Research Article Lactoferrin Decreases the Intestinal...

Research ArticleLactoferrin Decreases the Intestinal Inflammation Triggered bya Soybean Meal-Based Diet in Zebrafish

Pilar E Ulloa1 Camila J Soliacutes1 Javiera F De la Paz1 Trevor G S Alaurent1

Mario Caruffo1 Adriaacuten J Hernaacutendez2 Patricio Dantagnan2 and Carmen G Feijoacuteo13

1Departamento de Ciencias Biologicas Facultad de Ciencias Biologicas Universidad Andres Bello Republica 2178370146 Santiago Chile2Nucleo de Investigacion en Produccion Alimentaria Escuela de Acuicultura Facultad de Recursos NaturalesUniversidad Catolica de Temuco Avenida Rudecindo Ortega 02950 Casilla 15D 4780000 Temuco Chile3Interdisciplinary Center for Aquaculture Research 4070007 Concepcion Chile

Correspondence should be addressed to Carmen G Feijoo cfeijoounabcl

Received 6 February 2016 Accepted 18 April 2016

Academic Editor Senthamil Selvan

Copyright copy 2016 Pilar E Ulloa et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Intestinal inflammation is a harmful condition in fish that can be triggered by the ingestion of soybean meal Due to the positivecosts-benefits ratio of including soybean meal in farmed fish diets identifying additives with intestinal anti-inflammatory effectscould contribute to solving the issues caused by this plant protein This study evaluated the effect of incorporating lactoferrin (LF)into a soybean meal-based diet on intestinal inflammation in zebrafish Larvae were fed with diets containing 50 soybean meal(50SBM) or 50SBM supplemented with LF to 05 1 15 gkg (50SBM+LF05 50SBM+LF10 50SBM+LF15) The 50SBM+LF15diet was the most efficient and larvae had a reduced number of neutrophils in the intestine compared with 50SBM larvae and anindistinguishable number compared with control larvae Likewise the transcription of genes involved in neutrophil migration andintestinal mucosal barrier functions (mmp9 muc22 and 120573-def-1) were increased in 50SBM larvae but were normally expressedin 50SBM+LF15 larvae To determine the influence of intestinal inflammation on the general immune response larvae werechallenged with Edwardsiella tarda Larvae with intestinal inflammation had increased mortality rate compared to control larvaeImportantly 50SBM+LF15 larvae had a mortality rate lower than control larvae These results demonstrate that LF displays a dualeffect in zebrafish acting as an intestinal anti-inflammatory agent and improving performance against bacterial infection

1 Introduction

Intestinal inflammation in fish is a detrimental conditionthat affects growth and the ability to respond to pathogens[1] Preventing this pathology is of particular relevance forfish farming as small fish size andor high fish mortalitydrastically affect the competitiveness and profitability of theaquaculture industry

Most commercially important fish are carnivorous andrequire a high-protein diet usually provided through fish-meal [2] However increased aquaculture production haslimited fishmeal availability leading to the use of plantproteins in fish diets [2] Soybean meal which is widelyavailable and economical has a balanced amino acid profile

and contains a high amount of digestible proteins is currentlythe most common plant protein source used in fish feed[3] Studies in different fish species such as Atlantic salmon(Salmo salar) [4] rainbow trout (Oncorhynchus mykiss) [5]carp (Cyprinus carpio L) [6] Nile tilapia (Oreochromis niloti-cus) [7] gilthead seabream (Sparus aurata) [8] and zebrafish(Danio rerio) [9 10] have shown that the inclusion of soybeanmeal in the diet triggers intestinal inflammation [11ndash14]Nevertheless the advantages of soybean meal outweigh itsdisadvantageous effects to fish intestines

To optimize the use of this plant protein there is anongoing search to find dietary additives that could protectthe intestine from the effects of soybean meal Traditionallyadditives have been incorporated into fish diets to control

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2016 Article ID 1639720 10 pageshttpdxdoiorg10115520161639720

2 Journal of Immunology Research

diseases increase health and improve the stress response[1 15ndash19] However little focus has been given to the use ofadditives for controlling intestinal inflammation At presentthere are only two studies that evaluate the effects of addi-tives specifically mannan-oligosaccharide and 120573-glucans onsoybean meal-triggered intestinal inflammation in farmedfish [1 17] These investigations indicate that only mannan-oligosaccharide is able to decrease to varying degrees thealtered intestinal histology observed in fish fed with dietsincluding low amounts of soybean meal [1 17]

Lactoferrin (LF) is an abundant iron-binding glyco-protein secreted by epithelial cells and contributes to thecomposition of bodily fluids in mammals such as milk tearssaliva bile and pancreatic fluid [20 21] Specific LF receptorsare present on the surface of different tissues and cell typesincluding the gastrointestinal tract lungs neutrophils andeosinophils [22 23] Previous research has demonstrated thatthis glycoprotein has antimicrobial activity and can stimulatecytokine production enhance cell proliferation and regulatemucosal immunity [20 24ndash26] Due to these properties LFhas been used in prophylactic treatments for fish against dif-ferent infectious diseases [25 27ndash29] Furthermore LF exertsa potent anti-inflammatory effect in different tissues mainlyby reducing immune cell recruitment to inflammatory sitesDuring influenza virus infection LF reduces the numberof infiltrating leukocytes in bronchoalveolar lavage fluid inhumans [30] Likewise LF can reduce eosinophil infiltrationto the pigs small intestine in a mechanism independent ofcytokine [31 32] Moreover an in vivo study in rats andmice demonstrated that orally administered LF preventsintestinal injury triggered by nonsteroidal anti-inflammatorydrugs The authors related this effect to the attenuation ofneutrophil migration to the intestine [33] Despite thesevarious related studies there are currently no reports on thepossible role of LF as an intestinal protector in fish and less soon the possible protective effects of LF against soybean meal-induced intestinal inflammation in fish

Factors contributing to this lack of information are thehigh costs and long-term assays involved in evaluating theuse of additives in the aquaculture industry which is inaddition to the challenge ofworkingwithminimal precedentson the cellular and molecular processes in many farmedfish species Therefore an alternative research strategy is toperform preliminary studies in a model fish in which manydiets can be assessed in a short period and at low costsMoreover the selected fish model should facilitate under-standing the biological processes triggered by different dietsConsidering the extensive biological literature and power-ful biotechnological tools available for zebrafish (D rerio)this teleost fish is an ideal organism for immune-nutritionresearch [34]

One key advantage of zebrafish is the availability of trans-genic lines with certain fluorescently-labeled innate immunecells such as neutrophils [35] Since the hallmark of inflam-mation is neutrophil migration these cells can be used asinflammatory markers This strategy has been used beforeby Hedrera et al [9] who demonstrated that soybean mealconsumption by zebrafish results in inflammatory side effectssimilar to those observed in commercially farmed fish

A primary advantage of using transgenic fluorescentlylabeled zebrafish is that the inflammatory process can be veryquickly observed even before histological effects becomerecognizable

