Estrategias Ante mortem y Post mortem para el control de 

Salmonella spp. en pollo de engorda 

Guillermo Tellez D.V.M., MS., Ph.D.

University of Arkansas Division of AgricultureDepartment of Poultry ScienceJKS Poultry Health Laboratory

Ralph Waldo Emerson(25 – 05 ‐ 1803 – 27 – 04‐ 1882)

• ¿Qué es lo más difícil en el mundo?

PENSAR!

Salmonella

SalmonellaEnterobacteriaforma bacilarGram‐negativa,            no esporula,móvil

quimioheterótrofo, energía de reacciones de oxidación y reducción de 

fuentes orgánicas

Anaerobiofacultativo ‐

ATP porrespiración

aeróbica ‐ puedecambiar a 

fermentación

Super‐reino: Bacteria

Reino: Bacteria

Filo: Proteobacteria

Clase: Gammaproteobacteria

Orden: Enterobacteriales

Familia: Enterobacteriaceae

Género: Salmonella

Especies

S. bongoriS. enterica

subesp. enterica *subesp. salamaesubesp. arizonae

subesp. enterica *subesp. salamaesubesp. arizonae

subesp. diarizonaesubesp. houtenaesubesp. indica

subesp. diarizonaesubesp. houtenaesubesp. indica

Colonización in situ

• Brotes de salmonelosisrelacionados con el consumo de productosfrescos ha elevado el interés en lasinteraccionesSalmonella‐planta quepermiten la colonización de la planta

Incubación de Salmonella enterica marcadacon gfp en hojas de lechuga iceberg

• En la luz resultó en la agragción de bacteriascerca de los estomasabiertos e invasión haciael tejido de la hoja

• En contracte, la incubación en la oscuridad resultó en un patrón de de adhesiónescasa y muy pocainternalización en los estomas. 

Kroupitski et al. Ap and Env Mic. 2009;75(19):6076–6086.

• Estos resultadosimplican que el patógeno es atraídohacia los nutrientesrecién producidos porlas células activas en fotosíntesis

Kroupitski et al. Ap and Env Mic. 2009;75(19):6076–6086.

Estos hallazgos

• Sugieren la entradamecánica de Salmonellaal apoplasto de la planta

• E implica que los antígenos de Salmonellano son bien reconocidospor la inmunidad innatadel estoma, o que estepatógeno ha evolucionado mediospara evadirla. 

La internalización en las hojas

• Puede ofrecer unaexplicación parcial de la falla de los sanitizantespara erradicareficientemente los patógenos de origenalimentico de lasverduras.

En botánica Estoma (en inglés stoma, plural stomata), del griego “boca”

• Es un poro, encontrado en la epidermis de las hijas, tallos y otros órganos quese utiliza para controlar el intercambio gaseoso.

• El poro está delimitado porun par de célulasespecializadas[parénquima] conocidascomo células guarda queson responsables de regular el tamaño de la apertura

Complejo de estomas

• El término también se emplea colectivamentepara referirse a un complejo de estomascompleto, tanto el poroen sí como sus célulasguarda. 

Función de los estomas

• El aire que contiene CO2 y O2

entra a la planta a través de estas aperturasse usa para la respiración y fotosíntesis, respectivamente

Evolución de los estomas

• El registro fósil aporta pocainformación.

• Pueden haber evolucionadopor la modificación de los conceptáculos de los ancestros de las plantasparecidos a algas.

• Conceptáculos: Son cavidades especializadas de algas marinas y de aguadulce que contienen los órganos reproductores.

Conceptaculos

Su ruta evolutiva

• Es claro, sin embargo, que la evolución de los estomas pudo ocurrir al mismo tiempo que la de la cutícula serosa –estos dos rasgosconstituyeron unaventaja muy importantepara las primerasplantas terrestres.

La Salmonella sobrevive sobre o dentro de los tomates

• Desde la inoculación en la floración hasta la maduración.

• El tallo y flores del tomateson sitios donde la Salmonella puede adherirsey permanecer viable durante el desarrollo del fruto, y sirve como ruta o reservorio para la contaminación del frutomaduro. 

Guo X et al. Ap and Env Mic. 2001;67(10):4760–4764.

Reino animal

• La mayoría de los Filosanimales conocidos aparecenen el registro de fósiles comoespecies marinas de cerca de 542 millones de años.

• Los animales están dividios en varios sub‐grupos, incluidaslas aves, mamíferos, reptiles, peces e insectos. 

Salmonella: Mecanismos de Infección

Entrada de Salmonella

Sistema de Secreción Tipo III (TTSS, en inglés)• Forma principal en la que

Salmonella suministrafactores de virulencia al hospedero

• Constituído de 20 proteínas• Ensambladas en orden paso

a paso• PrgI es una estructura de

aguja extendida por unabase de proteína, forma un canal hacia el hospedero.

PrgI

Islas de patogenicidad de Salmonella

• SPI-1: Invasión

• SPI-2: Replicaciónintracelular

• SPI-3: SupervivenciaIntracelular

• SPI-4: Producción de toxinas

• SPI-5: Inflamación

Vesícula con Salmonella (VCS)• Después de la ingestión,

entra una VCS a través de la endocitosis mediadapor la bacteria

• Vive y se multiplica en la VCS

• Una forma de evadir la respuesta inmune del hospedero

Estrés e inmunidad

Los efectos del estréssobre el curso de unainfección se hanadjudicado por mucho tiempo al efecto directode las hormonasrelacionadas con el estrés sobre el sistemainmune y la fucnión de la barrera intestinal. 

Los animales para consumo pueden no tener una vidalibre de estrés, particularmente los que se crían

intensivamenteQuizá lo que fueinesperado es que la respuesta al estrésbacteriano impuesta porel medio del hospederosobre el organismo y la respuesta de adrenalinadel hospedero impuestapor la infección puedenpotenciar el crecimiento y virulencia del microorganismo.

Bacterias— patógeno comensal, obligado u oportunista

Viven en estréspermanente y regulan su expresión de genes, y, en el caso de patógenos potenciales, expresan genes de virulencia en respuestaal estrésmedioambiental. 

• Hace 112 años inició unanueva era en la endocrinología con la primera purificación de una hormona: adrenalina.

• Desde 1930, casiinmediatamente despuésde su primer uso, se reportaron casos de sepsis asociados con la adrenalina. 

Endocrinología Microbiana

Endocrinología Microbiana

• La teoría más aceptada paraexplicar la habilidad de lashormonas para influir el cursode una infección involucra la supresión del sistemainmune. 

• Hoy, sabemos que los microorganismos infecciososresponden igualmente al medioambienteneuroendócrino del hospedero. 

Adrenalina1. Incrementa el crecimiento

bacteriano

2. Une proteínas ligadores de hierro y entonces la bacteria usa el hierro para crecer. 

3. Está involucrada en el quorum sensing (autoinducción) de lasbacterias

4. Incrementa la expresión de adhesinas

5. Incrementa virulencia e invasión

Detección de Salmonella de tráquea en avicultura comercial

como herramienta epidemiológica

G. Tellez, G. Kallapura, J.D. Latorre, L.R. Bielke, A. Menconi, O.B. Faulkner, 

A. Wolfenden, and B.M. Hargis

Salmonella

Salmonella sp. Se piensacomúnmente que se transmite principalmentea través del contacto con individuos infectados y la ingestión de materialescontaminados con heces

Sin embargo, variosinvestigadores han

demostrado la importanciade la transmisión aérea de Salmonella como fuente de infección cruzada en la 

avicultura.

transmisión aérea de Salmonella comofuente de infección cruzada en la avicultura

• Baskerville A., et al.  1992Airborne infection of laying hens with Salmonella enteritidis phage type 4

• Nakamura M. et al. 1995• Intratracheal infection of chickens with Salmonella enteritidis and the effect of feed and

water deprivation• Lever M.S., et al.   1996

Cross‐infection of chicks by airborne transmission of Salmonella enteritidis PT4• Holt P.S. et al. 1998

Airborne horizontal transmission of Salmonella enteritidis in molted laying chickens• Gast R.K. et al. 1998

Airborne transmission of Salmonella enteritidis infection between groups of chicks in controlled‐environment isolation cabinets

• Harbaugh E. 2006Rapid aerosol transmission of Salmonella among turkeys in a simulated holding‐shedenvironment

• Basnet H.B., et al. 2008Reproduction of fowl typhoid by respiratory challenge with Salmonella Gallinarum

Desafío Intra‐Traqueal vs. Intra‐ Saco Aéreo Torácico

• Pollos de 7‐d inoculados IT o IAS con 105 ufc de S. Enteritidis (SE). 

• Sacrificio 24 h después, cultivo a partir de tráquea, tonsilas cecales, hígado y bazo para recuperación de SE por enriquecimientotoda la noche en caldotetrationato (CT).  

• Las muestra enriquecidas se sembraron por estría en agar XLD con novobiocina(NO) y ácido nalidíxico (NA). 

% pollos positivos SE

N=10

Transmisión Horizontal

• Sonda oral con ~1x105 SE (20 % inoculados) @ 3 d de edad (n=100)

• Pollos mezclados en un corral (80 % polloscontacto)

• Sacrificio y cultivo @ 10 d de edad

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Prevalencia de Salmonella sp. En tráqueas de pavosprocesados comercialmente

• Toma aséptica de tráquea y ciegos n=100, pavoscomerciales de 16‐ semanasde edad

• Las tráques se pinzaron en los extremos y se agregaron 20 mLde agua peptona e incubadas 8 h a 37 °C.

• El agua peptona de cadatráquea se recolectó y enriqueció con 20 mL de CT 2X e incubó toda la noche. 

• Se sembró por estría en agar XLD solo con NO.

