Estrategias de ante mortem y post mortem para el control de salmonella spp. en pollo de engorda
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Estrategias de ante mortem y post mortem para el control de salmonella spp. en pollo de engorda

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  • 1. 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 Agriculture Department of Poultry Science JKS Poultry Health Laboratory
  • 2. Ralph Waldo Emerson (25 – 05 ‐ 1803 – 27 – 04‐ 1882) • ¿Qué es lo más  difícil en el  mundo? PENSAR!
  • 3. Salmonella Salmonella Enterobacteria forma bacilar Gram‐negativa,             no esporula, móvil quimioheterótrof o, energía de  reacciones de  oxidación y  reducción de  fuentes orgánicas Anaerobio facultativo ‐ ATP por respiración aeróbica ‐ puede cambiar a  fermentación Super‐reino: Bacteria Reino: Bacteria Filo: Proteobacteria Clase: Gammaproteobacteria Orden: Enterobacteriales Familia: Enterobacteriaceae Género: Salmonella Especies S. bongori S. enterica subesp. enterica * subesp. salamae subesp. arizonae subesp. enterica * subesp. salamae subesp. arizonae subesp. diarizonae subesp. houtenae subesp. indica subesp. diarizonae subesp. houtenae subesp. indica
  • 4. Colonización in situ • Brotes de salmonelosis relacionados con el  consumo de productos frescos ha elevado el  interés en las interacciones Salmonella‐planta que permiten la  colonización de la  planta
  • 5. Incubación de Salmonella enterica marcada con gfp en hojas de lechuga iceberg • En la luz resultó en la  agragción de bacterias cerca de los estomas abiertos e invasión hacia el tejido de la hoja • En contracte, la  incubación en la  oscuridad resultó en un  patrón de de adhesión escasa y muy poca internalización en los  estomas.  Kroupitski et al. Ap and Env Mic. 2009;75(19):6076–6086.
  • 6. • Estos resultados implican que el  patógeno es atraído hacia los nutrientes recién producidos por las células activas en  fotosíntesis Kroupitski et al. Ap and Env Mic. 2009;75(19):6076–6086.
  • 7. Estos hallazgos • Sugieren la entrada mecánica de Salmonella al apoplasto de la planta • E implica que los  antígenos de Salmonella no son bien reconocidos por la inmunidad innata del estoma, o que este patógeno ha  evolucionado medios para evadirla. 
  • 8. La internalización en las hojas • Puede ofrecer una explicación parcial de la  falla de los sanitizantes para erradicar eficientemente los  patógenos de origen alimentico de las verduras.
  • 9. 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 que se utiliza para controlar el  intercambio gaseoso. • El poro está delimitado por un par de células especializadas [parénquima] conocidas como células guarda que son responsables de regular  el tamaño de la apertura
  • 10. Complejo de estomas • El término también se  emplea colectivamente para referirse a un  complejo de estomas completo, tanto el poro en sí como sus células guarda. 
  • 11. Función de los estomas • El aire que contiene CO2 y  O2 entra a la planta a través de  estas aperturas se usa para la respiración y  fotosíntesis,  respectivamente
  • 12. Evolución de los estomas • El registro fósil aporta poca información. • Pueden haber evolucionado por la modificación de los  conceptáculos de los  ancestros de las plantas parecidos a algas. • Conceptáculos: Son  cavidades especializadas de  algas marinas y de agua dulce que contienen los  órganos reproductores. Conceptaculos
  • 13. 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 rasgos constituyeron una ventaja muy importante para las primeras plantas terrestres.
  • 14. La Salmonella sobrevive sobre o  dentro de los tomates • Desde la inoculación en la  floración hasta la  maduración. • El tallo y flores del tomate son sitios donde la  Salmonella puede adherirse y permanecer viable  durante el desarrollo del  fruto, y sirve como ruta o  reservorio para la  contaminación del fruto maduro.  Guo X et al. Ap and Env Mic. 2001;67(10):4760–4764.
  • 15. Reino animal • La mayoría de los Filos animales conocidos aparecen en el registro de fósiles como especies marinas de cerca de  542 millones de años. • Los animales están dividios en  varios sub‐grupos, incluidas las aves, mamíferos, reptiles,  peces e insectos. 
  • 16. Salmonella: Mecanismos de Infección
  • 17. Entrada de Salmonella
  • 18. Sistema de Secreción Tipo III (TTSS, en inglés) • Forma principal en la que Salmonella suministra factores de virulencia al hospedero • Constituído de 20 proteínas • Ensambladas en orden paso a paso • PrgI es una estructura de aguja extendida por una base de proteína, forma un canal hacia el hospedero. PrgI
  • 19. Islas de patogenicidad de Salmonella • SPI-1: Invasión • SPI-2: Replicación intracelular • SPI-3: Supervivencia Intracelular • SPI-4: Producción de toxinas • SPI-5: Inflamación
  • 20. Vesícula con Salmonella (VCS) • Después de la ingestión, entra una VCS a través de la endocitosis mediada por la bacteria • Vive y se multiplica en la VCS • Una forma de evadir la respuesta inmune del hospedero
  • 21. Estrés e inmunidad Los efectos del estrés sobre el curso de una infección se han adjudicado por mucho  tiempo al efecto directo de las hormonas relacionadas con el  estrés sobre el sistema inmune y la fucnión de  la barrera intestinal. 
