Science & Solutions #4 Poultry

Issue 04 • January 2014 • Poultry A magazine of

Photo: Sebastian Kaulitzki

Zero Salmonella in 2014?

How organic acids can help you achieve your goal

Gut integrity for performance

Mycotoxins and poultry feed

Learn all about the important functions of the gut barrier complex in poultry

Which mycotoxins are the main culprits of reduced poultry performance?

Editorial Feed safety ďŹ rst Salmonella and mycotoxins are among the most common hazards in animal nutrition and tackling the dangers they pose is an ongoing effort. To date, science has discovered up to 400 mycotoxins in contaminated animal feedstuffs. Although their concentrations rarely exceed legal limits, the real concern lies in their co-occurrences and synergistic effects that harm the immune system and impair gut integrity, leading to growth retardation and performance losses.

Since the BIOMIN Mycotoxin Survey Program began in 2004, 24,000 samples have been analysed to provide our stakeholders and customers with information on the mycotoxin risk potential in different regions worldwide. Although mycotoxins are detected in about 80% of animal feeds, transmission to humans via animal products is rare, except in a few cases of aflatoxins carried over in milk.

In contrast, Salmonella in feed materials pose an even greater risk to humans than animals. Although some Salmonella strains directly impair poultry health, their main danger lies in the threat to human health through their transmission via poultry meat or eggs. In the EU, around 100,000 cases of this zoonotic disease are reported every year, and over 40,000 are reported in the US, although the actual number of infections may be at least thirty times greater.

At the gut level, Salmonella and mycotoxins interact – the latter impacts gut integrity and opens the gate to the invasion of pathogens, like Salmonella. From a research perspective, it is therefore very important to combine different strategies to manage these risks, such as conducting surveillance programmes on mycotoxins, developing new strategies for counteracting mycotoxins and Salmonella, and finding approaches to improve gut barrier function and gut health in animals.

Gerd Schatzmayr PhD Research Director

Science & Solutions • January 2014

Contents

Name, title position

Is zero Salmonella possible?

Minimise the risk of infections with the proven antimicrobial efficacy of organic acids.

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By Renata Breitsma, PhD

Gut integrity—the backbone of performance

The barrier function Besides its role in digestion and nutrient uptake, the gut also acts as the barrier that is responsible for a range of important immuno­ logical, physiological and physical defences in poultry.

By Raj Murugesan, DVM, PhD

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Mycotoxin update 2013

Which mycotoxins should we expect in poultry feed?

The latest BIOMIN Mycotoxin Survey reveals the most prevalent mycotoxins by region, and their potential effects on poultry health.

By Paula Kovalsky, PhD

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Science & Solutions is a monthly publication of BIOMIN Holding GmbH, distributed free-of-charge to our customers and partners. Each issue of Science & Solutions presents topics on the most current scientific insights in animal nutrition and health with a focus on one species (poultry, swine or ruminant) every quarter.

ISSN: 2309-5954 For a digital copy and details, visit: http://magazine.biomin.net For article reprints or to subscribe to Science & Solutions, please contact us: magazine@biomin.net Editor: Daphne Tan Contributors: Renata Breitsma, Paula Kovalsky, Raj Murugesan, Gerd Schatzmayr Marketing: Herbert Kneissl, Cristian Ilea Graphics: Reinhold Gallbrunner, Michaela Hössinger Research: Franz Waxenecker, Ursula Hofstetter, Mickaël Rouault Publisher: BIOMIN Holding GmbH

Industriestrasse 21, 3130 Herzogenburg, Austria

Tel: +43 2782 8030 www.biomin.net Printed in Austria by: Johann Sandler GesmbH & Co KG Printed on eco-friendly paper: Austrian Ecolabel (Österreichisches Umweltzeichen) ©Copyright 2014, BIOMIN Holding GmbH All rights reserved. No part of this publication may be reproduced in any material form for commercial purposes without the written permission of the copyright holder except in accordance with the provisions of the Copyright, Designs and Patents Act 1998.

All photos herein are the property of BIOMIN Holding GmbH or used with license.