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E. coli Salmonella sp.

%  tráqueas positivas

N=100

34/100 (34 %) 

97/100 (97 %)

Recuperación de Salmonella de tonsilas cecales

Se agregó CT  2X a la muestra restante y se siguó el mismoprocedimiento anterior

15/100 (15 % ) Tonsila vs. 34/100 (34 %) Tráquea

10^4 10^6 10^8IT 1,125 3,195 5,114Oral 1,799 1,858 5,989

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7,000UFC, Log10

 Ciego

‐Ton

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Recuperación de Salmonella Día‐8 

Pollos inoculados con Salmonella enteritidis (SE) – 1 semana – Intra‐traqueal u Oral, con 104, 106 o 108UFC/pollo. Cultivo 24 hrs post desafío. En las barras se expresa el Log10 SE/ gramo de contenido cecalcomo promedio ± error estándard P > 0.05. Las literales sobre las barras indican diferenciassignificativas P < 0.05

GrupoLog10  SE / gramo de contenido cecal

Hígado y bazo TráqueaCiego ‐ Tonsila

Cecal

Intra‐traqueal ‐ 10^4 1.125 ± 0.401c 6/12 (50%) a 8/12 (66.66%) a 5/12 (41.66%) c

Intra‐traqueal ‐ 10^6 3.195 ± 0.166b 10/12 (83.33%) ab 11/12 (91.66%) ab 12/12 (100%) a

Intra‐traqueal ‐ 10^8 5.114 ± 0.472a 11/12 (91.66%) b 12/12 (100%) b 12/12 (100%) ab

Oral ‐ 10^4 1.799 ± 0.384c 0/12 (0%)c 1/12 (8.33%) c 8/12 (66.66%) b

Oral ‐ 10^6 1.858 ± 0.400c 2/12 (16.66%) c 3/12 (25%) cd 8/12 (66.66%) b

Oral ‐ 10^8 5.989 ± 0.512a 1/11 (9.09%) c 5/11 (45.45%) d 11/11 (100%) ab

Evaluación de la infección Intra‐tracqueal de pollos conSalmonella enteritidis al día 8

Pollos inoculados con Salmonella enteritidis (SE) – 1 semana – Intra‐traqueal u Oral, con 104, 106 o108 UFC/pollo. Cultivo 24 hrs post desafío. En las barras se expresa el Log10 SE/ gramo de contenidocecal como promedio ± error estándard. Los datos de tonsilas cecales, tráchea, hígado y bazo seexpresan como pollos positivos/total para cada tejido (%). Literales en la misma columna indicandiferencias significativas, p < 0.05, N=12.

Aunque se sabe que el contacto directo con aves infectadas y el contactoindirecto con superficies medioambientalescontaminadas son factoresimportantes en la diseminación de Salmonellaen las parvadas, el papelpotencial de la transmisiónaerógena no estáclaramente definido

BAL

Salmonella parece penetrar a través de las células epiteliales en el TLAB (BALT ) para infectar células linfoides

Los resultados de estosestudios sugieren que la tráquea es un órgano viable para la recuperación de Salmonella

Reconfirma que el  movimiento aerógeno de Salmonella en las casetas esun punto de control relevantepara limitar la diseminaciónde la infección dentro de lasparvadas. 

¿Estamos a salvo?

¿Hay esperanza?

Recomendaciones para el Control

• Roedores• Aves silvestres• Insectos• Personal• Fomites• Medioambiente• Instalaciones• Equipo• Transporte

• Reproductores• Incubadora• Entrega• Parvadas de diferenteorigen

• Alimento/ProteínaAnimal

• Agua• Vacunación• Algunosprobióticos/EC

• Más….

Reducción de la Tasa Reproductiva de casos (R0)

• Prevenir la exposición al patógeno – difícil en el caso de Salmonella

• Reducir la exposición a nivel incapaz de causar la infección –difícil pero no imposible en el caso de Salmonella

• Reducir la tasa de diseminación del microorganismo al medioambiente (transmisiónhorizontal) o de los reproductores a través del huevo (transmission vertical) disminuye el R0

AntibióticosDietaProbióticosPrebióticosSimbióticos Ácidos OrgánicosExtractos de PlantasOtros

Herramientas para reducir el R0

La presión social ha llevadoa la creación de regulaciones para restringirel uso de antibióticos en la producción avícola y la ganadería. 

• Actualmente hay mayor interés público y científicorespecto a la administraciónterapéutica y sub‐terapéutica de antibióticosa los animales. 

Hay necesidad de evaluar el potencial de alternativas a los antibióticos para mejorar la resitencia a las enfermedades en la producción animal intensiva.

• Mejorar la resistencia a lasenfermedades de los animales criados sin antibióticos no solo beneficia su salud, bienestary eficiencia productiva, también es una estrategiaclave en el esfuerzo de mejorar la seguridadmicrobiológica de los productos avícolas. 

En los últimos 20 años

• Nuestro laboratorio ha trabajado en la identificación de candidatosa probióticos para la avicultura, los cualesrealmente puedendesplazar a Salmonella y otros patógenos entéricosque han colonizado el tractogastrointestinal de pollos y pollitas.

Floramax: Fabricado bajolicencia exclusiva de la Universidad de Arkansas 

• El trabajo intensivo permitió la identificación de 11 LAB (del género o relacionado con Lactobacillus en el productoFloraMax B‐11® que fueroneficaces en el tratamiento de pollos y pollas infectados con Salmonella.

Vicente, J.S. et al. 2007 J. of Applied Poult. Res. 16: 471‐476.

• La selección de parvadasinfectadas con Salmonellaantes del sacrificio, demostró que el tratamiento de dichasparvadas con B11, aproximadamente 2 semanas antes del sacrificio, puede reducirmarcadamente la recuperaciónmedioambiental de Salmonella en pavos y pollos comerciales.  

-

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Figure 3. Effect of Beneficial Bacteria Alone or in Combination with an acidifier on Salmonella Isolation in Commercial Turkey Houses

a,b

Control OrganicAcidifier

B11 Acidifier+ B11

aa

aa

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After trta,b

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Ácidos Orgánicos

Incremento de la productividad y reducción de costos de producción

• Ensayos comercialesde gran escalaindican que la administraciónapropiada de estamezcla de probióticos a pavos y pollos. 

• Vicente J. et al., 2006• Torres A. et al., 2007• Torres A. et al., 2007

100

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+ 7 d + 14 d + 21 d + 26 d

Age (days)

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g)

Control Probiotic in Drinking WaterProbiotic in Drinking Water + Lactose Probiotic in Feed + Lactose

a a a aa a

ab

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bbaa

a a

a

b b

b75.6g

124.5g173.6g

Mayor Resistencia a infecciones porSalmonella spp.

• La extensiva investigaciónde laboratorio y de campo realizados con este cutivo LAB ha demostrado el desarrolloacelerado de la microbiota normal en pollos y pavos. 

• Tellez  G. et al., 2001• Farnell M. et al., 2006• Tellez G. et al., 2006• Vicente J. et al., 2007• Higgins S. et al., 2007• Higgins J. et al., 2007• Wolfenden A., 2008• Higgins J. et al., 2009• Vicente J. et al., 2009• Higgins S., et al., 2011• Tellez G. et al., 2012

Higgins J. et al., 2007Poult. Sci. 86:1662–1666

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* *

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Actividad Bactericida BAL

Redes de Genes a partir del Ingenuity Pathway AnalysisPara comparar grupos de tratamiento SE y SE+B11

Higgins S. et al., 2011

Grupos de tratamiento SEGrupos de tratamiento SE+B11

Vacuna Compañía

MeganVac1 (ST, SH, SE) Lohmann Animal Health

Megan Egg Lohmann Animal Health

Salmune CEVA

Poulvac SE Pfizer

Poulvac ST Pfizer

Gallivac SE Merial

CEVAC  SG‐9R CEVA

Salmune CEVA

AVIPRO VAC T LAH

Vacunas contra Salmonellaautorizadas actualmente

Los órganos linfoides secundarios pueden sub‐dividirse en sistema inmune Sistémico (***) y Mucosal

NALTBALT

GALT

RALT

Mucosal

***

***

***

#

#

Superficies mucosas

• Gastrointestinal• Respiratoria• Tracto urogenital

• Representan un áreamuy grande de exposición a agentesexógenos, incluidoslos microorganismos

Sistema Inmune Mucosal común

¿Porqué es importante tener esarespuesta immune mucosal? 

Estos sitios anatómicos son las principalesáreas de interacción del cuerpo con el medio externo y con patógenospotenciales

IngestiónInhalaciónInseminación

Tejido linfoide asociado a mucosas–MALT‐

• Difiere fundamentalmente de la respuesta immune sistémica en:

• El isotipo principal en las secreciones de la mucosa esla IgA secretoria

• La mayoría de las células productoras de anticuerpos y linfocitos T efectores están en el MALT

• Sitios linfoides inductor y efector separados

El intestine es el órgano inmunológicomás grande en el cuerpo

• Comprende 70‐80% de lascélulas productoras de inmunoglobulinas

• Produce más IgA secretoria (SIgA) (50‐100 mg/kg peso corporal / día) que la producción total de IgG en el cuerpo (30 mg/kg/día). 

Desarrollo de las vacunas mucosales2006 Nov 2;126(21):2818‐21 

• La vacuna active oral contra la polio fue la primera mucosal aceptada para usogeneral. Desdeentonces, se handesarrollado vacunassimilares contra la fiebre tifoidea, cólerae infección porrotavirus. 