  • 22. Los animales para consumo pueden no tener una vida libre de estrés, particularmente los que se crían intensivamente Quizá lo que fue inesperado es que la  respuesta al estrés bacteriano impuesta por el medio del hospedero sobre el organismo y la  respuesta de adrenalina del hospedero impuesta por la infección pueden potenciar el crecimiento y  virulencia del  microorganismo.
  • 23. 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 respuesta al estrés medioambiental. 
  • 24. • Hace 112 años inició una nueva era en la  endocrinología con la  primera purificación de  una hormona: adrenalina. • Desde 1930, casi inmediatamente después de su primer uso, se  reportaron casos de  sepsis asociados con la  adrenalina.  Endocrinología Microbiana
  • 25. Endocrinología Microbiana • La teoría más aceptada para explicar la habilidad de las hormonas para influir el curso de una infección involucra la  supresión del sistema inmune.  • Hoy, sabemos que los  microorganismos infecciosos responden igualmente al  medioambiente neuroendócrino del  hospedero. 
  • 26. Adrenalina 1. 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 las bacterias 4. Incrementa la expresión de  adhesinas 5. Incrementa virulencia e invasión
  • 27. 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
  • 28. Salmonella Salmonella sp. Se piensa comúnmente que se  transmite principalmente a través del contacto con  individuos infectados y la  ingestión de materiales contaminados con heces
  • 29. Sin embargo, varios investigadores han demostrado la importancia de la transmisión aérea de  Salmonella como fuente de  infección cruzada en la  avicultura.
  • 30. transmisión aérea de Salmonella como fuente de infección cruzada en la avicultura • Baskerville A., et al.  1992 Airborne 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. 2006 Rapid aerosol transmission of Salmonella among turkeys in a simulated holding‐ shedenvironment • Basnet H.B., et al. 2008 Reproduction of fowl typhoid by respiratory challenge with Salmonella Gallinarum
  • 31. 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 enriquecimiento toda la noche en caldo tetrationato (CT).   • Las muestra enriquecidas se  sembraron por estría en  agar XLD con novobiocina (NO) y ácido nalidíxico (NA).  % pollos positivos SE N=10
  • 32. Transmisión Horizontal • Sonda oral con ~1x105 SE  (20 % inoculados) @ 3 d  de edad (n=100) • Pollos mezclados en un  corral (80 % pollos contacto) • Sacrificio y cultivo @ 10 d  de edad 0 10 20 30 40 50 60 70 80 90 100 Cecal Tonsils Trachea Challenged Contacts % pollos positivos a SE N=100
  • 33. Prevalencia de Salmonella sp. En tráqueas de pavos procesados comercialmente • Toma aséptica de tráquea y  ciegos n=100, pavos comerciales de 16‐ semanas de edad • Las tráques se pinzaron en los  extremos y se agregaron 20 mL de agua peptona e incubadas 8  h a 37 °C. • El agua peptona de cada tráquea 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. 0 10 20 30 40 50 60 70 80 90 100 E. coli Salmonella sp. %  tráqueas positivas N=100 34/100 (34 %)  97/100 (97 %)
  • 34. Recuperación de Salmonella de tonsilas cecales Se agregó CT  2X a la  muestra restante y se  siguó el mismo procedimiento anterior 15/100 (15 % ) Tonsila vs. 34/100 (34 %) Tráquea
  • 35. 10^4 10^6 10^8 IT 1,125 3,195 5,114 Oral 1,799 1,858 5,989 0,000 1,000 2,000 3,000 4,000 5,000 6,000 7,000 UFC, Log10 Ciego‐TonsilaCecal Recuperación de Salmonella Día‐8  Pollos inoculados con Salmonella enteritidis (SE) – 1 semana – Intra‐traqueal u Oral, con 104, 106 o 108 UFC/pollo. Cultivo 24 hrs post desafío. En las barras se expresa el Log10 SE/ gramo de contenido cecal como promedio ± error estándard P > 0.05. Las literales sobre las barras indican diferencias significativas P < 0.05
  • 36. Grupo Log10  SE / gramo de  contenido cecal Hígado y bazo Tráquea Ciego ‐ 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 o 108 UFC/pollo. Cultivo 24 hrs post desafío. En las barras se expresa el Log10 SE/ gramo de contenido cecal como promedio ± error estándard. Los datos de tonsilas cecales, tráchea, hígado y bazo se expresan como pollos positivos/total para cada tejido (%). Literales en la misma columna indican diferencias significativas, p < 0.05, N=12.
  • 37. Aunque se sabe que el  contacto directo con aves  infectadas y el contacto indirecto con superficies  medioambientales contaminadas son factores importantes en la  diseminación de Salmonella en las parvadas, el papel potencial de la transmisión aerógena no está claramente definido
  • 38. BAL Salmonella parece penetrar a través de las células epiteliales en el  TLAB (BALT ) para infectar células linfoides
  • 39. Los resultados de estos estudios sugieren que la  tráquea es un órgano viable  para la recuperación de  Salmonella Reconfirma que el   movimiento aerógeno de  Salmonella en las casetas es un punto de control relevante para limitar la diseminación de la infección dentro de las parvadas. 
  • 40. ¿Estamos a salvo?
  • 41. ¿Hay esperanza?