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Even as safety standards in global food supply are improving, Salmonella infections continue to inflict huge losses in terms of productivity on farms, recalls from supermarkets and medical costs for patients.

Minimising Salmonella should thus be a priority at every poultry farm.

B

n o Salm

acteria of the genus Salmonella are Gram-negative, facultative anaerobic bacteria belonging to the family Enterobacteriaceae. In poultry, two serotypes, Salmonella Gallinarum and Salmonella Pullorum, are known to produce clinical diseases such as fowl typhoid and pullorum disease. These two strains are the main cause of high mortality rates in poultry flocks that lead to substantial losses for farm owners. The export of contaminated breeding flock is suspended until the flock becomes free of Salmonella while the eradication of Salmonella is costly and may even lead to the closure of poultry units.

Many other serotypes such as Salmonella Typhi­ murium and Salmonella Enteritidis may infect internal organs, followed by contamination of poultry products such as meat and eggs. Food poisoning with

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Salmonella infected products becomes a true hazard to human health.

Poultry are usually infected through contaminated feed, infected breeder flocks, lack of effective biosecurity on farms, inadequate hygiene control during harvesting, transporting of broiler chickens and cross-contamination of carcasses during slaughter and processing. Different stress factors can reduce the efficacy of immune systems, creating favourable conditions for the bacteria to work further damage.

It is very hard to keep Salmonella under control because it is a faecal-oral infection and birds can excrete bacteria via their faeces for several months without showing any symptoms of the disease. Salmonella strains are capable of surviving for six or more years in the environment and biosecurity is therefore a major factor towards reducing the risk of Salmonella in poultry farms.

Science & Solutions • January 2014

Photo: khemporn tongphay

I s ze r

a l l e

Renata Breitsma

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Director of Competence Center, Acids

os

Organic acids and their antimicrobial efficacy The addition of organic acids has been shown to contribute to environmental and feed hygiene as well as a healthier gastrointestinal tract (GIT).

In their non-dissociated form, orga­nic acids can penetrate the bacterial cell wall and disrupt the metabolic processes of bacteria by altering their internal pH. This forces bacterial­cells to expend considerable resources in bringing their internal pH back to a normal, metabolically optimal level.

The effort made to rebalance internal cellular pH is an energy-draining process which can eventually stop the growth of the bacteria or even kill it. The anion part of the acid is trapped inside the bacteria because it does not diffuse freely through the cell wall. The accumulation of anions becomes toxic for the bacteria, interrupting enzymes and DNA synthesis.

Combining different organic acids and using acid blends instead of singular organic acids has become increasingly common over the last few decades. This is because using combinations of organic acids instead of single acids broadens their spectrum of activity. It has been shown that the antimicrobial effect of a blend of formic and propionic acids against Salmonella and E. coli (Biotronic® SE /forte) increased by up to 24% compared to the efficacy of individual acids.

In addition, there is a growing body of evidence that some essential oils or their phytochemical constituents as well as permeabilizing substances can act synergistically with organic acids. The permeabilizers generally do not have a bactericidal effect but are able to weaken the outer membrane of Gram-negative bacteria and facilitate the action of other antimicrobials against the bacteria.

However, different permeabilizers act differently with various antimicrobial substances. When combining antimicrobial substances and permeabilizers, it is of great importance that they interact in a manner that increases the permeability of a pathogen’s cell membrane. Such a

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synergistic effect has been confirmed in Biotronic® Top3, a product where the inclusion of a permeabilizing substance, Biomin® Permeabilizing Complex, increased the antimicrobial effect of a blend of organic acids and the phytochemical, cinnamaldehyde.

Raw materials and compound feed Contaminated feed is a known source of Salmonella infections and a potential route of disease transmission to animal and human populations. Heat treatment, usually during conditioning, pelleting or extrusion has been shown to be an effective way of reducing microbial loads in feed; however, absolute “feed sterility” cannot be achieved.

Moreover, these methods do not prevent feed recontamination, which is a true hazard in the feed production chain, especially in the finished product sector, cooling area for pellets, bulk transport to the farm and storage of the feed in the farm silos.