18a. Dinasty

Ventajas de la ruta de inmunizaciónmucosal

• Induce inmunidad protectora en el sitio de infección• Induce inmunidad sistémica y mucosal• Efectiva en presencia de anticuerpos maternos• No hay reacción de inyección, No necesita agujas• Fácil administración (vacunas orales combinadas con el alimento)

Incluso si la inmunización mucosal no elimina totalmente la infección

• Los anticuerpos de la mucosa limitan el gradode repicación y diseminación del patógeno; por lo tanto, reduce la carga ambientaldel mismo y minimizadramáticamente la tasa de infección en la parvada y la transmisión de la enfermedad. 

También

• El diseño de sistemas de administración que se enfoquen en la respuesta immune para conferir unarespuesta balanceada o una que se orientada ya sea a Th1 o Th2

• Depende del patógeo de interés, se puede dirigir la respuesta conforme se necesite para maximizar la protección y reducir las consecuencias de la infeccióncon la mayoría de los patógenos. 

Nuevos enfoques

• Identificación de antígenos protectores conservados(normalmente no inmunogénicos)

• Desarrollo de plataformas efectivas de aplicación mucosal.

Moléculas inmuno‐estimulantes CD154 & HMGB1

• CD154– Glicoproteína Tipo II– Se une al CD40 en LB & LT activados

CD40 CD154

• HMGB1– Proteína de Grupo B1 de altamovilidad.

– Citocina mediadora de la inflamación

– Se une al TLR4 y activa la liberación de citocinas de los macrófagos

Nueva tecnología de Adyuvantes

• La modificación de un polisacárido de origennatural permite la adhesion a las célulaspresentadoras de antígeno en la mucosa – y union química a antígenos autógenos

• Antígenos seleccionadosexperimentalmenteofrecen resultadosalentadores

Conclusiones

• En 2014, no hay “balas de plata” disponibles• Algunos Probióticos dan eficacia y consistenciasimilar a la de los antibióticos, y probablementemejor que las vacunas disponibles comercialmente. 

• Las tecnologías nuevas y emergentes de las vacunaspueden dar mejoras significativas

• Algunos adyuvantes recientes de actividad mucosal pueden mejorar la eficacia de las vacunas inactivadasautógenas.

• La producción de animals para consumolibres de estrés, en un medio limpio, puedetener implicacionespara prevenir la adquisición y transmission potencialde patógenos de origenalimentario. 

Equipo de Integridad Intestinal 2001‐2013• B. M. Hargis, D.V.M.,Ph.D.• G.Tellez, D.V.M., Ph.D.• L. Bielke, Ph.D.• M. Morgan, Microbiologist• N. Pumford, Ph.D.• O. Faulkner, Ph.D.• A. M. Donoghue, Ph.D.• D.J. Donoghue, Ph.D.

1. L. Bielke, Ph.D. (2006)2. A. Torres, Ph.D. (2006)3. R. Jarquim, MS (2006)4. J.L. Vicente, Ph.D. (2007)5. S. Higgins, Ph.D. (2007)6. J. Higgins, Ph.D. (2007)7. S. Henderson, MS (2007)8. F. Solis, Ph.D., (2007)9. A. Wolfenden, MS (2008)10. S. Layton, Ph.D. (2009)11. R. Wolfenden, Ph.D. (2009)12. G. Gaona, Ph.D. (2009)13. S. Shivaramaiah, Ph.D. (2011)

1. A. Menconi, Ph.D.2. J.D. Latorre, Ph.D. 3. G. Kallapura, Ph.D.4. C. Pixley,  Ph.D. 5. A. Wolfenden, Ph.D.

Current Graduate Students

International Journal of Poultry Science 12 (6): 318-321, 2013ISSN 1682-8356© Asian Network for Scientific Information, 2013

Corresponding Author: Guillermo Tellez, Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA

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Effect of Chitosan as a Biological Sanitizer for Salmonella Typhimurium andAerobic Gram Negative Spoilage Bacteria Present on Chicken Skin

Anita Menconi , Xochitl Hernandez-Velasco , Juan David Latorre , Gopala Kallapura ,1 2 1 1

Neil R. Pumford , Marion J. Morgan , B.M. Hargis and G. Tellez1 1 1 1

Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA1

Department de Medicina y Zootecnia de Aves, Facultad de Medicina Veterinaria y Zootecnia,2

Universidad Nacional Autonoma de Mexico, Mexico D.F., 04510

Abstract: Two experiments were conducted to evaluate the effect of chitosan as a biological sanitizer onchicken skin during storage. For experiment 1 (two trials) five skin samples of equal size were dipped intoa solution containing 10 cfu/mL of Salmonella Typhimurium (ST) for 30s. Skin samples were then removed6

and dipped into a solution containing PBS or 0.5% chitosan for 30s. In experiment 2, aerobic Gram negativespoilage bacteria were used as indicators instead of ST. In both experiments, all samples were placed inindividual bags and kept at 4°C. In experiment 1, dipping ST contaminated skin samples in a solution of0.5% chitosan reduced (p<0.05) the recovery of ST by 24 h. In experiment 2, 0.5% chitosan treatment solutionreduced (p<0.05) the presence of spoilage-causing psychrotrophic bacteria below detectable levels. Theseresults suggest that 0.5% chitosan has a potential for use in an intervention technology for the control offoodborne pathogens on the surface of chicken skin contaminated with bacteria during storage.

Key words: Salmonella, chitosan, chicken skin, sanitizer

INTRODUCTIONChickens contain large numbers of microorganisms intheir gastrointestinal tract and on their feathers and feet;therefore, storage quality of fresh chicken is partiallydependent on the bacteria present on the integumentprior to slaughter (Ramirez et al., 1997; Northcutt et al.,2003). Pathogenic microorganisms present in chickencarcasses after processing and throughout scaldingand picking can contaminate equipment and othercarcasses (Hargis et al., 1995; Byrd et al., 1998; Sarlinet al., 1998; Corrier et al., 1999b; Zhang et al., 2013).Pathogenic bacteria such as Salmonella enterica andCampylobacter spp. are able to attach to skin andpenetrate in skin layers or feather follicles (Zhang et al.,2013), facilitating their presence on chicken skin andcarcass during poultry processing (Chaine et al., 2013).Critical control point determination at broiler processinghas become very important, especially because of therecent attention on Hazard Analysis and Critical ControlPoints (HACCP) for reduction of microbial contaminationof meat and poultry (Rose et al., 2002). For all these MATERIALS AND METHODSreasons, strategies to reduce bacterial contamination on Bacterial strain and chitosan: A poultry isolate ofpoultry carcasses are important. However, most of the Salmonella enterica serovar Typhimurium (ST), selectedbacterial reduction strategies for poultry comprise the for resistance to Nalidixic Acid (NA) (Catalog No. N-4382,use of antimicrobial chemicals in rinses or washes and Sigma, St. Louis, MO 63178), was used for alltheir efficacy is reduced by the presence of organic experiments. The amplification and enumerationmatter (Zhao et al., 2009). Therefore, it grows the need protocol for the isolate have been previously describedof biological sanitizers in the processing plant to prevent (Tellez et al., 1993). Briefly, ST was grown in tryptic soycarcass to carcass cross-contamination by pathogenic broth (TSB, Catalog No. 22092, Sigma, St. Louis, MO

bacteria and to lower the potential of foodbornediseases.Interest in chitosan, a biocompatible polymer derivedfrom shellfish, as a biological sanitizer arises fromreports showing several beneficial effects such asantimicrobial and antioxidative activities in foods (No etal., 2002; Friedman and Juneja, 2010). The use ofchitosan in industry, agriculture and medicine is welldescribed (Rabea et al., 2003; Senel and McClure, 2004;Friedman and Juneja, 2010). The antimicrobial activitiesof chitosan against foodborne pathogens has beenbroadly investigated in the food industry (Singla andChawla, 2001; No et al., 2002; Senel and McClure, 2004;Petrovich et al., 2008; El-Hadrami et al., 2010; Kong etal., 2010; Vargas and Gonzalez-Martinez, 2010).Therefore, the objective of the present study was toevaluate the effect of chitosan as a biological sanitizerfor Salmonella and aerobic Gram negative spoilagebacteria on chicken skin during storage at 4°C.

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63178) for approximately 8 h. The cells were washed 0.5% chitosan (N = 30) for 30s and drained off.three times with 0.9% sterile saline by centrifugation Control and treated samples were placed in(1,864 x g) and the approximate concentration of the individual sample bags and kept in a refrigerator atstock solution was determined spectrophotometrically at 4°C. At 1 h, 24 h, 3, 6, 9 and 12 days, 5 control and625 nm. The stock solution was serially diluted and 5 treated skin samples were homogenized withinconfirmed by colony counts of three replicate samples sterile sample bags using a rubber mallet. Sterile(0.1 mL/replicate) spread plated on brilliant green agar saline (5 mL) was added to each sample bag and(BGA, Catalog No. 278820, Becton Dickinson, Sparks, hand stomached. Serial dilutions were spreadMD 21152) plates containing 25 µg/mL novobiocin (NO, plated on MacConkey agar (Becton, Dickinson andCatalog No. N-1628, Sigma, St. Louis, MO 63178) and Co. Sparks, MD, USA). Each sample was plated as20 µg/mL NA. triplicate. The plates were incubated at 37°C for 24

Chitosan: Deacetylated 95% food grade chitosan was enumerated respectively. The identification ofobtained commercially (Paragon Specialty Products, individual colonies with different morphology onLLC Rainsville, AL) and used in all experiments. The MacConkey agar was determined using the API-20Echitosan molecular weight was 350 kDa with viscosity of test kit for the identification of enteric Gram-negative800 mPas and particle size of 100 US mesh (sieve size bacteria (BioMerieux, Inc., Hazelwood, MO).0.152 mm). Chitosan was prepared by dissolving it in asolution containing 0.5% (w/v glacial acetic acid (Catalog Statistical analysis: The Most Probable Number methodNo. J41A08, Mallinckrodt Baker Inc, Phillipsburg, NJ was used to obtain the lowest possible detection limit:08865). 0.5 log cfu/square cm in the enumeration of ST and

Chicken skin samples: As described by Sarlin et al. bacteria per square cm were converted to log10 numbers(1998), raw chicken skin was used as an alternative to and analyzed using Analysis of Variance (ANOVA) withother sampling methods (whole carcass rinse further separation of significantly different means usingprocedure, excised skin sampling, or skin swabs) in all Duncan’s Multiple Range test using SAS (SAS Institute,experiments. Chicken thighs were purchased from a 2002). Significant differences were reported at (p<0.05).local super market and a strip of skin (approximately 2by 2 cm) was aseptically collected using forceps andscissors.

