MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

Miljø- og Fødevareudvalget 2016-17
MOF Alm.del Bilag 492
Offentligt

AARHUS

UNIVERSITET

DCA - NATIONALT CENTER FOR FØDEVARER OG JORDBRU G

Til Fødevarestyrelsen

Genfremsendelse af bestillingen:

”Vidensyntese

om muligheder for fore-

byggelse af fravænningsdiarré hos grise”.

Fødevarestyrelsen har i en bestilling fremsendt d. 4. maj

2016

via Miljø- og Fødeva-

reministeriets forskningsbank bedt DCA

–

Nationalt Center for Fødevarer og Jord-

brug

–

om at udarbejde en vidensyntese om muligheder for forebyggelse af fravæn-

ningsdiarré hos grise i såvel økologisk og konventionel produktion.

Bestillingen blev leveret den 24. april 2017, og Fødevarestyrelsen har efterfølgende

bedt om at få lavet ændringer til bestillingen. I denne version er der derfor rettet en

fejl i referencerne, og udtrykket cost-benefit er erstattet med cost-effectiveness, som

bruges af andre danske forskere.

Den reviderede vidensyntese, der følger nedenfor, er jf projektbeskrivelsen udarbej-

det på engelsk med et dansk resumé. Den er udarbejdet af Sektionsleder Charlotte

Lauridsen, Seniorforsker Ole Højbjerg, Videnskabelig assistent Hanne Kongsted og

Seniorforsker Nuria Canibe alle fra Institut for Husdyrvidenskab, Aarhus Universitet.

Besvarelsen

er udarbejdet som led i ”Aftale mellem Aarhus Universitet og Fødevare-

ministeriet om udførelse af forskningsbaseret myndighedsbetjening m.v. ved Aarhus

Universitet, DCA

–

Nationalt Center for Fødevarer og Jordbrug, 2016-2019 (punkt

BH203 i Aftalens Bilag 2 samt B14 i Arbejdsprogram 2017 for Ydelsesaftale for Hus-

dyrproduktion, 2017-2020)”.

DCA - Nationalt Center for

Fødevarer og Jordbrug

Ulla Sonne Bertelsen

Specialkonsulent

Dato . 26.04.2017

Direkte tlf.: 87 15 7685

Mobiltlf.:

E-mail: [email protected]

Afs. CVR-nr.: 31119103

Reference: khr

Journal 2017-760-000114

Venlig hilsen

Klaus Horsted og Ulla Sonne Bertelsen

DCA - Nationalt Center for

Fødevarer og Jordbrug

Aarhus Universitet

Blichers Allé 20

8830 Tjele

Tlf.: 8715 6000

Fax: 8715 6076

E-mail: [email protected]

http://dca.au.dk/

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

A critical review on alternatives to antibiotics and pharmacological zinc

for prevention of diarrhoea in pigs post-weaning

Charlotte Lauridsen, Ole Højberg, Hanne Kongsted and Nuria Canibe

Department of Animal Science, Aarhus University, Blichers Allé 20, Foulum, 8830 Tjele

Dansk resumé

Diarre associeret med

Escherichia coli

(E.

coli)

forekommer de første 7-10 dage efter fravænning og

kan behandles med antibiotika, men problematikken omkring udvikling af resistens betyder, at

forbruget af antibiotika skal reduceres. Som alternativ anvendes medicinsk zink (Zn) i form af

zinkoxid , som ordineres med 2500 ppm Zn i 14 dage efter fravænning hos både konventionelle og

økologiske besætninger. Anvendelsen af zinkoxid bevirker en øget mængde zink i svinegyllen, og i

forbindelse med MRSA risikovurderingen er det blevet anbefalet, at zink bør anvendes mere

restriktivt. Der er derfor behov for at finde alternativer til både antibiotika og medicinsk zink til

forebyggelse af fravænningsdiarre hos grise.

På denne baggrund har Fødevarestyrelsen anmodet DCA – Nationalt Center for Fødevarer og

Jordbrug om at foranledige udarbejdelse af en vidensyntese ved Aarhus Universitet. Rapporten er led

i ’Aftale om forskningsbaseret myndighedsbetjening mellem AU og Fødevareministeriet’. Formålet

var at undersøge muligheder for forebyggelse af fravænningsdiarre hos grise i såvel økologisk som

konventionel produktion baseret på litteratur fra både ind- og udland. Et andet formål var at

fremlægge rammer og oversigt af muligheder for forebyggelse af fravænningsdiarre, der kunne danne

udgangspunkt for en cost-effectiveness vurdering, som skulle gennemføres sideløbende ved IFRO,

Københavns universitet.

Vores litteraturgennemgang tager udgangspunkt i de ændringer, der forekommer i tarmkanalen

omkring fravænning, efterfulgt af en beskrivelse af patogenese og diagnosticering af

fravænningsdiarre, samt af de faktorer, der kan fremme/hæmme fravænningsdiarre forårsaget af

coli,

såsom genetik, management og miljø samt ernæring og fodring. Blandt disse faktorer er der kun

et begrænset antal undersøgelser, der har angivet effektiviteten af en given intervention eller dennes

potentielle effektivitet i relation til forebyggelse af fravænningsdiarre, og undersøgelserne er

fortrinsvist gennemført med henblik på at finde alternativer til antibiotika. I litteraturen er medicinsk

zink hidtil betragtet som et effektivt alternativ til antibiotika. På baggrund af vores

litteraturundersøgelse konkluderede vi, at 1) reduktion af foderets proteinindhold; 2) øgning i

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DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

fravænningsalder, og 3) optimering af miljøets hygiejnestatus for de fravænnede grise er strategier,

som kunne være relevante at underkaste en cost-benefit analyse. Strategierne er af interesse for både

konventionel og økologisk griseproduktion, men der er nogle særlige udfordringer, der gør, at alle tre

strategier måske ikke er lige relevante i de to produktionsformer: En begrænsning af foderets

proteinindhold medfører overvejelse af foderets aminosyresammensætning i forhold til dyrets vækst.

Det er ikke tilladt i økologisk svineproduktion at tilsætte syntetiske aminosyrer. I økologisk

svineproduktion anvendes i forvejen en højere fravænningsalder (7 uger), hvorfor 5 i stedet for 4

ugers fravænningsalder ikke er relevant. Det skal her nævnes, at pludselig fravænning muligvis er det

mest udfordrende for grisen, uanset om alderen er 4 eller 7 uger. Gradvis fravænning (forlænget

dieperiode) er derfor relevant at undersøge nærmere med henblik på reduktion af fravænningsdiarre

i relation til økologisk svineproduktion. Vi har beskrevet effekten af organiske syrer og fermenteret

vådfoder på forekomsten af fravænningsdiarre, samt muligheden for anvendelse af antistoffer

oprenset fra blodplasma eller æggepulver, men det er vores vurdering, at der skal tilvejebringes

yderligere forskning for at udvikle produkterne og vurdere deres effekt, førend de underkastes en

cost-effectiveness analyse.

1. Summary

Post-weaning diarrhoea (PWD) is a significant enteric disease causing considerable economic losses

for the pig industry. Among several etiological risk factors, enterotoxigenic

Escherichia coli

(ETEC)

is considered to be a major cause, i.e. colibacillosis. After being routinely used for several decades as

growth promoters and to control bacterial disease outbreaks in piglets, the use of antibiotics at sub-

therapeutic concentrations has been banned in the European Union since 1 January 2006 due to the

increasing prevalence of resistance to antibiotics in pigs. The removal of in-feed antibiotics from

piglet diets has negative economic consequences as it dramatically increases the rate of morbidity

and mortality due to ETEC as well as the use of antibiotics for therapeutic purposes. One of the used

substitutes for in-feed antibiotics in some European countries, including Denmark, in which this

practice is allowed, is pharmacological levels of dietary zinc (2500 ppm during the first two weeks

post-weaning following veterinary prescription), but its high excretion has a negative impact on the

environment. Thus, alternatives to antibiotics and to high levels of heavy metals that can control

ETEC infections in piglets post-weaning will be of great advantage, also because of the risk of cross

resistance between antibiotics and heavy metals. A number of nutritional, genetic and management

strategies have been reported in the literature as alternatives to in-feed antibiotics to prevent PWD,

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DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

but, in most studies, the reduction of PWD has not been addressed as a primary response parameter.

The present review addresses the possible mode of action by which nutrition and feed additives,

breeding and rearing/management may potentially prevent and/or reduce ETEC diarrhoea in pigs (in

conventional and organic production systems) during the post-weaning period, and as such, could be

considered as alternative strategies to in-feed antibiotics and pharmacological zinc oxide (ZnO). An

important objective of our knowledge synthesis is to propose strategies, which could be considered

relevant for further evaluation in a cost-effectiveness analysis. Our study of the literature revealed

that few experiments have actually demonstrated the efficiency of a given intervention or its potential

efficacy to reduce PWD; either because PWD was not a primary parameter of the study, the study

was not designed to evaluate effect on PWD, or the given intervention was not available on a

commercial basis. Our critical review therefore limited the available potential interventions for further

cost-effectiveness analysis. In conclusion, this review pointed out that the following strategies:

reduced dietary protein level, increased weaning age, and optimized hygienic status of weaning

facilities for the pigs post-weaning would have an effect on the occurrence of PWD. Besides, there

were a few more dietary factors which gained our special interest in terms of their potential in limiting

PWD in pigs, i.e. organic acids and antibodies, but these alternatives require more development and

research to pinpoint their efficacy in relation to prevention of PWD.

2. Review methodology

We performed a multiple database search to ensure comprehensive article retrieval in CABI and ISI

Web of Science. The main key words were: pigs, piglets, weaning, diarrhoea, ETEC,

E. coli,

nutrition,

feed, feed additives, management, genetics, environment, risk factors, post-weaning diarrhoea,

diarrhoea, hygiene, age, antibodies, feeding strategy, organic acid and liquid feed. The following

criteria were applied to the literature selection: 1) Peer-reviewed journal articles in English were

included; 2) Articles or chapters in an edited book were selectively included; 3) Grey literature such

as PhD theses and dissertations were selectively included; 4) Published reports from the official

organisation such as The Danish Integrated Antimicrobial Resistance Monitoring and Research

Programme (DANMAP) and European Food Safety Authority (EFSA) were selectively included;, 5)

In vitro and in vivo investigations related to pigs were included; 6) In vitro and in vivo studies on

humans, rodents and other animal species were selectively included, provided that the aims were to

investigate the cell/host pathogen interactions and/or enteric infections; 7) Enteric diseases related to

presence of

Brachyspira pilosicoli

and

Lawsonia intracellularis

were excluded. In addition, we used

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the references from the articles obtained by this method to check for additional relevant material.

Furthermore, we included Danish material available at SEGES Pig Research Centre

(http://vsp.lf.dk/Publikationer.aspx) and DCA (National Centre for Food and Agriculture)

(www.dca.au.dk).

3. Introduction

Diarrhoea is a common clinical condition in newborn and newly-weaned piglets (Lalles et al., 2007).

At its core, diarrhoea is simply an altered movement of ions and water that allows an osmotic gradient.

Under normal conditions, the gastrointestinal tract has tremendous capacity to absorb fluid. However,

enteric pathogens can alter the balance towards net secretion, leading to diarrhoeal disease (Hodges

and Gill, 2010). Diarrhoea is manifested as an increase in the water content of faeces and (or) as an

increased daily passage of faeces. Diarrhoea may result from nutritional imbalance causing irritation

and/or increased luminal osmotic forces; however, post-weaning diarrhoea (PWD) is primarily

caused by enteric infection by Enterotoxigenic

Escherichia coli

(ETEC), also called enteric

colibacillosis, appearing mostly within the first two weeks post-weaning (Fairbrother et al., 2005).

The prevalence of PWD creates a major challenge not only for the pig farmer because of its economic

impact but also for the society as diarrhoea results in decreased animal welfare and high use of

antibiotics and zinc (Zn) with the consequent risk of developing bacterial antibiotic resistance (WHO,

2012). Antibiotic resistance causes a major threat to human health. The ongoing increase in multi-

drug resistant bacterial infections (Blair et al., 2015) has emphasised an increased need for prudent

use of antibiotics and has put major focus on the antibiotic consumption in production animals.

According to the Danish Authorities (DANMAP, 2015), pigs consume 76% of the total antibiotic

mass consumed by production animals, horses, pets and fish while only contributing with 43% to the

live biomass. Pigs during the post-weaning period consume the largest amount of antibiotics

compared with sows and suckling piglets, and finishers (DANMAP, 2015). In addition to treatment

with antibiotics, dietary levels of zinc oxide (ZnO) at a high level can prevent PWD in weaner piglets

(Owusu-Asiedu et al., 2003); (Pluske, 2013). In Denmark, the use of ZnO prescribed by veterinarians

has increased over the last decade. From 2005-2011, there was a threefold increase in the use of Zn

and ZnO reported to VetStat. Since 2011, the use of pharmacological zinc for pigs has continued to

increase steadily (Erratum, 2015): from 471 tons (2010) to 525 tons (2015) of therapeutic zinc (as

ZnO) to pigs in Denmark (Erratum, 2015). It should, however, be noted that the consumption of

pharmacological zinc has decreased with approximately 4% from 2014 to 2015 (DANMAP, 2015).

