Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods / Resistência anti-helmíntica de Haemonchus contortus em rebanhos ovinos no Brasil: concordância de métodos in vivo e in vitro (RESISTA-Test©)

Abstract This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

Resumo Este estudo avaliou o status de resistência de Haemonchus contortus em rebanhos ovinos do estado de São Paulo, Brasil, através de um estudo comparativo entre o teste de redução da contagem de ovos nas fezes (TRCOF) e o teste de desenvolvimento larvar (TDL). Para o TRCOF, 35 ovinos foram selecionados em cada um dos cinco rebanhos e divididos em grupos tratados com: benzimidazol, levamisol, ivermectina, monepantel e controle. Fezes foram coletadas para OPG e coproculturas. O TDL foi realizado com tiabendazol-TBZ, levamisol-LEV, ivermectina aglicona-IVM-A e Zolvix-ZLV. Resistência a todos os fármacos foi detectada por meio do TRCOF em 100% dos rebanhos, exceto para ZLV (40% resistentes e 20% suspeitos de resistência). O LDT indicou resistência ao TBZ e IVM-A em todos os rebanhos; em 80% dos rebanhos ao LEV e 10% ao ZLV. Foi obtida concordância total entre os dois testes para TBZ e IVM (k = 1,0), enquanto para LEV (k = 0,8) e ZLV (k = 0,9) foram obtidas concordância substancial e quase perfeita, respectivamente. A concordância entre os testes foi significante, sendo possível usar o resultado do LDT para prever o do TRCOF, validando-se o primeiro como um teste diagnóstico rápido para criadores de ovinos no Brasil.

Helminth infections and hybridization between Haemonchus contortus and Haemonchus placei in sheep from Santana do Livramento, Brazil / Infecções helmínticas e hibridização entre Haemonchus contortus e Haemonchus placei em ovinos criados em Santana do Livramento, Brasil

Abstract The occurrence and intensity of helminth infections were evaluated in sheep from pastures shared with cattle. In 2015 and 2016, young male sheep acquired in Santana do Livramento, Rio Grande do Sul, Brazil, were finished in integrated crop-livestock system. We selected the 12 sheep that showed the highest number of nematode eggs per gram of faeces to search for worms in the gastrointestinal tract. Haemonchus contortus and Trichostrongylus colubriformis were the major parasites. H. contortus presented mean intensities of 1,159 and 257 worms in 2015 and 2016, respectively. T. colubriformis displayed mean intensities of 4,149 and 2,427 worms in 2015 and 2016, respectively. Of the 127 male specimens of Haemonchus spp. analysed by Polymerase Chain Reaction (PCR), 125 were H. contortus, one Haemonchus placei and one hybrid. Other species detected were Cooperia punctata , Cooperia pectinata, Cooperia spatulata, Cooperia curticei, Ostertagia ostertagi, Teladorsagia circumcincta , Trichostrongylus axei, Nematodirus spathiger , and Trichuris ovis. Twenty lambs presented cysts of Taenia hydatigena in the liver and mesentery. One lamb presented Coenurus cerebralis, the larval stage of Taenia multiceps, in the brain. In conclusion, sheep from pasture shared with cattle presented a high diversity of nematode species. H. contortus and H. placei co-infection occur with consequent hybridization.

Resumo A ocorrência de infecções helmínticas foi avaliada em ovinos que compartilhavam pastagem com bovinos. Em 2015 e em 2016, cordeiros machos foram adquiridos em Santana do Livramento, Rio Grande do Sul, para serem terminados em sistema de lavoura - pecuária (ILP). Em cada ano, 12 cordeiros que tinham maior contagem de ovos nas fezes foram abatidos para recuperação dos vermes. Haemonchus contortus e Trichostrongylus colubriformis foram as principais espécies registradas. H. contortus apresentou intensidade média de 1159 e 257 vermes em 2015 e 2016, respectivamente. T. colubriformis apresentou intensidade média de 4149 e 2427 parasitas em 2015 e 2016, respectivamente. De 127 machos de Haemonchus spp. analisados por "Polymerase Chain Reaction" (PCR), 125 foram identificados como H. contortus, um como Haemonchus placei e um como híbrido. Cooperia punctata, Cooperia pectinata, Cooperia spatulata, Cooperia curticei, Ostertagia ostertagi, Teladorsagia circumcincta , Trichostrongylus axei, Nematodirus spathiger e Trichuris ovis foram as outras espécies de nematódeos registradas. Vinte cordeiros apresentaram cistos de Taenia hydatigena no mesentério e no fígado. Um cordeiro apresentou no cérebro Coenurus cerebralis, o estágio larval de Taenia multiceps . Em conclusão, ovinos criados com bovinos apresentam grande diversidade de nematódeos. A co-infecção de H. contortus e H. placei favorece a produção de híbridos.

Immunodiagnosis of Haemonchus contortus infection in sheep by indirect enzyme linked immunosorbent assay [ELISA]

Indirect plate enzyme-linked immunosorbent assay was standardized and evaluated for its effectiveness in immunodiagnosis of haemonchosis in experimental and clinical cases in sheep by using somatic whole adult antigen of H. contortus. Plate ELISA was standardized using 5 mg/well antigen concentration with 1:100 and 1:1000 of sera and conjugate dilution. Indirect plate ELISA was able to demonstrate the antibody titre at different weeks post infection in experimental sheep. A comparison of plate ELISA on suspected field sera and faecal sample examination by floatation method revealed that 74 samples were found to be positive by ELISA but only ten by faecal examination. Sensitivity of plate ELISA was found to be 80.0%, whereas specificity was 21.42% indicating that this test is quite sensitive for clinical cases; an early diagnosis, however, lacks specificity