A multitude of steps must be taken to secure the hygienic status of feed. These steps include thermal treatment and the addition of compounds to control microorganisms in feed. Constant treatment with organic acids has a residual protective effect in feed. It helps to reduce the risk of feed recontamination and minimise the contamination of milling and feeding equipment.

The antibacterial efficacy of Biotro­nic® products in artificially contaminated feed was evaluated in a crop assay. The feed was contaminated with a high level of Salmonella Enteritidis and diluted with physiological saline solution. The cell count of Salmonella was determined after the contamination at 0, 1 and 2.5 hours of incubation under optimal conditions. Results showed that Salmonella replication grew steadily under optimal conditions while Salmonella counts declined in the group containing the acidifier (Figure 1).

The use of organic acids and their mixtures is viewed with caution by many feed mills. The corrosive nature of organic acids can lead to machinery damage, particular when a high proportion of the feeds are treated. The

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Is zero Salmonella possible?

The points of Salmonella control Hatchery and breeder houses

Feed raw materials and compound feed

Water

Farm environment

Table 1. Quantitative S. Eteritidis detection 5 and 10 days post­infection in the cecum Groups

Cecal samples log cfu Salmonella/g digesta 5 days post infection*

10 days post infection

2.25a

2.63a

0.87b

1.29b

Control Biotronic® Top3 1 kg/t of feed 105

*Infection level: colony forming units (cfu) S. Enteritidis; a,b significant difference P<0.05.

Biotronic® product line consists of buffered organic acids with the suppressed ability to corrode metal surfaces and is therefore suitable for use in feed mills.

Controlling Salmonella Some Salmonella serovars, e.g. Salmonella Enteritidis harbour innate properties to infect the poultry reproductive tract. The passage of an egg through the contaminated cloaca or in a Salmonella infected environment can cause outer egg shell contamination. Internal egg contamination is caused by shell penetration through cracks or by colonization of the reproductive tract and thus bacteria enters the forming egg.

In most European countries today, the implementation of Salmonella monitoring programmes has reduced Figure 1. Effect of acidification on Salmonella reduction in artificially contaminated feed.

Salmonalla count, cfu/ml

1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Salmonella contaminated feed ■ 0 hour Source: BIOMIN trials, 2013

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Salmonella contaminated feed with Biotronic®

■ 1 hour

■ 2.5 hours

Source: BIOMIN trials, 2013

the number of cases of human salmonellosis due to the consumption of contaminated eggs. Unfortunately, Salmonella infection has not been completely eliminated yet and the costs of hospitalisation and treatment can amount to €4,000 per patient.

The efficacy of Biotronic® on the reduction of S. Enteritidis in the GIT was evaluated in artificially inoculated SPF broilers. The control birds received a standard broiler diet, whereas the trial group had an additional supplementation of Biotronic® Top3 1 kg/t of feed. The results showed that counts of S. Enteritidis in the Biotronic® Top3 group were reduced by 1.4 and 1.3 log cfu/g digesta at five and 10 days post infection, respectively (Table 1).

Converting the log reduction in percentage, it was shown that the trial group had a 90% lower Salmonella count in the cecum compared to the control group.

Prevention Salmonella control is key to preventing the introduction of Salmonella in the farm. A significant number of pathogens are transported by feed and it is almost impossible for animals to consume zero Salmonella feed.

It is therefore important to use current technology and know-how to improve feed hygiene by reducing bacterial counts to a safe level for animals, preventing feed from recontamination and adopting proper farm management.

Using acidifiers in feed and water minimises Salmonella infection and promotes gut health, thereby enhancing poultry performance. The use of acidifiers can be seen as a management tool that effectively controls Salmonella, the main culprit of poultry disease.

Science & Solutions • January 2014

Raj Murugesan

Technical Specialist

Illu: anton_novik

Gut integrity — the backbone of performance

The barrier function

Livestock production today has to overcome major challenges, such as increased cost of feed ingredients and disease outbreaks, in order to increase production efficiently to feed the growing world population. In this context, gut health plays a critical role in influencing animal productivity; in other words, it is considered the “backbone of performance”.

