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www.aleidavillaespinosa.com
 Afectan gravemente la producción
y el bienestar animal ocasionando
importantes pérdidas económicas
en países tropicales ...
Piroplasmida
Theileria sp.
Th annulata
Th parva
Babesiasp.
B. bigemina
B. bovis
B. major
B. divergens
Anaplasmas Anaplasma...
 DIAGNÓSTICO DE RUTINA
HEMATOLOGÍA.
FROTIS SANGUÍNEO
DIAGNÓSTICO SEROLÓGICO
ELISA
IFAT.
IDENTIFICACIÓN MOLECULAR
PCR, NES...
 Introducir la PCR en tiempo Real en el
diagnóstico de rutina de Theileria. annulata,
Theileria parva, Babesia bigemina, ...
PUREZA
CUANTIFICACIÓN
110 casos con sospecha de hemoparásitos procedentes de España y
Portugal n= 280 muestras:
248 sangre...
qPCR cuantitativa
Diluciones seriadas (105
a 100
) de plásmidos específicos.
Cut Off ≤ 45
www.aleidavillaespinosa.com
qPCR...
Resultados de la qPCR
 El 52% (57/110) de los casos evaluados resultaron
positivos para hemoparásitos:
En sangre 51%
En s...
Resultados de la qPCR
Coexistencia de hemoparásitos: 37% (21/57) de los casos
positivos
- A. marginale y Th. annulata 21% ...
 El 91.2% (101/110) de los casos fueron
remitidos de 21 provincias españolas,
principalmente de las C.A. de Andalucía y
C...
 El 91.2% (101/110) de los casos fueron
remitidos de 21 provincias españolas,
principalmente de las C.A. de Andalucía y
C...
 El 91.2% (101/110) de los casos fueron
remitidos de 21 provincias españolas,
principalmente de las C.A. de Andalucía y
C...
 El 91.2% (101/110) de los casos fueron
remitidos de 21 provincias españolas,
principalmente de las C.A. de Andalucía y
C...
La linearidad de los ensayos nos permitió
identificar hasta 2x108
copias de
equivalente genómico de cada
hemoparásito/ml d...
 Para introducir la qPCR en el diagnostico de rutina en el sector veterinario
es de vital importancia contar con un preci...
 Hemos introducido la PCR en tiempo real en el diagnostico rutinario de las
principales enfermedades causadas por hemopar...
www.aleidavillaespinosa.com
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DIAGNOSTICO MOLECULAR DE LOS PRINCIPALES HEMOPARASITOS DEL GANADO BOVINO

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El objetivo de este trabajo fue Evaluar la PCR en Tiempo Real en el diagnóstico de rutina de Theileria. annulata, Theileria parva, Babesia bigemina, Babesia bovis, Anaplasma marginale, Anaplasma centrale y Anaplasma phagocitophylum en un formato multiparamétrico para el ganado bovino.

Published in: Health & Medicine
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DIAGNOSTICO MOLECULAR DE LOS PRINCIPALES HEMOPARASITOS DEL GANADO BOVINO

