance hall, a hygiene sluice that required a change of clothes and shoes had to be passed through. Flock 1 served as a control group and was watered by an own water-supply well, which was located directly on the farm.
This water was made free from iron by a deferrization unit (Remotector 2000; Remon water treatment, Marum, the Netherlands). Flock 2 received the same iron-free drinking water supplemented by 3 % of neutral electrochemical activated water as a water additive. Farm B was rearing for integration B.
Chicken were slaughtered at slaughterhouses D and E after batch depletion and at slaughterhouse F after main catching. The farm had 2 identical chicken houses, which were connected via a shared anteroom. In every chicken house 35, 000 chickens were kept. To gain access to the entrance hall, a hygiene sluice that required a change of clothes and shoes had to be passed through. There were 3 other broiler farms and 1 turkey farm within a radius of 4 km. The access road was not paved. The area in front of the barn (20 m x 80 m) was concreted. The supply with drinking water in flock 1
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occurred with water from an own water-supply well, which was made free from iron by a deferrization unit (Remotector 2000; Remon water treatment, Marum). Flock 1 served as a control group, and flock 2 received the same iron-free drinking water supplemented with 3 % neutral electrochemically activated water as a water additive.
Production of electrochemical activated water
Neutral EO water was generated using an Agrilyt-Generator (Schulz Systemtechnik GmbH, Visbek, Germany) equipped with a DEA-30 electrolytic cell operating at 24 V DC, 10 A and 30 l /h (Elliod GmbH, Berlin, Germany). The cell was divided into 2 chambers by a ceramic diaphragm for producing an acidic and a basic solution. To produce a salt solution, NaCl was used (8 kg/m3 Agrilyt).
To produce neutral Agrilyt, 5 to 10 % of the full amount of catholyte was mixed with anolyte. The generator consisted of a control cabinet for the electrical component, a control cabinet for the hydraulic components, and a reverse osmosis system. It was installed by the manufacturer and remained in the anteroom throughout the entire duration of the experiment. The device was directly connected to the dispenser at the water supply line, which was normally used for drug administration.
The solution was thus produced on site and was added at a concentration of 3 % directly into the drinking water. The neutral EO water solution had a pH of 6.2 to 7.5 and an ORP of 800 to 1,100 mV.
The amount of residual chlorine was measured using an ExStik CL200 chlorine tester (Extech instruments corporation, USA); pH and ORP were measured using an Exstik PH100 pH meter and ExStik RE300 ORP tester (Extech instruments corporation, USA).
Drinking water samples
Before the start of each rearing period at the anteroom, water samples (1 L) were taken before inflow to the drinking line (n=3).
For 3 rearing periods on day 0, 7, 14, 21, 28 and 35 samples (each 1 L) were taken from the drinking lines in both chicken houses (n= 36). The 1-L sample was a pooled sample of each 250 mL which were taken on the same day from 4 different drinking lines. The pH and ORP levels were measured.
Furthermore, the total viable cell (TVC) count and Escherichia coli counts were determined by transferring the water sample into a sterile 100-mL MircroFunnel (Pall Life Sciences, Dreieich, Germany) using membrane filters made of mixed cellulose esters with a pore size of 0.45 µm (GN-6 Metricel, PALL Life Sciences). The MicroFunnel was placed on the aluminium manifold (Pall Life Sciences) and a peristaltic pump drew the whole water sample (1 L) through an integrated system.
Subsequently, water samples were analyzed according to ISO 6222 and ISO 9308-1. After performing
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a serial dilution, all detected coliform bacteria were tested on their tryptophanase activity.
Tryptophanase-positive coliforms were recognized as E. coli.
The assessment of microbiological drinking water quality based on the German orientation guidelines for assessing hygienic quality of drinking water given to food producing animals (GERMAN MINISTRY OF FOOD AGRICULTURE AND CONSUMERS’ PROTECTION 2007; KAMPHUES et al. 2007). In these guidelines the quality of drinking water is specified by the absence of microbiological contaminants. To comply with these requirements drinking water should be free of Salmonella spp, Campylobacter spp. and E. coli ( in 100 mL). The total viable cells counts should not exceed 1,000 cfu/ mL at 37 °C and 10,000 cfu/mL at 20 °C. The determination of cfu at 22 °C is used to detect autochthonous water organisms. At 37 °C, organisms that could be facultative pathogens for birds are aimed to be isolated.