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Several reviews have been conducted on the treatment and control of ETEC infections in pigs

(Hodgson and Barton, 2009)and the possible intervention strategies to control diarrhoea in pigs (Halas

et al., 2007); (Amadori et al., 2012); (Heo et al., 2013); (Sugiharto et al., 2015a). However, little is

known regarding the real efficacy of the proposed alternative strategies and interventions. The

objective of the present review is to perform a knowledge synthesis based on existing literature, which

could form the basis for the selection of relevant interventions to study in a cost-effectiveness analysis

in order to identify economically feasible alternatives to in-feed antibiotics and zinc for

preventing/reducing PWD.

Changes in the gastrointestinal system around weaning

During the last three decades, research and field experience have focused on stressors encountered

by pigs around weaning in order to reduce them and thereby obtain good levels of production. A

weaning age of 22-28 days is practiced in many pig-producing countries, including Denmark. Many

scientists have considered the changes taking place around weaning concerning nutrition, housing,

health, and behavioural and environmental requirements of the young pig (Pluske et al., 2003), and

much recent research and commercial interests have focused on “gut health” and optimal immune

function. One of the major changes at weaning is nutrition, i.e. from sow milk to solid feed. Creep

feed is generally provided from around 14 days of age, but a significant creep feed intake is not

observed until approximately four weeks of age (Lucas and Lodge, 1961). When milk supply ceases

abruptly, the structure and function of the digestive tract begins to change within hours.

Alterations in the digestive tract may have long-term (one or more weeks) consequences and may

lead to poor absorption of nutrients in the small intestine, which is often associated with proliferation

of enterotoxigenic bacteria and/or increased microbial fermentation of nutrients. Many pigs

experience a so-called “growth check” or a fall in growth rate post-weaning, e.g. due to reduced feed

intake and infections. And the extent of the depression in growth depends on how rapidly the piglet

can cope with the challenges and regain homeostasis; for example is gut health highly dynamic, and

if the balance tips, the consequence is diarrhoea and in more severe cases probably enteric

inflammation. In the following, we will briefly describe how the intestinal microbiota, intestinal

barrier and immune function are affected by weaning.

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Microbiota

Several studies have investigated the changes in the gut ecosystem in relation to weaning (Jensen,

1998). Pigs were weaned and followed for 11 days. A steady decrease in pH and dry matter in rectal

samples was observed during the first week post-weaning. While lactic acid bacteria (LAB) were

significantly suppressed, the number of coliform bacteria increased in the intestine after weaning,

thus a reduced ratio lactobacilli:coliform bacteria was observed during the first week post-weaning

(Jensen, 1998). These alterations may result in an increased susceptibility of the pigs to colonization

by pathogenic bacteria such as ETEC, which typically colonize and proliferate in the distal jejunum

and ileum. 16S sequencing analyses have revealed that the intestinal microbial community is very

complex, and the majority of the bacteria colonizing the gastrointestinal tract in pigs have not been

characterised in detail (Leser et al., 2002; Kim et al., 2011a, 2012; Looft et al., 2014a)

It is clear from many studies that the profile of the microbiota in the intestine of pigs is affected by

the specific diet and nutrient content (Canibe and Jensen, 2003; Canibe et al., 2005); (Mikkelsen et

al., 2007); (Liu et al., 2013); (Rist et al., 2013); (Heinritz et al., 2013). As expected, antibiotic

treatments can also shift the intestinal microbiota (Kim et al., 2012); (Looft et al., 2012); (Looft et

al., 2014b). Another important factor for the microbial composition is the environment. Surprisingly,

it was demonstrated that exposure to a large variety of environmental microbes in early life (pigs

housed outdoor) did not generate greater diversity in the adult gut than in pigs housed indoor

(intensive) but rather led to a microbiota dominated by a limited number of phyla composed of

lactobacilli (Mulder et al., 2009). Farm management may also have an impact on the amount of

coli

excreted, as conventional herds had higher number per gram colon content than organic farms

(Kerouanton et al., 2014).

Jensen (Jensen, 1998)concluded that the microbiota is unstable during the first week post-weaning

(from 28 to 35 days of age) and that it takes two to three weeks from weaning before the fermentative

capacity in the hindgut is developed. (Kim et al., 2011b), on the other hand, reported continued

changes in the faecal microbiota composition when studying pigs from 10 to 22 weeks of age.

Intestinal barrier function and nutrient absorption

In comparison to other compartments of the GIT and its accessory organs, it is probably the small

intestine that is affected the most by weaning – largely irrespective of weaning age. Significant

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changes occur in the structure and function of the small intestine during the immediate post-weaning

period (Hopwood and Hampson, 2003), and it is generally accepted that there is a transient period of

villous atrophy and crypt hyperplasia after weaning, as reviewed by e.g. Heo et al. (Heo et al., 2013),

which could be ascribed to the reduction in feed intake. A reduced villous surface after weaning

implicates a reduction in intestinal absorptive capacity as well (Wijtten et al., 2011) as the presence

of villi helps to increase its surface area for digestion and absorption processes. In addition, the

process of weaning affects the small intestinal barrier function in pigs (Wijtten et al., 2011). In healthy

animals, antigen uptake is precisely regulated to train the immune system. The effect of weaning

stress or reduced feed intake is less clear in relation to the intestinal barrier function (Pluske, 2016).

Impaired intestinal barrier function or increased intestinal permeability may promote the translocation

of bacteria and the entering of allergenic compounds from the gut into the body, which increase the

immunological responses and the susceptibility to infections.

Immunity

The presence of commensal bacteria has a direct influence on the immune maturation, and the

microbial exposure influences expression of a large number of immune-related genes (Mulder et al.,

2011). Hence, host-independent factors in connection with weaning, such as nutrition and rearing

environment, which influence the gut microbiota may also influence the immune activation. Weaning

of pigs (between four and five weeks of age) suppressed a broad spectrum of adaptive immune

components in blood (T-lymphocyte subsets, B-lymphocytes, monocytes and granulocytes,

immunoglobulins, mannan-binding lectin) both immediately after weaning as well as after a lag

period of about one week; on the contrary, the innate immune system seems to be stimulated

immediately after weaning (Juul-Madsen et al., 2010). At the time considered to have highest

infection pressure, T-cells and TLR4+ cells were markedly enhanced, whereas the expression of SLA

1 did not seem to be affected by weaning (Juul-Madsen et al., 2010). In general, immune parameters

seem to be stabilised at an age of 8-10 weeks. Nutritional stress in terms of anorexia post-weaning

coupled with its consequences on gut morphology have been reported to be major contributor to local

intestinal inflammation during the immediate post-weaning period (McCracken et al., 1999).

However, it still remains to be answered what the most important factors are (i.e. deprivation of milk

intake and/or changes in nutrition, changes in microbial environment or natural age-dependent

developmental changes) for the immune activation at weaning.

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5. Pathogenesis and diagnosis of post-weaning diarrhoea

PWD is a multifactorial disease, and its pathogenesis is still unclear. Frequently described, this is a

condition of weaned pigs characterised by frequent discharge of watery faeces during the first two

weeks after weaning. This condition is typically associated with faecal shedding of haemolytic ETEC

that proliferate in the ileum. However, ETEC is detected in both healthy and diarrhoeic pigs upon

weaning. Pigs with

E. coli

PWD typically have watery diarrhoea that lasts from one to five days, but

some pigs may die suddenly without observed diarrhoea. Those pigs may have an accumulation of

fluid in the intestine and ETEC invasion in blood and tissues leading to sepsis. The pathophysiological

mechanisms of infectious diarrhoea have been described by Hodges and Gill (Hodges and Gill,

2010)and involve alterations in ion transport and tight junctions. In brief, by the use of fimbriae,

ETEC adheres to receptors of the small intestinal enterocytes where they produce one or more

enterotoxins such as heat-labile toxins (LTs) or heat-stable (STs) toxins, which activate the cAMP

and the cGMP systems. The LT toxins increase secretion of sodium, chloride and hydrogen carbonate

ions into the lumen, whilst the ST toxins reduce the absorption of liquid and salts. This leads to

hypersecretion of water and electrolytes to the small intestine that exceeds the ability of the colon to

reabsorb, causing diarrhoea and dehydration, and results in reduced feed intake, reduced nutrient

digestibility, reduced growth and even death. Another toxin, the shiga toxin Stx, is characteristic of

shiga toxin-producing

E. coli

strains causing porcine oedema disease (ED) but may also be present

in the ETEC strain (Hodgson and Barton, 2009).

To elicit diarrhoea in piglets, ETEC must first adhere to specific porcine enterocyte receptors for the

respective fimbriae (Heo et al., 2013); (Fairbrother et al., 2005). This adhesion facilitates colonization

of the distal jejunum and proximal ileum mucosa by ETEC and enables them to transmit enterotoxins.

The fimbriae are thin and flexible structures with a diameter of about 2-4 nm whereas pili are more

rigid structures with a diameter of about 7-8 nm and have an axial hole (Degraaf and Mooi, 1986),

and both contain an adhesive lectin subunit. The biosynthesis of fimbria is influenced by several

factors, and the pathogenic bacteria only fully express virulence factors like fimbria when the

conditions are appropriate for adherence and subsequent colonization (Verdonck et al., 2004).

Fimbriae designated F4 and F18 are the types that are commonly found on ETEC from PWD in pigs.

In case of F4 fimbriae, expression is optimal at a temperature of 37 °C, a pH ranging from 6.5 to 8.0

and at the end of the exponential growth. F18 fimbria are typically associated with diarrhoea in

nursing pigs whereas F4 fimbriae are associated with diarrhoea in nursing pigs as well as in weaned

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pigs. Frydendahl (Frydendahl, 2002) identified genes for F4 and F18 in 92.7% of all ETEC from

PWD. Recent results have shown that among diarrhoeic pools observed in 51% of the pens in batches

of nursery pigs (10-66 days after weaning),

E. coli

F4 and F18 were detected in 13% and 11%,

respectively (Weber et al., 2015)Adhesion of F18-fimbriated bacteria to the small intestine of

susceptible pigs is mediated by the minor fimbrial subunit FedF (Coddens et al., 2008)Adhesion of

F4-fimbriated bacteria to the small intestine is mediated by the fimbrial subunit FaeG adhesion

(Verdonck et al., 2004).

Specific serotypes of ETEC with particular sets of virulence genes, i.e. ETEC expressing F4 or F18

fimbriae, have been associated with diarrhoea in pigs as well as in other mammals, including humans

(Nagy and Fekete, 1999); (Fairbrother et al., 2005). The most frequent ETEC serotypes causing PWD

in pigs are O serotypes (8, 138, 139, 141, 147, 149 and 157), and the predominant serotype of

E. coli

associated with PWD in pigs worldwide is O149 (Fairbrother et al., 2005); (Heo et al., 2013). This

serotype was first detected in Denmark in 1966 and was still dominant in 2002 (Frydendahl, 2002).

Dam and Knox (1974) were able to trace the spread of O149 ETEC from its first appearance on four

farms in 1966 to its rapid spread over the entire country by 1969.

In the recent yearly report (Veterinærinstituttet, 2015), F4 and F18 appeared as the most predominant

in faecal and intestinal samples from pigs from Danish herds, and O149 was the predominant

hemolytic serotype of

E. coli

followed by O141, O139 and O138.

Diagnosis of post-weaning diarrhoea and enteric inflammation

Diarrhoea is the main cause of treatment with antibiotics in Danish pig production (Pedersen, 2014).

Whereas the classical weaning diarrhoea associated with

E. coli

within the first two weeks post-

weaning was a frequent diagnosis, the large consumption of antibiotics is not used for treatment of

PWD but for treatment of diarrhoea among pigs appearing later than two weeks post-weaning

(Pedersen, 2014). In Denmark, this may probably be ascribed to the use of pharmacological levels of

ZnO as prevention against diarrhoea appearing during the first weeks post-weaning. It is generally

accepted that

E. coli, Brachyspira pilosicoli

and

Lawsonia intracellularis

cause this diarrhoea and

that they appear in combination during infections in around half of the Danish herds (Pedersen, 2014).

By using qPCR on faecal and intestinal samples of pigs sent for diagnosis at the Veterinary Institute,

Denmark, it was detected that 196 out of 840 samples were positive for

E. coli

F4; 379 out of 840

samples were positive for

E. coli

F18 (F107); 599 out of 833 were positive for Lawsonia

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intracellularis; and 256 out of 834 were positive for Brachyspira pilosicoli (Veterinærinstituttet,

2015). A recent study (Weber et al., 2015) concluded that diarrhoeic status of pigs was a poor

indicator of intestinal infection with

E. coli

and subclinical infections.

Many studies conducted in research or under commercial settings have reported the impact of the

treatments under investigation on diarrhoea but often without being a primary response parameter.