This study evaluated the effects of LF on intestinal inflam-mation induced by a soybean meal-based diet in zebrafishBy using the Tg(BACmpoGFP)i114 transgenic zebrafish lineneutrophil migration as part of the intestinal inflammatoryprocess was monitored in vivo To complement this data thetranscriptional levels of different markers related to mucosalbarrier functions as matrix metallopeptidase 9 mucin 22and beta-defensin 1 (mmp9 muc22 and 120573-def-1) and lipidabsorption like the fatty-acid-binding proteins 2 and 6 (fabp2and fabp6) were evaluated Finally the influence of intestinalinflammation on the immune response to Edwardsiella tardainfection was assessed

2 Material and Methods

21 Zebrafish Strains andMaintenance Zebrafish weremain-tained and raised at the Laboratory of Developmental Biol-ogy Universidad Andres Bello Chile according to stan-dard protocols [37] The Tg(BACmpoGFP)i114 transgeniczebrafish line was used [35] All embryos were collectedthrough natural spawning according to Kimmel et al [38]Eggs were incubated in petri dishes at 28∘C for three days inthe E3 medium (5mM NaCl 017mM KCl 033mM CaCl

2

and 033mM MgSO4 with methylene blue equilibrated to

pH 70) [39] Embryonic and larval stages are expressed inhours postfertilization (hpf) or days postfertilization (dpf)All animal-handling procedures were approved by the Com-mittee of Animal Bioethics of the Universidad Andres Bello

22 Experimental Diets Five diets were formulated andprepared (Table 1) The positive control diet contained 50soybean meal (50SBM) while the negative control dietcontained fishmeal as the primary protein source (100FM)[9] Additionally a diet normally used for zebrafish larvae(ZFP sera Micron Heinsberg Germany) was used as asecond negative control To evaluate the intestinal protectiveeffect of LF three batches of the 50SBM diet were supple-mented with bovine LF obtained from milk (Lactoferrin100 S60 Natural Healthy Concepts Appleton WI USA)in concentrations of 05 10 or 15 gkg (50SBM+LF0550SBM+LF10 and 50SBM+LF15 resp) [40] Likewise onebatch of the 100FM diet was supplemented with 15 gkg ofbovine LF (100FM+LF15) All diets were supplemented witha standard vitamin and mineral premix and formulated tobe isoenergetic isonitrogenous and isolipidic Each feed dietwas prepared by cooking-extrusion in a twin screw extruder(CLEXTRAL BC-21 Clextral Firminy France) with a 2mmdiameterThe resulting moist pellets were oven-dried at 60∘Cfor approximately eight hours and then coated with fish oilaccording to the formulation for each diet using a laboratoryvacuum coater (Dinnissen Model VC10 Sevenum Nether-lands) The pellets were subsequently crumbled screened tothe appropriate particle size (75120583m) and stored at minus20∘Cuntil use in the feeding trials

Journal of Immunology Research 3

Table 1 Ingredients and nutrient composition of experimental diets

100FM 50SBM Different doses of LF incorporated into diets50SBM+LF05 50SBM+LF10 50SBM+LF15 FM+LF15

Ingredients gkgFishmeal 610 250 250 250 250 610Soybean meal 0 500 500 500 500 0Wheat grain meal 255 115 115 115 115 255Starch 45 45 45 45 45 45Fish oil 30 60 595 59 585 285Vitamineral mix1 30 30 30 30 30 30Cellulose 30 0 0 0 0 30Lactoferrin 0 0 05 1 15 15Total 1000 1000 1000 1000 1000 1000Analytical composition (dry base )Dry matter 953 935 940 940 9304 9427Total proteins 464 435 434 434 4346 4440Total lipids 78 84 76 76 678 639Ash 126 97 98 98 825 976Gross energy (MJkg) 200 203 202 202 202 2001As recommended by the National Research Council 1993 [36]

Table 2 Primer sequences used for amplification of specific genes through RT-qPCR

Gene Forward primer Reverse primer Amplicon(bp) Gene ID Efficiency

muc22 ACACGCTCAAGTAATCGCACAGTC TCAGCGAGTGTTTGGCTCACTT 137 XM 002667543 182mmp9 CATTAAAGATGCCCTGATGTATCCC AGTGGTGGTCCGTGGTTGAG 142 NM 2131231 189120573-def-1 CTCCTTGTCGTACTAGCATTGCAC ACACACTCCTTGTCTGCAAACACC 99 NM 0010815531 186fabp2 TCAACGGGACCTGGAAAGTC CCCATTTGTTCCATGAACTTCTC 61 NM 1314311 186fabp6 CTCCGCTCAATCAACACCAA TGAGATTCGGTTTCCCACTTG 59 NM 0010020761 193120573-actin TTCTGGTCGTACTACTGGTATTGTG ATCTTCATCAGGTAGTCTGTCAGGT 144 NM 1310311 199rpl13120572 TCTGGAGGACTGTAAGAGGTATGC AGACGCACAATCTTGAGAGCAG 148 NM 2127841 194Primers for mmp9 muc22 120573-def-1 fapb2 and fabp6 were designed using AmplifX v140 Two reference genes 120573-actin and ribosomal protein L13a (rpl13120572)were used [10]

23 Experimental Feeding Period Experimental feeding wasperformed as previously described by Hedrera et al [9]Briefly 45 larvae were fed two times daily from 5 to 8 dpfThelast feed was given at least 14 h before larval fixing to promoteintestine emptying

24 Immunohistochemistry and Sudan Black B StainingImmunohistochemistry was performed as previouslydescribed by Feijoo et al [41] The following antibodies wereused rabbit anti-Green Fluorescent Protein (anti-GFP) (Catnumber A11122 Invitrogen Carlsbad CA USA) and anti-rabbit peroxidase (Cat number A8275 Sigma St Louis MOUSA) Additionally Sudan Black B staining was performedfollowing the manufacturerrsquos protocol (Cat number 3801Sigma-Aldrich St Louis MO USA)The neutrophils presentin the intestine were quantified within a defined area thatincluded the mid and posterior intestine At least 28 larvaewere analyzed per diet in three independent experiments

25 Quantitative Polymerase Chain Reaction (qPCR) Larvaefed with the control and experimental diets were sampledat the end of each treatment for total RNA extraction TotalRNA was extracted from a pool of sim60 larval intestines perdiet The whole intestine was dissected from larvae anes-thetized in tricaine methanesulfonate using sterile instru-ments Samples were stored in the RNAlater solution andthen homogenized in the TRIzol Reagent (Cat number15596-026 Invitrogen Carlsbad CA USA) according to themanufacturerrsquos instructions The corresponding cDNA weresynthesized from 25 120583g of total RNA using SuperScript IIReverse Transcriptase (Cat number 100004925 InvitrogenCarlsbad CA USA) and Oligo-dt primers Primer sequencesand the efficiencies are shown in Table 2 qPCR was per-formed with the ABI 7300 Real-Time PCR system usingthe Maxim SYBR GreenROX qPCR Master Mix (2x) (Fer-mentas Waltham MA USA) following the manufacturerrsquosinstructions A 15 120583L reaction volume was used containing


1 120583L of 10-fold diluted cDNA The PCR was run with a ten-minute activation and denaturation step at 95∘C followed by40 cycles of 30 s at 95∘C 30 s at 57ndash60∘C and 30 s at 72∘CReaction specificity was verified using melting curve analysisand the absence of primer dimmers Standard curves wereobtained for each pair of primers by plotting Ct values againstthe log