  • 42. Recomendaciones para el Control • Roedores • Aves silvestres • Insectos • Personal • Fomites • Medioambiente • Instalaciones • Equipo • Transporte • Reproductores • Incubadora • Entrega • Parvadas de diferente origen • Alimento/Proteína Animal • Agua • Vacunación • Algunos probióticos/EC • Más….
  • 43. 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 (transmisión horizontal) o de los reproductores a través del  huevo (transmission vertical) disminuye el R0
  • 44. Antibióticos Dieta Probióticos Prebióticos Simbióticos Ácidos Orgánicos Extractos de Plantas Otros Herramientas para reducir el R0
  • 45. La presión social ha llevado a la creación de  regulaciones para restringir el uso de antibióticos en la  producción avícola y la  ganadería.  • Actualmente hay mayor  interés público y científico respecto a la administración terapéutica y sub‐ terapéutica de antibióticos a los animales. 
  • 46. 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 las enfermedades de los  animales criados sin  antibióticos no solo  beneficia su salud, bienestar y eficiencia productiva,  también es una estrategia clave en el esfuerzo de  mejorar la seguridad microbiológica de los  productos avícolas. 
  • 47. En los últimos 20 años • Nuestro laboratorio ha  trabajado en la  identificación de candidatos a probióticos para la  avicultura, los cuales realmente pueden desplazar a Salmonella y  otros patógenos entéricos que han colonizado el tracto gastrointestinal de pollos y  pollitas.
  • 48. Floramax: Fabricado bajo licencia exclusiva de la  Universidad de Arkansas  • El trabajo intensivo permitió la  identificación de 11 LAB (del  género o relacionado con  Lactobacillus en el producto FloraMax B‐11® que fueron eficaces en el tratamiento de  pollos y pollas infectados con  Salmonella.
  • 49. Vicente, J.S. et al. 2007  J. of Applied Poult. Res. 16: 471‐476. • La selección de parvadas infectadas con Salmonella antes del sacrificio,  demostró que el  tratamiento de dichas parvadas con B11,  aproximadamente 2  semanas antes del  sacrificio, puede reducir marcadamente la  recuperación medioambiental de  Salmonella en pavos y  pollos comerciales.   - 10 20 30 40 50 60 70 80 90 100 Figure 3. Effect of Beneficial Bacteria Alone or in Combination with an acidifier on Salmonella Isolation in Commercial Turkey Houses a,b Control Organic Acidifier B11 Acidifier + B11 a a a a PercentSalmonellapositiveswabs a Before trt After trt a,b b c Ácidos Orgánicos
  • 50. Incremento de la productividad y  reducción de costos de producción • Ensayos comerciales de gran escala indican que la  administración apropiada de esta mezcla de  probióticos a pavos y  pollos.  • Vicente J. et al., 2006 • Torres A. et al., 2007 • Torres A. et al., 2007 100 300 500 700 900 1,100 1,300 Initial (11 days of hatch) + 7 d + 14 d + 21 d + 26 d Age (days) Bodyweight(g) Control Probiotic in Drinking Water Probiotic in Drinking Water + Lactose Probiotic in Feed + Lactose a a a a a a a b bb bbaa a a a b b b75.6g 124.5g 173.6g
  • 51. Mayor Resistencia a infecciones por Salmonella spp. • La extensiva investigación de laboratorio y de  campo realizados con  este cutivo LAB ha  demostrado el desarrollo acelerado 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
  • 52. Higgins J. et al., 2007 Poult. Sci. 86:1662–1666 0 20 40 60 80 100 6 h 12 h 19 h 24 h Control Treated Porcentajede recuperacióndeSE * Significativamentemenor que el testigo (p<0.05) * *
  • 53. Hours post-treatment 6 h 12 h 19 h 24 h Log10 cfuofSalmonellaenteritidis 0 1 2 3 4 Control Treated * * * * Significantly less than control (p<0.05)
  • 54. Actividad Bactericida BAL
  • 55. Redes de Genes a partir del Ingenuity Pathway Analysis Para comparar grupos de tratamiento SE y SE+B11 Higgins S. et al., 2011 Grupos de tratamiento SE Grupos de tratamiento SE+B11
  • 56. 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 Salmonella autorizadas actualmente
  • 57. Los órganos linfoides secundarios pueden sub‐dividirse en  sistema inmune Sistémico (***) y Mucosal NALT BALT GALT RALT Mucosal ** * *** ** * # #
  • 58. Superficies mucosas • Gastrointestinal • Respiratoria • Tracto urogenital • Representan un área muy grande de  exposición a agentes exógenos, incluidos los microorganismos
  • 59. Sistema Inmune Mucosal común
  • 60. ¿Porqué es importante tener esa respuesta immune mucosal?  Estos sitios anatómicos son las principales áreas de interacción del cuerpo con el  medio externo y con patógenos potenciales Ingestión Inhalación Inseminación
  • 61. Tejido linfoide asociado a mucosas –MALT‐ • Difiere fundamentalmente de la respuesta immune  sistémica en: • El isotipo principal en las secreciones de la mucosa es la IgA secretoria • La mayoría de las células productoras de anticuerpos y  linfocitos T efectores están en el MALT • Sitios linfoides inductor y efector separados
  • 62. El intestine es el órgano inmunológico más grande en el cuerpo • Comprende 70‐80% de las células 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). 