Microbiological procedures:

C Experiment 1: Two trials were conducted. In eachtrial, skin samples (N = 20) were dipped into aphosphate buffered saline (PBS) solutioncontaining 10 cfu/mL of ST for 30 seconds. Skin8

samples were then removed, drained off anddipped for an additional 30s into a solutioncontaining PBS (control; N = 10) or 0.5% chitosan(N 1 ). Control and treated samples were placed inindividual sample bags and kept in a refrigerator at4°C. At one or twenty four hours, five control and fivetreated samples were removed from the refrigeratorand cultured for ST recovery. Briefly, skin sampleswere homogenized within sterile sample bagsusing a rubber mallet. Sterile saline (5 mL) wasadded to each sample bag and hand stomached.Serial dilutions were spread plated on BGA platescontaining 25 µg/mL of NO and 20 µg/mL of NA.Each sample was plated as triplicate. The plateswere incubated at 37°C for 24 h then viable colonieswere observed and enumerated.

C Experiment 2: Skin samples were dipped into asolution containing either PBS (control; N = 30) or

h and then viable colonies were observed and

aerobic Gram negative bacteria. Colony forming units of

RESULTS AND DISCUSSIONSalmonella is one of the most widespread bacterialspecies in poultry and it is often associated withfoodborne illness (Bailey et al., 2002; Lynch et al., 2006).Cross-contamination by Salmonella in birds andcarcasses may occur during transportation andprocessing (Cason et al., 1997; Corrier et al., 1999a).Therefore, the poultry industry has the challenge ofmonitoring and controlling Salmonella at all productionlevels (Hargis et al., 1995; Corrier et al., 1999a;Mikolajczyk and Radkowski, 2002). In the present study,dipping ST contaminated skin samples for 30 s in asolution of 0.5% chitosan was able to significantlyreduce the recovery of ST cfu/square cm after 24 h inboth trials (Table 1). The presence of spoilage bacteriain food products is an important economic problem.Therefore, an inexpensive and safe treatment to preventspoilage is needed. Chitosan has been shown to be aneffective antimicrobial, especially antibacterial. As shownin Table 2, 0.5% chitosan was effective in reducing totalaerobic mesophilic Gram negative bacteria (spoilagebacteria) to undetectable levels. The primary spoilagebacteria in the control group of experiment 2 wereidentified as Escherichia coli, Enterobacter aerogenesand Pseudomonas aeruginosa using the API-20E test kitfor enteric Gram-negative bacteria (bioMerieux, Inc.,Hazelwood, MO). The concentration of P. aeruginosa inthe control group increased from 7.5 x10 - 1.5x106 8

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Table 1: Salmonella Typhimurium (log10 cfu±standard error)/square cm ofchicken skin treated with 0.5% chitosan solution in experiment1

Trial 1 Trial 2Dipping ---------------------------------- ---------------------------------treatment 1 h 24 h 1 h 24 hControl 6.57±0.11 6.03±0.02 6.78±0.06 7.36±0.06a a a a

Chitosan (0.5%) 6.23±0.03 5.81±0.06 7.06±0.08 6.6±0.17a b a b

Values within columns with different lowercase superscripts differsignificantly (p<0.05)

Table 2: Aerobic Gram negative bacteria (log10 cfu±standarderror)/square cm of chicken skin treated with 0.5%chitosan solution in experiment 2

Sampling time Control Chitosan (0.5%)1 h 1.31±0.83 Undetectable levelsa

24 h 1.20±0.73 Undetectable levelsa

3 days 4.70±0.31 Undetectable levelsa

6 days 6.25±0.21 Undetectable levelsa

9 days 7.12±0.11 Undetectable levelsa

12 days 8.15±0.11 Undetectable levelsa

Values within columns with different lowercase superscripts differsignificantly (p<0.05)

cfu/square cm from 6-12 days stored at refrigerationtemperatures (data not shown). The decreased growthas shown in Table 2 indicates that chitosan was veryeffective in controlling this and possible other spoilagebacteria. These results are in agreement with thosepublished by Darmadji and Izumimoto (1994) whodescribed the effectiveness of chitosan on storagestability of minced beef. Solutions of chitosan at 0.5-1.0% were able to inhibit the growth of spoilage bacteriaon red meat after 10 days of storage at 4°C (Darmadjiand Izumimoto, 1994). The antimicrobial activity and film-forming characteristic of chitosan makes it a potentialsource of food preservative, increasing quality and shelflife of different types of foods (Darmadji and Izumimoto,1994; Ouattar et al., 2000; No et al., 2007; Friedman andJuneja, 2010; Suman et al., 2010; Vargas and Gonzalez-Martinez, 2010). The mechanism of the antimicrobialactivity of chitosan has not yet been fully elucidated;nevertheless different hypotheses have been proposed.The most realistic hypothesis is that chitosan is able tochange cell permeability due to interactions between thepositive charges of its molecules and the negativecharges of the bacterial cell membranes (No et al.,2007; Friedman and Juneja, 2010). Other hypothesesinclude the chelation of metals and essential nutrients,inhibiting bacterial growth (Rabea et al., 2003). Zhengand Zhu (2003) had also suggested that high molecularweight chitosan could be able to form a polymermembrane around the bacterial cell, preventing it fromreceiving nutrients. On the other hand, Zheng and Zhu(2003) also proposed that the low molecular weightchitosan could enter the bacterial cell through pervasion,disrupting the physiological activities of the bacterium.

Conclusion: The results of these experiments suggestthat dipping raw chicken skin in a 0.5% solution ofchitosan can reduce populations of Salmonella

Typhimurium, thus enhancing general food safety andmaybe shelf life of chicken meat. Moreover, these resultsalso suggest that a solution of 0.5% chitosan can extendthe shelf life of chicken meat as well as causedecreased growth of Gram negative spoilage bacteria.Future research will be directed at determining the effectof these organic compounds on the texture, color,oxidative stability, pH and consumer acceptance ofchicken meat with treatment combinations that exhibitedthe most effective antibacterial activity.

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Cason, J.A., J.S. Bailey, N.J. Stern, A.D. Whittemore andN.A Cox, 1997. Relationship between aerobicbacteria, Salmonella and Campylobacter on broilercarcasses. Poult. Sci., 76: 1037-1041.

Chaine, A., E. Arnaud, A. Kondjoyan, A. Collignan and S.Sarter, 2013. Effect of steam and lactic acidtreatments on the survival of Salmonella Enteritidisand Campylobacter jejuni inoculated on chickenskin. Int. J. Food Microbiol., 162: 276-282.

Corrier, D.E., J.A. Byrd, B.M. Hargis, M.E. Hume, R.H.Bailey and L.H. Stanker, 1999a. Survival ofSalmonella in the crop contents of market-agebroilers during feed withdrawal. Avian Dis., 43: 453-460.

Corrier, D.E., J.A. Byrd, B.M. Hargis, M.E. Hume, R.H.Bailey and L.H. Stanker, 1999b. Presence ofSalmonella in the crop and ceca of broiler chickensbefore and after preslaughter feed withdrawal.Poult. Sci., 78: 45-49.

Darmadji, P. and M. Izumimoto, 1994. Effect of chitosanin meat preservation. Meat Sci., 38: 243-254.

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Friedman, M. and V.K. Juneja, 2010. Review ofantimicrobial and antioxidative activities ofchitosans in food. J. Food Prot., 73: 1737-1761.

Hargis, B.M., D.J. Caldwell, R.L. Brewer, D.E. Corrier andJ.R. Deloach, 1995. Evaluation of the chicken cropas a source of Salmonella contamination for broilercarcasses. Poult. Sci., 74: 1548-1552.

Kong, M., X.G. Chen, K. Xing and H.J. Park, 2010.Antimicrobial properties of chitosan and mode ofaction: a state of the art review. Int. J. FoodMicrobiol., 144: 51-63.

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Mikolajczyk, A. and M. Radkowski, 2002. Salmonella Sci., 77: 1253-1257.spp. on chicken carcasses in processing plants in SAS Institute Inc., 2002. SAS user’s guide: statistics.Poland. J. Food Prot., 65: 1475-1479. SAS Institute Inc., Cary, N.C.