10of five different dilutions of a cDNA mix solution

for all analyzed samples Real-time PCR efficiency (119864) wascalculated from a standard curve according to the equation119864 = 10(minus1slope) [42] Relative expression was calculatedwith the Pfaffl method [42] and the 50SBM diet was used asa calibrator

26 cDNA Cloning Probe Synthesis and Whole-Mount InSitu Hybridization The fabp2 gene was cloned using thefollowing primers F-51015840-CGACCGCAATGAGAACTACGA-GAA-31015840 and R-51015840-CTCACAGGTGCAAATGACACGA-31015840(gene ID NM 1314311) from a 9 dpf cDNA library A 529base pair cDNA fragment was cloned into the pGEM-T easyvector (Promega Madison WI USA) which was digestedwith the ApaI restriction enzyme The anti-sense riboprobewas then synthesized using the Sp6 RNA polymerase Thefabp6 clone was kindly provided by Oehlers et al [43] Insitu hybridization was performed as previously described byJowett and Lettice [44]

27 Edwardsiella tarda Challenge Edwardsiella tarda FL60was kindly provided by Dr Phillip Klesius (USDA Agri-cultural Research Service Aquatic Animal Health ResearchUnit)The E tarda culture was grown as previously describedbyHarvie et al [45] andVan Soest et al [46] with somemod-ifications Briefly E tarda was grown overnight at 28∘C in atrypticase soy broth medium with agitation The overnightculture was diluted to 1 100 in fresh trypticase soy brothmedium and incubated at 28∘C to reach 108 CFUmL Theculture was pelleted by centrifugation at 1500 g for five min-utes washed with water from the aquarium and repelletedto recover the bacteria The clean E tarda was suspended inwater from the aquarium to reach 108 CFUmL After fourdays of feeding a group of 30 larvae were challenged for 5 hin 200mL of the E tarda water media and were subsequentlytransferred to a tank with new clean water Each respectivediet was resumed for the remainder of the trial period andlarvae mortality was monitored every 12 h for four days

28 Statistical Analysis and Imaging The data were analyzedusing a nonparametric Kruskal-Wallis one-way ANOVAandDunnmultiple comparisons testThe data were normallydistributed (the DrsquoAgostino and Pearson normality test) butvariance was not homogenous (the Brown-Forsythe test)Survival data were analyzed using Kaplan-Meier and groupdifferences were analyzed by the log-rank test using theBonferroni correction for multiple comparisons All analyseswere performed using Prism 4 (GraphPad Software LaJolla CA USA) Significance was established for all analysesat 119875 lt 005 Lateral view photographs of larvae weretaken in an Olympus SZX16 stereoscope with a QImaging

MicroPublisher 50 RVT camera Images were processed withPhotoshop CS4 or ImageJ v144

3 Results

31 Effect of Soybean Meal Diet Supplemented with Lactofer-rin on Intestinal Inflammation To determine if LF exertedan intestinal protector effect by preventing or decreasinginflammation this additive was incorporated to the soybeanmeal-based diet (50SBM) thereby generating three exper-imental diets (50SBM+LF05 50SBM+LF10 and 50SBM+LF15) The 50SBM diet was used as a positive control thattriggers inflammation while the 100FM and ZFP diets wereused as negative controls [9] To determine the extent ofintestinal inflammation the amount of neutrophils presentin the intestine was quantified (Figure 1)

Confirming previously published data the results indi-cated a clear increase in the number of neutrophils situatedin the intestine of larvae fed with a 50SBM diet comparedto larvae fed with the ZFP and 100FM diets (Figures 1(a)ndash1(c) and 1(i)) In larvae fed with the 50SBM+LF05 diet thenumber of neutrophils located in the intestine showed nosignificant differences compared with 50SBM larvae (Figures1(d) 1(c) and 1(i)) On the other hand larvae fed with the50SBM+LF10 and 50SBM+LF15 diets presented a decreasednumber of neutrophils in the intestine compared to the50SBM group and more importantly these larvae wereindistinguishable from those fed the 100FM and ZFP diets(Figures 1(e) 1(f) and 1(i)) Of significance the amount ofintestine-located neutrophils in larvae fed the 50SBM+LF15diet was similar to larvae fed the 100FM+LF15 diet (Figures1(f) 1(g) and 1(i))

To corroborate immunohistochemistry data Sudan BlackB staining was performed to specifically label leukocytesTotal concordance was found between the results obtainedwith the two techniques (Supplementary Figure 1 in Sup-plementary Material available online at httpdxdoiorg10115520161639720)

Since LF concentrations of 10 and 15 gkg exerted similareffects subsequent analyseswere performedusing the 50SBMdiet supplemented by 15 gkg of LF (ie 50SBM+LF15)

32 Effect of SoybeanMeal Diet Supplementedwith Lactoferrinon Genes Related to Intestinal Mucosal Function To evaluatethe effect of LF on genes involved in intestinal mucosalfunction the transcripts of muc22 120573-def-1 and mmp9 weredetermined through qPCR The relative expressions of thesegenes in response to the different diets were compared againstexpressions in larvae fed with the 50SBM diet (Figure 2dotted line) As expected in larvae fed with the ZFP and100FM control diets the transcriptional levels of muc22 120573-def-1 and mmp9 were significantly lower than in larvae fedwith the 50SBM diet (119875 lt 0001) This same result wasobserved in larvae fed the 100FM+LF15 diet Notably themRNA levels in larvae fed with the 50SBM+LF15 diet werecomparable to those observed in the ZFP and 100FM groupsand were significantly lower than those in larvae fed with the50SBM diet (119875 lt 0001)


ZFP

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

100

FM50

SBM

Diets

0

5

10

15

20

25

ZFP

100

FM

50

SBM

50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15

Num

ber o

f neu

troph

ilsd

efine

d ar

ea

Figure 1 Effect of different lactoferrin doses on neutrophil migration to the intestine (andashg) Lateral view of mid and posterior intestineof Tg(BACmpoGFP)i114 larvae of 9 dpf after four days of feeding with different diets (ZFP 100FM 50SBM 50SBM+LF05 50SBM+LF1050SBM+LF15 and 100FM+LF15) Neutrophils were quantified through immunohistochemistry against GFP (white arrows) (h) Larvascheme with the intestinal region of interest demarcated with a red rectangle (i) The experiments were conducted with at least 28 larvaeper treatment in three different assays Statistical analysis was performed by comparing data sets with the 50SBM diet through one-wayANOVAThe graph is a representation of three different results lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 Bar scale = 200 120583m