  • 63. Desarrollo de las vacunas mucosales 2006 Nov 2;126(21):2818‐21  • La vacuna active oral  contra la polio fue la  primera mucosal  aceptada para uso general. Desde entonces, se han desarrollado vacunas similares contra la  fiebre tifoidea, cólera e infección por rotavirus.  18a. Dinasty
  • 64. Ventajas de la ruta de inmunización mucosal • 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)
  • 65. Incluso si la inmunización mucosal no  elimina totalmente la infección • Los anticuerpos de la  mucosa limitan el grado de repicación y  diseminación del  patógeno; por lo tanto,  reduce la carga ambiental del mismo y minimiza dramáticamente la tasa de  infección en la parvada y  la transmisión de la  enfermedad. 
  • 66. También • El diseño de sistemas de administración que se  enfoquen en la respuesta immune para conferir una respuesta 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 infección con la mayoría de los patógenos. 
  • 67. Nuevos enfoques • Identificación de antígenos protectores conservados (normalmente no inmunogénicos) • Desarrollo de plataformas efectivas de aplicación mucosal.
  • 68. 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 alta movilidad. – Citocina mediadora de la  inflamación – Se une al TLR4 y activa la  liberación de citocinas de los  macrófagos
  • 69. Nueva tecnología de Adyuvantes • La modificación de un  polisacárido de origen natural permite la  adhesion a las células presentadoras de  antígeno en la mucosa – y  union química a  antígenos autógenos • Antígenos seleccionados experimentalmente ofrecen resultados alentadores
  • 70. Conclusiones • En 2014, no hay “balas de plata” disponibles • Algunos Probióticos dan eficacia y consistencia similar a la de los antibióticos, y probablemente mejor que las vacunas disponibles comercialmente.  • Las tecnologías nuevas y emergentes de las vacunas pueden dar mejoras significativas • Algunos adyuvantes recientes de actividad mucosal  pueden mejorar la eficacia de las vacunas inactivadas autógenas.
  • 71. • La producción de  animals para consumo libres de estrés, en un  medio limpio, puede tener implicaciones para prevenir la  adquisición y  transmission potencial de patógenos de origen alimentario. 
  • 72. 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
  • 73. International Journal of Poultry Science 12 (6): 318-321, 2013 ISSN 1682-8356 © Asian Network for Scientific Information, 2013 Corresponding Author: Guillermo Tellez, Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA 318 Effect of Chitosan as a Biological Sanitizer for Salmonella Typhimurium and Aerobic 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 on chicken skin during storage. For experiment 1 (two trials) five skin samples of equal size were dipped into a 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 negative spoilage bacteria were used as indicators instead of ST. In both experiments, all samples were placed in individual bags and kept at 4°C. In experiment 1, dipping ST contaminated skin samples in a solution of 0.5% chitosan reduced (p<0.05) the recovery of ST by 24 h. In experiment 2, 0.5% chitosan treatment solution reduced (p<0.05) the presence of spoilage-causing psychrotrophic bacteria below detectable levels. These results suggest that 0.5% chitosan has a potential for use in an intervention technology for the control of foodborne pathogens on the surface of chicken skin contaminated with bacteria during storage. Key words: Salmonella, chitosan, chicken skin, sanitizer INTRODUCTION Chickens contain large numbers of microorganisms in their gastrointestinal tract and on their feathers and feet; therefore, storage quality of fresh chicken is partially dependent on the bacteria present on the integument prior to slaughter (Ramirez et al., 1997; Northcutt et al., 2003). Pathogenic microorganisms present in chicken carcasses after processing and throughout scalding and picking can contaminate equipment and other carcasses (Hargis et al., 1995; Byrd et al., 1998; Sarlin et al., 1998; Corrier et al., 1999b; Zhang et al., 2013). Pathogenic bacteria such as Salmonella enterica and Campylobacter spp. are able to attach to skin and penetrate in skin layers or feather follicles (Zhang et al., 2013), facilitating their presence on chicken skin and carcass during poultry processing (Chaine et al., 2013). Critical control point determination at broiler processing has become very important, especially because of the recent attention on Hazard Analysis and Critical Control Points (HACCP) for reduction of microbial contamination of meat and poultry (Rose et al., 2002). For all these MATERIALS AND METHODS reasons, strategies to reduce bacterial contamination on Bacterial strain and chitosan: A poultry isolate of poultry carcasses are important. However, most of the Salmonella enterica serovar Typhimurium (ST), selected bacterial 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 all their efficacy is reduced by the presence of organic experiments. The amplification and enumeration matter (Zhao et al., 2009). Therefore, it grows the need protocol for the isolate have been previously described of biological sanitizers in the processing plant to prevent (Tellez et al., 1993). Briefly, ST was grown in tryptic soy carcass to carcass cross-contamination by pathogenic broth (TSB, Catalog No. 22092, Sigma, St. Louis, MO bacteria and to lower the potential of foodborne diseases. Interest in chitosan, a biocompatible polymer derived from shellfish, as a biological sanitizer arises from reports showing several beneficial effects such as antimicrobial and antioxidative activities in foods (No et al., 2002; Friedman and Juneja, 2010). The use of chitosan in industry, agriculture and medicine is well described (Rabea et al., 2003; Senel and McClure, 2004; Friedman and Juneja, 2010). The antimicrobial activities of chitosan against foodborne pathogens has been broadly investigated in the food industry (Singla and Chawla, 2001; No et al., 2002; Senel and McClure, 2004; Petrovich et al., 2008; El-Hadrami et al., 2010; Kong et al., 2010; Vargas and Gonzalez-Martinez, 2010). Therefore, the objective of the present study was to evaluate the effect of chitosan as a biological sanitizer for Salmonella and aerobic Gram negative spoilage bacteria on chicken skin during storage at 4°C.