It is often difficult to assess “diarrhoea” per se in commercial husbandry systems as it is associated

with a high level of variability, and the studies use different measures as indicators of diarrhoea, e.g.

frequency of treatment with antibiotics, faecal score, or number of days with diarrhoea. Diarrhoea

index can be measured as proportion of days with diarrhoea in relation to total number of days, but

information on whether or not the diarrhoea encompassed in the index required antibiotic treatment

has not consistently been given (Madec et al., 1998). Trials performed in commercial settings, the

frequency of diarrhoea may be given as “number of days with antibiotic treatments”; however, it may

vary among the published studies how the decision was taken for antibiotic intervention.

In experimentally-induced diarrhoea, in which diarrhoea is induced by inoculation with

E. coli

(Sugiharto et al., 2014); (Sorensen et al., 2009), faecal samples are obtained frequently from each

individual pig, and the faecal samples are scored according to the Bristol scale (1=hard, dry and

cloddy, 2=firm, 3=soft with shape, 4=soft and liquid, 5=watery and dark, 6=watery and yellow,

7=foamy and yellow; a faecal consistency score >3 was defined as a clinical sign of diarrhoea). The

aim of this experimental model is to induce a mild diarrhoea in pigs, which can be cleared by the pig

itself. In parallel with diarrhoea score, faecal samples can be analysed for dry matter, and there is a

good relationship between the diarrhoea score and the dry matter. ETEC diarrhoea is typically

associated with faecal shedding of large numbers

of β-haemolytic

E. coli

serotypes that proliferate

in the small intestine of pigs (Fairbrother et al., 2005); (Heo et al., 2013). Quantitative analyses of the

E. coli

content in the faecal samples are therefore relevant as this is an indicator of the burden of the

inoculum.

In many studies aiming to identify dietary and management tools to reduce diarrhoea in pigs, it is not

indicated how diarrhoea was assessed, and the experiments have not been designed to study diarrhoea.

It requires a large sample size to determine the prevalence of diarrhoea and to test the effect of a given

intervention on the outcome of diarrhoea (Weber et al., 2015; Weber et al., 2017). However, many

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studies have been performed to understand the mechanisms of the immune system and the function

of the microbiota in order to prevent the enteric diseases and to develop tools to control it. In the

following section, we will therefore describe how genetics, management and nutrition can influence

the infection with ETEC.

6. Factors predisposing/influencing PWD caused by ETEC

It is clear from the above that many factors related to the gastrointestinal changes post-weaning

can influence the presence of ETEC infection and diarrhoea. In order to identify alternatives to in-

feed antibiotics and Zn for prevention of ETEC-associated diarrhoea, it is important to understand

how genetic, management, environment and/or nutritional factors can influence the interactions

between the host and the pathogen, and this starts with the harbouring of the ETEC to its infection of

the host, which can subsequently lead to diarrhoea and intestinal inflammation. However, diarrhoea

in pigs after weaning is a multifactorial disease (Pluske, 2013), and changes around weaning, i.e. the

weaning-related stress and weakened immune competence, weaning age, feeding regimen and the

presence of other infectious agents (such as rotavirus) are critical for the diarrhoea to develop

(Fairbrother et al., 2005).

Genetics

To induce diarrhoea, fimbriae-designated F4 or F18 must bind to the specific receptor on the host

intestinal epithelium (Heo et al., 2013). In pigs, only the ones with positive F4 or the specific receptor

F18R on the intestinal enterocytes are susceptible to ETEC-diarrhoea, whereas pigs lacking these

receptors are resistant (Nagy and Fekete, 1999); (Fairbrother et al., 2005). Knowledge regarding the

distribution of the receptors in a herd or an experimental model for PWD is important to take into

account when assessing the efficacy of a given treatment against ETEC PWD in pigs (Niewold et al.,

2007). A DNA marker-based test has been employed to identify whether pigs lack or possess the F4

or F18R receptor (Fairbrother et al., 2005). The Mucin 4 gene on porcine chromosome 13 is proposed

as a candidate gene for the production of the specific ETEC F4ab/ac receptor (Jørgensen et al., 2004)

(Jensen et al., 2006)

while the α-(1,2)-fucosyltransferase

(FUT1) gene localised on the porcine

chromosome 6 has been proposed as a key gene for controlling the expression of

E. coli

F18R (Bao

et al., 2012). With the DNA-based marker test allowing genotyping for F4ab/ac ETEC

resistance/susceptibility, three genotypes were observed and were called resistant (RR), susceptible

heterozygote (SR) and susceptible homozygote (SS). It should be noted that, in other reviews,

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(Hodgson and Barton, 2009) it was concluded that there is no definitive simple test for F4 resistance.

In addition to the mucin 4 gene, the transmembrane mucin MUC13 appears to be significantly

associated with susceptibility and resistance to F4ab/ac, as the MUC13 gene is closely linked to the

genes encoding F4ab and F4ac receptors.

It seems obvious that there are host-specific factors that can influence the susceptibility to ETEC

adhesion, and breeding programs with F4R negative pigs could result in prevention of F4ab/ac-

induced ETEC diarrhoea in pig population. Such a screening has been applied in the Danish breeding

program, and although the Danish pig population is considered genetically F4 ab/ac resistant, the

incidence of diarrhoea has not been effectively reduced according to the authors’ knowledge.

Targeting a multifactorial production disease, such as PWD, by selection based on single-genes is

probably not efficient, because several factors beyond the receptor on the surface of the intestinal

epithelium of the pig are related to diarrhoea.

Management and environment

Weaning is probably one of the most stressful events in a pig’s life. Besides the social challenge

associated with separation from the sow, there are a number of stressors attributed to management

and environment, which can enhance PWD, for example the different physiological environments

(room, building, farm, water supply) and increased exposure to pathogens. In nature, pigs are weaned

from the sow over a period of up to 12-18 weeks of age, where they experience a gradual reduction

in the amount of contact with their dam, a reduction in suckling frequency, and a gradual change of

diet from milk to solid or semisolid food(s) (Jensen and Stangel, 1992). In commercial production

systems, pigs are abruptly separated from the sow at 4 weeks of age (conventional systems) or 7

weeks (organic systems). It seems clear from the literature, and in accordance with our own

experiences, that age at weaning influence PWD, and that early weaning (< 21 days of age) is by far

more challenging than weaning at 28 days of age (Kil and Stein, 2010).

The hygiene level, humidity, group size and stocking procedure were found to be important factors

leading to risky or secure profiles (Madec et al., 1998). Weaning piglets into a dirty environment may

expose them to pathogenic bacteria, thereby increasing the risk of PWD, and weaning pigs into a

clean environment improves growth performance compared with weaning pigs into a dirty

environment (Le Floc'h et al., 2006; Le Floc'h et al., 2009). Deterioration of sanitary conditions in

post-weaning facilities has been shown to impair nutrient utilization and pig growth performance by

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inducing a moderate inflammatory response (Le Floc'h et al., 2006). In his review, Pluske (Pluske,

2016)concluded that at weaning, and largely independent of weaning age, but dependent on diet type,

diet form and the rearing environment, the piglet is subjected to myriads of changes (nutritional,

psychological, environmental). These changes can further modify the overall structure and function

of the gastrointestinal tract, such that the newly weaned pig endures a ‘growth check’ while exposing

it to a greater disease and health risk.

The litter-size at birth may influence the individual pig susceptibility to PWD weaning. The use of

hyperprolific sow lines has increased the number of piglets per litter, and has made the piglets within

a litter more heterogeneous, and immunologically fragile (Lauridsen and Matte, 2017). In addition,

the transfer of antibodies from the sow to the piglet influences the robustness of the suckling pig and

probably also during the weaning period, and limited provision of colostrum because of large litter

size per se may therefore influence the susceptibility to PWD. Although not of relevance for the

modern pig lines of today, it should be noted that data from a Danish epidemiological study

comprising 48,931 litters in 89 sow herds, conducted from 1976 to 1982, showed that the incidence

of PWD increased with litter size at weaning, as a litter of 11-12 piglets at weaning had a 1.2 times

higher risk of getting PWD than litters with 8-10 piglets (Svensmark et al., 1989), and this variation

as well as the weight within a litter at weaning may also influence the susceptibility to PWD.

Feeding and Nutrition

According to the previous sections, the multifactorial nature of PWD is obvious and dependent on

the complex interaction between the microbial population and the immune system including host

factors for the ETEC infection. Many reviews have been published on nutritional approaches to

control ETEC infections in pigs, and in this context, it is important to differentiate between dietary

factors that may promote ETEC infections and those that may limit them. One of the major factors of

importance is the level of feed intake, both before weaning and post-weaning, because of its influence

on intestinal barrier function and maturity. This means that the low feed intake by the piglets after

weaning is consistently associated with short intestinal villi, which reduces the absorptive area.

Suckling pigs start eating feed (‘creep feed’) from about 14 days of age, but only few g per day until

about 21 days of age. Subsequently the feed intake is significantly increased, and a Danish study

showed that suckling piglets weaned at day 33 of age would triple the feed intake (0.2 to 0.6

kg/pig/day) during the last week in the farrowing stable (Callesen and Thorup, 2004). Pigs

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characterised by being ‘eaters’ have better initial post-weaning performance than ‘non-eaters’

(Pluske, 2016). Sørensen et al. (Sorensen et al., 2009) revealed that a feed intake of less than 200 g

during the first day after weaning seems to be associated with a relatively high incidence of a PWD

in pigs weaned at week 7 and challenged with

E. coli

F4. Another study (van der Meulen et al., 2010)

showed, however, that although pigs weaned at 7 weeks of age (compared to 4 weeks of age) had

improved post-weaning feed intake and reduced weaning stress, no improvements in functional

characteristics of the small intestine was observed. Hence, it is not obvious that increased feed intake

would be reflected in more mature gut, i.e. enhanced intestinal morphology and digestive function.

This is probably because such data are prone to a large variation, and according to Heo et al. (Heo et

al., 2013) it is rather difficult to compare data on intestinal morphology from different experiments

because of differences in the age, breed, diets and experimental conditions.

Furthermore, the critical role of feed intake is a focus point in relation to the feeding behaviour of the

post-weaned pig. Following abrupt weaning, the piglet has to adapt its feeding and drinking behaviour

very rapidly. However, in general, it fails to do this and, as a result, there is a dramatic reduction in

dry matter intake that does not recover until the second week post-weaning (Brooks and Tsourgiannis,

2003). Besides the great variation in the feeding behaviour of pigs pre-weaning (in terms of creep

feed intake), there is also a great variation immediately post-weaning. It is often difficult to assess the

individual feed intake, but interesting data have been presented by Brooks and Tsourgiannis (Brooks

and Tsourgiannis, 2003): The apparent smooth transition in feed intake as could be observed as an

average for the population was actually a statistical artefact: Very large variations in the feeding

pattern of individual pigs were observed, as half the pigs in the group had a peak feed intake on day

2 followed by a reduction that those pigs had not recovered from by the end of the first week. And

almost half the piglets had erratic feed intakes with peak intakes occurring on days 4, 5 or 6 (Brooks

and Tsourgiannis, 2003). Our own experience with pigs entering our

E. coli

challenge model confirms

that not only is the variation between litters large, but also within a litter in terms of feed intake

pattern. Generally speaking, however, pigs weaned from 3-4 weeks of age have lower feed intake

than pigs weaned at 4-5 weeks of age. According to the authors’ knowledge, there is a lack of

knowledge regarding the importance of heterogeneous litters at birth and their treatment during the

suckling period (such as cross-fostering and provision of milk replacer) in terms of feed intake for

the individual pig post-weaning and the relationship to gut maturity. However, it should be noted that

the gut epithelial tissue is the most rapidly growing tissue in the body, and that many nutrients

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required for gut growth are absorbed directly from the gut lumen. Pluske et al. (Pluske et al., 1997)

have demonstrated that a continuous supply of nutrients is essential in order to maintain the enteric

villus structure post-weaning. However, suckling pigs must consume milk to satisfy their need for

total volumetric fill, but they may fail to discriminate between the separate drives of hunger and thirst

in the early post-weaning period, and may consequently be prone to drink water to get the gut fill

(Brooks and Tsourgiannis, 2003). Overall, a sharp reduction in feed intake post-weaning may have

tremendous consequences for the enteric development.

7. Prevention of weaning diarrhoea

As introduced earlier, a prominent objective of our present knowledge synthesis was to propose

strategies that could be considered relevant for further study in a cost-effectiveness analysis. While

our study of existing literature has revealed many attempts and research focusing on the alternatives

to antimicrobial usage, less focus has been given to the identification of alternatives to the use of

pharmacological levels of ZnO, which has actually been proposed, also recently, as an alternative

strategy to in-feed antibiotics in the prevention of PWD (Heo et al., 2013).