Zootechnical parameters
The measurement of feed consumption was made by a feed weigher installed in the chicken house.
The feed consumption for every single compartment was determined. During the whole rearing period, mortality rate and BW were documented daily. The BW was daily evaluated by a computer-controlled weigher in each chicken house, which was able to calculate the average BW of the whole flock by the number of weighing per day. The feed conversion rate (FCR) was calculated for the whole duration of every rearing period.
Medical treatment
The evaluation of drug use was based on the evaluation method of the German antibiotic monitoring system, which is in force from April 2014. For every group on both farms, the number of used daily doses per population (nUDDPopulation), which represents the frequency of treatment days with the average number of active agents, was calculated. It is based on the number of used daily doses (nUDD).
Statistical analyses
Analyses were carried out with SAS 9.3 (Statistical Analysis System, Cary, USA). The significance level was set at P = 0.05. Drinking water samples from the control and test group were analyzed using a 2-factorial ANOVA for showing differences of TVC and E. coli counts. One-half of the detection limit was used for statistical analyses in water sample in which E. coli could be not detected. In this
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way, the biological variability could be considered. A 2-factorial ANOVA was even used to compare the means of BW, FCR and mortality rate of control and treated groups.
RESULTS
On farm A in the control groups, the mean water pH was 6.92 and the mean ORP level was 489 mV for 18 water samples from the drinking lines within 3 rearing periods. In treated groups on farm A, a mean pH of 6.73 and a mean ORP level of 803 mV were detected for 18 water samples. On farm B, a mean pH of 7.9 and a mean ORP of 631 mV for 18 water samples were measured from drinking lines within 3 rearing periods. In treated groups on this farm, the mean pH was 7.5 and ORP value increased by 814 mV on average for 18 water samples. Values of ORP were considerably higher in EO-water- treated groups on both farms. In comparison with the control group, Table 1 shows a reduction of TVC in drinking water of the treated groups on both farms. From day 21 on both farms, the EO water treatment led to reduced TVC counts (P < 0.05). Furthermore, the counts of E. coli in drinking water samples were reduced (P < 0.05; Table 1).
Table 1. Total viable cells (TVC; log10 cfu/mL) detected at an incubation temperature of 37°C for detecting facultative pathogens and Escherichia coli counts in drinking water samples1,2
TVC counts (36°C) E.coli counts
Day Control EO water
SD P-value Control EO water
SD P-value
Farm A 0 1.37 0.32 0.65 0.184 n.d. n.d. - -
7 4.43 2.48 1.11 0.163 1.32 n.d. 0.28 0.019
14 4.25 2.17 0.70 0.068 1.35 n.d. 0.33 0.034
21 4.73 1.21 0.21 0.002 2.05 n.d. 0.05 0.001
28 4.60 1.75 0.53 0.023 1.75 n.d. 0.16 0.004
35 5.13 2.14 0.41 0.012 1.73 n.d. 0.37 0.030
Farm B 0 3.23 1.55 1.20 0.229 1.65 n.d. 0.44 0.032
7 3.48 1.75 0.90 0.143 1.93 n.d. 0.13 0.002
14 4.96 1.10 1.40 0.078 2.34 0.54 0.39 0.030
21 4.60 2.29 0.19 0.004 2.13 0.39 0.45 0.042
28 4.56 1.98 1.26 0.129 0.91 n.d. 0.75 0.069
35 4.33 2.45 0.16 0.005 2.48 n.d. 0.72 0.042
1detection limit: 1 organism per 100 ml
2 According to each day of sampling, water samples of three rearing periods (n = 3) were log-transformed and represented as means with standard deviation.
n.d.: not detected
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Body weight and feed conversion rate are shown in Table 2. Due to variable slaughter dates on both farms (36 to 40 d) the longest common grow-out period (36 d) was chosen as the final weight to obtain reasonably comparable values. Feed conversion was adjusted accordingly. Concerning BW, a trend toward a positive influence of EO water on the treated group was observed at d 30. At d 36, a lower BW in the treated group was observed in 3 rearing cycles of the whole experiment.