  • 74. Int. J. Poult. Sci., 12 (6): 318-321, 2013 319 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 at stock solution was determined spectrophotometrically at 4°C. At 1 h, 24 h, 3, 6, 9 and 12 days, 5 control and 625 nm. The stock solution was serially diluted and 5 treated skin samples were homogenized within confirmed 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 spread MD 21152) plates containing 25 µg/mL novobiocin (NO, plated on MacConkey agar (Becton, Dickinson and Catalog No. N-1628, Sigma, St. Louis, MO 63178) and Co. Sparks, MD, USA). Each sample was plated as 20 µg/mL NA. triplicate. The plates were incubated at 37°C for 24 Chitosan: Deacetylated 95% food grade chitosan was enumerated respectively. The identification of obtained commercially (Paragon Specialty Products, individual colonies with different morphology on LLC Rainsville, AL) and used in all experiments. The MacConkey agar was determined using the API-20E chitosan molecular weight was 350 kDa with viscosity of test kit for the identification of enteric Gram-negative 800 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 a solution containing 0.5% (w/v glacial acetic acid (Catalog Statistical analysis: The Most Probable Number method No. 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) with other sampling methods (whole carcass rinse further separation of significantly different means using procedure, 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 2 by 2 cm) was aseptically collected using forceps and scissors. Microbiological procedures: C Experiment 1: Two trials were conducted. In each trial, skin samples (N = 20) were dipped into a phosphate buffered saline (PBS) solution containing 10 cfu/mL of ST for 30 seconds. Skin8 samples were then removed, drained off and dipped for an additional 30s into a solution containing PBS (control; N = 10) or 0.5% chitosan (N 1 ). Control and treated samples were placed in individual sample bags and kept in a refrigerator at 4°C. At one or twenty four hours, five control and five treated samples were removed from the refrigerator and cultured for ST recovery. 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 BGA plates containing 25 µg/mL of NO and 20 µg/mL of NA. Each sample was plated as triplicate. The plates were incubated at 37°C for 24 h then viable colonies were observed and enumerated. C Experiment 2: Skin samples were dipped into a solution 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 DISCUSSION Salmonella is one of the most widespread bacterial species in poultry and it is often associated with foodborne illness (Bailey et al., 2002; Lynch et al., 2006). Cross-contamination by Salmonella in birds and carcasses may occur during transportation and processing (Cason et al., 1997; Corrier et al., 1999a). Therefore, the poultry industry has the challenge of monitoring and controlling Salmonella at all production levels (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 a solution of 0.5% chitosan was able to significantly reduce the recovery of ST cfu/square cm after 24 h in both trials (Table 1). The presence of spoilage bacteria in food products is an important economic problem. Therefore, an inexpensive and safe treatment to prevent spoilage is needed. Chitosan has been shown to be an effective antimicrobial, especially antibacterial. As shown in Table 2, 0.5% chitosan was effective in reducing total aerobic mesophilic Gram negative bacteria (spoilage bacteria) to undetectable levels. The primary spoilage bacteria in the control group of experiment 2 were identified as Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa using the API-20E test kit for enteric Gram-negative bacteria (bioMerieux, Inc., Hazelwood, MO). The concentration of P. aeruginosa in the control group increased from 7.5 x10 - 1.5x106 8
  • 75. Int. J. Poult. Sci., 12 (6): 318-321, 2013 320 Table 1: Salmonella Typhimurium (log10 cfu±standard error)/square cm of chicken skin treated with 0.5% chitosan solution in experiment 1 Trial 1 Trial 2 Dipping ---------------------------------- --------------------------------- treatment 1 h 24 h 1 h 24 h Control 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 differ significantly (p<0.05) Table 2: Aerobic Gram negative bacteria (log10 cfu±standard error)/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 differ significantly (p<0.05) cfu/square cm from 6-12 days stored at refrigeration temperatures (data not shown). The decreased growth as shown in Table 2 indicates that chitosan was very effective in controlling this and possible other spoilage bacteria. These results are in agreement with those published by Darmadji and Izumimoto (1994) who described the effectiveness of chitosan on storage stability of minced beef. Solutions of chitosan at 0.5- 1.0% were able to inhibit the growth of spoilage bacteria on red meat after 10 days of storage at 4°C (Darmadji and Izumimoto, 1994). The antimicrobial activity and film- forming characteristic of chitosan makes it a potential source of food preservative, increasing quality and shelf life of different types of foods (Darmadji and Izumimoto, 1994; Ouattar et al., 2000; No et al., 2007; Friedman and Juneja, 2010; Suman et al., 2010; Vargas and Gonzalez- Martinez, 2010). The mechanism of the antimicrobial activity of chitosan has not yet been fully elucidated; nevertheless different hypotheses have been proposed. The most realistic hypothesis is that chitosan is able to change cell permeability due to interactions between the positive charges of its molecules and