Desenvolvimento e validação de um teste de diagnóstico para monitoramento da resistência anti-helmíntica em rebanhos ovinos
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Data
2020-05-29
Autores
Gainza, Yousmel Alemán
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Universidade Estadual Paulista (Unesp)
Resumo
O teste de desenvolvimento larvar (TDL) pode ser uma ferramenta de diagnóstico da resistência anti-helmíntica de rebanhos, permitindo seu monitoramento e ajustes de manejo. O objetivo deste estudo foi desenvolver e validar um teste de diagnóstico in vitro da resistência de Haemonchus contortus a anti-helmínticos comerciais e validar os resultados a campo. A eficácia do tiabendazol (TBZ), levamisol (LEV), ivermectina-monossacárida (IVM-M), monepantel (MPT) e Zolvix® (ZLV) foi avaliada no TDL em isolados de H. contortus susceptível (Echevarria1991-HcEc91) e resistente (Botucatu-HcBot), em placas de cultura de 24 e 96 poços. Testes complementares foram posteriormente realizados com a ivermectina aglicona (IVM-A). Para validação do TDL, realizou-se o teste de redução da contagem de ovos nas fezes (TRCOF) em cinco rebanhos ovinos com anti-helmínticos comerciais dos mesmos grupos químicos (grupos de 7 animais com OPG ≥ 200). TDL e coproculturas também foram realizadas com as amostras dos rebanhos (HcRebanhos). H. contortus foi o parasita majoritário em todas as fazendas (média de 74%). O fator de resistência (FR) definido para os isolados foi superior a 3, indicando que o teste foi capaz de diferenciar HcEc91 e HcBot para TBZ, LEV, MPT, ZLV, IVM-A, e de maneira menos consistente para IVM-M. A similaridade das curvas dose-resposta entre as placas e interação placa*concentração (R2 de 98,4 a 99,0% para HcEc91/HcBot e de 99,4 a 99,0% para HcRebanhos), bem como baixas diferenças na eficácia média (0,02 a 2,26% para HcEc91/HcBot e 0,02 a 4,90% para HcRebanhos) para todos os anti-helmínticos, exceto para MPT, indicaram concordância confiável do TDL em ambas as placas, tanto para a avaliação de HcEc91/HcBot quanto para HcRebanhos. No último caso, a detecção de resistência pelo ZLV foi mais clara e mais estável do que pelo MPT. A adaptação do teste para placas de 96 poços resultou em economia de pelo menos 51.9%. Por meio do TRCOF, detectou-se resistência a todos os grupos químicos em todos os rebanhos, exceto para ZLV (40% resistentes e 20% suspeitos de resistência). Os resultados do TDL nos rebanhos indicaram resistência a TBZ (100%), LEV (80%), ZLV (20%), IVM-M (0%) e IVM-A (100%). Foi obtida concordância total quanto aos resultados de ambos os testes para TBZ e IVM-A (k=1,00). Já para LEV e ZLV, apesar de haver divergência de resultados em dois rebanhos, esta não foi estatisticamente diferente para ambas as drogas (P= 0,077 e P= 0,197, respectivamente), obtendo-se concordância substancial (k= 0,8 e 0,6, respectivamente). Os dados do presente estudo indicam que os resultados dos dois testes são comparáveis, validando, portanto, esse teste como uma opção para o diagnóstico da resistência anti-helmíntica para produtores de pequenos ruminantes.
