A new in vitro test to evaluate the resistance level against acaricides of the cattle tick, Rhipicephalus (Boophilus) microplus
2011-7-1, Lovis, Leonore
In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The larval tarsal test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus (Boophilus) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC50) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo. The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than the in LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.
Evaluation of acaricide resistance in the cattle tick, 'Rhipicephalus (Boophilus) microplus', using a new in vitro test and molecular tools
, Lovis, Leonore, Betschart, Bruno, Sager, Heinz
Rhipicephalus (Boophilus) microplus est une tique des bovins à un hôte qui se trouve dans les zones tropicales et subtropicales. Cet ectoparasite a un impact économique très important sur l’élevage bovin dans toutes les régions où il se trouve. Au Brésil, par exemple, les pertes dues à son impact direct et indirect ont été estimées à 2 milliards de dollars US en 2000. Le contrôle des populations de tiques des bovins repose essentiellement sur l’utilisation d’acaricides. L’utilisation intensive de ce moyen de lutte a eu pour conséquence le développement de résistances à la majorité des classes d’acaricides disponibles sur le marché. Un suivi local de la résistance aux acaricides est essentiel afin que les éleveurs puissent recevoir des informations sur les composés auxquels les populations de tiques présentes dans leur établissement sont résistantes et être guidés dans le choix de composés de remplacement à disposition. A une échelle globale, le suivi de la résistance permet d’observer sa progression afin d’essayer de ralentir son développement et d’allonger la durée d’utilisation des composés.
Les tests in vitro sont des méthodes très utiles pour détecter la résistance des tiques. La FAO recommande deux d’entre eux, l’un utilisant des larves, nommé Larval Packet test (LPT), l’autre utilisant des adultes, appelé Adult Immersion Test (AIT). Chaque test a ses avantages et ses inconvénients : le LPT est un test laborieux qui prend beaucoup de temps alors que le AIT nécessite une grande quantité de tiques. Ces désavantages limitent le nombre de composés et de doses pouvant être testés, limitant ainsi l’information obtenue. Pour surmonter ces difficultés, nous avons développé un nouveau test, nommé Larval Tarsal Test (LTT), qui permet de tester de nombreux composés en peu de temps et avec un minimum de tiques. Dans ce test, des œufs de tiques sont distribués dans des puits de plaques de microtritation préalablement traités avec les acaricides voulant être testés. Les œufs sont incubés jusqu’à l’éclosion des larves, qui sont ainsi exposées aux composés. La résistance des souches de tiques est alors évaluée en fonction de la mortalité des larves écloses. La capacité du LTT à détecter la résistance a été comparée au LPT, un des tests recommandés par la FAO. Pour cela, une souche de tiques sensible ainsi qu’une souche résistante de référence ont été exposées à neuf composés de cinq classes principales d’acaricides : les organophosphorés (OP), les pyréthroïdes de synthèse (SP), les amidines, les lactones macrocycliques (ML) et les phénylpyrazoles. Le LTT a permis d’obtenir de bonnes courbes de dose-réponse, il s’est montré aussi sensible que le LPT et, à mortalités équivalentes, a nécessité des doses de composés nettement plus basses que le LPT.
Ayant démontré que le LTT est un test adéquat, des populations de tiques provenant d’Argentine, d’Afrique du Sud et d’Australie ont été envoyées en Suisse pour évaluer leur résistance à l’aide du LTT. Ces tests ont confirmé l’intérêt du LTT à être utilisé pour détecter la résistance dans des populations provenant du terrain. Afin que ce test puisse être effectué dans d’autres laboratoires, le LTT a été modifié de sorte que l’infrastructure nécessaire à sa réalisation soit simple. Suite à cela, le LTT a été utilisé dans deux laboratoires brésiliens, pour tester des populations de tiques locales. Quelques-unes de ces populations ont aussi été testées avec le LPT pour permettre des comparaisons supplémentaires. A nouveau, le LTT s’est monté très efficace en termes de temps et adéquat pour détecter la résistance aux acaricides des populations de terrain avec, cette fois-ci, une sensibilité même plus élevée que le LPT. Ces études ont également permis de fournir des données supplémentaires sur la situation de la résistance aux acaricides dans les pays étudiés. Ainsi, elles ont permis de détecter les premiers cas de résistance de la tique des bovins à l’amitraz en Argentine, elles ont montré l’ampleur de la résistance au fipronil au Brésil et mis en évidence l’importance de la résistance aux OP et aux SP dans les différents pays ainsi. De plus, elles ont permis de détecter quelques cas isolés de résistance ou de suspicion de résistance aux ML au Brésil, en Argentine et en Afrique du Sud.