the negative charges of the bacterial cell membranes (No et al., 2007; Friedman and Juneja, 2010). Other hypotheses include the chelation of metals and essential nutrients, inhibiting bacterial growth (Rabea et al., 2003). Zheng and Zhu (2003) had also suggested that high molecular weight chitosan could be able to form a polymer membrane around the bacterial cell, preventing it from receiving nutrients. On the other hand, Zheng and Zhu (2003) also proposed that the low molecular weight chitosan could enter the bacterial cell through pervasion, disrupting the physiological activities of the bacterium. Conclusion: The results of these experiments suggest that dipping raw chicken skin in a 0.5% solution of chitosan can reduce populations of Salmonella Typhimurium, thus enhancing general food safety and maybe shelf life of chicken meat. Moreover, these results also suggest that a solution of 0.5% chitosan can extend the shelf life of chicken meat as well as cause decreased growth of Gram negative spoilage bacteria. Future research will be directed at determining the effect of these organic compounds on the texture, color, oxidative stability, pH and consumer acceptance of chicken meat with treatment combinations that exhibited the most effective antibacterial activity. REFERENCES Bailey, J.S., N.A. Cox, S.E. Craven and D.E. Cosby, 2002. Serotype tracking of Salmonella through integrated broiler chicken operations. J. Food Prot., 65: 742- 745. Byrd, J.A., D.E. Corrier, M.E. Hume, R.H. Bailey, L.H. Stanker and B.M. Hargis, 1998. Effect of feed withdrawal on Campylobacter in the crops of market-age broiler chickens. Avian Dis., 42: 802- 806. Cason, J.A., J.S. Bailey, N.J. Stern, A.D. Whittemore and N.A Cox, 1997. Relationship between aerobic bacteria, Salmonella and Campylobacter on broiler carcasses. Poult. Sci., 76: 1037-1041. Chaine, A., E. Arnaud, A. Kondjoyan, A. Collignan and S. Sarter, 2013. Effect of steam and lactic acid treatments on the survival of Salmonella Enteritidis and Campylobacter jejuni inoculated on chicken skin. 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 of Salmonella in the crop contents of market-age broilers 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 of Salmonella in the crop and ceca of broiler chickens before and after preslaughter feed withdrawal. Poult. Sci., 78: 45-49. Darmadji, P. and M. Izumimoto, 1994. Effect of chitosan in meat preservation. Meat Sci., 38: 243-254. El-Hadrami, A., L.R. Adam, I. El-Hadrami and F. Daayf, 2010. Chitosan in plant protection. Mar. Drugs, 8: 968-987. Friedman, M. and V.K. Juneja, 2010. 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  • 76. Int. J. Poult. Sci., 12 (6): 318-321, 2013 321 Lynch, M., J. Painter, R. Woodruff and C. Braden, 2006. Sarlin, L.L., E.T. Barnhart, D.J. Caldwell, R.W. Moore, J.A. Surveillance for foodborne-disease outbreaks- Byrd, D.Y. Caldwell, D.E. Corrier, J.R. Deloach and United States, 1998-2002. Centers for Disease B.M. Hargis, 1998. Evaluation of alternative Control and Prevention. Morbidity and Mortality sampling methods for Salmonella critical control Weekly Report Surveillance Summaries, 55:1-42. point determination at broiler processing. Poult. 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 applications Antibacterial 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 No, H.K., S.P. Meyers, W. Prinyawiwatkul and Z. Xu, pharmaceutical and biological aspects-an update. 2007. Applications of chitosan for improvement of J. Pharm. Pharmacol., 53: 1047-1067. quality and shelf life of foods: A review. J. Food Sci., Suman, S.P., R.A. Mancini, P. Joseph, R. Ramanathan, 72: R87-100. M.K. Konda, G. Dady and S. Yin, 2010. Packaging- Northcutt, J.K., M.E. Berrang, J.A. Dickens, D.L. Fletcher specific influence of chitosan on color stability and and 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 dietary Poult. 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 Leghorn Holley, 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. Recent films prepared with chitosan. Int. J. Food Microbiol., patents on food applications of chitosan. Recent 62: 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 of 2008. Chitosan: structure, properties, use in hot water spray on broiler carcasses for reduction of medicine and stomatology. Stomatologiia, 87: 72- loosely attached, intermediately attached and tightly 77. 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 of Biomacromolecules, 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 acid M.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 antimicrobial market 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 and W.O. James, 2002. Testing for Salmonella in raw meat and poultry products collected at federally inspected establishments in the United States, 1998 through 2000. J. Food Prot., 65: 937-947.
  • 77. 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 92:2216–2220 http://dx.doi.org/10.3382/ps.2013-03172 Received March 8, 2013. Accepted May 11, 2013. 1Corresponding author: gtellez@uark.edu ©2013 Poultry Science Association Inc. 2216
  • 78. 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 Samples Forceps 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 Strains A 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
  • 79. 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 Analysis In 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. RESULTS Table 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:H7 Control L. monocytogenes OAM 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.