Table 2.Means of BW and feed conversion ratio (FCR) after batch depletion (d 30) and after main catching (d 36) of 2 farms with 3 rearing periods each
1Bodyweight on average of 40 000 broiler chicken at Farm A and 35 000 broiler chicken at Farm B EO = electrolyzed oxidizing
Figure 1 shows the mortality rate of birds on both farms. There were just 2 periods that showed higher mortality rates on farm B. In these cases the reason was due to the insufficient quality of the chicks on arrival or the day-old chicks were exposed to intensive stress in transit because of failure in the ventilation system.
The calculated therapy frequency is shown in Table 3. Within 3 rearing periods on farm A, the control group had to be treated 3 times. In the first rearing period at d 24 a lot of chickens showed lameness, joint inflammation and femoral head necrosis in the control stable. In the following rearing period at d 32 lameness and many cases of femoral head necrosis had to be treated in both the control and treated group. In the last rearing period at d 25, similar problems occurred again in the control group. Thus, the EO water-treated group required 2 treatments less than the control group. The mean calculated therapy frequency showed a reduction of 1.99 points on farm A.
Rearing period
Farm A Farm B
I II III SD P-
Value I II III SD P-
Value Bodyweight 30 days1, g
10.80 0.607 36.29 0.843
Control 1573 1531 1623 1539 1451 1491
EO water 1581 1509 1649 1475 1486 1500
Bodyweight 36 days1, g
18.90 0.393 18.75 0.616
Control 2082 2049 2077 1972 1942 1994
EO water 2040 2041 2097 1982 1977 1976
FCR2,
kg of feed/kg of gain
0.01 0.270 0.01 1.000
Control 1,64 1,63 1,64 1,60 1,61 1,61
EO water 1,61 1,62 1,64 1,61 1,62 1,59
12 In the first rearing period on farm B, the control group had to be treated because of lameness, joint inflammationand femoral head necrosis at d 19. After batch depletion, watery, frothy diarrhea and wet litter developed a high degree of dysbacteriosis. In this case, both groups had to be treated. The following rearing period started with transport-stressed day-old chicks that had great difficulties in achieving sufficient mobility for starting food and water intake after reaching the chicken house.
Therefore, from d 2 onward, higher mortality and infection of the yolk sacs caused by E.coli followed. To prevent excessive mortality, chicks in the control and treated group received antibiotic treatment. After batch depletion (at d 30), an infection with Ornithobacterium rhinotracheale was detected by PCR after significant clinical respiratory symptoms. Both groups had to be treated. The final rearing period on this farm again started with an E. coli yolk sac infection that became evident at d 2.
The control group received antibiotic
treatment. In 3 rearing periods on farm B, five antibiotic treatments were used in the control group.
The test group with EO water was treated 3 times. Subsequently, the therapeutic frequency in the test group was 3.4 points lower.
Table 3. Average frequency (d) of antibiotic therapy number of used daily doses per population 1
Farm A Farm B
Control 3.66 8
EO water 1.67 4.6
1Values are the means of three rearing periods.
Figure 1. Cummulative mortality rate
0 0,5 1 1,5 2 2,5 3
0 7 14 21 28 35
Cmulative mortality rate, %
Day of rearing
Farm A
0 0,5 1 1,5 2 2,5 3
0 7 14 21 28 35
Cumulative mortality rate, %
Day of rearing
Farm B
Control group EO group
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DISCUSSION
Analyses of the effects of EO water have been carried out mainly with regard to the disinfecting effect.
Thus, PARK et al. (2008) showed the improvement of a higher quality of vegetables by reducing surface bacterial loads. Furthermore, EO water was used to disinfect a variety of other products and surfaces in the food industry (AYEBAH u. HUNG 2005; HUANG et al. 2008) as well as in animal production (JIROTKOVÁ et al. 2012; BODAS et al. 2013). A particular advantage is its biodegradation and harmlessness to health (MORITA et al. 2011).The disinfecting effect of EO water and its resulting improvement of water quality were confirmed in this study. A good water quality and adequate amounts of water were considered prerequisites for the health and performance of food- producing animals (KAMPHUES u. SCHULZ 2002). Drinking water of food-producing animals should be free from E.coli (in 100 mL) and the TVC counts should be < 1,000 CFU/ml at 37°C and not more than 10,000 CFU/mL at 20 °C (GERMAN MINISTRY OF FOOD AGRICULTURE AND CONSUMERS’ PROTECTION 2007).
