preliminary results of a larval resistance test to ivermectins using boophilus microplus reference...
TRANSCRIPT
610
Preliminary Results of a Larval Resistance Test to Ivermectins Using
Boophilus microplus
Reference Strains
EFRAÍN BENAVIDES
a
AND ALVARO ROMERO
Programa Nacional de Epidemiología Veterinaria (EpiVet). Corporación Colombiana de Investigación Agropecuaria, CORPOICA-CEISA. Santafé de Bogotá, Colombia.
INTRODUCTION
Insecticides continue to be the primary means of controlling ectoparasites in live-stock. In Colombia, intensive use of these materials has led to development of resis-tance, in
Boophilus microplus,
to all currently used pesticides: organophosphates,carbamates, synthetic pyrethrods, and amidines.
1
New chemicals, such as the mac-rocyclic lactones, are available for the control of ectoparasites, but there are concernsabout the possible development of resistance to these new products.
2
Early resis-tance detection is an essential tool in resistance management.
2
For this reason, an
invitro
test for detecting resistance to macrocyclic lactones is required, with the char-acteristics of being easy to perform, inexpensive, and efficient.
3
MATERIALS AND METHODS
A protocol suggested by Jim Nolan (personal communication, 1995) was stan-dardized. Briefly, a group of approximately 500 tick larvæ from the susceptibleYeerongpilly strain were submerged for two minutes in 2 ml of commercial inject-able ivermectin, dissolved in a triton-ethanol mixture in demineralized water at ninedifferent dilutions. The contents were drained onto filter paper, allowed to dry, thenplaced inside a white paper package, and finally stored in an incubator (27
°
C, 90
%
R.H.). Mortality response was determined after 48 hours by counting the number ofdead and live larvæ. The whole process was repeated for the Montecitos multiresis-tant strain.
1
Abbot’s formula was applied if mortality in controls was between 0–5
%
,and data were discarded if mortality in control was above 5
%
. Percentage mortalitywas plotted against concentration on logarithmic probit graph paper.
4
The LC
50
,LC
99
, and LC
99.9
values were determined by applying regression equation analysisto the probit transformed data.
a
Centro de Investigación en Salud y Producción Animal. CEISA. Apartado Aéreo39144. Bogotá, Colombia. Telefax: (+57-1)-3686252.
611BENAVIDES & ROMERO: LARVAL RESISTANCE TO IVERMECTINS
RESULTS AND DISCUSSION
The concentrations used in the tests ranged from 0.39 to 100 p.p.m. At all con-centrations evaluated, higher mortalities were observed in the susceptible strain (seeT
ABLE
1). The LC
50
for the susceptible ticks was 2.46 p.p.m. (95
%
confidence lim-its: 2.1–2.8) and for Montecitos strain was 3.5 p.p.m. (95
%
CL: 2.2–4.8). Althoughthe differences are not significant, the greater degree of variability observed in theMontecitos strain is evident; the resistance factor of only 14.2. Differences betweencurves are shown in F
IGURE
1. There were no survivors when larvæ of both suscep-tible and resistant strains were exposed to the following dilutions: 25 p.p.m., 50
FIGURE 1. Modified larval test using ivermectin (1%) contained in 1% ethanol +0.02% Triton X-100 in demineralized water, comparing a field multiresistant strain, Mon-tecitos, with the susceptible Yeerongphilly strain.
T
ABLE
1. Results from the modified larval immersion test in two
B. microplus
reference strains, using commercial (injectable) macrocyclic lactones (Ivermectin 1%)contained in a triton-ethanol mixture in demineralized water
Yeerongpilly Montecitos
N
a
a
N
is the number of replicates of each test.
5 5
LC
50
p.p.m. (95
%
CL) 2.46 (2.1–2.8) 3.5 (2.2–4.8)
LC
99
p.p.m. (95
%
CL) 7.8 (7.0–8.6) 10 (7.5–12.5)
LC
99.9
p.p.m. (95
%
CL) 11.7 (10.6–12.8) 14.2 (10.8–17.7)
Slope (95
%
CL) 5.12292 (4.79495–5.45089) 5.24334 (4.22559–6.26109)
R
2
(95
%
CL) 0.94242 (0.91687–0.96797) 0.96998 (0.94457–0.99539)
612 ANNALS NEW YORK ACADEMY OF SCIENCES
p.p.m., and 100 p.p.m. This fact could be used to reduce the number of test concen-trations needed in a standardized test.
There is little information available about a test to detect resistance to macrocy-clic lactones in
B. microplus
ticks.
3
In our study, when the LC
50
values of two strainswere compared, little difference was observed between the Yeerongpilly (suscepti-ble) strain and the Montecitos strain, multiresistant to other pesticides. Our experi-ment describes the initial establishment of a base line for the susceptible referencestrain compared with a hypothetical resistant strain. This test is not yet recommend-ed by the FAO as a standardized protocol, and for this reason, further research isneeded to compare the behavior of local and reference strains in different locations.
REFERENCES
1. B
ENAVIDES
E., A. R
OMERO
& C. S
ANCHEZ
. 1997. Resultados preliminares de la evalu-ación de una cepa de campo
Boophilus microplus
multirresistente a diferentes acari-cidas. Brazilian J. Vet. Parasitol.
6
(2): 130.2. K
UNZ
S.E. & D.H. K
EMP
. 1994. Insecticides and acaricides: resistance and environ-mental impact. Rev. Sci. Tech. Office Intern. Epizoo.
13:
1249–1286.3. K
EMP
D.H., G.A. S
ABATINI
, S. H
UGHES
, J. H
ANSEN
& A. N
ARI
. 1998. Test to deter-mine LC
50
and discriminating doses for macrocyclic lactones against the cattle tick,
Boophilus microplus.
FAO Animal Production and Health Division, Rome.4. F
INNEY
, D.J. 1947. Probit Analysis. Cambridge University Press.