  • 80. 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). DISCUSSION In 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
  • 81. 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. REFERENCES Bilgili, S. F., D. E. Conner, J. L. Pinion, and K. C. Tamblyn. 1998. Broiler skin color as affected by organic acids: Influence of con- centration and method of application. Poult. Sci. 77:752–757. Bourassa, D. V., D. L. Fletcher, R. J. Buhr, M. E. Berrang, and J. A. Cason. 2004. Recovery of salmonellae from trisodium phosphate-treated commercially processed broiler carcasses after chilling and after seven-day storage. Poult. Sci. 83:2079–2082. Dickson, J. S., G. R. Siragusa, and J. E. Wray Jr. 1992. Predicting the growth of Salmonella Typhimurium on beef by using the temperature function integration technique. Appl. Environ. Mi- crobiol. 58:3482–3487. Harris, K., M. F. Miller, G. H. Loneragan, and M. M. Brashears. 2006. 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  • 82. International Journal of Poultry Science 12 (2): 72-75, 2013 ISSN 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 pathogens in the poultry industry. Direct acidification of drinking water with organic acids could significantly reduce the amount of recoverable Salmonella Typhimurium (ST) from the crop and cecal tonsils when used during the pre-slaughter feed withdrawal period. In the present study, in vitro and in vivo evaluations were conducted to 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. The two 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). All treatments reduced the number of ST recovered from crop contents at 24 h. This new formulation of OAM has great potential as a crop sanitizer and will be further evaluated under conditions similar to commercial chickens. Key words: Salmonella, organic acid, chickens INTRODUCTION Salmonella enterica causes an estimated 1.4 million cases of foodborne illnesses annually in the United States, resulting in over 15,000 hospitalizations (Voetsch et al., 2004a,b). Poultry and poultry products have been identified by some researchers as the most important source of transmission of Salmonella to the human population (Lynch et al., 2006). Increased pressure by consumers and regulatory agencies for reduced or even elimination of the use of antibiotics in food producing animals has created a need to find alternatives to maintain healthy and productive animals. These pressures are a challenge for the poultry industry for controlling Salmonella not only at the farm level, but also within processing and manufacturing plants (Hargis et al., 1995; Corrier et al., 1999a; Hinton et al., 2000; MATERIALS AND METHODS Mikolajczyk and Radkowski, 2002). An alternative to Salmonella amplification: A primary poultry isolate of antibiotics is the use of certain organic acids. Direct Salmonella Typhimurium (ST) was used in these acidification of the water with organic acids could experiments. This isolate was selected for resistance to significantly reduce the amount of recoverable nalidixic acid (NA) . For these experiments, ST was Salmonella 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 % sterile withdrawal period (Van Immerseel et al., 2006; Alali et saline by centrifugation (3,000 x g), and the approximate al., 2010; Vandeplas et al., 2010); however, previous concentration of the stock solution was determined research has suggested that administration of OA spectrophotometrically at 625 nm. The stock solution during the pre-slaughter feed withdrawal period could was serially diluted and confirmed by colony counts of lead to carcass shrinkage (Byrd et al., 2001). While this three replicate samples (0.1 mL/replicate) that were evidence was shown when using lactic acid alone, spread plated on brilliant green agar (BGA) plates Optimizer 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 not discouraged (Jarquin et al., 2007; Wolfenden et al., 2007; Vicente et al.,2007a,b,c). Organic acids are a readily available energy source for both the chicken and the bacteria. Therefore, it is important that the organic acids be administered in high enough concentrations to be bactericidal in the presence of organic matter, and low enough to be voluntarily consumed by the birds. In the present study, we compared a commercially available water acidifier (Optimizer , Pacific Vet Group,® Fayetteville, AR 72703), versus a new formulation of organic acid mix (OAM) to reduce Salmonella Typhimurium in the crop and cecal tonsils of broiler chicks. 1 2 3 4
  • 83. 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 were as the concentration of Salmonella (in cfu/mL) for in vitro experiments and total colony-forming units for in vivo challenge experiments. Experimental Design - in vitro crop assay: An assay previously described (Barnhart et al., 1999) was used with modifications. Briefly, 1.25g of unmedicated chick starter feed was measured into 13×100 mm borosilicate tubes and autoclaved. The feed was suspended in 4.5 mL sterile saline and inoculated with 0.5 mL of a Salmonella Typhimurium culture containing approximately 10 cfu/mL. The tubes were treated with4 either: 1) saline as a control; 2) OAM, having a final concentration of acetic, citric and propionic acids at 0.031 % or; 3) OAM, having a final concentration of acetic, citric and propionic acids at 0.062 %. Each sample was run as triplicate, each treatment had 5 replicates, and the entire assay was repeated in 2 additional trials. After administering the treatment, the tubes were vortexed and incubated at 37EC for 30 minutes and an additional 6 h. The tubes were then agitated and 20 µL of the content was serially diluted and plated as triplicates on BGA containing novobiocin and nalidixic acid. Typical ST colonies were counted after 24 h of incubation. Experimental design with chickens: In experiment 1, 64 day-of-hatch broiler chicks were obtained from a local hatchery. Chicks were randomized and challenged with 2 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 drinking water 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, having a final concentration of acetic, citric and propionic acids at 0.062%. Chicks were housed in brooder batteries with food 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 harvested separately. Salmonella recovery procedures have been previously described by our laboratory and were followed with some modifications (Tellez et al., 1993). Briefly, crop and cecal tonsils were enriched in 10 mL of tetrathionate broth overnight at 37EC. Following