7.1. Mode of action and use of antimicrobials and zinc

The most effective way to control PWD in piglets is still the use of antibiotics which function by

attacking various essential processes in the bacterial cell. In 2012, 43% of the antimicrobials

prescribed for pigs were used in nursery pigs (7-30 kg live weight), of which 75% were prescribed

for oral treatment of gastrointestinal disease. With regard to treatment against

E. coli,

several

antibiotics may function (Nielsen, 2012), but according to DANMAP (DANMAP, 2015),

tetracyclines have been one of the most commonly used antimicrobials in the Danish pig production

for a decade, and are almost entirely used for the treatment of gastrointestinal disease in weanling

pigs and finishers. In 2014, the Danish pig producers committed themselves to reduce the

consumption of tetracyclines by 50% by the end of 2015.Athough the use of tetracyclines has been

reduced since 2014 it is, however, of some concern that the use of colistin in pigs has increased,

especially from 2014 to 2015 (DANMAP, 2015), because colistin is of increasing importance as a

last resort antimicrobial in human medicine.

Since the late 1980s, the practice of adding pharmacological amounts of ZnO to the pig feed has

become widespread in commercial pig production, and the use of ZnO in the Danish pig production

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has increased markedly over the last 10 years (DANMAP, 2015). Although the recommended dietary

level of Zn for weaner pigs is 100 mg/kg (NRC 2012), pharmacological levels (up to 2,500 to 3,000

mg Zn/kg diet) have been used to ameliorate and (or) prevent PWD for 2 weeks post-weaning. This

practice was initially stimulated by a report (Poulsen, 1989) showing that 3,000 mg Zn from ZnO

resulted in a 60% reduction of the diarrhoea incidence when compared with lower dietary Zn levels

(addition of 0, 100, 200, or 1,000 ppm Zn as ZnO). In a subsequent publication, (Poulsen and Larsen,

1995) showed in a study using 216 piglets from 36 litters that a supplement of 2,500 ppm Zn for two

weeks after weaning (at 28 days) reduced the incidence of non-specific post-weaning diarrhoea by up

to 50%, and had a growth promoting action: observations that have also been supported by more

recent reports (Cho et al., 2015); (Kaevska et al., 2016); (Stensland et al., 2015); (Trckova et al.,

2015). A large Danish trial with nursery pigs (approximately 3200) in a herd suffering from

E. coli

0149 post-weaning also demonstrated a reduction in the number of diarrhoea treatments by

supplementation of 2500 ppm Zn in combination with organics acids (Johansen, 2007).

Although ZnO is regarded as a potent prophylaxis against the development of PWD in piglets, zinc

excretion in high amounts represents a hazard to the environment (Heo et al., 2013); (Sales, 2013).

Moreover, Zn and other heavy metals are involved in antibiotic cross-resistance phenomena, which

is why the practice of using ZnO for weaners is prohibited in some European countries, and is prone

to be banned in the EU entirely. Nevertheless, in the quest for alternatives to antibiotics for preventing

or treating PWD, there may be important lessons to learn from the ZnO-based mechanisms involved

in stabilising the gut environment of piglets in connection with the weaning period.

The exact modes of action in relation to physiology of pigs for the effects of feeding pharmacological

levels of Zn have not been fully elucidated. The effect has, however, been connected with improved

feed intake by piglets (Broom et al., 2003); (Hahn and Baker, 1993), a crucial response parameter for

all feed supplements aiming to improve the health and performance of weaners (Campbell et al.,

2013), as well as with an improved physiological Zn status (plasma Zn level) of piglets. It has thus

been suggested that Zn as well as copper additives may prevent the development of a physiological

Zn deficiency (Carlson et al., 1999; Carlson, 2003); (Hill et al., 2001); (Poulsen and Larsen, 1995).

Other reports have found changes in some pancreatic enzymes and hormonal status (Hedemann et al.,

2006), (Li et al., 2006), and new studies show that Zn accumulation in the liver or pancreas of weaning

pigs after feeding high dietary ZnO for 4 weeks changed the expression of genes and proteins related

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to energy and amino acid metabolism, but also increased oxidative stress reactions in these organs

(Bondzio et al., 2013); (Pieper et al., 2015).

Reported effects of, in particular, ZnO on the gastrointestinal microbiota point towards the

involvement of non-physiological mechanisms. Højberg et al. (Hojberg et al., 2005), using 2,500

mg/kg Zn as ZnO, showed a reduced bacterial activity in the digesta from the gastrointestinal tract

compared with that in animals receiving 100 mg/kg ZnO, most likely reflecting a reduced load of

bacteria present in the gastrointestinal tract of animals receiving the high ZnO level. Data like these

indicate a major influence of ZnO dose on the gastrointestinal tract microbiota and show that, besides

a potential promotion of feed intake, high dietary ZnO doses may further render more energy

available for the host animal by generally suppressing the commensal gut microbiota. This has

actually also been suggested as one of the working mechanisms behind the effect of antibiotic growth

promoters (Collier et al., 2003); (Gaskins et al., 2002). It should be emphasised, though, that it might

not only be a question of energy availability for the host animal. Commensal bacteria, like lactic acid

bacteria and certain clostridia, may also utilise essential feed components, such as amino acids, and

may impair lipid/fat digestibility by hydrolysing bile salts (Knarreborg et al., 2002a; Knarreborg et

al., 2002b).

A decrease in the total number of anaerobes and lactobacilli and an increase in the number of

coliforms have been observed in ileum samples from animals fed a ZnO-amended diet (H›jberg et

al., 2005); (Jensen, 1987). Thus, a direct inhibition of potential pathogens, as represented by

enterobacteria or coliforms, has often not been reported in post-weaning piglets receiving high ZnO

levels (Jensen, 1987); (Hojberg et al., 2005); (Broom et al., 2006); (Vahjen et al., 2010), even though

ZnO has been shown to reduce the susceptibility of pigs to

E. coli

infection (Mores et al., 1998). High

doses of dietary ZnO have, however, been shown to support a large diversity of

coliforms/enterobacteria in weaned piglets (Katouli et al., 1999), (Vahjen et al., 2011). Thus, more

specific modes of action than growth inhibition of an entire bacterial group may apply, and the impact

of dietary ZnO on the diversity of the coliform community may prevent the blooming of, e.g., specific

pathogenic ETEC strains. Indeed,

in vitro

studies have shown that members of different bacterial

groups, commensals as well as pathogens, show a wide range of sensitivity to ZnO (Liedtke and

Vahjen, 2012). Thus, the growth inhibiting effect in vivo may be governed by a more selective growth

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inhibition, i.e., the presence of sensitive and resistant species or strains within the different microbial

genera.

A recent study by Vahjen et al. (Vahjen et al., 2016)demonstrated that the level of specific

E. coli

toxin genes was actually decreased by a high ZnO dose (3000 ppm). However, this study also

demonstrated a more detailed time lapse, where the

E. coli

was generally inhibited from 3-4 days

after weaning, concomitantly with an increase in the faecal ZnO level, demonstrating that the piglets

had started to eat. However, the

E. coli

numbers started to increase again 8-14 days after weaning:

the period on which many published studies have focused. This scenario may indicate an adaptation

E. coli

to ZnO.

Dietary doses of CuSO

(at 175 mg/kg) inhibited the coliforms, and thus potential pathogens as well,

but overall the observed effect of CuSO

was limited compared to that of ZnO. In studies with both

high levels of Zn and high levels of Cu, both were efficacious individually in terms of growth

promotion, but they were not additive when they were added in combination in diets for weaning

pigs. In the study by Poulsen (Poulsen and Larsen, 1995), the severity of diarrhoea seemed to be more

pronounced among litters fed high copper (at high levels of zinc), although it should be noted that the

results were confounded with the litter number. Hill et al. (Hill et al., 2001) reported that the

improvements in performance with high Zn levels could be additive to antibiotics.

It may be concluded that it remains to be answered in detail how the growth-promoting and diarrhoea-

reducing effects of excess dietary Zn are exerted. However, it seems that high dietary ZnO has a

positive impact on the stability and diversity of the gastrointestinal microbiota, which contributes to

an increased colonization resistance against pathogens, and thereby resistance to diarrhoeal

infections, as well as better growth performance of the piglets (Katouli et al., 1999); (Vahjen et al.,

2011); (Pieper et al., 2012).

7.2. Interventions to limit predisposition to PWD

Until 2006, in-feed antibiotics (such as Zincbacitracin, Virginiamycin and Salinomycin) have been

frequently used in pig production to promote growth performance, and their mode of action has been

reviewed by Jensen (Jensen, 1998). Several reviews have been conducted with the aim to present

alternatives to in-feed antibiotics on health and growth, and these have included reports on feeding-

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and management strategies, and the use of feed additives (see for instance (Pluske, 2013); (Halas et

al., 2007); (Heo et al., 2013); (Papatsiros et al., 2013)).As a consequence of the ban on the use of

growth-promoting antibiotics, as well as a general reduction of the prophylactic and therapeutic use

of antibiotics in the swine production, high doses of dietary ZnO are used in pig nutrition. In the

present section, it should be emphasised that, although we have initially focused the aim of our

knowledge synthesis on identifying alternatives to antibiotics and high levels of zinc for prevention

of diarrhoea in pigs post-weaning, we need to stress that the modes of action by which antibiotics and

high doses of zinc are functioning are not exactly clear, and knowledge on this would form the basis

for more efficient selection of alternative in future research.

7.2.1. Nutrition

Apart from pharmacological levels of ZnO, some feeds and feed ingredients have been suggested to

possess immunomodulatory and antimicrobial properties. In a reviewed by Sugiharto et al. (Sugiharto

et al., 2015a), dairy-based products gained increasing interest as a potential nutritional tool to prevent

piglets from developing diarrhoea due to ETEC. Bovine colostrum, milk and milk fractions such as

whey and casein contain several bioactive components with antimicrobial and immunomodulatory

properties, but the knowledge related to the application of the dairy-based products to prevent ETEC

infection and post-weaning diarrhoea is very limited. Recently, a review was conducted on the

immunomodulating effects of probiotics, prebiotics and synbiotics for pig gut health, but so far only

a limited number of studies are available in which the clinical end-point has been diarrhoea score

(Roselli et al., 2017). Likewise, micronutrients, such as vitamins (E, D, and C) and some trace

elements (selenium), confer gut epithelial protection due to their potent anti-inflammatory and anti-

oxidant capacities (Lauridsen, 2010); (Dalgaard et al., 2017). Plant extracts and essential oils have

been investigated for their use in animal nutrition, in particular for exploiting their antimicrobial, anti-

inflammatory, anti-oxidative and anti-parasite properties (Namkung et al., 2004); (Brambilla and De

Filippis, 2005); (Costa et al., 2007); (Liu et al., 2008); (Magi et al., 2006). The addition of plant-based

bioactive additives to pig feed as a possible way to inhibit or reduce the growth of gastrointestinal

pathogens has been reported both

in vivo

and

in vitro

in several studies (Pluske, 2013), (Roca et al.,

2014), (Bontempo et al., 2014), (Michiels et al., 2012). However, in vivo effects of the additives are

sparse, probably because the concentration of antibacterial compounds in the raw material is often

too low to control bacteria

in vivo.

Further, sensory accept of feed additives by piglets may be a

challenge if effective concentrations are too high. However, while widely researched in other animal

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species and humans in relation to enteric diseases, further research is needed to elucidate the

effectiveness of these feed ingredients and micronutrients (as well as dairy-based products, pro- and

synbiotics, plant extracts, essential oils) to prevent ETEC infections and PWD in piglets before being

considered as strategies to the prevention of PWD. Among the more promising strategies, we will

consider the potential effect of reducing the dietary crude protein level; the supplementation of

organic acids; the use of fermented liquid feed; and the use of dietary antibodies as described in the

following.

7.2.1.1. Restriction of protein level

There is a considerable body of research dating back to the 1950s and 1960s which has implicated an

association between protein level and diarrhoea after weaning (Pluske, 2013), and as also stated in

their review (Kil and Stein, 2010), crude protein is the most important nutrient associated with

digestive disorders in the pig. Upon consumption of a meal, pH in the stomach is increased whereby

proliferating of

E. coli

is facilitated. In addition, feed ingredients such as soya bean meal, fishmeal

and milk powder, which are typically used in feed for weaned piglets, also have a high buffering

capacity which can also increase stomach pH and thereby limit pepsin activity (Manners, 1976).