33 Effect of Soybean Meal Diets Supplemented with Lacto-ferrin on Genes Related to Intestinal Lipid Absorption Toevaluate the effect of a 50SBM diet and LF supplementationon the expression of genes related to the lipid absorptionprocess in the intestine in situ hybridization and qPCRwere performed for the markers fabp2 and fabp6 at 9 dpfThe fabp2 expression was restricted to the anterior intestinewith a reduction towards the mid and posterior intestine(Figures 3(a)ndash3(e) red dotted line) In contrast fabp6 wasexpressed at the mid and posterior intestine (Figures 3(g)ndash3(k)) The expression of fabp6 in the defined area was similarbetween larvae fed with the different diets (Figures 3(g)ndash3(k) red continued line) Likewise qPCR analysis revealed asignificant upregulation in the expression of fabp2 in control

diets (ZFP 100FM and 100FM+LF15) in comparison to thosefed with 50SBM (Figure 3(f)) The transcriptional expressionof fabp6 between control and experimental larvae did notvary except in larvae fed the ZFP diet where significantupregulation was observed (119875 lt 001) (Figure 3(l))

34 Effect of Intestinal Inflammation on Immune Performanceagainst Edwardsiella tarda To determine if intestinal inflam-mation affected the immune performance of the differentlarvae groups a challenge assay was performed using theenterobacteria E tarda (Figure 4) The challenged larvae fedwith the 50SBM diet presented significantly higher mortalityrates than those fed with the 100FM or ZFP diets (119875 lt001) However larvae fed the 50SBM+LF15 diet showed


00

05

10

15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

ZFP 100FM 50SBM+LF15

Dietsmuc22120573-def-1

mmp9

lowastlowast

lowastlowast

lowastlowast



lowastlowast lowastlowastlowast

lowastlowastlowastlowastlowastlowastlowast lowastlowastlowastlowast

LF15100FM+

Figure 2 Effect of lactoferrin on transcriptional levels of mucosalbarrier functional markers Transcription levels of muc22 120573-def-1andmmp9 were quantified by qPCR Quantification was performedfrom a pool of sim60 intestines of larvae of 9 dpf after four days offeedingwith different diets (ZFP 100FM 50SBM 50SBM+LF15 and100FM+LF15) All data were normalized with 120573-actin and rpl13120572The data from the different diets were compared to the 50SBM diet(dotted lines) lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 lowastlowastlowastlowast119875 lt 00001

a drastic decrease in accumulated mortality at the end ofthe experiment compared to 50SBM (119875 lt 001) and mor-tality rates were even lower in the 50SBM+LF15 groupthan in larvae fed with the 100FM or ZFP diets Similarlylarvae fed with the 100FM+LF15 diet also showed reducedaccumulated mortality compared to the 100FM diet (119875 lt001) The mortality of larvae fed with the 50SBM+LF15or 100FM+LF15 diets was comparable and no significantdifferences existed between these groups

4 Discussion

This is the first study to provide evidence that orally admin-istered LF protects the fish intestine from the inflammatoryeffect induced by soybean meal This observation widens theopportunity for using this plant protein in the fish nutritionindustry

The obtained results suggest that LF reduces neutrophilrecruitment to the intestine and that this effect is dose-dependent In line with this attenuated neutrophil migrationthere was a downregulation in the transcription of mmp9an enzyme that degrades the basement membrane to facil-itate cell migration and infiltration to affected tissue [47]Moreover neutrophils are the major contributors of MMP-9during intestinal inflammation in mammals [48 49]

The effect of LF as an intestinal protector has beenpreviously reported in rodents Dial et al [33] found that LFprotects the intestine of rats andmice from the effects of non-steroidal anti-inflammatory drugs The authors speculatedthat LF couldmodulate neutrophil function attenuating neu-trophil migration to the intestine However the mechanismby which LF inhibits leukocyte migration is still unknownThere is evidence that this protein reduces the integrin-dependent adherence of eosinophils [50] and inhibits

the expression of adhesion molecules such as E-selectin andICAM-1 in the vascular endothelium [51] Considering thatthe presence of these adhesion molecules on the surface ofthe endothelium is a key step during leukocyte recruitment toaffected sites the absence or low levels of adhesion moleculescould possibly explain the effect observed in the presentstudy on neutrophil migration On the other hand LF canalso regulate cytokine production inmiceTherefore anotherpossible scenario is that by modulating cytokine levelsLF decreases neutrophil migration to the intestine Datasupporting this hypothesis indicate that LF can suppress theproinflammatory cytokines tumor necrosis factor alfa [52]and IL-1 [53] in addition to promoting the anti-inflammatorycytokine IL-18 in the gut [54] Moreover it is possible thatthe decreased neutrophil migration triggered by LF is partlya consequence of the inhibition of different proinflammatorycytokines

Regarding the effect of intestinal inflammation on sur-vival rate the challenge assay with E tarda results clearlyindicated that an inflammatory process significantly affectsthe immune response against pathogens The present resultsshowed that the survival rate of larvae fed with soybean mealwas almost half of that observed in larvae fed with fishmeal(19 and 35 resp) Similar results have been reported inadult zebrafish specimens Specifically oxazolone-inducedintestinal inflammation resulted in treated zebrafish beingmore susceptible to E tarda infection than healthy zebrafish[55] Therefore it is not the soybean meal per se thataffects the immune response against pathogens but ratherintestinal inflammation Moreover in a mouse colitis modelwith concomitant intestinal inflammation infection withSalmonella enterica was facilitated [56]

The present results further suggest that the effect of LFon the intestine is not limited to preventing intestinal inflam-mation but that LF can also influence larvae performanceagainst pathogen This was evidenced in the challenge assaywhen comparing the survival rates of larvae fed diets with orwithout LF In the case of larvae fed with fishmeal that didnot have intestinal inflammation the survival rate increasedfrom 35 to 47 when LF was incorporated Interestinglytranscriptional analyses of muc22 and 120573-def-1 in larvae fedwith LF were indistinguishable from control larvae Theseresults indicate that LF did not improve the mucosal barrierfunction of the host immune response Defensins including120573-def-1 are crucial antimicrobial peptides that protect theintestine against infection as a result of antibacterial andimmunomodulatory properties [55 57 58] Likewise mucinssuch as muc22 form part of the mucosal defense systempresent in themucous gel layer that covers the luminal surfaceof the gastrointestinal tract This viscoelastic protective bar-rier forms the first line of defense to the external environment[59] Additionally in a chemically induced zebrafishmodel ofintestinal inflammation the mucus layer increases [60]

Therefore the presently observed increase in survivalrate could be the result of LF directly acting against theinvading bacteria Indeed LF has an antimicrobial effectagainst a broad spectrum of bacteria mainly Gram-negativebacteria present in the gut [61] By inhibiting the overgrowthandor colonization of bacterial pathogens LF could promote


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

ZFP 100FM 50SBM+LF15Diets

100FM+LF15

(l)


100FM+LF15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

lowastlowast

lowastlowast

lowastlowast

fabp2

fabp2

fabp6

fabp6ZF

P100

FM50

SBM

50

SBM+

LF15

100

FM+

LF15

Figure 3 Effect of lactoferrin on lipid absorption markers (andashe) fabp2 and (gndashk) fabp6 mRNA expression pattern was analyzed by whole-mount in situ hybridization Lateral view of larvae of 9 dpf after four days of feeding with different diets (ZFP 100FM 50SBM 50SBM+LF15and 100FM+LF15) (andashe) fabp2 expression was restricted to anterior intestine (red dotted line) (gndashk) fabp6 expression was observed in thewhole intestine with a stronger expression in the mid and posterior gut (red continued line) (f and l) The transcriptional levels of fabp2and fabp6 were quantified by qPCR Data were normalized with 120573-actin and rpl13120572 and compared to 50SBM diet (dotted line) lowast119875 lt 005lowastlowast119875 lt 001