enrichment, each sample was streaked for isolation on BGA plates containing 25 µg/mL NO and 20 µg/mL NA. The plates were incubated at 37EC for 24 h and examined for the presence or absence of the antibiotic resistant ST. Ceca were weighed and then homogenized within sterile sample bags using a rubber mallet. Sterile saline (4X5 weight to volume) was added to each sample bag and hand stomached with the cecal contents. Dilutions were spread plated on BGA plates containing 25 g/mL NO and 20 µg/mL NA. The plates were incubated at 37EC for 24 h and cfu of ST per ceca were determined. obtained from a local hatchery. Chicks were randomized and challenged with 2 x 10 cfu/mL of ST. The chicks5 were then held in chick boxes for 1 h and then randomly assigned to 1) untreated control or continuous treatment in the drinking water 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, having a final concentration of acetic, citric and propionic acids at 0.062 %. Chicks were housed in brooder batteries with food and water ad libitum. At 24 hr post-challenge, chicks were humanely killed by CO inhalation and crops were aseptically2 harvested, weighed and were homogenized within sterile sample bags using a rubber mallet. Sterile saline (4X weight to volume) was added to each sample bag and hand stomached with the crop contents. Dilutions were spread plated on BGA plates containing 25 µg/mL NO and 20 µg/mL NA. The plates were incubated at 37EC for 24 h and cfu of ST per crop were determined. Following this, crops were enriched with a 2X solution of tetrathionate broth overnight at 37EC. Following enrichment, each sample was streaked for isolation on BGA plates containing 25 µg/mL NO and 20 µg/mL NA. The plates were incubated at 37EC for 24 h and examined for the presence or absence of the antibiotic resistant ST. Statistical analysis: The incidence of Salmonella recovery within experiments was compared using the chi-square test of independence (Zar, 1984) testing all possible combinations to determine significant (P<0.05) differences between control and treated groups. Cecal cfu data were converted to log10 cfu numbers and then compared using the GLM procedure of SAS (SAS Institute, 2002) with significance reported at P < 0.05. RESULTS AND DISCUSSION Salmonella colonization of poultry flocks can occur via horizontal transmission (Bailey et al., 2002; Kim et al., 2007; Alali et al., 2010; Vandeplas et al., 2010). Once cecal tonsil colonization is established, the bacterium is consistently shed in the feces (Bailey et al., 2002; Foley et al., 2008). Feed Withdrawal induces pecking of the contaminated litter which may contaminate the crop (Corrier et al., 1999c) and if the crop is ruptured during processing, Salmonella may contaminate raw poultry products (Corrier et al., 1999b). Because the crop is more likely to rupture than the ceca, the crop represents an important source of Salmonella contamination to carcasses (Hargis et al., 1995; Corrier et al., 1999a). Table 1 summarizes the results of effect of OAM on ST in an in vitro crop assay. In 3 independent trials, the 0.031% OAM reduced ST by 6 h and the 0.062 % OAM was also efficacious. However, when 0.062 % OAM was tested in chickens, it had a similar effect as Optimizer® showing a significant reduction in total number of ST
  • 84. 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 assay Trial 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 chicks during 24 hours period Crop Cecal tonsils Log ST/gram10 Enrichment Enrichment of ceca Treatment culture culture content Control 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 enrichment culture is expressed as positive/total chickens for each tissue sampled (%). * Indicates significant difference at P < 0.05. ** Indicates significant 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 differ significantly (P < 0.05). Table 3: Experiment 2, effect of Optimizer or organic acids mix® (OAM) on Salmonella Typhimurium (ST) infection in broiler chicks during 24 hours period Crop Log ST/gram10 enrichment of crop Treatment culture content Control 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 acid Data of enrichment culture is expressed as positive/total chickens for each tissue sampled (%). Log ST / gram of crop content is expressed as mean ± standard10 error. Values within columns with different lowercase superscripts differ significantly (P < 0.05). positive chickens in cecal tonsils (Table 2), and reducing the number of ST in the crop (Table 3) when compared with controls. In the present study, Optimizer® reduced ST colonization in both crop and ceca (Tables 2 and 3) as has been previously reported (Jarquin et al., 2007; Wolfenden et al., 2007). In experiment 1, treatment with OAM in the drinking water caused a significant reduction (P<0.05) in ST recovery from cecal tonsils when compared with the controls (OA treated = 19% vs. controls = 87%). Also, treatment with OAM reduced 2.21 logs of ST when compared with controls (Table 2). While any of the treatments reduced recovery of ST from the crop by enrichment, all treatments reduced the number of ST recovered from crop content at 24 h (Table 3). The organic acids used in this study (citric, acetic and propionic) as well as others have been shown to be individually effective in reducing Salmonella in vitro (Van Immerseel et al., 2006). The biocidal efficacy and the effect on virulence of Salmonella differ with each organic acid treatment and each organic acid has a unique effect on bacteria normally present in the crop and gastrointestinal 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 hydrophobicity could be factors that effect biocidal activity (Van Immerseel et al., 2006). For these reasons, a mixture of organic acids was tested to reduce ST crop contamination. Further studies are being conducted to evaluate these new formulations of OAM during the pre- slaughter feed withdrawal period in commercial chickens to evaluate water consumption and bactericidal activity against Salmonella in the crop. REFERENCES Alali, W.Q., S. Thakur, R.D. Berghaus, M.P. Martin and W.A. Gebreyes, 2010. Prevalence and Distribution of Salmonella in Organic and Conventional Broiler Poultry Farms. Foodborne Pathog. Dis. Bailey, J.S., N.A. Cox, S.E. Craven and D.E. Cosby, 2002. Serotype tracking of Salmonella through integrated broiler chicken operations. J. Food Prot., 65: 742- 745. Barnhart, E.T., L.L. Sarlin, D.J. Caldwell, J.A. Byrd, D.E. Corrier and B.M. Hargis, 1999. 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