Hence, excessive crude protein intake by weaned pigs can lead to an increase in microbial

fermentation of undigested protein, and this is a contributing factor to development of PWD. In

Table

we have listed examples of studies in which reduction of the dietary protein level influenced the

diarrhoea incidence in piglets post-weaning. Hence, one of our recommended alternative

interventions to limit diarrhoea incidence is the use of low-protein diets, i.e. containing less than 18%

crude protein. This strategy is not without challenges because reduction of the concentration of crude

protein for weaned pigs may influence the growth performance because some of the indispensable

amino acids may be present in concentrations below the requirement for maximum growth. However,

the loss in growth of piglets on low-protein diets in comparison with piglets on diets with a normal

protein content may be compensated on the longer term, as the lifetime performance in some studies

did not differentiate (Wellock et al., 2009); (Kim et al., 2011b). The use of crystalline amino acids

(lysine, methionine, threonine, tryptophan, isoleucine and valine) in low-protein diets to maintain the

balance of the required amino acids for the weanling pigs and thereby pig performance (e.g. (Lordelo

et al., 2008)). This approach is not a feasible approach in organic pig production because the use of

synthetic amino acids is not allowed in organic feed for livestock (EC 889/2008), and one of the major

problems in the formulation of feed for organic pigs is the lack of high quality protein sources to meet

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the amino acid requirements to replace soya bean products, (EGTOP, 2015). Use of lysine,

methionine and threonine is commonly used in the conventional pig feed production, but crystalline

sources of tryptophan, isoleucine and valine are, however, very expensive. It is beyond the scope of

this review to propose nutritional strategies for maximising growth performance while limiting crude

protein, but recently published research is available (e.g. (Almeida et al., 2017). In this context, it

should be mentioned that the study by (Kim et al., 2011b) showed no negative impact on lifetime

performance or carcass characteristics of pigs provided low-protein diets without crystalline essential

amino acids but it reduced the clinical expression of PWD. Hence, a strategy of using low-protein

diets to contribute to a limitation of the development of PWD should encourage to further research

and economic evaluation.

7.2.1.2. Use of dietary antibodies

Passive immunisation, i.e. the administration of antibodies (immunoglobulins) in order to protect

against infection, is an old idea and seems to constitute a real and widely applicable alternative to

antibiotics in modern animal production (Hedegaard and Heegaard, 2016)and also in prevention of

PWD in pigs. A good example is the administration of colostrum feed supplements in which

antibodies derived from the dam provide protection against infectious agents in the suckling offspring

which underline the role of colostrum provision in the disease protection (Theil et al., 2014). Recently,

passive immunisation using chicken egg yolk immunoglobulin (Igγ) has become an attractive

approach with considerable attention, as it possesses a variety of advantages over mammalian IgG

such as convenience, high yield and cost-effectiveness. Oral administration

of specific chicken Igγ

has been shown to be effective against a variety of intestinal pathogens, especially diarrhoeal

pathogens, including ETEC. Based on a systematic review and meta-analysis, Diraviyam et al.

(Diraviyam et al., 2014) demonstrated beneficial

effects of Igγ in controlling and preventing the

diarrhoea in domesticated animals, hence supporting the opinion that Igγ is useful for prophylaxis

and treatment of gastrointestinal infection by oral immunisation as an alternative strategy to

antibiotics.

Use of blood plasma for prevention of PWD has received interest, especially in countries using an

early weaning age. Besides being an easy-digestible protein source with a good amino acid profile,

the content of immunoglobulins is also considered to enhance the immunity of pigs. Use of 5% blood

plasma (DAKA Porcine Plasma) in comparison with addition of 2500 ppm Zn to pigs weaned at a

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weight below 9 kg in a Danish trial showed a positive influence on productivity and a reduction in

diarrhoea treatments (Maribo, 2009).

However,

Danish

Pig

Production,

SEGES

recommends

not

use

blood

plasma

(http://svineproduktion.dk/aktuelt/nyheder/2015/11/121115_kontrol-med-blodplasma-bliver-en-del-

af-danish-ordningen) because of the risk for porcine epidemic virus diarrhoea. Results of a study with

dietary specific antibodies in spray dried plasma powder showed protection against ETEC, and in F4

receptor positive piglets, the spray dried immune plasma powder reduced diarrhoea and decreased

ETEC excretion (Niewold et al., 2007). In an experimental model for PWD, we recently investigated

the oral administration of purified porcine immunoglobulin G from pooled natural pig plasma on

enteric infection. We observed that the oral immunoglobulin product accelerated clearance of feacal

hemolytic bacteria in pigs challenged with

E. coli

in comparison with pigs not receiving the

immunoglobulin product (Hedegaard et al., 2017). The effect of this dietary intervention, which was

observed during the seven days post-weaning, was equal to or better than that of dietary ZnO in

reducing diarrhoea symptoms and in clearing faecal hemolytic bacteria for 14 days post-weaning.

Thus, provision of dietary antibodies based on blood plasma and eggs to pigs seem to be a promising

alternative strategy to reduce the use of antibiotics and ZnO. The observations listed above warrant

future experiments to investigate the efficacy in prevention of PWD. However, the products are not

yet commercially available in Denmark, and will therefore not be suggested for economic evaluation..

7.2.1.3. Organic acids

A number of reviews can be found in the literature dealing with feeding strategies to control post-

weaning diarrhoea in pigs, in which addition of organic acids to the diet is considered as a valid one

(Mroz, 2001); (Metzler et al., 2005); (Heo et al., 2013); (Vondruskova et al., 2010)

Organic acids have been used for decades in feed preservation, protecting feed from microbial and

fungal destruction or to increase the preservation effect of fermented feed, e.g. silages. In particular

formic acid and propionic acid have been used extensively for this purpose.

A number of studies with piglets have shown that several organic acids have a positive influence on

growth performance (reviewed by (Partanen and Mroz, 1999); (Maribo, 2014). Some studies have

shown an impact on post-weaning diarrhoea/faecal score (e.g., (Maribo et al., 2000a); (Johansen,

2007); (Tsiloyiannis et al., 2001); (Callegari et al., 2016); (Partanen et al., 2007). As described above,

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high diarrhoea incidence at weaning is triggered by a number of factors. An insufficient production

of hydrochloric acid and digestive enzymes, and the feeding of pre-starter diets with high protein

content and high acid binding capacity are some of the factors made responsible for problems at

weaning. Organic acids are believed to have their beneficial impact of piglet diarrhoea through

improving some of these aspects.

Single or mixtures of acids and/or their salts have been used in piglet diets, i.e., formic, acetic,

propionic, butyric, lactic, sorbic, fumaric, malic, tartaric, citric, and benzoic acid. Organic acids

together with other additives, like essential oils, herbs or phosphoric acid have also been used.

Mode of action

Although several hypotheses have been proposed, the exact mode of action behind the positive effects

of organic acids on growth performance and diarrhoea frequency in piglets has not been established.

The proposed mechanisms behind the positive effects of organic acids in weaners are related to the

gastrointestinal microbiota, and nutritional aspects:

•

Antimicrobial activity. The antimicrobial activity of organic acids can be due to a pH reduction

and/or to a more direct antibacterial effect. Different aspects of the working mechanism of organic

acids with respect to their antibacterial activity are given in the review articles of Cherrington

al.

(Cherrington et al., 1991)and Russell (Russel, 1992). The antibacterial activity of organic acids

is related to the reduction of pH, as well as their ability to dissociate, which is determined by the

-value of the respective acid, and the pH of the surrounding milieu. The antibacterial activity

increases with decreasing pH-value. Organic acids are lipid soluble in the undissociated form, in

which they are able to enter the microbial cell. However, carrier-mediated transport mechanisms

seem to be also involved in the membrane transport. Once in the cell, the acid releases the proton

in the more alkaline environment, resulting in a decrease of intracellular pH. This influences

microbial metabolism inhibiting the action of important microbial enzymes and forces the bacterial

cell to use energy to release protons, leading to an intracellular accumulation of acid anions. This

accumulation depends on the pH gradient across the membrane. The acid anion seems to be very

important regarding the antibacterial effect of organic acids and their salts. Several investigations

have shown a strong bactericidal effect of organic acid without significantly decreasing the pH-

value in the GI-tract. Generally lactic acid bacteria are able to grow at relatively low pH, which

means that they are more resistant to organic acids than other bacterial species, e. g.

E. coli.

explanation for this may be that gram-positive bacteria have a high intracellular potassium

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concentration, which provides a counteraction for the acid anions (Russell and Diez-Gonzalez,

1997).

A reduction in the bacteria numbers in the proximal GI-tract would both lower the risk of infections

by inhibiting pathogenic bacteria proliferation, but also reduce the microbial competition with the

pig for nutrients.

The in vitro antimicrobial activity of various organic acids used in piglet nutrition has been

demonstrated (Knarreborg et al., 2002c); (Naughton and Jensen, 2001). (Knarreborg et al., 2002c)

testing the impact of various organic acids on growth/death of coliform bacteria in stomach

contents observed differences among the acids in the following order (from more to less

antibacterial): benzoic acid > fumaric acid > lactic acid > butyric acid > formic acid > propionic

acid. When testing the impact in small intestine content, only benzoic acid, formic acid and butyric

acid showed a bactericidal effect. Benzoic acid was superior to the other acids tested in exhibiting

a bactericidal effect on coliform as well as lactic acid bacteria in both stomach and small intestinal

content. Potasium diformate reduced the number of coliforms in stomach and small intestine

contents, too. In similar experiments carried out with

Salmonella typhimurium

in stomach content,

it was found that the bactericidal effect of organic acids decreased in the following order: benzoic

acid > sorbic acid > lactic acid > propionic acid > formic acid > acetic acid (Naughton and Jensen,

•

Increased proteolytic activity. Dietary acidification (reduced buffering capacity) with organic

acids lowers gastric pH, resulting in increased activity of proteolytic enzymes and gastric retention

time. Pepsinogen is converted to pepsin, and pepsin is more active at low pH. Due to the lower

rate of gastric emptying, large protein molecules may be better hydrolysed, which will have a

•

Increased pancreatic secretions. Dietary supplementation with organic acids can stimulate

endocrine and exocrine pancreatic secretion. Intestinal acidification increases the contents of

serum secretion, which stimulates exocrine pancreatic secretion and bile secretion (Partanen and

Mroz, 1999). Thaela

et al.

(Thaela et al., 1998) showed that supplementation of 2.5% lactic acid

in the diets of weaning pigs increased the volume and protein content of pancreatic juice, as well

as the secretion of trypsin and chymotrypsin. However, the amount of bicarbonate produced in the

animal intestine was not affected by the supplementation with lactic acid. Therefore, according to

the authors, it is unlikely that the stimulation of pancreatic secretion was caused by decreased

beneficial effect on protein digestion.

2001).

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dietary and gastric pH, but probably by the direct action of the acid (Thaela et al., 1998); (Costa et

•

Improved physiology of the intestinal mucosa. Organic acids may act on the villi, maintaining

their integrity, promoting an increase in the number of cells and preventing its flattening, as well

as serving as a substrate in the intermediary metabolism of the citric acid-cycle (Partanen and

Mroz, 1999). In (Costa et al., 2013), 54). This effect is more proved regarding SCFA, whereas

little is known on the impact of dietary acidification on pig gut morphology (Partanen and Mroz,

•

Organic acids can serve as substrates in the intermediary metabolism. Kirchgessner & Roth

(Kirchgessner and Roth, 1988) have proposed that organic acids may stimulate intermediary

•

Organic acids may improve the absorption of minerals, particularly Ca and P. Further, organic

acids also appear to influence the retention of minerals (Partanen and Mroz, 1999)

Where do organic acids have their action?

As mentioned above, organic acids exert their antimicrobial action both in the feed and in the GI-tract

of the animal. Following dietary intake, organic acids are only recovered from the proximal part of

the pigs GI-tract (stomach and small intestine). This is in agreement with observations that the

strongest effect of organic acids with respect to digesta pH and antimicrobial activity are found in the

stomach and the small intestine.

Impact on diarrhoea frequency in piglets

A compilation of studies conducted by SEGES, Denmark, in which the impact of dietary organic acid

addition on diarrhoea frequency, or more specifically, on the treatment frequency against diarrhoea,

were reported is shown in

Table 2.

A total of 35 studies are presented, in which several acids and

mixtures of acids, or mixtures of acids and other additives were tested.

Only two studies showed a significant impact of the added organic acids on frequency of treatment

for diarrhoea: a study in which feed added 2% benzoic acid was fed during two weeks from weaning

at 4 weeks, followed by offering feed added 1% benzoic acid the next 4 weeks (Maribo et al., 2000a);

and a study in which feed added 1% lactic+1% formic+0.5% benzoic was offered during six weeks

from weaning at four weeks of age (Johansen, 2007). In the first study, there was a reduction in the

treatment frequency (indicating the proportion of piglets treated) from 117 in the control group to 60

metabolism resulting in improved energy or protein/amino acid utilization

1999)

al., 2013).

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in the group fed with the diet containing benzoic acid. The number of

Enterobacteriaceae

and lactic

acid bacteria in stomach, small intestine, caecum and mid-colon was also measured. The number of

Enterobacteriaceae

was not affected by adding benzoic acid, whereas that of lactic acid bacteria was

significantly lower in all segments in the benzoic acid group. In the study of (Johansen, 2007), there

was a reduction from a mean of 8.7 treatment days per piglet in the control group to 6.9 in the 2.5%

organic acid group. Furthermore, one study showed tendency to reduction of treatment frequency

((Maribo, 1998). Adding 0.2% of a product containing a mixture of lactic, formic, citric, and

propionic acid tended (P = 0.09) to reduce the treatment frequency from 44% in the control group to

3.2% in the acid-added group.

It has to be emphasized that in these studies, the impact of the organic acids on treatment against post-

weaning diarrhoea was measured as a secondary response parameter, that is, the studies were not

dimensioned to test this effect. A high number of animals would have been needed in order to

appropriately test this hypothesis. Furthermore, it has also to be kept in mind that the dose of the acids

reported in the table is that of the product used not of the pure acid, which means that the dose of the

pure acid is lower and varies somewhat from product to product.