No, H.K., N.Y. Park, S.H. Lee and S.P. Meyers, 2002. Senel, S. and S.J. McClure, 2004. Potential applicationsAntibacterial activity of chitosans and chitosan of chitosan in veterinary medicine. Adv. Drug Deliv.oligomers with different molecular weights. Int. J. Rev., 56: 1467-1480.Food Microbiol., 74: 65-72. Singla, A.K. and M. Chawla, 2001. Chitosan: some

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Northcutt, J.K., M.E. Berrang, J.A. Dickens, D.L. Fletcher specific influence of chitosan on color stability andand N.A. Cox, 2003. Effect of broiler age, feed lipid oxidation in refrigerated ground beef. Meat Sci.,withdrawal and transportation on levels of coliforms, 86: 994-998.Campylobacter, Escherichia coli and Salmonella on Tellez, G., C.E. Dean, D.E. Corrier, J.R. Deloach, L.carcasses before and after immersion chilling. Jaeger and B.M. Hargis, 1993. Effect of dietaryPoult. Sci., 82: 169-173. lactose on cecal morphology, pH, organic acids and

Ouattar, B., R.E. Simard, G. Piett, A. Begin and R.A. Salmonella enteritidis organ invasion in LeghornHolley, 2000. Inhibition of surface spoilage bacteria chicks. Poult. Sci., 72: 636-642.in processed meats by application of antimicrobial Vargas, M. and C. Gonzalez-Martinez, 2010. Recentfilms prepared with chitosan. Int. J. Food Microbiol., patents on food applications of chitosan. Recent62: 139-148. Pat. Food, Nutr. Agric., 2: 121-128.

Petrovich, I., L.A. Grigor'iants, A.N. Gurin and N.A. Gurin, Zhang, L., P. Singh, H.C. Lee and I. Kang, 2013. Effect of2008. Chitosan: structure, properties, use in hot water spray on broiler carcasses for reduction ofmedicine and stomatology. Stomatologiia, 87: 72- loosely attached, intermediately attached and tightly77. attached pathogenic (Salmonella and

Rabea, E.I., M.E.T. Badawy, C.V. Stevens, G. Smagghe Campylobacter) and mesophilic aerobic bacteria.and W. Steurbaut, 2003. Chitosan as antimicrobial Poult. Sci., 92: 804-810.agent: applications and mode of action. Zhao, T., P. Zhao and M.P. Doyle, 2009. Inactivation ofBiomacromolecules, 4: 1457-1465. Salmonella and Escherichia coli O157:H7 on lettuce

Ramirez, G.A., L.L. Sarlin, D.J. Caldwell, Jr.C.R. Yezak, and poultry skin by combinations of levulinic acidM.E. Hume, D.E. Corrier, J.R. Deloach and B.M. and sodium dodecyl sulfate. J. Food Prot., 72: 928-Hargis, 1997. Effect of feed withdrawal on the 936.incidence of Salmonella in the crops and ceca of Zheng, L.Y. and J.F. Zhu, 2003. Study on antimicrobialmarket age broiler chickens. Poult. Sci., 76: 654- activity of chitosan with different molecular weights.656. Carbohydr. Polym., 54: 527-530.

Rose, B.E., W.E. Hill, R. Umholtz, G.M. Ransom andW.O. James, 2002. Testing for Salmonella in rawmeat and poultry products collected at federallyinspected establishments in the United States,1998 through 2000. J. Food Prot., 65: 937-947.

INTRODUCTION The poultry and beef industries have the challenge of

controlling Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes within processing and manu-facturing facilities (Dickson et al., 1992; Harris et al., 2006; Lynch et al., 2006; Laury et al., 2009; Zhao et al., 2009). Poultry and poultry products have been identi-fied by some researchers as the most important source of transmission of Salmonella to humans (Lynch et al., 2006). Contamination by Salmonella on live animals and carcasses can occur during transportation and pro-cessing (Bourassa et al., 2004; Parveen et al., 2007). A

2007 study reported that 88% of chicken carcasses were contaminated with Salmonella, and 80% of the isolates were resistant to one or more antibiotics (Parveen et al., 2007). Chickens contain large numbers of bacteria in their gastrointestinal tract, feathers, and feet; there-fore, fecal bacteria are present on chicken carcasses im-mediately after processing (Ramirez et al., 1997; North-cutt et al., 2003). Consequently, acceptable methods of intervention are needed to decrease populations of spoilage bacteria and foodborne enteropathogens. An-timicrobial chemicals are commonly used during pro-cessing to reduce pathogen loads on carcasses, and the most common antimicrobial treatment used for decon-tamination of poultry meat is chlorine (sodium hypo-chlorite; Mountney and O’Malley, 1965). As reported by Mountney and O’Malley (1965), chlorine was effec-tive in reducing Salmonella and Campylobacter by only as much as 1 to 2 log10 on poultry carcasses. Although

Effect of different concentrations of acetic, citric, and propionic acid dipping solutions on bacterial contamination of raw chicken skin

A. Menconi ,* S. Shivaramaiah ,* G. R. Huff ,† O. Prado ,‡ J. E. Morales ,§ N. R. Pumford ,* M. Morgan ,* A. Wolfenden ,* L. R. Bielke ,* B. M. Hargis ,* and G. Tellez *1

* Department of Poultry Science, and † Poultry Production and Product Safety Research Unit, USDA, Agricultural Research Service, Poultry Science Center, University of Arkansas, Fayetteville 72701;

‡ Laboratorio de Producción Avícola, Facultad de Medicina Veterinaria y Zootecnia, Universidad de Colima, Tecomán, Colima 28100; and § Departamento de Producción Agrícola y Animal,

Universidad Autónoma Metropolitana, México D. F. 04960

ABSTRACT Bacterial contamination of raw, processed poultry may include spoilage bacteria and foodborne pathogens. We evaluated different combinations of or-ganic acid (OA) wash solutions for their ability to re-duce bacterial contamination of raw chicken skin and to inhibit growth of spoilage bacteria and pathogens on skin during refrigerated storage. In experiment 1, raw chicken skin samples were dipped into a suspension of either 108 cfu/mL of Salmonella Typhimurium, Esch-erichia coli O157:H7, or Listeria monocytogenes for 30 s and then immersed in PBS or an OA wash solution mixture of 0.8% citric, 0.8% acetic, and 0.8% propionic acid (at equal wt/vol concentrations) for an additional 30 s. In experiment 2, three different concentrations of the OA wash solution (0.2, 0.4, and 0.6% at equal wt/vol concentrations) were tested against chicken skin samples contaminated with Salmonella Typhimurium.

Viable pathogenic bacteria on each skin sample were enumerated after 1 and 24 h of storage at 4°C in both experiments. In experiment 3, skin samples were ini-tially treated on d 1 with PBS or 2 concentrations of the OA mixture (0.4 and 0.8%), and total aerobic bac-teria were enumerated during a 2-wk storage period. In all experiments, significant (P < 0.05) differences were observed when skin samples were treated with the OA wash solution and no spoilage organisms were recovered at any given time point, whereas increasing log10 num-bers of spoilage organisms were recovered over time in PBS-treated skin samples. These results suggest that 0.2 to 0.8% concentrations of an equal-percentage mix-ture of this OA combination may reduce pathogens and spoilage organisms and improve food safety properties of raw poultry.

Key words: organic acid , foodborne pathogen , skin rinse , chicken , shelf-life

2013 Poultry Science 92 :2216–2220http://dx.doi.org/ 10.3382/ps.2013-03172

Received March 8, 2013. Accepted May 11, 2013. 1 Corresponding author: gtellez@uark.edu

© 2013 Poultry Science Association Inc.

2216

this may be enough to eliminate Salmonella from most poultry carcasses, chlorine may bind to organic matter and be ineffective. In fact, the continued lack of decline in rates of foodborne illness (MMWR, 2011; Scallan et al., 2011) indicates that chlorine treatment of carcasses in the processing facility is not effectively reducing the incidence of Salmonella contamination. Moreover, fail-ure to optimize the disinfectant properties of chlorine (improper pH, concentration, or composition of incom-ing water) may reduce its efficacy. Chlorine treatment may also cause offensive and harmful odors due to the production of chlorine gas and trichloramines (North-cutt et al., 2005, 2008; Hinton et al., 2007). Because of these reasons, alternative methods to disinfect poultry carcass are needed. Studies using organic acids (OA) to spray or dip poultry carcasses have shown as much as 3 log10 of Salmonella reduction (Bilgili et al., 1998; Vasseur et al., 1999; Kubena et al., 2001; Hinton and Ingram, 2005; Lu et al., 2005; Harris et al., 2006; Van Immerseel et al., 2006). A specific example was the use of 2% lactic acid sprayed on chicken carcasses by Yang et al. (1998), which resulted in a 2 log10 cfu per carcass reduction of Salmonella.

In this regard, the use of OA may be a viable alterna-tive to avoid hazards associated with chlorine. There-fore, the objectives of these studies were to determine the effects of a mixture of different concentrations of OA rinse solutions at reducing foodborne pathogens and spoilage organisms on the surface of contaminated raw chicken skin during storage at 4°C.

MATERIALS AND METHODS

Chicken Skin SamplesForceps and scissors were used to aseptically remove

strips of skin (approximately 2 cm × 2 cm) from chick-en thighs (Sarlin et al., 1998) purchased from a local supermarket.

Bacterial StrainsA poultry isolate of Salmonella enterica subspecies

enterica serovar Typhimurium was used for all experi-ments. An enterohemorrhagic Escherichia coli O157:H7 strain, negative for sorbitol fermentation, as well as a laboratory strain of L. monocytogenes, were obtained from the Biomass Research Center and USDA Food Safety Laboratory (University of Arkansas, Fayette-ville). The amplification and enumeration protocol for these isolates has previously been described (Tellez et al., 1993).

Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes Culture Preparation

A frozen aliquot of each pathogen was inoculated into 10 mL of brain heart infusion (BHI) broth (Dif-co, Sparks, MD) and incubated at 37°C for 24 h in a

shaking incubator (New Brunswick Scientific, Edison, NJ) at 200 rpm. After 24 h, 10 mL of fresh BHI was inoculated with 10 μL of this culture, vortexed, and incubated at 37°C for 18 h at 10 × g to ensure that the bacterial culture was in the exponential growth phase. Finally, 10 mL of fresh BHI was inoculated with 20 μL of the 18 h culture to obtain a concentration of approxi-mately 108 cfu/mL.