05 12 24 36 48 60 72 84 96

20

40

60

80

100

Hours after challenge

Accu

mul

ated

mor

talit

y (

)

ZFP challenge100FM challenge50SBM challenge

50SBM+LF15 challenge100FM+LF15 challenge

lowast

lowast


lowastlowast

Figure 4 Effect of lactoferrin on fish mortality after pathogen chal-lenge Tg(BACmpoGFP)i114 larvae were challenged with Edward-siella tarda after four days of feeding with different diets at 9 dpf(ZFP 100FM 50SBM 50SBM+LF15 and 100FM+LF15) Mortalitywas monitored immediately after the challenge and every 12 h overfour days until 13 dpf Statistical analysis was performed usingsurvival curve analysis with the log-rank test against the 100FM and50SBM diets lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowastlowast119875 lt 00001 Continuouslines represent challenged larvaewhile dotted lines represent controllarvae

a healthy condition This inhibitory action of LF couldbe achieved by the following three events (1) chelatingferric iron necessary for bacterial growth (2) destabilizingmicrobial membranes or (3) modifying microbial adherenceto host cells independent of the microbersquos iron-bindingproperties [62] Obviously the antimicrobial activities ofLF are not absolute and permit the growth of commensalbacteria [63]

5 Conclusions

The present study provides novel and relevant data regardingthe effects of LF on fish physiology especially in relation tointestinal inflammation In light of the obtained results thesupplementation of fish diets with LF appears to be a plausiblealternative to cope at least in part with two major problemscurrently affecting the aquaculture industry-soybean meal-triggered enteritis and pathogenic infections

Competing Interests

The authors declare no competing interests in relation to theresearch and authorship

Acknowledgments

This work was supported by the Fondo Nacional de Desar-rollo Cientifico y Tecnologico 3130664 awarded to PilarE Ulloa and Fondo Nacional de Desarrollo Cientifico y

Tecnologico 1140297 Direccion de Investigacion UNAB DI-483-14R and Fondo de Financiamiento de Centros de Inves-tigacion en Areas Prioritarias 15110027 awarded toCarmenGFeijooThe authors also thank Alex Oporto and Alex Cabrerafor manufacturing the experimental diets

References

[1] S Refstie G Baeverfjord R R Seim and O Elveboslash ldquoEffectsof dietary yeast cell wall 120573-glucans and MOS on performancegut health and salmon lice resistance in Atlantic salmon (Salmosalar) fed sunflower and soybean mealrdquo Aquaculture vol 305no 1ndash4 pp 109ndash116 2010

[2] Food and Agriculture Organization of the United Nations ElEstado Mundial de la Pesca y la Acuicultura FAO Rome Italy2012

[3] R L Naylor R W Hardy D P Bureau et al ldquoFeedingaquaculture in an era of finite resourcesrdquo Proceedings of theNational Academy of Sciences of the United States vol 106 no36 pp 15103ndash15110 2009

[4] HMundheimAAksnes andBHope ldquoGrowth feed efficiencyand digestibility in salmon (Salmo salar L) fed different dietaryproportions of vegetable protein sources in combination withtwo fish meal qualitiesrdquo Aquaculture vol 237 no 1ndash4 pp 315ndash331 2004

[5] F Medale T Boujard F Vallee et al ldquoVoluntary feedintake nitrogen and phosphorus losses in rainbow trout(Oncorhynchus mykiss) fed increasing dietary levels of soyprotein concentraterdquo Aquatic Living Resources vol 11 no 4 pp239ndash246 1998

[6] J Pongmaneerat T Watanabe T Takeuchi and S Satoh ldquoUseof different protein meals as partial or total substitution for fishmeal in carp dietsrdquo Nippon Suisan Gakkaishi vol 59 no 7 pp1249ndash1257 1993

[7] A Fontaınhas-Fernandes E Gomes M A Reis-Henriquesand J Coimbra ldquoReplacement of fish meal by plant proteins inthe diet of Nile tilapia digestibility and growth performancerdquoAquaculture International vol 7 no 1 pp 57ndash67 1999

[8] P Gomez-Requeni M Mingarro J A Calduch-Giner etal ldquoProtein growth performance amino acid utilisation andsomatotropic axis responsiveness to fish meal replacement byplant protein sources in gilthead sea bream (Sparus aurata)rdquoAquaculture vol 232 no 1ndash4 pp 493ndash510 2004

[9] M I Hedrera J A Galdames M F Jimenez-Reyes et alldquoSoybean meal induces intestinal inflammation in zebrafishlarvaerdquo PLoS ONE vol 8 no 7 Article ID e69983 2013

[10] P EUlloa AA Pena CD Lizama et al ldquoGrowth response andexpression of muscle growth-related candidate genes in adultzebrafish fed plant and fishmeal protein-based dietsrdquo Zebrafishvol 10 no 1 pp 99ndash109 2013

[11] D Knudsen P Uran A Arnous W Koppe and H FroslashkiaeligrldquoSaponin-containing subfractions of soybean molasses induceenteritis in the distal intestine of Atlantic salmonrdquo Journal ofAgricultural and Food Chemistry vol 55 no 6 pp 2261ndash22672007

[12] P AUran A A Goncalves J J Taverne-Thiele JW Schrama JA J Verreth and J H W M Rombout ldquoSoybean meal inducesintestinal inflammation in common carp (Cyprinus carpio L)rdquoFish and Shellfish Immunology vol 25 no 6 pp 751ndash760 2008

[13] D L Merrifield A Dimitroglou G Bradley R T M Bakerand S J Davies ldquoSoybean meal alters autochthonous microbial


populations microvilli morphology and compromises intesti-nal enterocyte integrity of rainbow trout Oncorhynchus mykiss(Walbaum)rdquo Journal of Fish Diseases vol 32 no 9 pp 755ndash7662009

[14] D Montero F Mathlouthi L Tort et al ldquoReplacement ofdietary fish oil by vegetable oils affects humoral immunity andexpression of pro-inflammatory cytokines genes in gilthead seabream Sparus auratardquo Fish and Shellfish Immunology vol 29no 6 pp 1073ndash1081 2010

[15] Y Staykov P Spring S Denev and J Sweetman ldquoEffect ofa mannan oligosaccharide on the growth performance andimmune status of rainbow trout (Oncorhynchus mykiss)rdquoAqua-culture International vol 15 no 2 pp 153ndash161 2007

[16] N Piaget A Vega and A S Toledo ldquoEffect of the applicationof 120573-glucans and mannan-oligosaccharides (120573G MOS) in anintensive larval rearing system of Paralichthys adspersus (Par-alichthydae)rdquo Investigaciones Marinas vol 35 no 2 pp 35ndash432007

[17] A Dimitroglou D L Merrifield P Spring J Sweetman RMoate and S J Davies ldquoEffects of mannan oligosaccharide(MOS) supplementation on growth performance feed utilisa-tion intestinal histology and gut microbiota of gilthead seabream (Sparus aurata)rdquo Aquaculture vol 300 no 1ndash4 pp 182ndash188 2010