A review on data on the effect of organic acid addition to the feed on piglet growth performance

obtained in the same studies shown in Table 1 reported that out of 58 studies, 19 showed significant

positive results (Maribo, 2014). The author concluded that, in general, inclusion of minimum 1% pure

acid in the feed is needed to give significant positive impact on growth performance.

A summary of results from studies testing the impact of dietary organic acids on faecal score or

diarrhoea frequency from the international literature is shown in

Table 3.

Here 22 studies are

presented, of which eight reported a significant beneficial impact of dietary organic acid addition.

Moreover, one study testing the impact of benzoic acid and thymol addition showed a tendency (P =

0.08) to a faecal score compared to a control diet (Diao et al., 2015). Another study (Partanen et al.,

2002) showed a tendency to a higher incidence of post-weaning diarrhoea in piglets fed 0.8% calcium

formate. The studies showing significant effects tested both mixtures of acids or acids/salts alone,

and included a number of different organic acids. The acid inclusion level tested in the studies

showing significant reduction of diarrhoea varied too, from as low as 0.1% to 1.6%. A few studies

reported the number of coliforms and lactic acid bacteria in the GI-tract or faeces, but these values

did not always follow the results on faeces scores.

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The relatively low number of studies showing significant impact on diarrhoea frequency/faecal score

can be due to a real lack of effect of the products but also to the set-up of the studies. In order to see

an effect of feeding organic acids (or any other additive or feeding strategy) on diarrhoea frequency,

a high number of replicates is needed. Many studies in the literature testing the impact of organic

acids on weaners have mainly focussed on effects on growth performance, digestibility of nutrients,

and microbiota composition and their metabolites in the GI-tract, and not on diarrhoea as a main

parameter (reviewed by (Partanen and Mroz, 1999) ; (Canibe et al., 2001); (Vondruskova et al., 2010);

(Suiryanrayna and Ramana, 2015); (Costa et al., 2013); (Metzler et al., 2005); (Maribo, 2014)The

impact of the acids on the number of coliform bacteria in the GI-tract or faeces has been used as an

indicator of their potential to reduce the risk of post-weaning diarrhoea (the fewer coliform bacteria,

the lower the risk for post-weaning diarrhoea). However, a direct relationship between these two

parameters has not been proven. Therefore, in order to establish more solidly the impact of addition

of organic acids to the feed on post-weaning diarrhoea, more studies designed to test the impact of

organic acids on this parameter are needed.

7.2.1.4. Fermented liquid feed

Fermented liquid feed is prepared by mixing water or another liquid, e.g., whey, with feed and

incubating the mixture for a certain period of time, at a certain temperature. During the initial hours

of fermentation, a blooming of coliform bacteria is typically observed followed by an increase in the

number of lactic acid bacteria, a reduced pH level, and, as a consequence, a reduced number of

coliform bacteria (Canibe and Jensen, 2012).

Several studies have shown that feeding piglets with FLF of good microbial quality, that is, with high

lactic acid bacteria counts (around 9 log cfu/g), low pH (around 4,5), and low counts of

Enterobacteriaceae (around 3 log cfu/g), reduces the number of coliforms along the GI-tract or in

faeces compared to feeding non-FLF or dry feed (Mikkelsen and Jensen, 2000); (Moran, 2001); (van

Winsen et al., 2001a; van Winsen et al., 2002; Hong et al., 2009). On the other hand, feeding non-

FLF (in practise, partially fermented) containing high numbers of coliforms, increases the number of

coliforms along the GI-tract of growing pigs (Canibe and Jensen, 2003). Therefore, animals fed with

partially FLF of suboptimal microbial quality will harbour higher numbers of coliforms in their GI-

tract, what could increase the risk of diarrhoea incidence.

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The main factors considered responsible for the bactericidal activity of FLF against

Enterobacteriaceae are the low pH and high concentration of lactic acid observed in the FLF and in

the stomach of the animals fed with FLF(van Winsen et al., 2001b); (Canibe and Jensen, 2003). Both

factors are known to reduce the survival of

Enterobacteriaceae

to a great extent, greater than that of

lactic acid bacteria (Knarreborg et al., 2002c).

Because diarrhoeic pigs harbour higher numbers of coliforms in the GI-tract than healthy subjects, a

reduction in the number of coliforms along the GI-tract itself by feeding FLF is considered as

indicator of a lower risk of post-weaning diarrhoea. However, to establish whether feeding FLF does

reduce post-weaning diarrhoea in piglets, studies designed to test this parameter showing an effect

are needed. Unfortunately, there are very few studies in the literature reporting this.

Six Danish studies (Table

have reported treatment for post-weaning diarrhoea in piglets fed with

FLF as compared to those fed dry feed or non-FLF. None of the studies showed an impact of feeding

FLF on this parameter. It is important to notice, though, that the incidence of diarrhoea in most of

these studies was very low, what makes detection of an improvement very difficult to achieve. In two

of the studies (Pedersen, 2011); (Maribo, 2012), the piglets received 2500 Zn in the feed the first two

weeks post-weaning, what is considered to reduce diarrhoea frequency.

Data on the impact of FLF on post-weaning diarrhoea reported from studies published in the

international peer-reviewed literature are shown in

Table 5.

All three studies are characterized by a

very low number of replicates, and two of them are challenge studies, in which animals were

challenged with

E. coli

O149:F4. Only one study showed some significant effects of feeding

fermented liquid whey as compared to non-fermented whey, i.e., a reduced number of pigs with

diarrhoea two days out of 12 days measured.

In summary, very few published studies have been designed to test the impact of FLF on post-weaning

diarrhoea, therefore, there is no basis to conclude whether feeding FLF reduces post-weaning

diarrhoea in piglets.

7.2.2. Management

A recent study had the hypothesis that well-managed 7-30 kg (weaners) productions had a set of

common keyfactors characterizing their management practices (Fertner et al., 2015). There was a

wide variation between the farmers regarding their perception concerning which management

parameters were the reasons for success in terms of low mortality, high daily weight gain and limited

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use of antimicrobials (Fertner et al., 2015). In this descriptive study, 10 out of the 11 Danish weaner

producing farms used zinc the first 2 weeks post-weaning. Although this study may enhance our

knowledge on the contribution of management parameters of importance for prevention of PWD, the

results may not be extrapolated to farms not using pharmacological levels of ZnO post-weaning.

While neonatal infection with

E. coli

can be prevented effectively by passive lactogenic immunity

obtained by vaccination of the sow, vaccines for prevention of infection with ETEC post-weaning

remain to be studied further (Melkebeek et al., 2013). This is not yet considered an efficient solution

in Denmark, either because the vaccines are not available commercially or because the use of oral

vaccines based on live bacteria for oro-gastric protection has several limitations as listed by

Hedegaard and Heegaard (Hedegaard and Heegaard, 2016) – one of the reasons being the lactogenic

protection at weaning. It should be noted, however, that oral immunisation of weaned piglets with

purified F4 fimbriae induced a protective mucosal immune response (Verdonck et al., 2007), and that

active immunizsation against the shiga-toxin producing

E. Coli

is possible (Ecoporc Shiga) thus

preventing oedema disease. An orally provided F4 fimbria subunit vaccine was shown to be able to

induce protection against F4 positive ETEC in an experimental model of PWD (Van den Broeck et

al., 1999), and in Canada a commercial vaccine (Coliprotec

) for oral use against PWD has been

marketed for some years (Melkebeek et al., 2013) and has also been approved for the European

market. So far, efficacy data on this vaccine is not available (Hedegaard and Heegaard, 2016). Our

focus in relation to management factors in the present paper has therefore been devoted to both ‘host-

specific’ factors ( i.e. the robustness of the pig at weaning in terms of the influence of age and weight),

and to ‘the hygienic level of the rearing environment’ post-weaning (as limiting the presence of ETEC

in the environment may limit the risk of ETEC related diarrhea).

7.2.2.1. Weaning age and weight

Our recommendation is to consider an increase in weaning age for further cost-effectiveness

evaluation.

Weaning at a low age has been shown to be associated with the presence of weaning diarrhoea (Madec

et al., 1998). In this cohort study, pigs above or below a weaning age of 26.5 days were compared,

and odds for diarrhoea was 12 times higher in herds with the low weaning age. In addition, weaning

age below 7.2 kg enhanced odds for diarrhoea 6.5 times compared with pigs weaned at age 8-9 kg

but was almost unchanged compared with pigs weaned at age 7.2-8.1 kg (Madec et al., 1998). A

Danish study compared weaning at 4 weeks (26 days of age) versus 5 weeks (33 days of age) and

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observed more ‘robust’ piglets post-weaning (less mortality and less disease treatments, (Callesen

and Thorup, 2004); 0.5 less diarrhoea treatment days, (Callesen and Thorup, 2005); and less diarrhoea

treatments in two out of the three herds, (Callesen and Thorup, 2006)) (Callesen and Thorup, 2004,

2006);(Thorup

et al., 2006).

It remains to be elucidated how weaning weight is related to the incidence of PWD, as most of the studies

included in the previous sections have been performed with indication of weaning age but not indication

of weaning weight. In practice, pigs of different litters are often mixed and moved around during the

suckling period to obtain a homogenous litter at weaning with regard to weaning weight. It should be

underlined that the weight of piglets at weaning does not reflect the age of piglets at weaning (Jensen and

Thodberg, 2013).

As example of the high variation, the following can be mentioned:

the average weight

in three different herds varied from 7.6 kg and 9.5 kg at age 24 and 33 days (Callesen and Thorup, 2004)6.6

kg and 7.9 at age 29 and 35 days (Callesen and Thorup, 2005)7.6 and 9.0 at age 28 and 33 days (Callesen

and Thorup, 2006). In the study by Thompson (Thomsson, 2008)it

was demonstrated that the mixing

and moving procedure of pigs of different relative size (small, medium and large) resulted in lower

BW gain compared to that of pigs weaned as whole litters, which was most pronounced in small pigs.

Mixed and moved pigs also had lower feed intakes and higher diarrhoea scores (small and large pigs).

It should also be mentioned that although pigs in organic production systems are weaned at 7 weeks of

age, at which age they are more mature and have a more stable microbiota, PWD is also seen in these

herds (however, the infectious aetiology may differ from the aetiology in conventional productions).

However, in organic production systems in which a weaning age of 10 or even 12 weeks is practiced

(personal communication Charlotte Lauridsen) it seems like pigs are less prone to PWD. This information

from practice may be partly supported by our preliminary results showing that pigs staying with the sow

until 10 weeks seem to be more robust than pigs being weaned at 7 weeks of age, whereas microbiota of

7 and 10 weeks old pigs were rather similar.

7.2.2.2. Weaning environment

Practical recommendation regarding the weaning environment is to clean the rearing area with soap

and to dry itout thoroughly between each batch of weaned pigs (personal communication Hanne

Kongsted). The hygiene level is a factor in a lot of epidemiological studies, and there is a tendency

to a reduced incidence of weaning diarrhoea (Laine et al., 2008)and diarrhoea between 15-40 kg

(Chase-Topping et al., 2007)in herds with strict hygiene procedures. In addition, single studies have

demonstrated a statistically significant relationship between hygiene status and diarrhoea incidence

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(Madec et al., 1998); (Pearce, 1999). In a descriptive study including 11 Danish herds characterised

by having a low antimicrobial usage, all herds had also a high hygiene level (Fertner et al., 2015).

Although there is a lack of systematic research on which of the factors included in the weaning

environment are the most important for predisposing pigs to PWD, our recommendation would be to

ensure that pigs are weaned to an environment with a high hygiene level, because proper cleaning

and drying would limit exposure to ETEC for the newly weaned pig.

8. Conclusion and Perspective

Several reviews have been conducted with the aim to present alternatives to in-feed antibiotics on

health and on growth of pigs, and these have included reports on feeding, nutrition and management

strategies and use of feed additives as well as breeding strategies. However, the intervention studies

have focused mainly on proxy measurements, i.e. indicators of gut health and/or parameters

indicating mode of action and the relation to gut health and/or the pathogenesis of ETEC. Attachment

of pathogenic bacteria to host tissues is a critical early step in the development of disease, including

ETEC associated post-weaning diarrhoea, but it may not necessarily lead to PWD. Likewise, it is a

critical point that although PWD would often impair growth of the pigs, measurable reduction of

growth or performance is not an indicator of PWD. The reason why several studies have included

proxy measurements rather than the clinical endpoint diarrhoea is probably that it is a challenging

disease to research. It requires either large trials, including many replicates, and/or inoculation

experiments, which are very laborious and require a special permission. An objective of our present

knowledge synthesis was to propose alternative strategies for prevention and treatment which could

be considered relevant for further studies in a cost-effectiveness analysis. Since very few studies have

actually demonstrated the efficacy of the given alternative intervention or their potential efficacy in

relation to incidence PWD, our review limited the available potential interventions. In conclusion,

this review pointed out that the following factors could limit the predisposition to PWD and as such

could be relevant to consider in a cost-effectiveness analysis for reducing the usage of zinc and

antimicrobials: restriction of dietary protein, increase in weaning age, and optimisation of the

hygienic status of the environment for the pigs post-weaning. Besides, there were a few more dietary

factors which gained our special interest in terms of their potential for limiting PWD in pigs, i.e.

organic acids, fermented liquid feed and antibodies. More studies designed to test the impact of the

dietary intervention on diarrhoea as primary parameters are needed.