OA Wash Solution

For use in these experiments, mixtures of equal con-centrations (wt/vol) of acetic (Mallinckrodt Chemicals, Phillipsburg, NJ), citric (Sigma, St. Louis, MO), and propionic (Sigma) acids were prepared. All of these ac-ids are considered generally recognized as safe (GRAS) and are commonly employed in the food industry (USDA Food Safety and Inspection Service, 2005).

Experimental Design

Experiment 1. Chicken skin samples were dipped into a suspension of 108 cfu/mL of Salmonella Ty-phimurium (n = 20), E. coli O157:H7 (n = 20), or L. monocytogenes (n = 20) for 30 s. Skin samples were then removed and dipped into a solution of PBS (con-trol; n = 30) or an OA wash solution (n = 30) of 0.8% final concentration of each of the acids for an additional 30 s. Control and treated samples were placed in indi-vidual sample bags and kept in a refrigerator at 4°C. At 1 and 24 h, 5 control and 5 treated samples were re-moved from the refrigerator and cultured separately for each pathogen. Briefly, skin samples were homogenized within sterile sample bags using a rubber mallet. Sterile saline (5 mL) was added to each sample bag and hand stomached. Serial dilutions were spread plated on bril-liant green agar (Becton, Dickinson and Co., Sparks, MD) plates containing 25 μg/mL of novobiocin (Sigma, St. Louis, MO) and 20 μg/mL of nalidixic acid (Sig-ma) for Salmonella Typhimurium; MacConkey Sorbitol Agar for E. coli O157:H7 (Becton, Dickinson and Co.); or Oxoid Listeria selective agar (EMD Chemicals Inc., Gibbstown, NJ) for L. monocytogenes. Each sample was plated in triplicate. The plates were incubated at 37°C for 24 h, and viable colonies were observed and enumer-ated.

Experiment 2. Skin samples (n = 40) were dipped into a suspension of 106 cfu/mL of Salmonella Ty-phimurium for 30 s. Skin samples were then removed and dipped into a solution of PBS (control; n = 10) or the OA wash solution at 0.2% (n = 10), 0.4% (n = 10), or 0.6% (n = 10) final concentration of each of the acids for an additional 30 s. Samples were placed in individual sample bags and kept in a refrigerator at 4°C. At 1 or 24 h, 5 control and 5 treated samples were removed from the refrigerator and cultured separately for Salmonella Typhimurium recovery as described in experiment 1.

2217BACTERIAL CONTAMINATION OF RAW CHICKEN SKIN

Experiment 3. Skin samples (n = 105) were dipped into a solution of 106 cfu/mL of Salmonella Typhimuri-um for 30 s. Skin samples were then removed and dipped into a solution of PBS (control; n = 35) or the OA wash solution at 0.4% (n = 35) or 0.8% (n = 35) final concentration of each acid for an additional 30 s. Control and treated samples were placed in individual sample bags and kept in a refrigerator at 4°C. At 1 h, 24 h, 3 d, 6 d, 9 d, 12 d, and 15 d, 5 control and 5 treated samples were removed from the refrigerator and cultured separately for Salmonella Typhimurium recov-ery as described in experiment 1.

Experiment 4. Skin samples were dipped into a solu-tion of PBS (control; n = 35) or the OA wash solution at 0.4% (n = 35) or 0.8% (n = 35) final concentration of each acid for an additional 30 s. Control and treated samples were placed in individual sample bags and kept in a refrigerator at 4°C. At 1 h, 24 h, 3 d, 6 d, 9 d, 12 d, and 15 d 5 control and 5 treated skin samples were homogenized within sterile sample bags using a rub-ber mallet. Sterile saline (5 mL) was added to each sample bag and hand stomached. Serial dilutions were spread plated on tryptic soy agar (Becton Dickinson and Co.) and MacConkey agar (Becton, Dickinson and Co.). Each sample was plated in triplicate. The plates were incubated at 37°C for 24 h, and viable colonies were observed and enumerated. Bacterial identification of different morphology colonies that grew on MacCo-nkey agar was determined using the API-20E test kit for the identification of enteric gram-negative bacteria (bioMerieux Inc., Hazelwood, MO).

Statistical AnalysisIn all experiments, for each foodborne pathogen or

psychotropic bacteria, the cfu/skin section in control or treated group, respectively, was analyzed using ANO-VA with further separation of significantly different means using Duncan’s multiple range test using SAS (SAS Institute Inc., 2002). Significant differences were reported at P < 0.05.

RESULTSTable 1 summarizes the effect of 0.8% OA wash so-

lution on chicken skin inoculated with Salmonella Ty-phimurium, E. coli O157:H7, or L. monocytogenes in experiment 1. The OA wash solution caused a 3.8 and

3.2 cfu/skin section log10 reduction in presumptive Salmonella Typhimurium and E. coli O157:H7, respec-tively, 1 h after cold storage. By 24 h, no Salmonella Typhimurium or E. coli O157:H7 were recovered from treated samples. For presumptive L. monocytogenes, there was a 1.85 and 2.87 cfu/skin section log10 reduc-tion at 1 and 24 h, respectively.

Table 2 summarizes the results of 3 additional con-centrations (0.2, 0.4, or 0.6%) of the same OA wash solution used as a sanitizing dip for raw chicken skin samples inoculated with Salmonella Typhimurium. All 3 concentrations were able to significantly reduce pre-sumptive Salmonella Typhimurium at both 1 and 24 h of storage, and no Salmonella Typhimurium were re-covered from skin dipped in 0.6% solutions after 24 h of storage. However, 0.6% OA mixture solution showed complete bactericidal activity against Salmonella Ty-phimurium by 24 h.

Table 3 summarizes the effect of the OA wash so-lution at a concentration of 0.4 or 0.8% on Salmo-nella Typhimurium skin rinse in experiment 3. At 1 h posttreatment, the 0.8% OA wash solution signifi-cantly reduced (P < 0.05) presumptive Salmonella Ty-phimurium cfu by 1.72 cfu/skin section log10 compared with control skin samples, whereas at a concentration of 0.4%, there was a numerical decrease in presump-tive Salmonella Typhimurium cfu (P > 0.05). However, both OA mixtures significantly reduced total presump-tive Salmonella Typhimurium cfu recovered at all other storage times (24 h, 3 d, 6 d, 9 d, 12 d, and 15 d). In all samples treated with either concentration of the OA wash solution, Salmonella was not detected at d 9, 12, and 15 posttreatment. In contrast, control skin samples

Table 1. Experiment 1: effect of rinsing chicken skin with an organic acid mixture (OAM) on recovery of presumptive Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes1

Time of sampling (h)

Control Salmonella

Typhimurium

OAM Salmonella

Typhimurium

Control E. coli

O157:H7

OAM E. coli

O157:H7Control

L. monocytogenesOAM

L. monocytogenes

1 6.0 ± 0.07a 2.20 ± 0.75b 7.57 ± 0.10a 4.32 ± 0.24b 7.39 ± 0.01a 5.54 ± 0.13b

24 6.90 ± 0.04a 0 ± 0b 7.12 ± 0.09a 0 ± 0b 7.21 ± 0.09a 4.34 ± 0.44b

a,bValues within rows for control or treated group for each foodborne pathogen, respectively, with different lowercase superscripts differ significantly (P < 0.05).

1Data expressed as log10 cfu/skin section mean ± SE. OAM = 0.8% acetic acid, 0.8% citric acid, and 0.8% propionic acid.

Table 2. Experiment 2: effect of 3 different concentrations of an organic acid mixture (OAM) rinse solutions on chicken skin inoculated with Salmonella Typhimurium1

Treatment 1 h 24 h

Control PBS 6.8 ± 0.04a,x 6.2 ± 0.09a,x

0.2% OAM 5.5 ± 0.18b,x 2.08 ± 1.2b,y

0.3% OAM 4.6 ± 0.09c,x 1.4 ± 0.87b,y

0.4% OAM 4.6 ± 0.17c,x 0.0 ± 0.0c,y

a–cValues within treatment columns, or x,yvalues within time of evalu-ation rows for each treatment with different superscripts differ signifi-cantly (P < 0.05).

1Data expressed as log10 cfu/skin section mean ± SE. OAM = acetic acid, citric acid, and propionic acid.

2218 MENCONI ET AL.

showed a numerical increase in Salmonella Typhimuri-um cfu at each day of sampling (Table 3).

The results of experiment 4, the effect of 0.4 or 0.8% OA wash solutions on total aerobic bacterial cfu skin section of chicken skin are summarized in Table 4. On tryptic soy agar, after 1 h of cold storage, the total number of aerobic bacteria detected was low in the con-trol samples. However, in both OA wash solutions, no bacteria were detected at this time of evaluation. At all other times of evaluation, control samples showed an increase in total cfu/skin section of chicken skin with a sharp increase between 3 and 6 d poststorage and was significantly different (P < 0.05) from both treated groups. Compared with control samples, the 0.4% OA wash solution showed a significant reduction (P < 0.05) in total cfu/skin section at 24 h and 3 d poststorage. At 6, 9, 12, and 15 d, no aerobic bacteria were recovered from skin samples treated with the 0.4% OA wash solu-tion. Interestingly, at all times of evaluation, no aerobic bacteria were recovered from skin samples treated with the 0.8% OA wash solution (Table 4).

Samples from both control and treated bags were plated on MacConkey agar for the detection of gram-negative bacteria associated with food spoilage. Both OA wash solutions inhibited to not detectable levels the growth of gram-negative bacteria at all times of evalu-ation. However, bacteria were recovered from the 24 h samples and these numbers increased exponentially in the control samples to levels that were too numerous to count at d 9, 12, and 15, being Escherichia ssp., Enterobacter spp., and Pseudomonas spp. among the

predominant bacterial flora on the broiler skin (data not shown).