Afin de compléter ce diagnostic in vitro de la résistance aux acaricides, une PCR multiplex a été mise au point pour détecter simultanément trois mutations ponctuelles connues comme étant impliquées dans la résistance aux SP chez R. (B.) microplus. Cette PCR a été utilisée pour tester les échantillons de tiques provenant du Brésil, d’Argentine, d’Afrique du Sud et d’Australie dont le phénotype avait été préalablement évalué avec le LTT, ainsi que quelques souches supplémentaires du Mexique. Il est apparu que les trois mutations ont des distributions géographiques distinctes et qu’elles résultent dans des phénotypes de résistance différents., Rhipicephalus (Boophilus) microplus is a one-host cattle tick which has a tropical and subtropical distribution. This major ectoparasite has a very important economic impact on cattle husbandry throughout its area of distribution. In Brazil, for example, the loss due to its direct and indirect effects was estimated at 2 billion US dollars in 2000. The control of cattle tick populations relies mainly on acaricides and the intensive use of such products has led to the development of resistance to most of the acaricide classes available on the market. Monitoring of resistance is essential on a local scale, so that producers can obtain information on the resistance pattern of the tick population established in their farm and advice on using alternative compounds. In addition, on a global scale, monitoring of resistance may help to slow down the development of resistance and to extend compound lifespan.
Bioassays are very useful tools to monitor resistance. The Food and Agriculture Organisation of the United Nations (FAO) recommends two in vitro tests: the Laval Packet Test (LPT) and the Adult Immersion Test (AIT). Each test has its own advantages and disadvantages: the LPT is a laborious and time-consuming test while the AIT requires many ticks for testing. These two flaws limit the number of compounds and doses which can be tested, and therefore the information which can be obtained. Hence, we developed a new test named Larval Tarsal Test (LTT), to overcome these limitations. The LTT is a time effective test requiring small numbers of ticks which is based on the distribution of tick eggs in pre-treated wells of microtiter plates. The plates are incubated until larvae hatch and get exposed to the acaricidal compounds. Then, the evaluation of the susceptibility of the ticks is based on the assessment of larva mortality. The ability of the LTT to detect resistance was compared to the FAO-recommended LPT by testing a susceptible and a resistant reference laboratory strain with 9 compounds of 5 major acaricide classes: organophosphates (OP), synthetic pyrethroids (SP), amidines, macrocyclic lactones (ML), and phenylpyrazols. The LTT provided satisfactory dose-response curves, was as sensitive as the LPT and required much lower doses of the acaricides to obtain equal mortality levels.
Having demonstrated the suitability of the LTT, this test was used to evaluate the acaricide resistance pattern of field populations from Argentina, South Africa and Australia, which were shipped to Switzerland for testing. This study proved the ability of the LTT to detect resistance in field populations and the test was subsequently modified to simplify the equipment required and to be carried out in other laboratories. The LTT was then carried out in two Brazilian laboratories testing tick field populations from that country and, some of these populations were also tested with the LPT for additional comparison. Again, the LTT proved to be a time-effective and appropriate test to detect acaricide resistance in field populations with, this time, an even higher sensitivity than the LPT. Furthermore, these studies provided additional data on the resistance in the four sampled countries. This study detected the first cases of resistance to amitraz in Argentina, found widespread fipronil resistance in Brazil, widespread OP and SP resistance in the different countries, and occasional cases of resistance or suspected resistance to ML in Brazil, Argentina and South Africa.
Finally, to complete this in vitro diagnosis of acaricidal resistance, a multiplex PCR was developed allowing the simultaneous detection of three single nucleotide substitutions known to confer resistance to SP in R. (B.) microplus. This assay was used to screen the tick samples originating from Brazil, Argentina, South Africa and Australia whose phenotype to SP had been determined by the use of the LTT, as well as some additional tick strains from Mexico. The three mutations were found to have distinct geographical distributions and to result in different resistance phenotypes.