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Gut integrity — the backbone of performance The barrier function

M

aintenance or enhancement of gut health is far more complicated in commercially raised food animals. Any factor that affects gut health will undoubtedly influence the animal as a whole and consequently change its nutrient uptake and requirements, resulting in altered performance.

The basis of gut health lies in its integrity, which is a highly complex process encompassing the macro- and micro-structural integrity of the gut, the balance of microflora, the status of gut-associated immune system and the energetic cost of metabolism. This article reviews the gut barrier function and its effect on performance.

The gut harbours more than 650 different species of bacteria, contains over 20 dif­ ferent hormones, digests and absorbs the vast majority of nutrients, and accounts for 20% of the body’s total energy expenditure, while being the largest immune organ in the body.

Gut barrier function The gastrointestinal tract is a vital organ with conflicting functions, and plays a major role in the digestion and absorption of dietary nutrients. At the same time, the gut constitutes the animal‘s most important barrier between the internal and external environments.

The ability of epithelium to control the uptake of molecules into the body is the “gut barrier function”. The gut barrier complex comprises the immunological, physiological and physical barriers.

Gut epithelium exerts an important immunological defence by the secretion of mucus into lumen to bind, dilute and wash away pathogens and noxious substances. The tight junctions, which interconnect the continuous epithelial cell layer, restrict both transcellular and paracellular permeation of molecules, thus constituting the principal component of physiological barrier.

In addition, the epithelium itself, consisting of the villi and crypts as well as the epithelial and endothelial cell components, forms the physical barrier by posing as a final blockade to the entry of

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external factors. With the coordination of these individual barriers, the gut barrier complex overcomes the challenges presented by stressors.

Stressors that could affect animal health and production include pathogens, endotoxins, mycotoxins, as well as processes that induce stress such as environment (hot or cold), feed withdrawal, feed change, vaccination, fasting and transportation.

Immunological barrier The epithelium covering mucus layer, comprised of mucins, is the initial point of contact between the host and gut microflora. Hence, mucins are the first line of defence against pathogens, and they communicate extensively with commensal and probiotic microbes. Mucins are glycoproteins, which are categorised into distinct families: gel-forming, soluble and membrane-bound.

Binding to epithelial cells is the primary step for many enteric pathogens to translocate into circulation as well as to affect net fluid and electrolyte secretion. Therefore, the interruption of entero-pathogenic adherence could provide therapeutic benefits to the host. By virtue of their negatively charged filamentous protruding structure, mucins act as a selective barrier protecting the epithelial cells.

Under normal circumstances, mucins allow only minute quantities of intact antigens to cross into the mucosa, where they interact with the mucosal immune system to down-regulate inflammation, otherwise known as oral tolerance. However, mucin synthesis from goblet cells is altered during stressful conditions to efficiently overcome the challenges.

Physiological barrier Epithelial tight junctions are the primary component of gut physiological barrier as they join epithelial and endothelial cells to each other and function as a ‘‘fence’’. The barrier function of tight junctions is the ability of epithelium and endothelium-lined surfaces to differen-

Science & Solutions • January 2014

Raj Murugesan

Technical Specialist

Tight Junction

Villus Epithelzelle

Microvilli

Parazellulärer Weg

Crypt of Lieberkühn Muscularis mucosa

Transzellulärer Weg

Figure 1. Transcellular and paracellular pathways in the intestinal epithelium.

tially restrict the passage of water, ions, and larger solutes, based on size and charge, through paracellular permeation.

The presence of or exposure to stressors compromises the integrity of the tight junctions, leading to increased ion conductance across the paracellular route (Figure 1). This condition, commonly known as “leaky gut”, essentially enables pathogens, endotoxins and mycotoxins to access the whole body including vital organs by reducing the integrity of the tight junctions and increasing their translocation into circulation.

