Monitoring benzimidazole resistance in Phyllosticta citricarpa using a molecular assay targeting mutations in codons 198 and 200 of the beta-tubulin gene

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is an economically important disease, which is effectively controlled by repeated fungicide applications to protect fruit from infection. Systemic fungicides such as benzimidazoles are widely used for controlling CBS in South Africa, but the molecular mechanisms of benzimidazole resistance in P. citricarpa had not been investigated. Analysis of the nucleotide sequence of the beta-tubulin gene in P. citricarpa revealed mutations inducing three amino acid replacements in benzimidazole-resistant isolates when compared to that of sensitive strains. Amino acid replacements in benzimidazole-resistant isolates included the change of glutamic acid to either alanine or lysine at codon 198 of the beta-tubulin gene and the change from phenylalanine to tyrosine at codon 200. All three mutations were previously implicated in benzimidazole resistance in several fungal pathogens. A polymerase chain reaction (PCR) assay was designed to amplify a portion of the beta-tubulin gene, which is subsequently sequenced to identify benzimidazole resistance in P. citricarpa. This PCR and sequence assay was found to be a more rapid and reliable method for detecting resistance compared to the fungicide-amended plate tests and is valuable for monitoring the occurrence of benzimidazole-resistant P. citricarpa and for assessment of the need for alternative CBS management practices.

Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. Methods Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. Conclusions We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Graphical Abstract

Molecular Detection of Benzimidazole Resistance in Haemonchus contortus Larvae of Goats in Chhattisgarh, India

Background: Benzimidazole resistance is one of the key problem in small ruminant production. A rapid, truthful and responsive system is required for detection benzimidazole resistance so that proper regulatory measure can be applied. Allele specific PCR is one of the tools to understand the mechanism and origin of benzimidazole resistance. Methods: A total 198 larvae of Haemonchus contortus were isolated from goats of Chhattisgarh region, central India were genotyped by allele specific polymerase chain reaction (AS-PCR). Faecal samples of goats were collected from three Government farms and adjoining field goats and were subjected for faecal culture, separately. DNA of third stage larva was used for nested PCR for amplification of β- tubulin gene. Restriction Fragment Length Polymorphism (RFLP) was applied on nested PCR product for species identification with RsaI enzyme. AS-PCR was applied on the nested-PCR product to know the genotypic and allelic frequency. Result: The nested PCR amplified product showed approximately 820 bp in all cases and PCR-RFLP revealed 462 bp, 211 bp and 147 bp fragments, which confirmed the species as H. Contortus. Frequency of resistant allele ('r') was 49.7% and 50.3% for susceptible allele ('S '). Frequency of homozygous resistant (rr), heterozygous susceptible (rS) and homozygous susceptible (SS) genotype were 33.83 per cent, 31.81 per cent and 34.34 per cent, respectively. The frequency of homozygous resistant (rr) genotype was low (19.61%) in field compare to farm (48.96%) indicating refugia in field region.

The first molecular identification of benzimidazole resistance in Haemonchus contortus from goats in Thailand

Background and Aim: Haemonchus contortus is one of the major trichostrongyloid nematodes affecting small ruminant production worldwide, especially in tropical and subtropical regions. Adult H. contortus suck the blood from the host abomasum leading to anemia and often death in heavily infected animals. The mainstay of parasitic control is an anthelmintic drug, but long-term drug use may cause drug resistance. The aim of this study was to examine benzimidazole resistance in H. contortus of goats from different regions in Thailand by detecting the frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene. Materials and Methods: A total of 121 H. contortus adults were obtained from 31 naturally infected out of 37 slaughtered goats from city abattoirs in five regions of Thailand. The frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene was detected following the allele-specific polymerase chain reaction protocol. Results: The overall genotype frequencies in Thailand were homozygous resistant (RR: 24%), heterozygous (SR: 44.6%), and homozygous susceptible (SS: 31.4%). The allele frequencies were resistant allele (R: 46%) and susceptible allele (S: 54%). The R allele frequency and the RR genotype varied from 30% to 65% and 0% to 43.9%, respectively. The frequency of R alleles was significantly higher in the southern region (0.65) as compared to northern (0.30, p=0.001), western (0.38, p=0.04), and central regions (0.30, p=0.03). The RR genotype was also significantly higher in the southern region (43.9%) versus the northern (0 %, p=0.001), western (11.8%, p=0.012), and central regions (17.4%, p=0.001). Conclusion: This is the first study of the detection of single-nucleotide polymorphisms in codon 200 of the β-tubulin isotype 1 gene of H. contortus from goats in Thailand. These findings are essential and imply that an integrated approach is needed for issues such as drug treatment, farm management, prevention, and control strategies. This is of interest to farmers, veterinarians, and the department of livestock.