[18] A Tahmasebi-Kohyani S Keyvanshokooh A NematollahiN Mahmoudi and H Pasha-Zanoosi ldquoEffects of dietarynucleotides supplementation on rainbow trout (Oncorhynchusmykiss) performance and acute stress responserdquo Fish Physiologyand Biochemistry vol 38 no 2 pp 431ndash440 2012

[19] M A Esteban A Rodrıguez A Cuesta and J MeseguerldquoEffects of lactoferrin on non-specific immune responses ofgilthead seabream (Sparus auratus L)rdquo Fish and ShellfishImmunology vol 18 no 2 pp 109ndash124 2005

[20] N Larkins ldquoPotential implications of lactoferrin as a therapeu-tic agentrdquo American Journal of Veterinary Research vol 66 no4 pp 739ndash742 2005

[21] D Legrand and JMazurier ldquoA critical review of the roles of hostlactoferrin in immunityrdquo BioMetals vol 23 no 3 pp 365ndash3762010

[22] M Elfinger C Maucksch and C Rudolph ldquoCharacterizationof lactoferrin as a targeting ligand for nonviral gene delivery toairway epithelial cellsrdquo Biomaterials vol 28 no 23 pp 3448ndash3455 2007

[23] Y A Suzuki V Lopez and B Lonnerdal ldquoMammalian lacto-ferrin receptors structure and functionrdquoCellular andMolecularLife Sciences vol 62 no 22 pp 2560ndash2575 2005

[24] D Legrand ldquoLactoferrin a key molecule in immune andinflammatory processesrdquo Biochemistry and Cell Biology vol 90no 3 pp 252ndash268 2012

[25] M Sakai T Otubo S Atsuta et al ldquoEnhancement of resistanceto bacterial infection in rainbow trout Oncorhynchus mykiss(Walbaum) by oral administration of bovine lactoferrinrdquo Jour-nal of Fish Diseases vol 16 no 3 pp 239ndash247 1993

[26] P PWard S Uribe-Luna andOM Conneely ldquoLactoferrin andhost defenserdquo Biochemistry and Cell Biology vol 80 no 1 pp95ndash102 2002

[27] I Kakuta and H Kurokura ldquoDefensive effect of orally adminis-tered bovine lactoferrin against Cryptocaryon irritans infectionof Red Sea Breamrdquo Fish Pathology vol 4 pp 289ndash290 1995

[28] J Kumari T Swain and P K Sahoo ldquoDietary bovine lactoferrininduces changes in immunity level and disease resistance in

Asian catfish Clarias batrachusrdquo Veterinary Immunology andImmunopathology vol 94 pp 1ndash9 2003

[29] B Lygren H Sveier B Hjeltness and RWaagboslash ldquoExaminationof the immunomodulatory properties and the effect on diseaseresistance of dietary bovine lactoferrin and vitamin C fed toatlantic salmon (Salmo salar) for a short-term periodrdquo Fish andShellfish Immunology vol 9 no 2 pp 95ndash107 1999

[30] K Yamauchi HWakabayashi K Shin andM Takase ldquoBovinelactoferrin benefits and mechanism of action against infec-tionsrdquo Biochemistry and Cell Biology vol 84 no 3 pp 291ndash2962006

[31] C Cooper E Nonnecke B Lonnerdal and J Murray ldquoThelactoferrin receptormaymediate the reduction of eosinophils inthe duodenum of pigs consumingmilk containing recombinanthuman lactoferrinrdquo BioMetals vol 27 no 5 pp 1031ndash1038 2014

[32] I Bournazou K J Mackenzie R Duffin A G Rossi and CD Gregory ldquoInhibition of eosinophil migration by lactoferrinrdquoImmunology and Cell Biology vol 88 no 2 pp 220ndash223 2010

[33] E J Dial A J Dohrman J J Romero and L M Licht-enberger ldquoRecombinant human lactoferrin prevents NSAID-induced intestinal bleeding in rodentsrdquo Journal of Pharmacyand Pharmacology vol 57 no 1 pp 93ndash99 2005

[34] P E Ulloa J F Medrano and C G Feijo ldquoZebrafish as animalmodel for aquaculture nutrition researchrdquo Frontiers in Geneticsvol 5 article 313 2014

[35] S A Renshaw C A Loynes D M Trushell et al ldquoA transgeniczebrafish model of neutrophilic inflammationrdquo Blood vol 108no 13 pp 3976ndash3978 2006

[36] National Research Council Nutritional Requirements of FishNational Academies Press Washington DC USA 1993

[37] M WesterfieldThe Zebrafish Book A Guide for the LaboratoryUse of the Zebrafish (Danio rerio) University ofOregon EugeneOre USA 5th edition 1994

[38] C B Kimmel W W Ballard S R Kimmel B Ullmann and TF Schilling ldquoStages of embryonic development of the zebrafishrdquoDevelopmental Dynamics vol 203 no 3 pp 253ndash310 1995

[39] M Brand M Granato and C Nusslein-Volhard ldquoKeepingand raising zebrafishrdquo in Zebrafish A Practical ApproachC Nusslein-Volhard and R Dahm Eds pp 7ndash37 OxfordUniversity Press New York NY USA 2002

[40] T L Welker Ch Lim M Yildirim-Aksoy and P H KlesiusldquoGrowth immune function and disease and stress resistanceof juvenile Nile tilapia (Oreochromis niloticus) fed graded levelsof bovine lactoferrinrdquo Aquaculture vol 262 no 1 pp 156ndash1622007

[41] C G Feijoo A Sarrazin M L Allende et al ldquoCysteine-serinerich nuclear protein 1 Axud1Csrnp1 is essential forcephalic neural progenitor proliferation and survival inzebrafishrdquo Developmental Dynamics vol 238 pp 2034ndash20432009

[42] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 2001

[43] S H Oehlers M V Flores K S Okuda C J Hall K ECrosier and P S Crosier ldquoA chemical enterocolitis model inzebrafish larvae that is dependent onmicrobiota and responsiveto pharmacological agentsrdquo Developmental Dynamics vol 240no 1 pp 288ndash298 2011

[44] T Jowett and L Lettice ldquoWhole-mount in situ hybridizationson zebrafish embryos using a mixture of digoxigenin- andfluorescein-labelled probesrdquo Trends in Genetics vol 10 no 3pp 73ndash74 1994


[45] E A Harvie J M Green M N Neely and A HuttenlocherldquoInnate immune response to Streptococcus iniae infection inzebrafish larvaerdquo Infection and Immunity vol 81 no 1 pp 110ndash121 2013

[46] J J Van Soest O W Stockhammer A Ordas G V BloembergH P Spaink andAHMeijer ldquoComparison of static immersionand intravenous injection systems for exposure of zebrafishembryos to the natural pathogen Edwardsiella tardardquo BMCImmunology vol 12 article 58 2011

[47] S OrsquoSullivan J F Gilmer and C Medina ldquoMatrix metallopro-teinases in inflammatory bowel disease an updaterdquo Mediatorsof Inflammation vol 2015 Article ID 964131 19 pages 2015