Physical barrier The gastrointestinal epithelium undergoes morphologic and physiologic changes immediately after birth/hatch, increasing the surface area for digestion and absorption which is essential to fully express the animal’s genetic potential for growth. The high plasticity of gut epithelium, such as changes in villus density, villus height, crypt depth and rate of epithelial turnover, allows for the response to challenges. Crypts are considered as villus factories as they contain stem cells, hence deeper crypts indicate faster tissue turnover and higher nutrient demand for new tissue.

However, increased tissue turnover to permit the renewal of villi and crypts is required in response to inflammation from pathogens or their toxins (Figure 2).

Reduced villus height and increased crypt depth (reduced ratio) lead to increased

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Figure 2. Villus and crypt in the intestinal tract.

Source: University of Waikato, 2011

endogenous secretion and reduced nutrient absorption, disease resistance and performance.

Summary There is a growing body of evidence that stressors can have an impact on several components of the intestinal barrier function and can adversely increase epithelial permeability. With the combination of the above-discussed barriers, the gut protects itself as well as the animal from various physical and physiological stressors. Gut integrity is compromised when the conditions are conducive for increased presence of or exposure to these stressors, which expose the animal to a variety of challenges. These could be either overt, clinical conditions or subtle, sub-clinical conditions.

The sub-clinical challenges happen on a day-to-day basis and do not exhibit by any symptoms. Animals channel their nutrients to overcome these challenges through various means such as the activation of immune system, which otherwise would have been used to increase growth and productivity. This loss has been estimated to be 10-12% of the nutrients absorbed at any given time. Thus, it is imperative to understand that even a small amount of stress could affect the animal’s productivity, and maintaining an integral and healthy gut is the primary step towards efficient production.

Stressors that could affect animal health and production in­ clude pathogens, endotoxins, myco­ toxins, as well as processes that induce stress such as environ­ ment (hot or cold), feed withdrawal, feed change, vaccination, fasting and transportation.

References are available upon request.

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Paula Kovalsky

Product Manager, Mycotoxin Risk Management

Mycotoxin update 2013 Which mycotoxins should we expect in poultry feed?

Mycotoxin contaminated grains are a global problem that leads to immunosuppression and an altered response to vaccination programmes in poultry. These effects result in a decline in animal performance and ultimately poorer profitability. Results from the latest BIOMIN Mycotoxin Survey 2013 show that deoxynivalenol is still the most prevalent mycotoxin worldwide.

Poultry uses almost half of world feed World compound feed production is approaching 1 billion tonnes per year according to the International Feed Industry Federation (IFIF). The poultry feed industry comprises 45% of the total market (Figure 1). Asia is the largest feed producing region and covers 38% of the world feed market (Figure 2).

Corn and wheat are the main grains used in the poultry industry, as well as rice (whole grain and/ or cracked) in many parts of Asia. The leading plant protein source is soybean meal. Due to rising prices for conventional commodities, the poultry industry has turned to the use of distiller’s dried grains with solubles (DDGS), which present a cost-effective and nutritious substitute.

Mycotoxins to watch out for The latest BIOMIN Mycotoxin Survey 2013 focused on the main feed ingredients for poultry (corn, wheat, soybean, rice and DDGS) analysed between January and September. Data showed that mycotoxin levels were above the detection limits in 73% of all samples tested. Figure 3 shows the percentage of samples tested positive (% pos.), where concentration levels exceed the level of detection, and average mycotoxin contamination levels of all samples tested positive in

Figure 1. World feed utilisation by species.

commodities according to the main producing regions.

The highest aflatoxin (Afla) and fumonisin (FUM) concentrations were observed in the USA for corn samples. In China, deoxynivalenol (DON) levels for wheat showed the highest overall concentration.

Mycotoxins reduce poultry performance Feed is the most important component in poultry production and represents up to 50% of total costs. Procuring high quality feed is an indispensable but challenging requisite due to the unavoidable contamination of grains with mycotoxins.

The effects of mycotoxins in the animal depend on the animal’s age, physiological state and nutrition given, and the type and quantity of mycotoxin uptake. Animals fed a mycotoxin-contaminated diet have reduced immune responses due to an alteration of the normal immune system.