DISCUSSIONIn general, carcass rinse applications that decrease

Salmonella by 2 log10 cfu/mL are considered effective because most carcasses are considered to have about 100 Salmonella cells (Jetton et al., 1992). Lactic acid and citric acid at concentrations of 1 to 3% have been shown to reduce E. coli O157:H7, Salmonella serotypes, and L. monocytogenes when sprayed on beef and poul-try carcasses by causing intracellular acidification (Vas-seur et al., 1999). According to Vasseur et al. (1999), citric acid showed to have the highest inhibitory effect because of its ability to diffuse through the cell mem-brane. In the same experiment, lactic acid decreased the ionic concentration within the bacterial cell mem-brane, leading to accumulation of acid within the cell cytoplasm, disruption of the proton motive force, and inhibition of substrate transport (Vasseur et al., 1999).

In these experiments, the blend of OA wash solution showed significant antibacterial activity against 3 food-borne pathogens commonly implicated in meat process-ing (Table 1). Additionally, we also found that lower concentrations of the OA wash solution are almost as effective as higher concentrations, and based on these experiments, we conclude that a concentration of 0.4% demonstrates optimum antibacterial/bactericidial ac-tivity (Tables 2, 3, and 4). Furthermore, the OA wash solution, when used at a concentration of 0.4%, was able

Table 3. Experiment 3: effect of 2 different concentrations of an organic acid mixture (OAM) rinse solution on chicken skin inoculated with Salmonella Typhimurium1

Sample time Control PBS 0.4% OAM 0.8% OAM

1 h 3.37 ± 0.20a,x 2.01 ± 0.83ab,x 1.65 ± 1.05b,x

24 h 3.55 ± 0.30a,x 1.26 ± 0.77bc,x 0 ± 0c,x

3 d 3.31 ± 0.30a,x 0 ± 0b,x 0.60 ± 0.60b,x

6 d 3.40 ± 0.31a,x 0.60 ± 0.60b,x 0 ± 0b,x

9 d 3.49 ± 0.33a,x 0 ± 0b,x 0 ± 0b,x

12 d 4.89 ± 0.32a,y 0 ± 0b,x 0 ± 0b,x

15 d 6.82 ± 0.15a,y 0 ± 0b,x 0 ± 0b,x

a–cValues within treatment rows, or x,yvalues within time of evaluation column for each treatment with different superscripts differ significantly (P < 0.05).

1Data expressed as log10 mean ± SE. OAM = acetic acid, citric acid, and propionic acid.

Table 4. Experiment 4: effect of 2 different concentrations of organic acid mixture (OAM) rinse solu-tions on total cfu/skin section of chicken skin plated on tryptic soy agar plates

Sample time Control PBS 0.4% OAM 0.8% OAM

1 h 0.60 ± 0.60a,x 0 ± 0b 0 ± 0b,x

24 h 1.62 ± 0.66a,x 0.60 ± 0.60ab,y 0 ± 0b,x

3 d 4.49 ± 0.39a,y 2.45 ± 1.51b,x 0 ± 0c,x

6 d 7.03 ± 0.37a,z 0 ± 0b,y 0 ± 0b,x

9 d 7.26 ± 0.19a,z 0 ± 0b,y 0 ± 0b,x

12 d 7.61 ± 0.23a,z 0 ± 0b,y 0 ± 0b,x

15 d 7.99 ± 0.27a,z 0 ± 0b,y 0 ± 0b,x

a–cValues within treatment rows, or x–zvalues within time of evaluation column for each treatment with different superscripts differ significantly (P < 0.05).

1Data expressed as log10 mean ± SE. OAM = acetic acid, citric acid, and propionic acid.

2219BACTERIAL CONTAMINATION OF RAW CHICKEN SKIN

to prevent recovery of aerobic food-spoilage bacteria up to 2 wk of storage at 4°C, indicating that one wash with this solution may enhance shelf-life of packaged meat significantly. Overall, the results of these experi-ments suggest that dipping raw chicken skin in an OA wash solution of citric, lactic, and propionic acids can greatly reduce populations of pathogenic bacteria, thus enhancing overall food safety and shelf life of chicken meat. Poultry meat quality is a concern when using different OA washes. In an earlier study, the quality effects of acetic, citric, lactic, malic, mandelic, or tar-taric acids at 0.5, 1, 2, 4, and 6% concentrations were tested on broiler carcasses, revealing that in simulated dip application, each of the acids decreased lightness and increased redness and yellowness values in the skin of broiler carcasses with increasing acid concentration (Bilgili et al., 1998). Therefore, future research will be directed at determining the effect of these OA on the texture, color, oxidative stability, pH, and consumer ac-ceptance of chicken meat with treatment combinations that exhibited the most effective antibacterial activity.

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International Journal of Poultry Science 12 (2): 72-75, 2013ISSN 1682-8356© Asian Network for Scientific Information, 2013

Corresponding Author: Guillermo Tellez, POSC O-114, Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701,USA

72

Effect of Organic Acids on Salmonella Typhimurium Infection in Broiler Chickens

A. Menconi , A.R. Reginatto , A. Londero , N.R. Pumford , M. Morgan , B.M. Hargis and G. Tellez1 2 2 1 1 1 1

Department of Poultry Science, University of Arkansas, Fayetteville AR 72701, USA1

Depto. de Medicina Veterinária - CCR Universidade Federal de Santa Maria, Brazil2

Abstract: An alternative to antibiotics is the use of certain organic acids for routinely encountered pathogensin the poultry industry. Direct acidification of drinking water with organic acids could significantly reduce theamount of recoverable Salmonella Typhimurium (ST) from the crop and cecal tonsils when used during thepre-slaughter feed withdrawal period. In the present study, in vitro and in vivo evaluations were conductedto compare a commercially available water acidifier (Optimizer ), versus two formulations of organic acid mix®

(OAM), made up of of acetic, citric and propionic acids at a final concentration of either 0.031% or 0.062%,to reduce Salmonella Typhimurium in the crop and cecal tonsils of broiler chicks during a 24 h period. Thetwo OAM showed better in vitro activity to reduce Salmonella when compared to control. In vivo, the OAM(0.062%) had a similar effect as Optimizer showing a significant reduction in total number of ST positive®

cecal tonsils, and reducing the number of ST in the crop when compared with controls (P < 0.05). Alltreatments reduced the number of ST recovered from crop contents at 24 h. This new formulation of OAM hasgreat potential as a crop sanitizer and will be further evaluated under conditions similar to commercialchickens.

Key words: Salmonella, organic acid, chickens

INTRODUCTIONSalmonella enterica causes an estimated 1.4 millioncases of foodborne illnesses annually in the UnitedStates, resulting in over 15,000 hospitalizations (Voetschet al., 2004a,b). Poultry and poultry products have beenidentified by some researchers as the most importantsource of transmission of Salmonella to the humanpopulation (Lynch et al., 2006). Increased pressure byconsumers and regulatory agencies for reduced or evenelimination of the use of antibiotics in food producinganimals has created a need to find alternatives tomaintain healthy and productive animals. Thesepressures are a challenge for the poultry industry forcontrolling Salmonella not only at the farm level, but alsowithin processing and manufacturing plants (Hargis etal., 1995; Corrier et al., 1999a; Hinton et al., 2000; MATERIALS AND METHODSMikolajczyk and Radkowski, 2002). An alternative to Salmonella amplification: A primary poultry isolate ofantibiotics is the use of certain organic acids. Direct Salmonella Typhimurium (ST) was used in theseacidification of the water with organic acids could experiments. This isolate was selected for resistance tosignificantly reduce the amount of recoverable nalidixic acid (NA) . For these experiments, ST wasSalmonella on the carcasses or in the crops and cecal grown in tryptic soy broth (TSB) for approximately 8 h.tonsils when used during the pre-slaughter feed The cells were washed three times with 0.9 % sterilewithdrawal period (Van Immerseel et al., 2006; Alali et saline by centrifugation (3,000 x g), and the approximateal., 2010; Vandeplas et al., 2010); however, previous concentration of the stock solution was determinedresearch has suggested that administration of OA spectrophotometrically at 625 nm. The stock solutionduring the pre-slaughter feed withdrawal period could was serially diluted and confirmed by colony counts oflead to carcass shrinkage (Byrd et al., 2001). While this three replicate samples (0.1 mL/replicate) that wereevidence was shown when using lactic acid alone, spread plated on brilliant green agar (BGA) platesOptimizer was developed as a combination of organic containing 25 µg/mL novobiocin (NO) and 20 µg/Ml®

acids used in combination at low individual nalidixic acid (NA). The colony-forming units of

concentrations so that water consumption was notdiscouraged (Jarquin et al., 2007; Wolfenden et al.,2007; Vicente et al.,2007a,b,c). Organic acids are areadily available energy source for both the chicken andthe bacteria. Therefore, it is important that the organicacids be administered in high enough concentrations tobe bactericidal in the presence of organic matter, andlow enough to be voluntarily consumed by the birds. Inthe present study, we compared a commerciallyavailable water acidifier (Optimizer , Pacific Vet Group,®

Fayetteville, AR 72703), versus a new formulation oforganic acid mix (OAM) to reduce SalmonellaTyphimurium in the crop and cecal tonsils of broilerchicks.

1

2

3

4

Int. J. Poult. Sci., 12 (2): 72-75, 2013

73

Salmonella determined by spread plating were reported In experiment 2, 80 day-of-hatch broiler chicks wereas the concentration of Salmonella (in cfu/mL) for in vitroexperiments and total colony-forming units for in vivochallenge experiments.

