Comparison of P-glycoprotein gene expression of two Haemonchus contortus isolates from Yucatan, Mexico, with resistant or susceptible phenotype to ivermectin in relation to a susceptible reference strain.

The variability in the expression of different P-glycoprotein (P-gp) genes in parasitic nematodes of ruminants such as Haemonchus contortus (Hco-pgp) may be caused by different factors including nematode biology, geographical region and anthelmintic pressure. This study analysed the relative expression level of 10 P-gp genes in two H. contortus (Hco-pgp) field isolates from Yucatan, Mexico: 1) PARAISO (IVM-resistant) and 2) FMVZ-UADY (IVM-susceptible). These isolates were compared with a susceptible reference isolate from Puebla, Mexico, namely "CENID-SAI". In all cases H. contortus adult males were used. The Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14 and 16) were analysed for each isolate using the RT-qPCR technique. The Hco-pgp expressions were pairwise compared using the 2-ΔΔCt method and a t-test. The PARAISO isolate showed upregulation compared to the CENID-SAI isolate for Hco-pgp 1, 3, 9, 10 and 16 (P < 0.05), and the PARAISO isolate showed upregulation vs. FMVZ-UADY isolate for Hco-pgp 2 and 9 (P < 0.05), displaying 6.58- and 5.93-fold differences (P < 0.05), respectively. In contrast, similar Hco-pgp gene expression levels were recorded for FMVZ-UADY and CENID-SAI isolates except for Hco-pgp1 (P <0.1), which presented a significant upregulation (6.08-fold). The relative expression of Hco-pgp allowed confirming the IVM-resistant status of the PARAISO isolate and the IVM-susceptible status of the FMVZ-UADY isolate when compared to the CENID-SAI reference isolate. Therefore, understanding the association between the Hco-pgp genes expression of H. contortus and its IVM resistance status could help identifying the genes that could be used as molecular markers in the diagnosis of IVM resistance. However, it is important to consider the geographic origin of the nematode isolate and the deworming history at the farm of origin.

Curcumin-PVP improves the in vitro efficacy of ivermectin against resistant and susceptible Haemonchus contortus.

Ivermectin (IVM) resistance in parasitic nematodes such as Haemonchus contortus has spurred a search for substances that help to recover its efficacy. One potential agent is the natural product curcumin (CUR). In this study, CUR was combined with polyvinylpyrrolidone (PVP) (CUR/PVP) to improve its solubility and biological applicability. This study determined the effect of CUR preincubation on the effective concentration 50% (EC50) of IVM in three H. contortus isolates with different susceptibilities to IVM. The IVM EC50 was determined for three H. contortus isolates with different IVM susceptibilities using the larval migration inhibition (LMI) test. The three isolates were (i) PARAISO (IVM resistant), (ii) FMVZ-UADY (IVM susceptible), and (iii) CENID-SAI INIFAP (reference IVM susceptible). The L3 of each isolate were preincubated for 3 h with one of three concentrations of CUR (µg curcumin/mL): CONC-1 (3.67), CONC-2 (5.67), or CONC-3 (8.48). Corresponding controls were performed without CUR. The EC50 of IVM was determined for each isolate after they were exposed to the different CUR concentrations. The EC50 of IVM differed between the isolates PARAISO > FMVZ-UADY > CENID-SAI INIFAP (P < 0.05). The CUR preincubation at CONC-1 did not decrease the EC50 of IVM for any of the three isolates, suggesting a hormetic effect. By contrast, CUR preincubation at CONC-2 or CONC-3 decreased the IVM EC50 for the PARAISO isolate (P < 0.05) compared with the reference isolate and reduced the EC50 of IVM for the FMVZ-UADY and CENID-SAI INIFAP isolates below the EC50 for the CENID-SAI INIFAP isolate without CUR preincubation. In conclusion, preincubation of H. contortus L3 with CUR reduced the EC50 of IVM for field isolates classified as resistant and susceptible to IVM. The CUR preincubation reduced the IVM resistance factor in the different isolates tested.

Phenotypic and genotypic characterisation of Haemonchus spp. and other gastrointestinal nematodes resistant to benzimidazole in infected calves from the tropical regions of Campeche State, Mexico.