The larval development test (LDT) can be a diagnostic tool for anthelmintic resistance in flocks, allowing monitoring and management adjustments. The aim of this study was to develop and validate an in vitro diagnostic test of Haemonchus contortus resistance to commercial anthelmintics and to validate the results at the farm level. The efficacy of thiabendazole (TBZ), levamisole (LEV), ivermectin-monosaccharide (IVM-M), monepantel (MPT) and Zolvix® (ZLV) were evaluated in LDT in susceptible (Echevarria1991-HcEc91) and resistant (Botucatu-HcBot) isolates, in 24 and 96 well culture plates. Complementary tests were subsequently performed with ivermectin aglycone (IVM-A). To validate the LDT, the fecal egg count reduction test (FECRT) was performed in five sheep flocks with commercial anthelmintics from the same chemical groups (groups of 7 animals with FEC ≥ 200). LDT and fecal cultures were also carried out with the samples of the flocks (HcFlocks). H. contortus was the major parasite on all farms (average of 74%). The resistance factor (RF) defined for the isolates was greater than 3, indicating that the test was able to differentiate HcEc91 and HcBot for TBZ, LEV, MPT, ZLV, IVM-A, and in a less consistent way for IVM-M. The similarity of the dose-response curves between the plates and plate*concentration interaction (R2 from 98.4 to 99.0% for HcEc91/HcBot and from 99.4 to 99.0% for HcFlocks), as well as low differences in efficacy mean (0.02 to 2.26% for HcEc91/HcBot and 0.02 to 4.90% for HcFlocks) for all anthelmintics, except for MPT, indicated a reliable agreement of the LDT in both plates, both for the evaluation of HcEc91/HcBot as for HcFlocks. In the latter case, resistance detection by ZLV was clearer and more stable than by MPT. The adaptation of the test to 96-well plates resulted in savings of at least 51.9%. Through FECRT, resistance to all chemical groups was detected in all flocks, except for ZLV (40% resistant and 20% suspected of resistance). LDT results in flocks indicated resistance to TBZ (100%), LEV (80%), ZLV (20%), IVM-M (0%) and IVM-A (100%). Total agreement was obtained regarding the results of both tests for TBZ and IVM-A (k = 1.00). For LEV and ZLV, although there was a divergence of results in two flocks, this was not statistically different for both drugs (P = 0.077 and P = 0.197, respectively), with substantial agreement (k = 0.8 and 0.6, respectively). The data from the present study indicate that the results of the two tests are comparable, thus validating this test as an option for the diagnosis of resistance for small ruminant farmers.
The larval development test (LDT) can be a diagnostic tool for anthelmintic resistance in flocks, allowing monitoring and management adjustments. The aim of this study was to develop and validate an in vitro diagnostic test of Haemonchus contortus resistance to commercial anthelmintics and to validate the results at the farm level. The efficacy of thiabendazole (TBZ), levamisole (LEV), ivermectin-monosaccharide (IVM-M), monepantel (MPT) and Zolvix® (ZLV) were evaluated in LDT in susceptible (Echevarria1991-HcEc91) and resistant (Botucatu-HcBot) isolates, in 24 and 96 well culture plates. Complementary tests were subsequently performed with ivermectin aglycone (IVM-A). To validate the LDT, the fecal egg count reduction test (FECRT) was performed in five sheep flocks with commercial anthelmintics from the same chemical groups (groups of 7 animals with FEC ≥ 200). LDT and fecal cultures were also carried out with the samples of the flocks (HcFlocks). H. contortus was the major parasite on all farms (average of 74%). The resistance factor (RF) defined for the isolates was greater than 3, indicating that the test was able to differentiate HcEc91 and HcBot for TBZ, LEV, MPT, ZLV, IVM-A, and in a less consistent way for IVM-M. The similarity of the dose-response curves between the plates and plate*concentration interaction (R2 from 98.4 to 99.0% for HcEc91/HcBot and from 99.4 to 99.0% for HcFlocks), as well as low differences in efficacy mean (0.02 to 2.26% for HcEc91/HcBot and 0.02 to 4.90% for HcFlocks) for all anthelmintics, except for MPT, indicated a reliable agreement of the LDT in both plates, both for the evaluation of HcEc91/HcBot as for HcFlocks. In the latter case, resistance detection by ZLV was clearer and more stable than by MPT. The adaptation of the test to 96-well plates resulted in savings of at least 51.9%. Through FECRT, resistance to all chemical groups was detected in all flocks, except for ZLV (40% resistant and 20% suspected of resistance). LDT results in flocks indicated resistance to TBZ (100%), LEV (80%), ZLV (20%), IVM-M (0%) and IVM-A (100%). Total agreement was obtained regarding the results of both tests for TBZ and IVM-A (k = 1.00). For LEV and ZLV, although there was a divergence of results in two flocks, this was not statistically different for both drugs (P = 0.077 and P = 0.197, respectively), with substantial agreement (k = 0.8 and 0.6, respectively). The data from the present study indicate that the results of the two tests are comparable, thus validating this test as an option for the diagnosis of resistance for small ruminant farmers.
Descrição
Palavras-chave
Diagnóstico, RESISTA-Test, Resistência anti-helmíntica, Nematoides gastrintestinais, Ovinos, Diagnosis, RESISTA-Test, Anthelmintic resistance, Gastrointestinal nematodes, Sheep