Experimental Design - in vitro crop assay: An assaypreviously described (Barnhart et al., 1999) was usedwith modifications. Briefly, 1.25g of unmedicated chickstarter feed was measured into 13×100 mm borosilicatetubes and autoclaved. The feed was suspended in 4.5mL sterile saline and inoculated with 0.5 mL of aSalmonella Typhimurium culture containingapproximately 10 cfu/mL. The tubes were treated with4

either: 1) saline as a control; 2) OAM, having a finalconcentration of acetic, citric and propionic acids at0.031 % or; 3) OAM, having a final concentration ofacetic, citric and propionic acids at 0.062 %. Eachsample was run as triplicate, each treatment had 5replicates, and the entire assay was repeated in 2additional trials. After administering the treatment, thetubes were vortexed and incubated at 37EC for 30minutes and an additional 6 h. The tubes were thenagitated and 20 µL of the content was serially dilutedand plated as triplicates on BGA containing novobiocinand nalidixic acid. Typical ST colonies were countedafter 24 h of incubation.

Experimental design with chickens: In experiment 1, 64day-of-hatch broiler chicks were obtained from a localhatchery. Chicks were randomized and challenged with2 x 10 cfu/mL of ST. The chicks were then held in chick5

boxes for 1 h and then randomly assigned to 1)untreated control or continuous treatment in the drinkingwater with: 2) Optimizer at commercial recommended®

doses; 3) OAM, having a final concentration of acetic,citric and propionic acids at 0.031 % or; 4) OAM, havinga final concentration of acetic, citric and propionic acidsat 0.062%. Chicks were housed in brooder batteries withfood and water ad libitum. At 24 hr post-challenge,chicks were humanely killed by CO inhalation and crop,2

both ceca and cecal tonsils were aseptically harvestedseparately. Salmonella recovery procedures have beenpreviously described by our laboratory and were followedwith some modifications (Tellez et al., 1993). Briefly, cropand cecal tonsils were enriched in 10 mL of tetrathionatebroth overnight at 37EC. Following enrichment, eachsample was streaked for isolation on BGA platescontaining 25 µg/mL NO and 20 µg/mL NA. The plateswere incubated at 37EC for 24 h and examined for thepresence or absence of the antibiotic resistant ST. Cecawere weighed and then homogenized within sterilesample bags using a rubber mallet. Sterile saline (4X5

weight to volume) was added to each sample bag andhand stomached with the cecal contents. Dilutions werespread plated on BGA plates containing 25 g/mL NOand 20 µg/mL NA. The plates were incubated at 37EC for24 h and cfu of ST per ceca were determined.

obtained from a local hatchery. Chicks were randomizedand challenged with 2 x 10 cfu/mL of ST. The chicks5

were then held in chick boxes for 1 h and then randomlyassigned to 1) untreated control or continuous treatmentin the drinking water with: 2) Optimizer® at commercialrecommended doses; 3) OAM, having a finalconcentration of acetic, citric and propionic acids at0.031 % or; 4) OAM, having a final concentration ofacetic, citric and propionic acids at 0.062 %. Chicks werehoused in brooder batteries with food and water adlibitum. At 24 hr post-challenge, chicks were humanelykilled by CO inhalation and crops were aseptically2

harvested, weighed and were homogenized withinsterile sample bags using a rubber mallet. Sterile saline(4X weight to volume) was added to each sample bagand hand stomached with the crop contents. Dilutionswere spread plated on BGA plates containing 25 µg/mLNO and 20 µg/mL NA. The plates were incubated at37EC for 24 h and cfu of ST per crop were determined.Following this, crops were enriched with a 2X solution oftetrathionate broth overnight at 37EC. Followingenrichment, each sample was streaked for isolation onBGA plates containing 25 µg/mL NO and 20 µg/mL NA.The plates were incubated at 37EC for 24 h andexamined for the presence or absence of the antibioticresistant ST.

Statistical analysis: The incidence of Salmonellarecovery within experiments was compared using thechi-square test of independence (Zar, 1984) testing allpossible combinations to determine significant (P<0.05)differences between control and treated groups. Cecalcfu data were converted to log10 cfu numbers and thencompared using the GLM procedure of SAS (SASInstitute, 2002) with significance reported at P < 0.05.

RESULTS AND DISCUSSIONSalmonella colonization of poultry flocks can occur viahorizontal transmission (Bailey et al., 2002; Kim et al.,2007; Alali et al., 2010; Vandeplas et al., 2010). Oncececal tonsil colonization is established, the bacterium isconsistently shed in the feces (Bailey et al., 2002; Foleyet al., 2008). Feed Withdrawal induces pecking of thecontaminated litter which may contaminate the crop(Corrier et al., 1999c) and if the crop is ruptured duringprocessing, Salmonella may contaminate raw poultryproducts (Corrier et al., 1999b). Because the crop ismore likely to rupture than the ceca, the crop representsan important source of Salmonella contamination tocarcasses (Hargis et al., 1995; Corrier et al., 1999a).Table 1 summarizes the results of effect of OAM on ST inan in vitro crop assay. In 3 independent trials, the0.031% OAM reduced ST by 6 h and the 0.062 % OAMwas also efficacious. However, when 0.062 % OAM wastested in chickens, it had a similar effect as Optimizer®showing a significant reduction in total number of ST

Int. J. Poult. Sci., 12 (2): 72-75, 2013

74

Table 1: Effect of organic acid mix (OAM) on Salmonella Typhimurium (ST) in an in vitro crop assayTrial 1 Trial 2 Trial 3------------------------------------------- ------------------------------------------- -------------------------------------------30 minutes 6 hours 30 minutes 6 hours 30 minutes 6 hours

Control (ST) 6.25±0.13 7.09±0.09 7.42±0.03 7.07±0.04 4.95±0.13 5.99±0.22a a a a a a

0.031% OAM 6.08±0.8 5.98±0.01 7.43±0.03 5.86±0.03 4.88±0.24 4.56±0.07a b a b a b

0.062% OAM ND ND 7.39±0.04 6.24±0.12 4.70±0.22 4.56±0.07a b b b

Organic acids mix= acetic, citric, and propionic acid. ND= Not determined. Data are expressed as log mean ± standard error. 10

Values within columns with different lowercase superscripts differ significantly (P < 0.05).

Table 2: Experiment 1, effect of Optimizer or organic acids mix (OAM)®

on Salmonella Typhimurium (ST) infection in broiler chicksduring 24 hours period

Crop Cecal tonsils Log ST/gram10

Enrichment Enrichment of cecaTreatment culture culture contentControl ST 15/16 (94%) 14/16 (87%) 2.43±0.35a

Optimizer 13/16 (81%) 3/16 (19%) ** 0.22±0.22® b

0.031% OAM 16/16 (100%) 12/16 (75%) 2.02±0.35a

0.062% OAM 13/16 (81%) 8/16 (50%) * 1.34±0.40a

Organic acids mix= acetic, citric, and propionic acid. Data of enrichmentculture is expressed as positive/total chickens for each tissue sampled(%). * Indicates significant difference at P < 0.05. ** Indicatessignificant difference at P < 0.001. Log ST/gram of ceca content data is expressed as mean ± standard10

error. Values within columns with different lowercase superscripts differsignificantly (P < 0.05).

Table 3: Experiment 2, effect of Optimizer or organic acids mix®

(OAM) on Salmonella Typhimurium (ST) infection inbroiler chicks during 24 hours period

Crop Log ST/gram10

enrichment of cropTreatment culture contentControl ST 20/20 (100%) 5.21 ± 0.31a

Optimizer 18/20 (90%) 3.73 ± 0.25® b

0.031% OAM 20/20 (100%) 3.96 ± 0.37b

0.062% OAM 18/20 (90%) 3.89 ± 0.22b

Organic acids mix= acetic, citric, and propionic acidData of enrichment culture is expressed as positive/total chickensfor each tissue sampled (%). Log ST / gram of crop content is expressed as mean ± standard10

error. Values within columns with different lowercase superscriptsdiffer significantly (P < 0.05).

positive chickens in cecal tonsils (Table 2), and reducingthe number of ST in the crop (Table 3) when comparedwith controls.In the present study, Optimizer® reduced ST colonizationin both crop and ceca (Tables 2 and 3) as has beenpreviously reported (Jarquin et al., 2007; Wolfenden etal., 2007). In experiment 1, treatment with OAM in thedrinking water caused a significant reduction (P<0.05) inST recovery from cecal tonsils when compared with thecontrols (OA treated = 19% vs. controls = 87%). Also,treatment with OAM reduced 2.21 logs of ST whencompared with controls (Table 2). While any of thetreatments reduced recovery of ST from the crop byenrichment, all treatments reduced the number of STrecovered from crop content at 24 h (Table 3). Theorganic acids used in this study (citric, acetic andpropionic) as well as others have been shown to beindividually effective in reducing Salmonella in vitro (VanImmerseel et al., 2006). The biocidal efficacy and theeffect on virulence of Salmonella differ with each organic

acid treatment and each organic acid has a unique effecton bacteria normally present in the crop andgastrointestinal tract (Furuse et al., 1991; Byrd et al.,2001; Castro Gonzalez et al., 2001; Kubena et al., 2001).Characteristics of organic acids such as chain length,side chain composition, pkA values and hydrophobicitycould be factors that effect biocidal activity (VanImmerseel et al., 2006). For these reasons, a mixture oforganic acids was tested to reduce ST cropcontamination. Further studies are being conducted toevaluate these new formulations of OAM during the pre-slaughter feed withdrawal period in commercialchickens to evaluate water consumption and bactericidalactivity against Salmonella in the crop.

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Catalog No. N-4382, Sigma, St. Louis, MO 63178 USA1

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