However, in this field experiment no continuous reduction of TVC counts could be shown during the respective rearing period (Table 1). This is related to the reentry of microorganisms from the environment of the birds. Furthermore, because of a low flow rate in water pipes and high temperatures at the beginning of the rearing period, in many cases the formation of a biofilm occurred during rearing. The organisms in biofilms form matrices which are irreversibly associated with a surface and enclosed in extracellular polymeric substances (DONLAN 2002; HALL-STOODLEY et al. 2004). Protection by the matrix and a slow uptake of antimicrobial agents are the supposed reasons for a higher resistance of bacteria in biofilms (ANWAR et al. 1992; BROWN u. GILBERT 1993;
DONLAN u. COSTERTON 2002). Most of the bacteria in a drinking water system are located at the surface of biofilms, whereas a small fraction is found in the water phase. Only this part is considered when sampling as commonly performed for routine quality control (FLEMMING et al. 2002).
The TVC counts and the counts of E. coli showed a decrease compared to the respective control group (Table 1). These results were consistent with studies of BODAS et al. (2013) that showed lower counts of TVC in drinking water of dairy ewes in a test period of 25 d. Studies of ZENG et al. (2010) on the disinfection mechanism of EO water revealed leakages of DNA and damage to membrane and nucleus as well as a decrease of dehydrogenase activities of E. coli and Staphylococcus aureus which resulted in inhibition to the respiration and anabolism.
However, not only the benefits of EO water on drinking water quality but also the health status of broiler chickens were investigated in this field experiment. Nevertheless, to make statements about the effects of EO water on the health status of chicken, it is necessary to consider the entire herd. To evaluate the herd health status, different parameters such as BW, FCR and mortality could be used.
14
Alongside mortality, DICKHAUS (2010) mentioned the use of drugs as a direct health indicator. The reduced use of antimicrobial substances contributes to better animal health (MEEMKEN et al. 2014).
In the present study performance-oriented data (BW, FCR, mortality) as well as drug use were examined. Even after 6 rearing periods no significant differences in the average BW and the FCR could be determined. Similar observations in poultry were only made in the study of BERK et al.
(2005), which was not a field trial. Therefore, it should be noted that the comparability is limited due to the housing conditions in experimental cages. The mortality rate was investigated after the first week and after main catching. Thus, lack of chick quality and frequently occurring young chick diseases could be considered separately from the rest of the rearing period. It might be assumed that EO water treated chicks may have a lower load of E.coli which resulted in a lower mortality rate.
Nonetheless, this had to be a hypothesis because the load of E.coli in chicks after leaving the hatchery was not measured and the results were not significant (P > 0.05). In contrast to results of this study, FASENKO et al. (2009) showed a significantly lower cumulative mortality for the first 14 d of rearing. However, it should be pointed out that FASENKO et al. (2009) sprayed EO water on eggs in the hatchery. Therefore, the bacterial load of eggs decreased already before hatching. Perhaps young chick diseases like yolk sac inflammations caused by E.coli are not clinically apparent. Adding EO water to the drinking water does not seem to affect existing infections but is obviously able to prevent new infections.
This prevention effect is also reflected by the drug use in this study. During the rearing periods, the influence of EO water on bird health was revealed through the reduced nUDDPopulation in comparison with the control group. The nUDDPopulation specifies the number of days that an animal of a population was treated with a single active agent on average during the rearing period (MERLE et al. 2012). The number of treatments with active agents in a rearing period represents in some way the number of diseases in the population and is therefore suitable for describing the animal health of a population (VAN RENNINGS et al. 2013). Against the backdrop of increasing evidence of resistance to antibiotics even in commensals (LEUSCHNER et al. 2010), the number of antibiotic treatments is a critical factor. It is the policy of the European Commission to prevent antibiotic resistances, which are considered to be one of the major emerging threats to human health (EUROPEAN COMMISSION 2011).
Each treatment of a specific disease also leads to the selection of the normal accompanying bacterial flora and promotes resistant organisms. The nUDDPopulation was used for quantification of antibiotic treatments in the 16th revision of the medicinal law in Germany which comes into force in 2014. Such a collection of data on used antimicrobial agents is even demanded by the European Union, which
15
seeks for a standardized solution of documentation on antibiotic agents (EUROPEAN MEDICINES AGENCY 2012).