Utilização do método Famacha no diagnóstico clínico individual de haemoncose em ovinos no Sudoeste do Estado de Minas Gerais / Utilization of the Famacha method in individual clinic diagnostic of haemonchosis in sheep of south-west of Minas Gerais State

O objetivo deste estudo foi observar a eficácia do método Famacha no diagnóstico clínico individual de haemoncose em ovinos provenientes do Sudoeste de Minas Gerais. Cinquenta ovelhas mestiças Santa Inês foram avaliadas durante 5 meses quanto à coloração da mucosa conjuntiva (método Famacha), volume globular (VG), ovos por grama de fezes (OPG), escore corporal e alterações clínicas. O escore Famacha observado variou de 1 a 3; o escore corporal de 2 a 5; o VG de 20 a 47 por cento; e o OPG de 0 a 7.750. As correlações entre os parâmetros avaliados, exceto Famacha com escore corporal, foram altamente significativas (P < 0,0001). O principal gênero identificado nas coproculturas foi Haemonchus (87 a 100 por cento do total de larvas, ± 93,5 por cento). Constatou-se redução de 87,33 por cento na utilização de anti-helmínticos na propriedade. Conclui-se que o método Famacha, associado a outros métodos de diagnóstico, é ferramenta útil na detecção da haemoncose em ovinos.

The aim of this study was to evaluate the efficacy of Famacha method in individual clinical diagnosis of haemonchosis in sheep from the south-west of Minas Gerais. Fifty crossbred Santa Inês ewes were evaluated during 5 months as for conjunctival mucous colour (Famacha method), Packed Cell Volume (PCV), Faecal Egg Counts (FEC), body score and clinic alterations. The score Famacha observed was from 1 to 3, the body score from 2 to 5, the PCV from 20 to 47 percent, and FEC from 0 to 7,750. The correlations between the parameters evaluated, except Famacha with body score, were highly significant (P < 0.0001). The main genus identified in the coprocultures was Haemonchus (87 - 100 percent of total larvae, ± 93.5 percent). We observed reduction of 87.33 percent in the use of anthelmintics on the property. It was concluded that the Famacha method, associated with other diagnostic methods, is a useful tool to detect hemonchosis in sheep.

Utilização do método Famacha no diagnóstico clínico individual de haemoncose em ovinos no Sudoeste do Estado de Minas Gerais / Utilization of the Famacha method in individual clinic diagnostic of haemonchosis in sheep of south-west of Minas Gerais State

O objetivo deste estudo foi observar a eficácia do método Famacha no diagnóstico clínico individual de haemoncose em ovinos provenientes do Sudoeste de Minas Gerais. Cinquenta ovelhas mestiças Santa Inês foram avaliadas durante 5 meses quanto à coloração da mucosa conjuntiva (método Famacha), volume globular (VG), ovos por grama de fezes (OPG), escore corporal e alterações clínicas. O escore Famacha observado variou de 1 a 3; o escore corporal de 2 a 5; o VG de 20 a 47%; e o OPG de 0 a 7.750. As correlações entre os parâmetros avaliados, exceto Famacha com escore corporal, foram altamente significativas (P < 0,0001). O principal gênero identificado nas coproculturas foi Haemonchus (87 a 100% do total de larvas, 93,5%). Constatou-se redução de 87,33% na utilização de anti-helmínticos na propriedade. Conclui-se que o método Famacha, associado a outros métodos de diagnóstico, é ferramenta útil nadetecção da haemoncose em ovinos.

The aim of this study was to evaluate the efficacy of Famacha method in individual clinical diagnosis of haemonchosis in sheep from the south-west of Minas Gerais. Fifty crossbred Santa Inês ewes were evaluated during 5 months as for conjunctival mucous colour (Famacha method), Packed Cell Volume (PCV), Faecal Egg Counts (FEC), body score and clinic alterations. The score Famacha observed was from 1 to 3, the body score from 2 to 5, the PCV from 20 to 47%, and FEC from 0 to 7,750. The correlations between the parameters evaluated, except Famacha with body score, werehighly significant (P < 0.0001). The main genus identified in the coprocultures was Haemonchus (87 - 100% of total larvae, 93.5%). We observed reduction of 87.33% in the use of anthelmintics on the property. It was concluded that the Famacha method, associated with other diagnostic methods, is a useful tool to detect hemonchosis in sheep.

Detection of anti-Haemonchus contortus antibodies in sheep by dot-ELISA with immunoaffinity purified fraction of ES antigen during prepatency.

Haemonchosis is a very common disease in small ruminants caused by H. contortus, a blood sucking parasite causing anaemia that may be fatal particularly to young animals. Therefore, detection of the infection during prepatent period is important for early treatment. Excretory-Secretory (ES) protein of H. contortus was purified through immunoaffinity chromatography. Dot -ELISA was performed with crude ES antigen as well as immunoaffinity purified fraction (F-1) with experimental and natural sera of sheep infected with H. contortus. Solid dot formation took place with 4 day, 1, 2 and 3 weeks post infection sera. Dot formation did not take place with negative control serum and uninfected control animal serum. When crude ES antigens was reacted to natural sheep sera having H. contortus infection, 60% sera samples showed solid dot formation whereas in F-1 fraction 75% of the sera samples showed solid dot indicating purified fraction was a more potent antigen. Crude ES and F-1 were also fractionated through SDS-PAGE. ES antigen revealed polypeptides in the range of 10 to 200 kDa of which 26, 32, 60 and 120 kDa were found more prominent. F-1 fraction on SDS-PAGE analysis revealed only four polypeptides of 26, 32, 60, and 120 of which 60 and 120 kDa were found to be most prominent. Results indicate that the purified fraction of ES antigen may be utilized for early diagnosis of haemonchosis. Further studies on cross antigenicity of this fraction with other nematode and trematode needs to be conducted.