Acaricide Resistance Mechanisms in Rhipicephalus microplus
2012-2-15, Lovis, Leonore
Acaricide resistance has become widespread in countries where cattle ticks, Rhipicephalus (Boophilus) microplus, are a problem. Resistance arises through genetic changes in a cattle tick population that causes modifications to the target site, increased metabolism or sequestration of the acaricide, or reduced ability of the acaricide to penetrate through the outer protective layers of the tick's body. We review the molecular and biochemical mechanisms of acaricide resistance that have been shown to be functional in R. microplus. From a mechanistic point of view, resistance to pyrethroids has been characterized to a greater degree than any other acaricide class. Although a great deal of research has gone into discovery of the the mechanisms that cause organophosphate resistance, very little is defined at the molecular level and organophosphate resistance seems to be maintained through a complex and multifactorial process. The resistance mechanisms for other acaricides are less well understood. The target sites of fipronil and the macrocyclic lactones are known and resistance mechanism studies are in the early stages. The target site of amitraz has not been definitively identified and this is hampering mechanistic studies on this acaricide.
PCR diagnosis of Opisthorchis viverrini and Haplorchis taichui infections in an endemic Lao community: a comparison of diagnostic methods for parasitological field surveys
2012-11-18, Lovis, Leonore
Opisthorchiasis is a major public health problem in Southeast Asia. Affected individuals often have mixed infections with the liver fluke Opisthorchis viverrini and minute intestinal flukes such as Haplorchis taichui. The usual methods of diagnosing these infections involve the demonstration of fluke eggs in stool samples under light microscopy, but sensitivity and specificity are low. We developed two PCR tests that detect and discriminate between O. viverrini and H. taichui infections. PCR tests were validated by stool samples from purged individuals. We then applied the PCR tests to estimate the prevalence of O. viverrini and H. taichui infections from a random sample of individuals selected from an endemic community in Khong District, Laos. PCR results were compared with those from the Kato-Katz (KK) method and the formalin-ether concentration technique (FECT). When validated with purge results, PCR tests of O. viverrini and H. taichui had a sensitivity of 93.7% (95% Confidence Interval (CI): 85.8 to 97.9%) and 73.3% (95% CI: 60.3 to 83.9%) and could detect as low as 0.75 pg DNA and 1.32 ng DNA, respectively. The PCR-determined community prevalence of O. viverrini and H. taichui infections was 63.9% (95% CI: 54.1 to 72.9%) and 30.6% (95% CI: 22.1 to 40.2%), respectively. Using PCR as the gold standard to detect O. viverrini, three KK thick smears performed comparably well whereas one KK smear and FECT were poorer (sensitivity of 91.4% (95% CI: 81.0 to 97.1%), 62.3% (95% CI: 49.8 to 73.7%) and 49.3% (95% CI: 37.0 to 61.6%), respectively). PCR may be a valuable and sensitive diagnostic tool, particularly for low intensity O. viverrini and H. taichui infections.
Distribution patterns of three sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus) microplus populations from North and South America, South Africa and Australia
2012-9-3, Lovis, Leonore
Resistance to synthetic pyrethroids (SP) in the cattle tick Rhipicephalus (Boophilus) microplus is widespread throughout its distribution area. Three single nucleotide substitutions identified in Domains II and III of the sodium channel gene of R. (B.) microplus are known to be associated with target site pyrethroid resistance. We developed a multiplex PCR using allele-specific primers to amplify wild type or mutated genotypes of the three mutations simultaneously. This assay was used to screen tick samples originating from Brazil, Argentina, Mexico, South Africa and Australia whose phenotype to flumethrin and cypermethrin had been determined by the use of the Larval Tarsal test (LTT) or the Larval Packet Test (LPT). These mutations were found to have distinct geographical distributions and result in different resistance phenotypes. The L64I Domain II mutation conferring resistance to several SP compounds was found in all the Brazilian, Argentinean and Australian populations and in one South African population, with frequencies between 38% and 100% in flumethrin and cypermethrin resistant populations, respectively. In contrast, this mutation was not found in samples from Mexico, while the Domain III mutation was found exclusively in this country. The G72V Domain II flumethrin-specific mutation was found in a single Australian population, at a very low frequency (6%). The homozygous resistant RR genotype of the L64I Domain II mutation correlated significantly with the survival rates at the discriminating doses of flumethrin and cypermethrin. This survey shows the widespread distribution of the L64I Domain II mutation and provides evidence of its geographic separation from the Domain III mutation.