Thus, many environmental influences in the husbandry of broiler chickens can have a large effect on health and welfare (ESTEVEZ 2007). Nevertheless, the use of EO water as a supplementary measure can be considered to permanently achieve a better drinking water quality and had no negative effects on animal health and performance. Furthermore, the use of EO water contributes to current European Union requirements regarding the reduction of using antibiotic substances.
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2.2 Effect of electrolyzed oxidizing water on reducing Campylobacter spp. in broiler chicken at primary production
1 WEK Veterinary Practice, Visbek, Germany
2 WEK Laboratory, Visbek, Germany
3 Institute for Food Quality and Safety, Faculty of Veterinary Medicine Hannover, Germany
Einfluss von elektrochemisch aktiviertem Wasser auf das Vorkommen von Campylobacter spp. bei Masthähnchen in der Primärproduktion
Eva-Maria Bügener1,3, Maximilian Casteel2, Andreas Wilms-Schulze Kump1, Günter Klein3
Abstract
In addition to numerous methods of controlling Campylobacter spp. during slaughter and processing, there are efforts to develop reduction strategies in primary production. In the present study drinking water was treated with a 3 % solution of neutral electrolyzed oxidizing water as water additive in two naturally Campylobacter colonized farms. The experiment was performed from day zero until main catching. On each farm three rearing periods were included in the study. Carcasses were examined for contamination after batch depletion and after main catching. In none of the treated water samples Campylobacter spp. could be detected. Drinking water samples in all control groups were positive on day 35 of the rearing period. In one case Campylobacter spp. could be detected in the water sample of a control group already at day 28. After main catching significant lower numbers of Campylobacter spp. were isolated from the carcasses in the test group at both farms. The permanent addition of electrochemically activated water seems to be an opportunity to reduce the carriage of Campylobacter spp. in poultry drinking water and appears to affect counts on carcasses.
Keywords: Electrolyzed oxidizing water, Campylobacter, broiler chicken, drinking water, carcasses
20 Zusammenfassung
Neben zahlreichen Verfahren zur Bekämpfung von Campylobacter spp. während der Schlachtung und Verarbeitung gibt es mehr und mehr Bemühungen, Strategien zur Senkung des Erregervorkommens in der Primärproduktion zu entwickeln. In diesem Feldversuch wurde in zwei Hähnchenmastbetrieben, bei jeweils auf natürliche Weise mit Campylobacter spp. kolonisierten Herden, Tränkwasser mit einer 3% igen Lösung von neutralem, elektrochemisch aktiviertem Wasser als Tränkwasserzusatz eingesetzt. Die Behandlung erfolgte von Tag Null der Mastperiode bis zum Hauptfang. Als Kontrollgruppe diente eine unter gleichen Bedingungen gemästete Herde gleicher Herkunft. In jedem Betrieb wurden 3 Mastdurchgänge in die Studie einbezogen. Nach dem Vorfang sowie nach dem Hauptfang wurden ganze Schlachtkörper hinsichtlich einer Kontamination des Endproduktes untersucht.
In keiner der Wasserproben der Versuchsgruppen wurden Campylobacter spp. nachgewiesen.
Die Tränkwasserproben in allen Kontrollgruppen waren am Tag 35 der Aufzucht positiv. In einem Fall wurden Campylobacter spp. bereits am 28. Masttag im Tränkwasser der Kontrollgruppe nachgewiesen. Nach dem Hauptfang konnte die Anzahl der Campylobacter spp. auf den Karkassen der Versuchsgruppe beider Betriebe signifikant gesenkt werden.
Somit scheint die permanente Zugabe von elektrochemisch aktiviertem Wasser zum Tränkwasser eine Möglichkeit darzustellen, die Campylobacterlast im Tränkwasser und auf Karkassen von Masthähnchen zu reduzieren.
Schlüsselwörter: Elektrochemisch aktiviertes Wasser, Campylobacter, Masthähnchen, Tränkwasser, Karkassen
Introduction
Poultry seems to be the main source of human infection with Campylobacter spp.
