Caracterization of the Boophilus microplus Suceptible Strain Centro Nacional de Servicios de Constatación en Salud Animal Director Subdirector Parasitología Coordinador Técnico Dr. Hugo Fragoso S. Dr. Fernando Parrodi L. Biol. Alejandrina Ortiz N. October 2005

Test Strain Reference Strain Porto Alegre Media Joya - México Source 1 Received October 2003CENAPA Joao Martins, CPVDF, Brasil Continous propagation > 4 years October 2005

October 2005 CENAPA 1st. infestation CENAPA, 1st. Eng. Females AIT (Drummond test) 1st. (probit) LPT CENAPA 2nd passage Propagation of Media Joya sample 1st sample G O G 1 Field sample – 2002 LPT (DD) - LIT LPT * Every 6 months the strain is toxicologically evaluated trough LPT - Probit

Active Ingredient Compound Coumaphos Chlorfenvinphos Diazinon Flumethrin Deltamethrin Cypermethrin Amitraz October 2005 STANDARD ACARICIDES

Active Ingredient (Compound) Coumaphos 200 ppm Chlorfenvinphos 300 ppm Diazinon 250 ppm Flumethrin 30 ppm Deltamethrin 25 ppm Cypermethrin200 ppm Amitraz200 ppm October 2005 FORMULED ACARICIDES

Protocol Products: Comercial formulation Concentratión: Recommended Comercial Concentration Female ticks:Fully engorged: <24 hrs after detachment Number: 40 ticks/concentration Immersión: 1 min. in 100 ml of comercial formulation Incubatión: 45 days at 28 ± 2° C, 80-90% RH. Evaluatión: Egg laying inhibition Egg fertility (hatchability) Percentage control October 2005 Adult Inmersion Test (Drummond)

Protocol Larvae: 7 days (maximum 14 days) Solvent: Trichloroethilen Solvent: High quality olive oil Filter paper: Whatman No X 8.5 cm. Impregnation: 0.67 ml. distributed on paper/ dried 12 hrs/ Concentration: 1:2 dilution Set-up: filter bags produced according FAO, transfer 100 larvae/ bag (duplicated) Incubation: 28 ±2° C, 80-90% RH, Pirethroids and phosphates incubated separately Evaluatión: Mortality counts 24 hrs p.a. October 2005 Larval Package Test

Make sure the susceptible strain is homogenous Use strain not older than 14 days in LPT Evaluate substance concentrations by Chromatography Personnel participating should by qualified Use lab. standard procedure October 2005 Critical factors controled in LPT and AIT

Percentage control found in B. microplus Susceptible and Porto Alegre, obtained by the Drummond test. October 2005 COMERCIAL PRODUCTC.C.R. %Strain % control B. microplus Susceptible- CENAPA B. microplus POA 1a. sample Coumaphos Cholrfenvinphos Clorpiriphos Flumethrin Deltamethrin Cypermethrin Permethrin+chlorfenvinphos Amitraz (Taktic)

Comparition of the Lethal concentrations 50 (CL50) in B. microplus; Susceptible and Porto Alegre obtained by the Stone technique. October 2005 ACTIVE INGREDIENT LETHAL CONCENTRATIONS 50 B. microplus Susceptible-CENAPA 2002 B. microplus POA Replica 1Replica 2 Coumaphos Cholrfenvinphos Diazinon Flumethrin Deltamethrin Cypermethrin

Comparation of the resistance indexes in B. microplus Susceptible and Porto Alegre, obtained by the Stone technique. October 2005 ACTIVE INGREDIENT RESISTANCE INDEX B. microplus Susceptible- CENAPA LC50 B. microplus POA Generation 1 Replica 1Replica 2 Coumaphos Cholrfenvinphos Clorpiriphos Flumethrin Deltamethrin Cypermethrin

Cenapa susceptible strain is completly susceptible in LPT for all products Cenapa susceptible strain is completely susceptible in Drummond test for all products The strain is very homogeneus, using LPT to evaluate the ticks trough 12 generations (4 years) has been no modified the original pattern. Compared with POA strain, CENAPA susceptible ticks are more susceptible October 2005 Summary

ACTIVE INGREDI ENT LETHAL 50 CONCENTRATIONS B. microplus Susceptible CENAPA 2002 B. microplus Susceptible CENAPA 2003 B. microplus Susceptible CENAPA 2004 B. microplus Susceptible CENAPA 2005 B. microplus POA 1 st. Generation Coumaphos Chlorfenvinpho s Diazinon Flumethrin Deltamethrin Cypermethrin Comparation of the Lethal concentrations 50 (CL50) in B. microplus Susceptible obtained by the Stone technique from

Probit lines of B. microplus “Media Joya” susceptible strain compared with POA strain Results with Coumaphos from 2002 to D.D. 0.2

Probit lines of B. microplus “Media Joya” susceptible strain compared with POA strain Results with Deltamethrin from 2002 to D.D. 0.09

ACTIV INGREDIENTD.D. ORGANOFOSFORADOS COUMAHOS0.2 CHLORFENVINPHOS0.2 DIAZINON0.08 CHLORPIRIFOS0.3 PIRETROIDES FLUMETRINA0.01 DELTAMETRINA0.09 CIPERMETRINA0.5 ORGANOCLORADOLINDANO0.1 DISCRIMINANT DOSIS Boophilus microplus Tick LPT STONE & HAYDOCK

Draft framework for CENAPA, Cuernavaca, Mexico Qualification of technical personal and laboratories 1.Standardization analytical procedure applying LPT in reference centers and subcenters. a. Utilize standardized FAO procedures b. Utilize larvae 7 to 14 days old c. Make Probit analysis with CENAPA discriminating doses (table 1) using 0.5 dilution factor. ( Coumaphos 0.2, 0.1, 0.05, 0.025, , , Cypermethrin 0.5, 0.25, 0.125, , , , ) d. Utilize Boophilus susceptible strain “Media Joya” e. Carry out 10 probits per product and and determine standard deviation and variation coefficient. f. Calculate CL50, 90 and 99 by Sigma software 2. Evaluate substance concentrations by Chromatography a. Utilize certified active ingredient with at least 97% purity 3. Perform Proficiency test (ring test) 1.Five sample of Boophilus tick egg mass will be sent from CENAPA to the reference center and subscenters. 2.LPT should be conducted by every laboratory and results to be sent to CENAPA.. 3.CENAPA will send a written report to each lab and a summary to FAO. October 2005

TICK CAMPAIGN IN MEXICO The National Program is suported by federal governement, state governement and cattle producers The governement stablish the rules and the operators decide how to work. The governement make evaluation if the state wants to have a recognition of the status Cenapa is the Nat. Ref. Cent. There are 4 small labs doing service to the cattle producers.

Ensenada Baño de Sto. Tomas Baño de San Isidro

DESARROLLO DE LA RESISTENCIA EN MÉXICO

Porcentajes de eficacia en garrapatas B. microplus resistentes al Amitraz Besuntol Garraban Ectogan Batestan Bayticol Permetrina Esteladon Asuntol % CCR 92.80Dursban 1.64Ethión 22.80Amitraz San AlfonsoIxodicida Sta. Luisa

Resistencia en Boophilus MEXICO, Enero Diciembre 2004