[48] P J Koelink S A Overbeek S Braber et al ldquoCollagendegradation and neutrophilic infiltration a vicious circle ininflammatory bowel diseaserdquo Gut vol 63 no 4 pp 578ndash5872014

[49] J Vandooren P E Van den Steen and G Opdenakker ldquoBio-chemistry andmolecular biology of gelatinase B or matrix met-alloproteinase-9 (MMP-9) the next decaderdquo Critical Reviews inBiochemistry and Molecular Biology vol 48 no 3 pp 222ndash2722013

[50] C S Curran and P J Bertics ldquoLactoferrin regulates an axisinvolving CD11b and CD49d integrins and the chemokinesMIP-1120572 and MCP-1 in GM-CSF-treated human primaryeosinophilsrdquo Journal of Interferon and Cytokine Research vol32 no 10 pp 450ndash461 2012

[51] S Baveye E Elass D G Fernig C Blanquart J Mazurier andD Legrand ldquoHuman lactoferrin interacts with soluble CD14and inhibits expression of endothelial adhesion molecules E-selectin and ICAM-1 induced by the CD14-lipopolysaccharidecomplexrdquo Infection and Immunity vol 68 no 12 pp 6519ndash65252000

[52] M Machnicki M Zimecki and T Zagulski ldquoLactoferrin regu-lates the release of tumour necrosis factor alpha and interleukin6 in vivordquo International Journal of Experimental Pathology vol5 pp 433ndash439 1993

[53] J R Zucali H E Broxmeyer D Levy and C Morse ldquoLacto-ferrin decreases monocyte-induced fibroblast production ofmyeloid colony-stimulating activity by suppressing monocyterelease of interleukin-1rdquoBlood vol 74 no 5 pp 1531ndash1536 1989

[54] T KuharaM Iigo T Itoh et al ldquoOrally administered lactoferrinexerts an antimetastatic effect and enhances production of IL-18in the intestinal epitheliumrdquo Nutrition and Cancer vol 38 no2 pp 192ndash199 2000

[55] X Liu X Chang H Wu J Xiao Y Gao and Y Zhang ldquoRoleof intestinal inflammation in predisposition of Edwardsiellatarda infection in zebrafish (Danio rerio)rdquo Fish and ShellfishImmunology vol 41 no 2 pp 271ndash278 2014

[56] B Stecher R Robbiani AWWalker et al ldquoSalmonella entericaserovar typhimurium exploits inflammation to compete withthe intestinal microbiotardquo PLoS Biology vol 5 no 10 pp 2177ndash2189 2007

[57] A Cederlund GH Gudmundsson and B Agerberth ldquoAntimi-crobial peptides important in innate immunityrdquo FEBS Journalvol 278 no 20 pp 3942ndash3951 2011

[58] P Garcıa-Valtanen A Martinez-Lopez M Ortega-Villaizan LPerez J M Coll and A Estepa ldquoIn addition to its antiviraland immunomodulatory properties the zebrafish 120573-defensin2 (zfBD2) is a potent viral DNA vaccine molecular adjuvantrdquoAntiviral Research vol 101 no 1 pp 136ndash147 2014

[59] A P Corfield N Myerscough R Longman P Sylvester SArul and M Pignatelli ldquoMucins and mucosal protection in

the gastrointestinal tract new prospects for mucins in thepathology of gastrointestinal diseaserdquo Gut vol 47 no 4 pp589ndash594 2000

[60] S H Oehlers M V Flores C J Hall K E Crosier and P SCrosier ldquoRetinoic acid suppresses intestinal mucus productionand exacerbates experimental enterocolitisrdquo Disease Models ampMechanisms vol 5 no 4 pp 457ndash467 2012

[61] I A Garcıa-Montoya T S Cendon S Arevalo-Gallegos andQ Rascon-Cruz ldquoLactoferrin a multiple bioactive protein anoverviewrdquo Biochimica et Biophysica ActamdashGeneral Subjects vol1820 no 3 pp 226ndash236 2012

[62] P Valenti and G Antonini ldquoLactoferrin an important hostdefence against microbial and viral attackrdquo Cellular and Molec-ular Life Sciences vol 62 no 22 pp 2576ndash2587 2005

[63] H Jenssen and R E W Hancock ldquoAntimicrobial properties oflactoferrinrdquo Biochimie vol 91 no 1 pp 19ndash29 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


diseases increase health and improve the stress response[1 15ndash19] However little focus has been given to the use ofadditives for controlling intestinal inflammation At presentthere are only two studies that evaluate the effects of addi-tives specifically mannan-oligosaccharide and 120573-glucans onsoybean meal-triggered intestinal inflammation in farmedfish [1 17] These investigations indicate that only mannan-oligosaccharide is able to decrease to varying degrees thealtered intestinal histology observed in fish fed with dietsincluding low amounts of soybean meal [1 17]

Lactoferrin (LF) is an abundant iron-binding glyco-protein secreted by epithelial cells and contributes to thecomposition of bodily fluids in mammals such as milk tearssaliva bile and pancreatic fluid [20 21] Specific LF receptorsare present on the surface of different tissues and cell typesincluding the gastrointestinal tract lungs neutrophils andeosinophils [22 23] Previous research has demonstrated thatthis glycoprotein has antimicrobial activity and can stimulatecytokine production enhance cell proliferation and regulatemucosal immunity [20 24ndash26] Due to these properties LFhas been used in prophylactic treatments for fish against dif-ferent infectious diseases [25 27ndash29] Furthermore LF exertsa potent anti-inflammatory effect in different tissues mainlyby reducing immune cell recruitment to inflammatory sitesDuring influenza virus infection LF reduces the numberof infiltrating leukocytes in bronchoalveolar lavage fluid inhumans [30] Likewise LF can reduce eosinophil infiltrationto the pigs small intestine in a mechanism independent ofcytokine [31 32] Moreover an in vivo study in rats andmice demonstrated that orally administered LF preventsintestinal injury triggered by nonsteroidal anti-inflammatorydrugs The authors related this effect to the attenuation ofneutrophil migration to the intestine [33] Despite thesevarious related studies there are currently no reports on thepossible role of LF as an intestinal protector in fish and less soon the possible protective effects of LF against soybean meal-induced intestinal inflammation in fish

Factors contributing to this lack of information are thehigh costs and long-term assays involved in evaluating theuse of additives in the aquaculture industry which is inaddition to the challenge ofworkingwithminimal precedentson the cellular and molecular processes in many farmedfish species Therefore an alternative research strategy is toperform preliminary studies in a model fish in which manydiets can be assessed in a short period and at low costsMoreover the selected fish model should facilitate under-standing the biological processes triggered by different dietsConsidering the extensive biological literature and power-ful biotechnological tools available for zebrafish (D rerio)this teleost fish is an ideal organism for immune-nutritionresearch [34]

One key advantage of zebrafish is the availability of trans-genic lines with certain fluorescently-labeled innate immunecells such as neutrophils [35] Since the hallmark of inflam-mation is neutrophil migration these cells can be used asinflammatory markers This strategy has been used beforeby Hedrera et al [9] who demonstrated that soybean mealconsumption by zebrafish results in inflammatory side effectssimilar to those observed in commercially farmed fish