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Table 1. Examples of studies showing effect of dietary protein level on post-weaning diarrhoea

Diarrhoea

female

pigs

weaned

infection/non-

infection with E. coli

0149.

diet

contained

antimicrobials

Protein lev

High (256 g CP/kg for

14 d pw), low (175 g

CP/kg for 7 d pw), low

(175 g CP/kg for 14 d).

LP diets were fortified

with crystalline Ile and

VAL to obtain ideal AA

pattern. From 14-28 d

all pigs were fed 213

g/kg

32 piglets, weaned at Control (167 g (20.1%

weeks, CP), and low (94 g

infection/non-

(12.0%)

digestible

infection with E. coli protein pr.kg

0149

diet

contained

antimicrobials.

150 pigs weaned at 3 Control diet of 17% CP

weeks, 5 treatments, (control); 2) 19% CP

no antibiotics

diets formulated with

more soy protein

concentrate (SPC19);

3) fish meal (FM19); 4)

23.7%

diets

formulated with more

soy

protein

concentrate (SPC23);

5) fish meal (FM23).

Measurement

Other parameters

Diarrhoea incidence Protein fermentation

(3 point scale).

Growth, feed intake

Shedding

β-

hemolytic

E.coli

Results

Reduction of diarrhea Heo et al. 2009

incidence (number of

days with diarrhoea

out of 14 days), and

increase in feacal DM

with LP diet compared

with HP.

Diarrhoea incidence Growth, feed intake

(6 point scale). DM.

Shedding

β-

hemolytic

E.coli

Protein

restriction Sørensen et al. 2009

decreased

feacal

score and increased

feacal DM, and weight

gain

tended

decrease.

Significantly

less Wu et al., 2015

diarrhoea incidence in

pigs on control (Low

protein).

conclusion, the 17%

CP diet without in-

feed

antibiotics

helped

improve

growth performance

and relief of diarrhoea

of 21 to 35 d-old

weaned

piglets.

Dietary CP level,

rather than its source

Diarrhoea incidence Morphology,

(4 point scale): Fecal inflammation,

consistency

was

assessed visually and

classified at 4 levels:

0, normal; 1, pasty; 2,

semi-liquid; and 3,

liquid.

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

4 groups: 2 LP Diarrhea treatment

treatments (18% CP,

130

digestible

protein/FEs) 2 HP (21%

CP, 155 % g digestible

protein/Fes).

Diets

were

based

‘expensive’

and

‘cheap’

feed

ingredients.

200 individually fed High

Diarrhea incidence

pigs, experimentally protein + antimicrobial

infected with

E. coli

F4 compound

diet

(HP + AMC,

230 g

crude protein (CP) with

2.5 g lincospectin and

3 g ZnO per kg feed),

High protein diet (HP,

230 g CP/kg), Reduced

protein + amino acid

supplemented

diet

(RP + AA, 185 g CP/kg

with added CEAA up to

Test in 2 herds (8272

pigs in total) with high

presence of

E. coli

(0138 eller 0149).

Piglets were weaned

at 28 days

Performance,

Mortality

(either fish meal or

soy

protein

concentrate),

has

significant

impacts

the

growth performance

and intestinal health

of 21 to 35 d-old

weaned piglets when

fed antibiotics-free

diets

Treatment

for Callesen

diarrhea was less in Johansen, 2006

LP-pigs (1.4 days)

corresponding

25% reduction in

treatment

frequency. But daily

gain was 28 g lower,

and pigs weighed 0.6

kg less after 3 weeks.

Pigs fed the HP diet Kim et al 2011

showed an increased

faecal score (P<0.05–

0.001),

diarrhoea

index (P<0.001), and

mean number of

therapeutic antibiotic

treatments (P<0.001)

compared with pigs

fed other diets.

and

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

HP level), and Reduced

protein diet (RP, 185 g

CP/kg without CEAA

supplementation).

1) In total 4 dietary experiments were included in this study.

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Table 2. Impact of organic acid addition to the feed on post-weaning diarrhoea in piglets (Danish studies).

Starting age Study length

Number of

Ref Treatment

Dose (%)

(d)

(d) pigs/treatment

control

479

fumaric acid

1.5

479

control

116

tylosin

40 ppm

116

etheric oils

0.01

116

fumaric acid, lactic acid, propionic acid, formic acid, citric

0.5

116

acid

formic acid+propionic acid

0.5

116

peppermint, juniper and garlic

0.05

116

control

27/28

34/41

250/163

2500 ppm

27/28

34/41

250/163

formic acid in water

0.2

27/28

34/41

250/163

Replicates

Diarrhoea, %

16.70

8.10

12 NS (values not given)

Faecal coliform bacteria Faecal lactic acid bacteria

(cfu/g)

20.4/3.0 - 38.0/9.8

(without formic acid)

17.5/2.2 - 28.7/27.8

(with formic acid)

164-25

175-13

zn+formic acid in water

control

orto-phosphoric acid, formic acid, acetic acid, propionic

acid, citric acid

tylosin

control

avilamycin

fumaric acid, malic acid, citric acid, ortho-phosphoric acid

herbs, spices, essential oils

herbs

formic acid, lactic acid, free fatty acids

control

tylosin

3 diets P-712, P-140 og P-120

control +methionine analogue

control+ garlic, herbs

control+ ammonium formate, formic acid, lactic acid,

fumaric acid, citric acid

control

avilamycin

formic acid, acetic acid, sorbic acid, propionic acid, lactic

lactic acid, fumaric acid, citric acid, formic acid, propionic

plant extract

etheric oils

control

lactic acid

2500ppm+0.2

1.4/1.2

40 ppm

0.20

0.02

0.5

0.04

40 ppm

27/28

23/26

26/24

34/41

41/37

48/35

250/163

240/178

157

159

152

755

787

1.2/0.7

0.70/0.5

176-13

NS (values not given)

16 NS (values not given)

47.3

12.9

18.1

30.4

11.1

43.1

2.9

40 ppm

0.5

0.05

0.01

5g/dl water;

6.4g/pig/d;

12.8g/pig/d

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Starting age Study length

Number of

(d)

(d) pigs/treatment

157

ca 28

ca. 28

ca 42

ca. 42

420

Ref

Treatment

control

avilamycin

citric acid, ortho-phosphoric acid

lactic acid, formic acid, citric acid, propionic acid

formic acid

formic acid, sodium formate, ortho-phosphoric acid, lactic

acid, citric acid

control

lactic acid

control

methionine hydroxy analogue

propionic acid, formic acid

propionic acid, formic acid, phytase

propionic acid, formic acid

control

Enterococcus faecium

organic acid mix1

organic acid mix2

garlic and cinnamon oil

control

calcium formate

sorbic acid

control

formic acid, lactic acid, phosphoric acid

yeast wall oligosaccharides

control

lactic acid

formic acid

control

phosphoric acid, citric acid, lactic acid, malic acid, tartaric

phosphoric acid, formic acid, lactic acid

ammonium formate, formic acid, lactic acid, fumaric acid,

tartaric acid, malic acid

control

formic acid, ammonium formate, potassium hydroxide

lysine

fructoligosaccharides

control

lactic acid+formic acid

benzoic acid

Dose (%)

40 ppm

0.8

0.2

1.25

0.65

Replicates

Diarrhoea, %

5.3

10.6

3.2*

25.9

0.6

128.3

Faecal coliform bacteria Faecal lactic acid bacteria

(cfu/g)

2.00/1.00

0.6

120 ppm

80 ppm

0.4

0.1/0.05

1.25

2.0

0.3

0.7

0.3/0.15

0.7

1.4

2.8

0.7

1.4

0.2

0.3

0.5

151

148

112

155

4.09

/8.15

/8.44

/8.53

8.31

/8.71

/8.89

/9.62

8.76

/9.21

/9.33

/9.39

3.97

/8.36

/8.28

/8.29

4.32 /7.33 /8.30 /8.21

8.77 /9.16 /9.03 /9.36

abc

4.25 /8.10 /7.33 /7.58

8.61 /8.91 /9.09 /9.20

8.73 /9.13 /9.25 /9.08

3.28 /8.01 /8.00 /8.15

3.62

/7.91

/8.29

/8.39

8.08

/8.80

/8.90

/9.12

0.6/0.5

1.2/1

4.3/3.9

1.25/0.5

0.7+0.7

2.0/1.0

168

117

113

3.12/6.29/6.23/6.56

3.20/5.81/6.05/6.16

3.15/5.13/5.83/5.93

7.75 /8.24 /8.95 /9.24

7.0

/8.27

/8.85

/9.12

5.58

/6.91

/7.74

/7.88

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Number of

Starting age Study length

(d) pigs/treatment

(d)

ca. 28

ca. 42

450

ca. 28

ca. 42

450

ca. 28

ca. 42

450

ca. 28

ca. 42

450

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 28

ca. 42

400

ca. 28

ca. 42

400

ca. 28

ca. 42

400

ca. 28

ca. 42

400

ca. 28

ca. 56

400

ca. 28

ca. 56

400

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 28

ca. 42

165

ca. 56

400

ca. 56

400

ca.28

ca.42

144

ca.28

ca.42

144

ca.28

8.6-13 kg

0.5+0.5

1.00

ca.42

144

226

Ref

Treatment

control

formic acid, phosphoric acid, lactic acid

control

flavours

formic acid

flavours+formic acid

control

formic acid, propionic acid, special lignin

control

lactic acid, phosphoric acid, salts of free fatty acids

control

etheric oils oregano+lactic acid, formic acid, butyric acid

etheric oils oregano

flavours

benzoic acid

control

citric acid

control

lactic acid+formic acid/lactic acid+formic acid

control

lactic+formic+mannan

oligosaccharide/lactic+formic+mannan-oligosaccharide

mannan-oligosaccharide

control

formic acid, sodium formate, phosphoric acid, lactic acid,

citric acid

formic acid, sodium formate, phosphoric acid, lactic acid,

citric acid

formic acid, sodium formate, phosphoric acid, lactic acid,

citric acid

ammoniated formic acid, sodium formate, phosphoric acid,

potassium sorbate, citric acid

control

lactic acid+formic acid

benzoic acid

Dose (%)

1.2/0.6

1.20

1.8/1.2

0.02

0.04

0.7

0.02+0.35

1.0

1.5

2.0

0.3

0.12/0.07+0.4

0.12/0.07

0.2

0.5

4.5/1

0.6+0.6/0.3+0.3

0.3+0.3+0.2/0.15+0

.15+0.05

0.4/0.1

0.3

0.6

0.9

0.6

Replicates

Diarrhoea, %

113

Faecal coliform bacteria Faecal lactic acid bacteria

(cfu/g)

9.6

1.3 d/pig

3.4/7.4

/6.7/7.3

3.4/6.9

/6.8/6.9

3.4/5.1

/6.3/7.0

3.4/6.7 /7.0/7.1

7.3/8.4/8.8/9.1

7.1/8.1/8.7/9.2

7.2/8.0/8.7/8.9

6.8/8.0/8.4/8.9

1.9 d/pig

1.1 d/pig

1 d/pig

0.8 d/pig

0.9 d/pig

0.5 d/pig

3.5/6.6/6.5/6.5

8.0/8.7/9.3/9.3

3.2/6.2/6.4/6.7

8.5/9.2/9.5/9.7

3.3/6.7/7.1/7.2

0.14 d/pig

0.08 d/pig

0.07 d/pig

7.1

8.4/9.1/9.3/9.2

6.5

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Starting age Study length

Number of

(d)

(d) pigs/treatment

8 kg

200

8 kg

200

ca.28

ca.42

ca.28

ca.42

ca.28

ca. 35

7.5

15.0

7.5+0.5

7.3 kg

0.30

180

4.1 d/pig

4.8 d/pig

ca.42

ca. 38

684

Ref

Treatment

control

potasium diformate+0.6% formic acid

control

lactic acid+formic acid+benzoic acid

lactic acid+formic acid+benzoic acid+Zn

control

chicory

chicory+benzoic acid

control

sodium butyrate

Dose (%)

0.6+0.6

1+1+0.5

1+1+0.5+2500 ppm

Replicates

Diarrhoea, %

1.5 d/pig

8.7 d/pig

6.9 d/pig

0.9 d/pig

2.4 d/pig

Faecal coliform bacteria Faecal lactic acid bacteria

(cfu/g)

Diarrhoea is in most cases reported as the frequency of antibiotic treatment against diarrhoea. Otherwise the units are stated.

Diarrhoea values from two farms.

and lactic acid bacteria in stomach, small intestine, caecum and colon.