(HERMANS et al. 2012b). In 2010 more than 210 000 humans in the European Union were infected. This number is rising since 2005. In most cases the infection of humans took place through contact of fecal contaminated fresh broiler meat (EFSA 2012). An important measure for consumers would be a more intensive compliance with the handling of poultry meat. Due to the sharp increase in human campylobacteriosis there are other measures needed to reduce the prevalence of Campylobacter spp. in meat processing and at farm level (KLEIN
21
2010). In primary production strict biosecurity measures should minimize the entry sources.
This does not seem to suffice. (HERMANS et al. 2011). Some studies deal with the use of non-biosecurity measures. The use of probiotics (GHAREEB et al. 2012) or organic acids (JANSEN 2012) in drinking water of chicken seem to be effective to stop or reduce the entry and colonization of Campylobacter. Electrolyzed oxidizing (EO) water as water additive might be promising because investigations for reduction of Campylobacter on eggshells, wash water of carcasses and in poultry processing have shown a bactericidal effect of EO water (PARK et al. 2002; KIM et al. 2005; FASENKO et al. 2009). EO water is a nontoxic sanitizer (RUSSELL 2003). The method is based upon an electrolytic process which takes place in a special made generator (Fig.1). Before the generator starts, a reverse osmosis process for water softening is conducted. To this softened water sodium chloride (NaCl) is added. The solution passes through an electrochemical cell containing an anode and a cathode. The two poles are separated by a ceramic diaphragm. By applying a direct current voltage, two different solutions can be obtained. From the cathode side, a solution with pH 10 – 11.5 and an oxidation-reduction potential (ORP) between -800 and -900 mV is produced. From the anode side, acidic EO water with pH between 2.3 and 2.7 and a high ORP (> 1000 mV) is generated (HSU 2005). The chlorine concentration depends on the amperage applied.
Increasing voltage and NaCl concentration results in a lower pH, higher ORP and higher residual chlorine of the acidic EO water, increasing the electrolyte flow rate causes a reversal of these trends because of shorter residence time in the electrolytic cell (EZEIKE u. HUNG 2004). Hypochlorous acid (LEN et al. 2000) and the ORP (LIAO et al. 2007) seem to be the main active components and closely related to the bactericidal effect of EO water. Numerous studies demonstrate that the acidic EO water is a promising possibility for disinfection of surfaces and objects in the food production (HRICOVA et al. 2008). It provides an opportunity for decontamination of eggshells in the hatchery, lettuce, spinach, cattle hides as well as fresh pork (BIALKA et al. 2004; FABRIZIO u. CUTTER 2004; BOSILEVAC et al.
2005; PARK et al. 2008) and shows its effectiveness at spraying carcasses in poultry processing (NORTHCUTT et al. 2007). The disadvantage of using acidic EO water is especially the corrosive effect on processing equipment.
The use of neutral EO water does not cause these disadvantages (EZEIKE u. HUNG 2004;
AYEBAH u. HUNG 2005) and the solution is more stable than the acid variant because
22
chlorine loss is significantly reduced at pH 6-9 (LEN et al. 2002). DEZA et al. (2003) showed that washing tomatoes inoculated with E. coli, Salmonella enteritidis and L. monocytogenes with neutral EO water reduced the bacterial number without prejudice for the surface of tomatoes and without sensory deviations. A bactericidal effect could also be achieved in the cleaning of kitchen cutting boards (DEZA et al. 2007). Some other studies have been conducted in evaluating the effects of EO water in animal production systems. Using neutral EO water as spray in the air of a layer breeding house resulted in dust retention, lower temperatures and lower mortality of chickens near the EO spray device (ZHENG et al. 2012).
Furthermore JIROTKOVÁ et al. (2012) showed that neutral EO water has no negative effects regarding color, pH and loss of water on poultry carcasses after adding it to drinking water in chicken houses while fattening. Until now there have been no studies conducted to determine the influence of EO water for reduction of Campylobacter across different production levels.
The aim of this study was therefore to investigate the influence of electrolyzed oxidizing water as water additive, on the occurrence of Campylobacter in broiler farms and its effects on the carcasses.