Mycotoxins have been shown to decrease the resistance of the animal to many pathogens, leading to higher susceptibility to diseases. DON and FUM predispose poultry to necrotic enteritis, one of the main diseases in the broiler industry caused by Clostridium perfringens, even at concentrations below the European guidance level (5 ppm for DON and 20 ppm for FUM).

Figure 2. World feed production by region.

500

400

Million tonnes

Million tonnes

400 300 200 100 0 Poultry Ruminant Source: IFIF, 2013

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Pig Aquaculture

300 200 100 0 North America Latin America

Europe Middle East+Africa

Asia

Source: IFIF, 2013

Science & Solutions • January 2014

Corn USA Afla ZEN % pos.

67% 30% Average [ppb] 89 9

DON 40% 212

FUM OTA 100% 13% 6226 1

Europe Afla % pos.

69% Average [ppb] 12

Corn ZEN 32% 47

DON 61% 525

FUM 90% 1692

OTA 58% 7

% pos.

21% Average [ppb] 1

Wheat 11% 17

73% 983

6% 190

17% 2

DON 92% 919

FUM 32% 1257

OTA 7% 6

91% 1877

4% 564

7% 3

China Afla % pos.

6% Average [ppb] 36 Brazil Afla % pos.

17% Average [ppb] 4

Corn ZEN 40% 224

DON 40% 321

FUM 91% 3193

OTA 0% 0

% pos.

0% Average [ppb] 0

Corn ZEN 65% 353 Wheat 39% 44

Results of the BIOMIN Myco­ toxin Survey 2013 show that DON is still the most prevalent mycotoxin world­ wide. DON and fumonisins pre­ dispose poultry to necrotic enteritis, one of the main diseases in the broiler industry.

Worldwide Soybean Afla ZEN DON FUM OTA % pos.

29% 33% 23% 20% 29% Average [ppb] 2 24 383 192 2

Afla % pos.

42% Average [ppb] 14

Rice ZEN DON FUM OTA 50% 37% 32% 24% 114 141 218 2

Afla % pos.

67% Average [ppb] 7

DDGS ZEN DON FUM OTA 60% 77% 84% 21% 87 1038 2756 12

Figure 3. Occurrence of mycotoxins by region, commodity and type of toxin shown as % of positive values above the limit of detection (% pos.) and average mycotoxin contamination levels (ppb) of all samples tested positive in the main producing regions (for corn and wheat) and worldwide (for soybean, rice and DDGS). The survey provides insights on the occurrence of the most important mycotoxins in agriculture and animal production and covers aflatoxins (Afla), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FUM) and ochratoxin A (OTA).

Summary The world demand for poultry keeps rising despite high feed and production prices. The Food and Agriculture Organization estimates that poultry output will hit 106 million tonnes in 2013. Demands for quality poultry are directly linked to demands for quality feed.

As the effects of mycotoxins in poultry are often subclinical, they are frequently overlooked by farm technicians. Mycotoxin risk management is crucial to eliminate

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Figure 4. Percentage of broilers with necrotic enteritis (NE) lesions out of a set of 90 birds per treatment.

60 % % of broilers with NE lesions

Broilers infected with C. perfringens and fed a DONor FUM-contaminated diet showed an increase in necrotic enteritis lesions compared to the control (Figure 4). DON also affects the animal’s response to vaccination programmes resulting for instance in reduced titres of infectious bronchitis virus (IBV) antibodies and Newcastle disease humoral antibodies in broilers.

The ingestion of mycotoxins leads to an overall performance decline. Decreased egg production, egg quality and hatchability as well as higher mortality and feed conversion ratios are some effects of mycotoxins that may result in high economic losses.

50 % 40 % 30 % 20 % 10 % 0%

C. perfringens + C. perfringens + C. perfringens + C. perfringens + control feed fumonisins fumonisins and DON DON Source: Antonissen, 2013

the effect of fungal toxins and their toxicity and should be carried out on a continuous basis through a proper mycotoxin risk management tool which uses different strategies. The Mycofix® product line combines the three strategies of adsorption, biotransformation and bioprotection, which systematically targets the most common and agriculturally relevant mycotoxins.

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