Coliforms

*p<0.10 with respect to the control treatment.

1) (Jakobsen, 1985); 2) (Jørgensen, 1995); 3) (Jørgensen, 1996); 4) (Callesen, 1997a); 5) (Callesen, 1997b); 6) (Callesen, 1997); 7) (Callesen, 1998); 8) (Jørgensen, 1998); 9) (Maribo, 1998); 10) (Maribo, 1999);

11) (Callesen, 1999); 12) (Maribo and Olsen, 1999a); 13) (Maribo and Olsen, 1999b); 14) (Maribo and L.E., 2000); 15) (Maribo et al., 2000b); 16) (Maribo et al., 2000c); 17) (Hansen, 2000); 18)(Maribo et al.,

2000a); 19) (Maribo, 2000); 20) (Maribo, 2001); 21) (Maribo and Jensen, 2001); 22) (Maribo, 2002); 23) (Maribo, 2003a); 24) (Maribo, 2003); 25) (Maribo, 2003b); 26) (Maribo, 2003c); 27) (Maribo, 2004); 28)

(Jørgensen, 2004); 29) (Maribo, 2007); 30) (Johansen, 2007); 31) (Maribo, 2010); 32) (Jørgensen, 2013).

abc

Values

with different superscript within study are different P < 0.05.

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Table 3. Impact of organic acid addition to the feed on post-weaning diarrhoea in piglets.

Ref

Treatments

control

HCl

fumaric acid

sodium formate

tylosin

B. cereus Toyoi

control

sodium hydrogencarbonate

calcium formate

calcium formate+sodium hydrogencarbonate

formic acid

formic acid+sodium hydrogencarbonate

control

sorbic acid

control

formic acid

diformate product A

diformate product B

control_low energy

potassium diformate_low energy

control_high energy

potassium diformate_high energy

control_low energy

potassium diformate_low energy

control_high energy

potassium diformate_high energy

control barley

potassium diformate barley

control wheat

potassium diformate wheat

control corn

potassium diformate corn

control mix

potassium diformate mix

control

lincomycin+spectinomycin

propionic acid

lactic acid

formic acid

malic acid

citric acid

fumaric acid

Dose (%)

1.4

1.8

40 ppm

0.5x10

cfu/g

1.8

1.8+2

1.25

1.25+2

1.2

1.8

2.4

0.85

0.65

1.30

1.95

0.65

1.30

1.95

(7.25 kg)

1.8

(9.08 kg)

1.8

(8.6 kg)

1.8

1.6

1.2

1.5

Age start

(d)

Length of

study (d)

Number of

pigs/treatment

Replicates

Diarrhoea

frequency 23.4/0.4%

11.1/2.4

24.6/4

13.1/1.2

7.1/0

18.7/0.8

frequency 24.2/3.6%

29.8/7.1

19.4/4.8

22.1/4.8

15.9/0

23/5.6

frequency 5.3%

6.6

frequency 8.2%

6.7

8.7

6.5

6.3

8.4

5.8

5.4

20 days(4%)

3 (0.6%)

28 (5.5%)

10 (2%)

330 days(87%)

236 (31%)

362 (47.9)

317 (42%)

6 days (1.2%)

2 (0.4%)

4 (0.8%)

16 (3.2%)

2 (0.4%)

9 (1.8%)

7 (1.4%)

score 5.63

1.7

4.41

1.94

2.5

3.49

3.21

Faecal coliform

bacteria (log cfu/g)

Faecal lactic acid

bacteria (log cfu/g)

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DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Ref

Treatments

control

lactic acid

formic acid

calcium formate

sodium benzoate

spray-dried plasma prot

pea protein isolate (PPI)

PPI+egg yolk antibody

PPI+ZnO

PPI+fumaric acid

PPI+carbadox

control

carbacol,cinnamaldehyde,capsicum oleoresin

formic acid

formic acid+ (carbacol,cinnamaldehyde,capsicum oleoresin)

formic acid+(carbacol,cinnamaldehyde,capsicum oleoresin)

control

50 ppm carbadox

anis oil, citrus oil, oregano oil, flavours + phosphoric acid, lactic acid

control (contains 3000 ppm ZnO)

lactic acid,formic acid,phosphoric acid

lactic acid,acetic acid,phosphoric acid

lactic acid,formic acid,phosphoric acid

control

free calcium formate

fat-protected calcium formate

control

avilamycin

formic acid

formic acid,propionic acid,potassium sorbate

formic acid,propionic acid,sodium benzoate

formic acid in diatomaceous earth

control

inulin

benzoic acid

benzoic+inulin

control

B. Cereus Toyoi

benzoic acid

B. Cereus Toyoi+benzoic acid

control (water pH 8)

water pH 4 (lactic,formic,propionic,acetic (1%cu +0.01%zn))

water pH 5 (lactic,formic,propionic,acetic (1%cu +0.01%zn))

water pH 6 (lactic,formic,propionic,acetic (1%cu +0.01%zn))

Dose (%)

0.8

Age start

(d)

28/38

Length of

study (d)

30/20

Number of

pigs/treatment

Replicates

Diarrhoea

index 4.1

4.5

1.5

8.3

6.1

score 1.6

2.7

1.3

1.4

1.3

1.1

pens 2

score 3.2

3.43

score 2.6

2.5

2.4

2.7

4.4 days

2.9

3.1

score 9.6

5.4

7.8

9.5

6.6

2.6 days

1.2

2.9

0.8

score 0.322

0.096

0.132

0.064

frequency 2.1%

2.1

1.9

1.5

Faecal coliform

bacteria (log cfu/g)

Faecal lactic acid

bacteria (log cfu/g)

3000 ppm

0.2

55 ppm

150 ppm

300 ppm

0.5

0.5+150 ppm

0.5+300 ppm

50 ppm

0.4/0.2+0.4/0.

1.2/0.9

1.5/1.1

1.2

26 or 36

40 ppm

0.8

0.6

1.86

0.5

0.5+8

10 cfu/kg

0.5

10 cfu/kg+0.5

ca.289

5.9

6.2

5.6

5.8

5.9

5.3

7.3

7.9

8.7

7.9

7.6

7.9

34-24

8.4

7.7

7.6

8.3

8.0

7.9

7.8

7.9

7.4

0.8

0.4

0.1

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Ref

Treatments

control

challenged

challenged+dried bovine colostrum

challenged+pineapple stem with bromelain

challenged+yeast preparation from S. cerevisiae

challenged+(formic acid,ammonium formate,acetic acid)

challenged+thyme essential oil

control

sodium butyrate

control

benzoic +thymol

benzoic + thymol

control (with 40ppm colistin and 3680/3000 ppm ZnO)

fumaric acid, malic acid, lemon essential oil

phosphoric acid, citric acid, malic acid, fumaric acid

formic acid,phosphoric acid,propionic acid, its salts

control

calcium formate,calcium lactate,citric acid,medium chain fatty acids

control

ZnO

lactic acid, formic acid, citric acid

bacteriophages

Dose (%)

Age start

(d)

Length of

study (d)

Number of

pigs/treatment

Replicates

0.20

0.10

0.70

0.015

0.1

0.1+0.01

0.1+0.02

0.2+0.01

0.07

0.02

0.85

0.5

0.3

(7.27 kg)

2500 ppm

0.2

0.1

Diarrhoea

score 3.1

2.5

2.8

2.3

2.9

2.3

2.5

incidence 14.97%

10.73

(a)

score 30

9.6/9.05/9.15 /8.56

(bc)

12.3

8.7/9.07/7.71 /7.77

(b)

17.2

8.62/9.3/7.56 /7.55

(c)

7.67

8.48/8.94/7.85 /7.91

frequency 62.5%

6.5

20.83

6.4

33.33

6.9

6.5

frequency ca.27%

ca.28

ca.20

score 1.63

1.24

1.35

1.26

7.05

6.93

6.60

6.58

Faecal coliform

bacteria (log cfu/g)

Faecal lactic acid

bacteria (log cfu/g)

6.8 /8.47/7.2/8.57

7.88 /8.75/7.88/8.92

7.54

/8.63/7.64/8.78

8.53

/8.74/7.89/8.53

8.3

8.1

8.2

8.05

8.5

8.6

8.59

Coliform

bacteria and lactic acid bacteria: ileum day 14, caecum day 14, ileum day 42, caecum day 42.

1) (Eidelsburger et al., 1992a); 2) (Eidelsburger et al., 1992b); 3) (Kirchgessner et al., 1995); 4) (Roth et al., 1996); 5) (Paulicks et al., 2000); 6) (Tsiloyiannis et al., 2001); 7) (Partanen et al., 2002); 8) (Owusu-

Asiedu et al., 2003); 9) (Manzanilla et al., 2004); 10) (Kommera et al., 2006); 11) (de Freitas et al., 2006); 12) (Bosi et al., 2007); 13) (Partanen et al., 2007); 14)(Halas et al., 2009); 15) (Papatsiros et al., 2011);

16) (De Busser et al., 2011); 17) (Spitzer et al., 2014); 18) (Fang et al., 2014); 19) (Diao et al., 2015); 20) (Callegari et al., 2016); 21) (Wang et al., 2016); 22)(Hosseindoust et al., 2017).

abc

Values

with a different superscript within study are different P < 0.05.

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Table 4. Impact of feeding fermented liquid feed on post-weaning diarrhoea in piglets (Danish studies)

Ref

Treatment

Weight start

(kg)

Age start (d)

Length of

No.

study, d pigs/treatment

Diarrhoea

Replicates frequency

dry feed

liquid feed

fermented liquid feed

dry feed

partially fermented feed

fermented liquid feed

dry feed+20% maize

dry feed+20% fermented maize

liquid feed with cereals/dry feed

liquid feed with ferm cereals/dry feed

liquid feed

liquid feed+50% fermented cereals

fermented liquid feed/dry feed

fermented liquid feed+lactic acid bacteria

/dry feed

fermented liquid feed+Kazachstania

exigua

/dry feed

dry feed

dry feed+15% fermented rapeseed cake product

dry feed+11% standard rapeseed cake

Frequency of antibiotic treatment against diarrhoea.

ca.42

9.8

ca.28-35

6.9

7.6

8.4

ca. 63

303

300

302

291

288

291

480

843

3860

3868

387

546

559

551

1.2

1.1

0.1

ca. 35

0.47

Treatment describes the feed during the first three weeks of the study and the remaining four-five weeks.

Lactobacillus rossiae/L. sp. CS1/L. siligionis +Lactobacillus sanfranciscensis/L. sanfrancisco/L. lindneri.

1) (Pedersen, 2001a); 2) (Pedersen, 2001b); 3) (Pedersen, 2006); 4) (Pedersen, 2009); 5) (Pedersen, 2011); 6) (Maribo, 2012).

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

Table 5. Impact of feeding fermented liquid feed on post-weaning diarrhoea in piglets

Age start

Weight start Length of study,

No.

Ref.

Treatments

(d)

(kg)

d pigs/treatment Replicates

7.5

liquid feed+whey+dextrose

liquid feed+fermented whey+dextrose

dry+0.1% lincomycin

liquid feed+whey+dextrose

7.9

liquid feed+fermented whey+dextrose

dry+0.1% lincomycin

7.9

dry feed

fermented liquid feed+5.5x10

cfu/ml

Pediococcus acidilactici

dry feed

dry feed+5.5x10

cfu/ml

Pediococcus

fermented liquid feed+5.5x10

cfu/ml

Pediococcus acidilactici

no challenged-no whey permeate

challenged-no whey permeate

challenged+ whey permeate

challenged+fermented whey permeate 1

challenged+fermented whey permeate 2

challenged+fermented whey permeate 3

6.3

Lactic acid bacteria

Diarrhoea Coliform bacteria (cfu/g)

(log cfu/g)

7 /5 pigs

8/8.1/7.6

7.8/7.6/7.3

NS (values not

given)

7.5/6.5/7.5

7.2/6.6/6.7

7/7.2/7.5

5.7/7.43/7.68/7.97

6.01/7.75/7.48/7.53

5.6/7.11/7.4/7.43

5.15/6.99/7.52/7.1

5.61/6.91/6.77/6.92

5.34/6.87/7.41/6.99

9.1/8.2/9

9.2/8.1/9.3

8.8/9.2/9

9.1/9.4/8.8

8.6/8.9/9

7.84

11 days

Challenge study with E. coli.

Diarrhoea values indicate number of pigs with diarrhoea on day 7 and 9 post-challenge.

Coliform bacteria in stomach, small intestine, caecum and colon. P

diet

= 0.07.

Coliforms and lactobacilli in faeces on days 8, 15 and 22.

1)(Amezcua et al., 2007); (Missotten et al., 2015); 3) (Sugiharto et al., 2015b).

Values with a different superscript within study are different P < 0.05.

MOF, Alm.del - 2016-17 - Bilag 492: Vidensyntese og cost-effectiveness vurdering vedr. zink og rapport om kontrolkampagne om zink

DCA - Nationalt Center for Fødevarer og Jordbrug, 26. april 2017

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