The aim of this study was to identify the presence of anthelmintic resistance to benzimidazole (BZ) in gastrointestinal nematodes (GIN) from naturally infected calves in the tropical regions of Campeche State of Mexico. The faecal egg count reduction test (FECRT) was conducted at 10 livestock farms localised in the Carmen, Candelaria, Champotón, Escárcega and Palizada municipalities of Campeche. The assessed anthelmintic was albendazole. The trial period was between August and November 2012. Infected calves were allocated into two groups, control and treated, on each farm. The number of eggs excreted per g of faeces was estimated by the McMaster technique at 0 and 14 days pre- and post- treatment, respectively. Recovered infective larvae (L3) (pre- and post-treatment) were identified using taxonomic keys and a genomic DNA (gDNA) template from a pool of L3 species prior to BZ treatment. Additionally, BZ-resistance polymorphisms in Haemonchus were determined by Allele Specific PCR (AS-PCR) at codon 200 and by end-point PCR at codons 200, 198 and 167 from isotype 1 of the ß-tubulin gene. Morphological identification revealed Haemonchus, Cooperia, Trichostrongylus, Ostertagia and Oesophagostumum L3 species before BZ treatment, and Haemonchus and Cooperia L3 species after treatment. Additionally, of the GIN populations, three exhibited BZ resistance, and seven were BZ-susceptible by FECRT. Molecular analysis identified mutations in Haemonchus populations on nine farms at codon 200 (TTC to TAC) by AS-PCR, while no changes were observed at 167 (TTC to TAC) or 198 (GAA to GCA) codons in any population. In conclusion, resistance to BZ was determined in Haemonchus and Cooperia nematodes in infected cattle in five tropical regions of Campeche State.

Primer informe en México sobre la presencia de resistencia a ivermectina en bovinos infectados naturalmente con nematodos gastrointestinales / First report in Mexico on ivermectin resistance on naturally infected calves with gastrointestinal nematodes

The aim of this work was to determine the presence of ivermectin anthelmintic resistance in gastrointestinal parasitic nematodes in young cattle naturally infected in the state of Campeche, Mexico. This research was performed based on a survey of sanitary management to 35 cattlemen for the selection of suspicious herds with anthelmintic resistance problems. Calves from six to 12 months age were selected because no anthelmintic treatment had been given for at least 60 days and because they showed more than 200 eggs per gram of feces (epg) before treatment. Two groups of calves from seven to 13 each were randomly chosen; control group received no treatment, and treated group received ivermectin at a dose of 0.2 mg per kg of body weight, subcutaneously. Fourteen days after treatment, the epg number was determined and infective larvae genera were taxonomically identified. Data were analyzed using RESO program for anthelmintic resistance. The results indicated a concerning problem of ivermectin anthelmintic resistance in the evaluated cattle of five ranches with levels higher than 60%. The nematode genera identified before treatment were: Cooperia spp, Haemonchus spp and Oesophagostomum sp, and after treatment Cooperia spp still remained in five ranches, followed by Oesophagostomum sp and Haemonchus spp for two of the evaluated ranches. This research shows, for the first time in the state of Campeche, anthelmintic resistance in young cattle, as well as presence of gastrointestinal nematodes resistant to ivermectin.

El objetivo del presente trabajo fue detectar resistencia a ivermectina en nematodos gastrointestinales en bovinos jóvenes infectados naturalmente en Campeche, México. El trabajo se llevó a cabo con base en una encuesta de manejo sanitario realizada a 35 ganaderos para la selección de hatos sospechosos de problemas de resistencia antihelmíntica. Becerros de seis a 12 meses de edad fueron seleccionados debido a que no habían recibido tratamiento antihelmíntico en un periodo mínimo de 60 días y porque presentaron cuentas parasitarias mayores de 200 huevos por gramo de heces (hpg), antes del tratamiento. Se formaron dos grupos de bovinos al azar, de siete a 13 animales cada uno; el grupo testigo no recibió tratamiento, y grupo tratado recibió 0.2 mg por kg de peso de ivermectina, vía subcutánea. Catorce días después del tratamiento, se determinó el número de hpg y se identificaron los géneros de larvas infectantes. Los datos se analizaron en el programa RESO para resistencia antihelmíntica. Los resultados indicaron serios problemas de resistencia a ivermectina en el ganado vacuno evaluado en cinco ranchos con niveles mayores de 60%. Los nematodos identificados antes del tratamiento, Cooperia spp, Haemonchus spp y Oesophagostomum sp, y después del tratamiento fueron Cooperia spp en los cinco ranchos, seguido por Oesophagostomum sp y Haemonchus spp para dos de los ranchos evaluados. Este trabajo muestra evidencia, por primera vez en Campeche, de resistencia a ivermectina en bovinos jóvenes, así como presencia de tres géneros de nematodos gastrointestinales resistentes a ivermectina.