  1. 1. www.aleidavillaespinosa.com
  2. 2.  Afectan gravemente la producción y el bienestar animal ocasionando importantes pérdidas económicas en países tropicales y subtropicales, aunque se están convirtiendo en un grave y creciente problema en muchos países de Europa.  Son un grupo de patógenos (protozoarios y ricketssias) transmitidos por diferentes especies de garrapatas y otros insectos que causan enfermedades que tienen características clínicas similares.  La Piroplasmosis y la Anaplasmosis son las de mayor relevancia, estando su prevalencia y distribución geográfica asociadas con los vectores www.aleidavillaespinosa.com
  3. 3. Piroplasmida Theileria sp. Th annulata Th parva Babesiasp. B. bigemina B. bovis B. major B. divergens Anaplasmas Anaplasmasp. A. marginale A. centrale A. phagocytophylum www.aleidavillaespinosa.com
  4. 4.  DIAGNÓSTICO DE RUTINA HEMATOLOGÍA. FROTIS SANGUÍNEO DIAGNÓSTICO SEROLÓGICO ELISA IFAT. IDENTIFICACIÓN MOLECULAR PCR, NESTED-PCR, PCR EN TIEMPO REAL (qPCR) RLB www.aleidavillaespinosa.com
  5. 5.  Introducir la PCR en tiempo Real en el diagnóstico de rutina de Theileria. annulata, Theileria parva, Babesia bigemina, Babesia bovis, Anaplasma marginale, Anaplasma centrale y Anaplasma phagocitophylum en un formato multiparamétrico para el ganado bovino. www.aleidavillaespinosa.com
  6. 6. PUREZA CUANTIFICACIÓN 110 casos con sospecha de hemoparásitos procedentes de España y Portugal n= 280 muestras: 248 sangres en EDTA 23 sueros 9 tejidos Kit Genómico mini LGD 500 LabTurbo” y el equipo automático de extracción “LabTurbo 36 Compact System C3620” (Taigen Bioscience Corporation). www.aleidavillaespinosa.com CONTROL DE CALIDAD Quawell UV Q500 Software 4.0
  7. 7. qPCR cuantitativa Diluciones seriadas (105 a 100 ) de plásmidos específicos. Cut Off ≤ 45 www.aleidavillaespinosa.com qPCR PARA HEMOPARÁSITOS CADA MUESTRA DE DNA FUE INVESTIGADA PARA 6 HEMOPARÁSITOS (1rxn/patógeno), UTILIZANDO OLIGOS/SONDAS ESPECÍFICAS BAJO EL MISMO PROTOCOLO TERMICO PERFIL MULTIPARAMÉTRICO MiniOpticon System Bio Rad, Software CFX manager 2.0). 1 2 3 4 95.0 C 10:00 95.0 C 00:10 60.0 C 01:00 GO TO 2 49 X
  8. 8. Resultados de la qPCR  El 52% (57/110) de los casos evaluados resultaron positivos para hemoparásitos: En sangre 51% En suero 17% En tejidos (hÍgado y bazo) 22% www.aleidavillaespinosa.com 0 50 100 150 200 250 300 Sangre Suero Hig/Bazo 248 23 9 51 % 17 % 22 % muestras Tipo de muestra Total muestras % Positivos
  9. 9. Resultados de la qPCR Coexistencia de hemoparásitos: 37% (21/57) de los casos positivos - A. marginale y Th. annulata 21% (12/52) - Th. annulata, A.marginale y B. bigemina en un 14% (8/57). Th. annulata 37% (34/93) A. marginale 29% (26/90) B. bigemina 17% (15/90) B. bovis 2.2% (2/90) Th parva 0% (0/55) A. phagocytophylum 0% (0/54) A. centrale 0% (0/67) www.aleidavillaespinosa.com
  10. 10.  El 91.2% (101/110) de los casos fueron remitidos de 21 provincias españolas, principalmente de las C.A. de Andalucía y Cataluña. Comunidad Total Nro. casos Autonoma casos Positivos Cadiz 12 9 Cordoba 8 3 Huelva 1 1 Jaen 2 0 Sevilla 5 3 28 16 Huesca 2 1 Teruel 1 1 Zaragoza 2 1 5 3 Cantabria Santander 10 2 Castilla La Mancha Avila 2 2 Burgos 1 1 Palencia 2 0 5 3 Barcelona 18 10 Lleida 4 0 Tarragona 1 0 23 10 Extemadura Caceres 4 3 A. Coruña 2 2 Lugo 6 2 8 4 Madrid Madrid 8 1 Valenciana Castellon 4 3 6 5 Andalucia Aragón Castilla y Leon Cataluña Galicia Provincia Ciudad Real www.aleidavillaespinosa.com
  11. 11.  El 91.2% (101/110) de los casos fueron remitidos de 21 provincias españolas, principalmente de las C.A. de Andalucía y Cataluña. www.aleidavillaespinosa.com
  12. 12.  El 91.2% (101/110) de los casos fueron remitidos de 21 provincias españolas, principalmente de las C.A. de Andalucía y Cataluña. Comunidad Total Nro. casos Autonoma casos Positivos Cadiz 12 9 Cordoba 8 3 Huelva 1 1 Jaen 2 0 Sevilla 5 3 28 16 Huesca 2 1 Teruel 1 1 Zaragoza 2 1 5 3 Cantabria Santander 10 2 Castilla La Mancha Avila 2 2 Burgos 1 1 Palencia 2 0 5 3 Barcelona 18 10 Lleida 4 0 Tarragona 1 0 23 10 Extemadura Caceres 4 3 A. Coruña 2 2 Lugo 6 2 8 4 Madrid Madrid 8 1 Valenciana Castellon 4 3 6 5 Andalucia Aragón Castilla y Leon Cataluña Galicia Provincia Ciudad Real www.aleidavillaespinosa.com
  13. 13.  El 91.2% (101/110) de los casos fueron remitidos de 21 provincias españolas, principalmente de las C.A. de Andalucía y Cataluña. www.aleidavillaespinosa.com
  14. 14. La linearidad de los ensayos nos permitió identificar hasta 2x108 copias de equivalente genómico de cada hemoparásito/ml de sangre, suero o gramo de tejido con una eficiencia ≥ 95%. www.aleidavillaespinosa.com CUANTIFICACIÓN El valor umbral (Cq) de los Controles Positivos oscilo entre 20-35 ciclos, mientras que las muestras presentaron Cq entre 15 y 40 ciclos. El limite inferior de detección de hemoparásitos en los ensayos (6 qPCR) varió entre 2x100 a 2x101 copias/5µl
  15. 15.  Para introducir la qPCR en el diagnostico de rutina en el sector veterinario es de vital importancia contar con un precio accesible para el ganadero, la agrupación de las muestras y la conformación de paneles como el realizado en el presente estudio permite racionalizar recursos logrando un diagnóstico rápido y económico de estos patógenos.  Una ventaja adicional del formato multiparamétrico es que no existe competencia de múltiples cebadores por el ADN de la muestra por lo que no ocurre agotamiento del ADN muestra sobretodo en las infecciones mixtas.  El formato multiparamétrico permite la inclusión de otros ensayos de qPCR para completar el diagnóstico diferencial de hemoparásitos utilizando simultáneamente las mismas condiciones y ciclos térmicos. Ej. Leptospira patógenas, BLV, Erlichia ruminatum, trypanosoma spp. entre otras. www.aleidavillaespinosa.com
  16. 16.  Hemos introducido la PCR en tiempo real en el diagnostico rutinario de las principales enfermedades causadas por hemoparásitos en el ganado bovino mediante el uso del formato multiparamétrico.  El alto porcentaje de caso positivos encontrados en el presente estudio en España y Portugal confirman la presencia de varias especies de hemoparásitos y la importancia de estas enfermedades en Europa.  La PCR cuantitativa es una excelente herramienta para monitorear el tratamiento de estas hemoparasitosis y para la titulación de las cepas empleadas en la elaboración de vacunas. www.aleidavillaespinosa.com
  17. 17. www.aleidavillaespinosa.com

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