In vitro diagnosis of the first case of amitraz resistance in Rhipicephalus microplus in Santo Tomé (Corrientes), Argentina
2012-10-1, Lovis, Leonore
In Argentina, the cattle tick Rhipicephalus microplus has already developed resistance to organophospates and synthetic pyrethroids. However, no cases of amitraz resistance have ever been recorded in this country despite its heavy use. A recent failure of amitraz to control ticks in a farm located in Santo Tomé, province of Corrientes, resulted in the collection of samples for acaricide resistance diagnosis. The modified Drummond adult immersion test (AIT) and the larval tarsal test (LTT) were performed separately in Argentina and Switzerland to evaluate efficacy of amitraz and other acaricides. The AIT showed that oviposition in the Santo Tomé field isolate was not inhibited when it was challenged to 250 and 500 ppm amitraz, and 50 ppm deltamethrin. However, oviposition was reduced by 90.6% when this field isolate was challenged to a combination of 400 ppm ethion and 100 ppm cypermethrin. To confirm the results obtained with the AIT, 2 additional tick samples were collected and shipped to Switzerland for resistance diagnosis of amitraz, cypermethrin and flumethrin, using the LTT. With this bioassay, the resistance ratios of the 2 field isolates were 32.5 and 57.0 for amitraz and between 5.9 and 27.2 for the synthetic pyrethroids. Both in vitro bioassays confirmed amitraz and synthetic pyrethroid resistance in the Santo Tomé samples. These results account for the first evidence of amitraz resistance in R. microplus in Argentina.
Use of the Larval Tarsal Test to determine acaricide resistance in Rhipicephalus (Boophilus) microplus Brazilian field populations
2012-9-10, Lovis, Leonore
Acaricide resistance of the cattle tick Rhipicephalus (Boophilus) microplus is widespread in most of the countries where this parasite is present. Bioassays are used to diagnose the level and pattern of resistance in tick populations. In the present study, we describe a detailed protocol of the Larval Tarsal Test (LTT) using simplified equipment and data on the resistance of 17 tick field populations originating from 5 Brazilian states. Nine acaricidal compounds from 5 major classes were tested: organophosphates (OP), synthetic pyrethroids (SP), macrocyclic lactones (ML), phenylpyrazols (PYZ) and amidines. For comparison, four of the tick populations were also tested with the Larval Packet Test (LPT) with one compound per class. The most common resistances were to SP, amitraz and OP, with frequencies of 94%, 88% and 82%, respectively. Resistance to PYZ was also found to be widespread (65%), suggesting a rapid development of fipronil resistance in Brazil. One case of ML resistance and 2 cases of suspected ML resistance were identified with the LTT. The LTT led to higher resistance ratios to all compounds than the LPT, reflecting its high sensitivity to detect resistance. Finally, the LTT allowed testing a larger number of compounds and doses with reduced labour in comparison to the LPT and turned out to be a reliable bioassay to detect resistance in field populations.
Efficacy of Praziquantel against Schistosoma mekongi and Opisthorchis viverrini: A Randomized, Single- Blinded Dose-Comparison Trial
2012-8-5, Lovis, Leonore
Background: Schistosomiasis and opisthorchiasis are of public health importance in Southeast Asia. Praziquantel (PZQ) is the drug of choice for morbidity control but few dose comparisons have been made. Methodology: Ninety-three schoolchildren were enrolled in an area of Lao PDR where Schistosoma mekongi and Opisthorchis viverrini coexist for a PZQ dose-comparison trial. Prevalence and intensity of infections were determined by a rigorous diagnostic effort (3 stool specimens, each examined with triplicate Kato-Katz) before and 28–30 days after treatment. Ninety children with full baseline data were randomized to receive PZQ: the 40 mg/kg standard single dose (n = 45) or a 75 mg/kg total dose (50 mg/kg+25 mg/kg, 4 hours apart; n = 45). Adverse events were assessed at 3 and 24 hours posttreatment. Principal Findings: Baseline infection prevalence of S. mekongi and O. viverrini were 87.8% and 98.9%, respectively. S. mekongi cure rates were 75.0% (95% confidence interval (CI): 56.6–88.5%) and 80.8% (95% CI: 60.6–93.4%) for 40 mg/kg and 75 mg/kg PZQ, respectively (P = 0.60). O. viverrini cure rates were significantly different at 71.4% (95% CI: 53.4–84.4%) and 96.6% (95% CI: not defined), respectively (P = 0.009). Egg reduction rates (ERRs) against O. viverrini were very high for both doses (.99%), but slightly lower for S. mekongi at 40 mg/kg (96.4% vs. 98.1%) and not influenced by increasing diagnostic effort. O. viverrini cure rates would have been overestimated and no statistical difference between doses found if efficacy was based on a minimum sampling effort (single Kato-Katz before and after treatment). Adverse events were common (96%), mainly mild with no significant differences between the two treatment groups. Conclusions/Significance: Cure rate from the 75 mg/kg PZQ dose was more efficacious than 40 mg/kg against O. viverrini but not against S. mekongi infections, while ERRs were similar for both doses.