Figure 1: Schematic diagram of a generator for the production of electrochemical activated water
23 Material and Methods
Rearing farms
As part of a field trial, two broiler farms were examined for three rearing periods under conventional production conditions. The selection of farms was carried out according to criteria in consideration of type of chicken house, animals and biosecurity. Chicken houses on each farm were built in the same year and identical in size. Equipment for water and feed was the same and could be individually operated. Two chicken flocks were examined from the same breeder flock. In both houses, chicken got consistently the same feed. Another criterion was natural colonization of Campylobacter before the experiment, which was confirmed by the Status Quo investigations. These investigations took place in the period before starting the experiment to the same extent as in the test series. Farm A was rearing for integration A.
Chicken were slaughtered at slaughterhouse A and B after batch depletion and at slaughterhouse C after main catching. The farm had two identical stables, which were connected via a shared entrance hall. Within a radius of 3 km there was no other broiler farm.
The access road was paved. Both houses were stabled with 40 000 chicken. For gaining access to the entrance hall a hygiene system which required a change of clothes and shoes had to be passed though. The supply with drinking water in flock 1 occurred with water from an own well, which was made free from iron by a deferrization unit (Remotector 2000; Remon water treatment, Marum, Netherlands). Flock 2 got drinking water supplemented by 3 % of neutral electrochemical activated water as water additive. Farm B was rearing for integration B. Chicken were slaughtered at slaughterhouse D and E after batch depletion and at slaughterhouse F after main catching. The farm had two identical stables, which were connected via a shared anteroom. There were three other broiler farms and one turkey farm within a radius of 4 km. The access road was not paved. The area in front of the barn (20 m x 80 m) was concreted. Both houses were stabled with 35 000 chickens. The supply with drinking water occurred with water from an own well, which was made free from iron by a deferrization unit (Remotector 2000; Remon water treatment, Marum). Flock 1 served as a control group and flock 2 got drinking water supplemented with 3 % neutral electrochemically activated water as water additive.
24 Production of electrochemical activated water
Neutral EO water was generated using a Agrilyt-Generator (Schulz Systemtechnik GmbH, Visbek, Germany) equipped with a DEA-30 electrolytic cell operating at 24 V DC, 10 A and 30 l /h (Elliod GmbH, Berlin, Germany). The cell was divided in two chambers by a ceramic diaphragm for producing an acidic and a basic solution. To produce a salt solution NaCl was used (8 kg/m3 Agrilyt). In order to produce neutral Agrilyt, five to ten percent of the full amount of Catholyte was mixed with Anolyte. The generator consisted of a control cabinet for the electrical component, a control cabinet for the hydraulic components and a reverse osmosis system. It was installed by the manufacturer, and remained in the anteroom throughout the entire duration of the experiment. The device was directly connected to the dispenser at the water supply line, which is normally used for drug administration. The solution was thus produced on site and was added at a concentration of three percent directly into the drinking water. The neutral EO water solution had a pH of 6.2 to 7.5 and an ORP of 800–1100 mV. The amount of residual chlorine was measured using ExStik CL200 chlorine tester (Extech instruments corporation, USA), pH and ORP were measured using an Exstik PH100 pH meter and ExStik RE300 ORP tester (Extech instruments corporation, USA).
Presence of Campylobacter spp.
To acknowledge the presence of Campylobacter spp. in each group in every rearing period water samples before influent to the drinking line (n= 1), sock swabs (n= 2), cloacal swabs at day 25 (n=15) and cloacal swabs at day 35 (n= 15) were taken. The samples were examined for thermophilic Campylobacter qualitatively according to ISO 10272-1:2002. Before the start of each rearing period water samples (1 liter) were taken before influent to the drinking line. The water was examined for Campylobacter by transferring the water sample in sterile 100 ml MicroFunnel (PAL Life Science) using membrane filters made of mixed cellulose esters with a pore size of 0.45 µm (GN-6 Metricel, PALL Life Sciences). The MicroFunnel was placed on the aluminium manifold (PAL Life Science) and a peristaltic pump drew the whole water sample (1 liter) through an integrated system. Subsequently the membrane filter was cultured in 90 ml of Bolton broth (CM 0983, supplement SR 0183 and SR 048, Oxoid).
The broth was incubated for 48 h ± 2h at 42°C ± 0,5 °C under microaerophilic conditions (5
% O2, 10 % CO2, 85 % N2) in gas jars (Campygen 2.5 L; Oxoid). After enrichment, 10 μl of
