Phenotypic and genotypic approaches for detection of anthelmintic resistant sheep gastrointestinal nematodes from Brazilian northeast

Araújo-Filho, José Vilemar de; Ribeiro, Wesley Lyeverton Correia; André, Weibson Paz Pinheiro; Cavalcante, Géssica Soares; Santos, Jéssica Maria Leite dos; Monteiro, Jomar Patrício; Macedo, Iara Tersia Freitas; Oliveira, Lorena Mayana Beserra de; Bevilaqua, Claudia Maria Leal

doi:10.1590/S1984-29612021048

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Revista Brasileira de Parasitologia Veterinária

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Original Article • Rev. Bras. Parasitol. Vet. 30 (2) • 2021 • https://doi.org/10.1590/S1984-29612021048 copy

Phenotypic and genotypic approaches for detection of anthelmintic resistant sheep gastrointestinal nematodes from Brazilian northeast

Abordagens fenotípica e genotípica para detecção de resistência anti-helmíntica em nematoides gastrintestinais de ovinos no nordeste do Brasil

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The aim of this study was to characterize the anthelmintic resistance (AR) of a sheep gastrointestinal nematode population, named Caucaia, from northeastern Brazil. Phenotypic tests performed were: egg hatch (EHT), larval development (LDT) and fecal egg count reduction (FECRT). Benzimidazoles (BZs) genotypic evaluation was by frequency of single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A, and for levamisole (LEV), by frequency of resistance alleles of Hco-acr-8 gene. The primers were designed specifically for Haemonchus contortus. Effective concentrations 50% (EC50) for BZs (EHT), and for macrocyclic lactones (MLs) and LEV (LDT) were 1.02 µg/mL, 1.81 ng/mL and 0.04 µg/mL, respectively. Resistance ratios for MLs and LEV were 0.91 and 3.07, respectively. FECRT efficacies of BZs, MLs, monepantel (MPTL) and LEV were 52.4; 87.0; 94.5 and 99.6%, respectively. qPCR for BZs demonstrated resistance allele frequencies of 0%, 26.24% and 69.08% for SNPs E198A, F200Y and F167Y, respectively. For LEV, 54.37% of resistance alleles were found. There was agreement between EHT, FECRT and qPCR for BZs, and agreement between LDT and qPCR for LEV. Thus, based on higher sensitivity of qPCR, and phenotypic evaluation, the Caucaia population was considered resistant to BZs, MLs, LEV and suspect for MPTL.

Keywords:
Nematode; small ruminants; anthelmintic resistance; diagnosis; qPCR

Anthelmintics	Day 0	Day 7	Day 14	Day 21
Albendazole
Mean epg	2,246±1,928^a	1,067±1,682^b	2,317±3,756^a	1,600±2,287^a
Efficacy (CI)	-	52.4 (-15-86)	0	28.7 (-59-80)
Ivermectin
Mean epg	1,993±1,510^a	786±1,030^b	276±281^b	523±645^b
Efficacy (CI)	-	61.8 (11-87)	87.0 (73-96)	72.5 (42-91)
Levamisole
Mean epg	2,107±1,959^a	7±18^b	64±123^b	46±90^b
Efficacy (CI)	-	99.6 (99-101)	96.9 (92-100)	97.7 (94-101)
Monepantel
Mean epg	1,988±1,389^a	143±202^b	106±156^b	103±143^b
Efficacy (CI)	-	92.7 (82-100)	94.5 (86-100)	93.0 (85-99)

Assay	Drug	EC50/Efficacy (E) Caucaia	*EC50 ISE H. contortus* isolate**	Resistance Ratio (RR)^a
EHT	Thiabendazole	EC50 = 1.02 µg/mL (0.58-3.24)^b	-	-
LDT	Ivermectin	EC50 = 1.81 ng/mL (0.35-5.62)^b	EC50 = 1.974 ng/mL (1.37-2.67)^b	0.91
LDT	Levamisole	EC50 = 0.04 µg/mL (0.035-0.057)^b	EC50 = 0.013 µg/mL (0.009-0.018)^b	3.07
FECRT	Albendazole	E = 52.4% (-15-86)^b	-	-
	Ivermectin	E = 87.0% (73-96)^b	-	-
	Levamisole	E = 99.6% (99-101)^b	-	-
	Monepantel	E = 94.5% (86-100)^b	-	-

Treatment	*H. contortus*		Trichostrongylus spp.		Oesophagostomum spp.		Total
Treatment	Larvae (n)	%	Larvae (n)	%	Larvae (n)	%	Total
No treatment	91	91.0	8	8.0	1	1.0	100
ABZ – D7	88	88.0	10	10.0	2	2.0	100
ABZ – D14	94	94.0	6	6.0	0	0.0	100
ABZ – D21	92	92.0	7	7.0	1	1.0	100
IVM – D7	89	89.0	9	9.0	2	2.0	100
IVM – D14	83	83.0	16	16.0	1	1.0	100
IVM – D21	88	88.0	12	12.0	0	0.0	100
LEV – D7	39	73.58	11	20.75	3	5.66	53^a
LEV – D14	61	78.20	17	21.79	0	0.0	78^a
LEV – D21	82	82.0	16	16.0	2	2.0	100
MPTL - D7	82	82.0	7	7.0	11	11.0	100
MPTL – D14	79	79.0	7	7.0	14	14.0	100
MPTL – D21	77	77.0	5	5.0	18	18.0	100

qPCR target	H. contortus - Caucaia		Resistance allele (%)	Resistant homozygous (%)	Proportion heterozygous at both positions (%)	ISE Isolate		Resistance allele (%)
	Ct^a ± SD of each allele					Ct^a ± SD of each allele
	Sensitive	Resistant				Sensitive	Resistant
SNP – F200Y	30.82 ± 0.63	32.31 ± 0.87	26.24	6.88	16.53	24.33 ± 0.49	30.13 ± 0.24	1.76
SNP – F167Y	29.36 ± 0.10	28.20 ± 0.70	69.08	47.72	16.53	26.02 ± 0.38	-	-
SNP – E198A	29.36 ± 0.10	-	-	-	-	23.80 ± 0.04	-	-
Hco-acr-8	29.20 ± 0.13	28.94 ± 0.42	54.37	29.56	49.61	24.63 ± 0.12	24.37 ± 0.19	38.5

Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br

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[1] *Corresponding author: Claudia Maria Leal Bevilaqua. E-mail: claudiamlb@yahoo.com.br