A primary advantage of using transgenic fluorescentlylabeled zebrafish is that the inflammatory process can be veryquickly observed even before histological effects becomerecognizable

This study evaluated the effects of LF on intestinal inflam-mation induced by a soybean meal-based diet in zebrafishBy using the Tg(BACmpoGFP)i114 transgenic zebrafish lineneutrophil migration as part of the intestinal inflammatoryprocess was monitored in vivo To complement this data thetranscriptional levels of different markers related to mucosalbarrier functions as matrix metallopeptidase 9 mucin 22and beta-defensin 1 (mmp9 muc22 and 120573-def-1) and lipidabsorption like the fatty-acid-binding proteins 2 and 6 (fabp2and fabp6) were evaluated Finally the influence of intestinalinflammation on the immune response to Edwardsiella tardainfection was assessed

2 Material and Methods

21 Zebrafish Strains andMaintenance Zebrafish weremain-tained and raised at the Laboratory of Developmental Biol-ogy Universidad Andres Bello Chile according to stan-dard protocols [37] The Tg(BACmpoGFP)i114 transgeniczebrafish line was used [35] All embryos were collectedthrough natural spawning according to Kimmel et al [38]Eggs were incubated in petri dishes at 28∘C for three days inthe E3 medium (5mM NaCl 017mM KCl 033mM CaCl

2

and 033mM MgSO4 with methylene blue equilibrated to

pH 70) [39] Embryonic and larval stages are expressed inhours postfertilization (hpf) or days postfertilization (dpf)All animal-handling procedures were approved by the Com-mittee of Animal Bioethics of the Universidad Andres Bello

22 Experimental Diets Five diets were formulated andprepared (Table 1) The positive control diet contained 50soybean meal (50SBM) while the negative control dietcontained fishmeal as the primary protein source (100FM)[9] Additionally a diet normally used for zebrafish larvae(ZFP sera Micron Heinsberg Germany) was used as asecond negative control To evaluate the intestinal protectiveeffect of LF three batches of the 50SBM diet were supple-mented with bovine LF obtained from milk (Lactoferrin100 S60 Natural Healthy Concepts Appleton WI USA)in concentrations of 05 10 or 15 gkg (50SBM+LF0550SBM+LF10 and 50SBM+LF15 resp) [40] Likewise onebatch of the 100FM diet was supplemented with 15 gkg ofbovine LF (100FM+LF15) All diets were supplemented witha standard vitamin and mineral premix and formulated tobe isoenergetic isonitrogenous and isolipidic Each feed dietwas prepared by cooking-extrusion in a twin screw extruder(CLEXTRAL BC-21 Clextral Firminy France) with a 2mmdiameterThe resulting moist pellets were oven-dried at 60∘Cfor approximately eight hours and then coated with fish oilaccording to the formulation for each diet using a laboratoryvacuum coater (Dinnissen Model VC10 Sevenum Nether-lands) The pellets were subsequently crumbled screened tothe appropriate particle size (75120583m) and stored at minus20∘Cuntil use in the feeding trials



















3 Results







ZFP

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

100

FM50

SBM

Diets

0

5

10

15

20

25

ZFP

100

FM

50

SBM

50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15




50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15

Num

ber o

f neu

troph

ilsd

efine

d ar

ea






00

05

10

15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)



mmp9

lowastlowast

lowastlowast

lowastlowast





LF15100FM+



4 Discussion









(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)


100FM+LF15

(l)


100FM+LF15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

lowastlowast

lowastlowast

lowastlowast

fabp2

fabp2

fabp6

fabp6ZF

P100

FM50

SBM

50

SBM+

LF15

100

FM+

LF15



05 12 24 36 48 60 72 84 96

20

40

60

80

100


Accu

mul

ated

mor

talit

y (

)



lowast

lowast


lowastlowast



5 Conclusions


Competing Interests


Acknowledgments



References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































3 Results







ZFP

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

100

FM50

SBM

Diets

0

5

10

15

20

25

ZFP

100

FM

50

SBM

50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15




50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15

Num

ber o

f neu

troph

ilsd

efine

d ar

ea






00

05

10

15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)



mmp9

lowastlowast

lowastlowast

lowastlowast





LF15100FM+



4 Discussion









(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)


100FM+LF15

(l)


100FM+LF15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

lowastlowast

lowastlowast

lowastlowast

fabp2

fabp2

fabp6

fabp6ZF

P100

FM50

SBM

50

SBM+

LF15

100

FM+

LF15



05 12 24 36 48 60 72 84 96

20

40

60

80

100


Accu

mul

ated

mor

talit

y (

)



lowast

lowast


lowastlowast



5 Conclusions


Competing Interests


Acknowledgments



References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ZFP

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

100

FM50

SBM

Diets

0

5

10

15

20

25

ZFP

100

FM

50

SBM

50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15




50

SBM+

LF05

50

SBM+

LF10

50

SBM+

LF15

100

FM+

LF15

Num

ber o

f neu

troph

ilsd

efine

d ar

ea






00

05

10

15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)



mmp9

lowastlowast

lowastlowast

lowastlowast





LF15100FM+



4 Discussion









(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)


100FM+LF15

(l)


100FM+LF15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

lowastlowast

lowastlowast

lowastlowast

fabp2

fabp2

fabp6

fabp6ZF

P100

FM50

SBM

50

SBM+

LF15

100

FM+

LF15



05 12 24 36 48 60 72 84 96

20

40

60

80

100


Accu

mul

ated

mor

talit

y (

)



lowast

lowast


lowastlowast



5 Conclusions


Competing Interests


Acknowledgments



References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)



mmp9

lowastlowast

lowastlowast

lowastlowast





LF15100FM+



4 Discussion









(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)


100FM+LF15

(l)


100FM+LF15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

lowastlowast

lowastlowast

lowastlowast

fabp2

fabp2

fabp6

fabp6ZF

P100

FM50

SBM

50

SBM+

LF15

100

FM+

LF15



05 12 24 36 48 60 72 84 96

20

40

60

80

100


Accu

mul

ated

mor

talit

y (

)



lowast

lowast


lowastlowast



5 Conclusions


Competing Interests


Acknowledgments



References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)


100FM+LF15

(l)


100FM+LF15

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

Gen

e exp

ress

ion

(rel

ativ

e to50

SBM

)

25

20

15

10

05

00

lowastlowast

lowastlowast

lowastlowast

fabp2

fabp2

fabp6

fabp6ZF

P100

FM50

SBM

50

SBM+

LF15

100

FM+

LF15



05 12 24 36 48 60 72 84 96

20

40

60

80

100


Accu

mul

ated

mor

talit

y (

)



lowast

lowast


lowastlowast



5 Conclusions


Competing Interests


Acknowledgments



References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















05 12 24 36 48 60 72 84 96

20

40

60

80

100


Accu

mul

ated

mor

talit

y (

)



lowast

lowast


lowastlowast



5 Conclusions


Competing Interests


Acknowledgments



References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Lactoferrin Decreases the Intestinal...

Documents

Transcript of Research Article Lactoferrin Decreases the Intestinal...