Biology subjects

Jimenez Castro, P. D.

Publications and source records attributed to Jimenez Castro, P. D..

4 recordsLinked to original sources

bioRxiv2024

Investigation of risk factors associated with Ancylostoma spp. infection and the benzimidazole F167Y resistance marker polymorphism in dogs from the United States

Ancylostoma caninum is the most significant intestinal nematode parasite of dogs. We acquired fecal surveillance data using molecular diagnostics in a large population of dogs in the United States (US). A diagnostic test using real-time PCR (qPCR) for Ancylostoma spp. and allele-specific qPCR detecting the SNP F167Y was used in 885,424 canine fecal samples collected between March 2022 and December 2023. Overall, Ancylostoma spp. had a prevalence of 1.76% (15,537/885,424), with the highest observed in the South 3.73% (10,747/287,576), and the lowest in the West 0.45% (632/140,282). Within the subset of Ancylostoma spp.-detected dogs used for further analysis, the F167Y SNP had an overall prevalence of 14.2% with the highest in the West and the lowest in the Midwest (10.76%). The greyhound breed exhibited a higher prevalence of Ancylostoma spp. infections (17.03%) and a higher prevalence of the F167Y polymorphism (33.6%) compared to non-greyhound breeds (13.7% and 2.08%), respectively, but were not associated with the highest breed risk for the F167Y polymorphism. Sex did not influence hookworm infection nor F167Y polymorphism prevalence. Intact dogs had a prevalence of hookworm infection and F167Y polymorphism of 2.51% and 14.6%, respectively. Puppies showed increased prevalence of hookworms (3.70%) and the F167Y SNP (17.1%). Greyhounds, bluetick coonhounds, and boerboels had the highest relative risks (RR) for hookworm infection, while Cavalier King Charles spaniels, Havanese, and shiba inus had the lowest. The top and bottom three with the highest and lowest RR for the F167Y SNP were the old English sheepdog, American foxhound, and toy poodle Toy, and shih tzu, Maltese, and Australian cattle dogs, respectively. This study highlights the value of an accessible diagnostic qPCR test with fast turnaround times in unraveling the molecular epidemiology of hookworms and benzimidazole resistance, as well as explore potentially important risk factors associated with infection in medicalized dogs. Highlights- Greyhounds had the highest RR relative risk for Ancylostoma spp., but only fourth for the A. caninum F167Y polymorphism. - The highest prevalence of Ancylostoma spp. in the United States was in the South. - The West had the lowest prevalence for Ancylostoma spp., but the highest prevalence for the A. caninum F167Y SNP. - Puppies had the highest prevalence and AOR for Ancylostoma spp. and F167Y polymorphism.

molecular biology↗

bioRxiv2022

Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel isotype-1 β-tubulin benzimidazole resistance mutation.

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhounds in the USA are infected with A. caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A. caninum in greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 {beta}-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A. caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 {beta}-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistant A. caninum isolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans {beta}-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A. caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, often at high frequency, with prevalences of 49.7% (overall frequency 54.0%) and 31.1% (overall frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms. Author SummaryAlthough increasingly common in livestock, no reports of widespread anthelmintic resistance are confirmed in any companion animal or human gastrointestinal nematode parasite to date. The canine hookworm is a common intestinal zoonotic parasite of dogs with severe clinical impacts in young dogs, and for which control is dependent on regular anthelmintic use. We recently reported multiple anthelmintic drug resistance in A. caninum isolates from greyhounds derived from multiple locations in the USA likely caused by long standing intensive treatment regimens in kennels. In this study, we investigated benzimidazole resistance in A. caninum in pet dogs across the USA. We also identified and showed the functional significance of a novel benzimidazole isotype-1 {beta}-tubulin resistance mutation in A. caninum from greyhounds that has not been previously reported in the field for any organism. We then determined that this novel mutation, as well as a previously characterized resistance mutation, were present, often at high frequency, in many A. caninum populations across the USA. This study reports the first evidence of widespread drug resistance for any parasitic nematode of companion animals and illustrates the power of molecular approaches to rapidly assess anthelmintic resistance in a region.

genomics↗

bioRxiv2021

Multiple drug resistance in hookworms infecting greyhound dogs in the USA

The hookworm Ancylostoma caninum is the most prevalent nematode parasite of dogs. Recently, we confirmed multiple-drug resistance (MDR) in several A. caninum isolates to all anthelmintic drug classes approved for the treatment of hookworms in dogs in the United States (USA). Cases of MDR hookworms appear to be highly overrepresented in greyhounds, suggesting that the MDR worms evolved on racing greyhound farms/kennels. The aims of this study were to evaluate the range of drug-resistant phenotypes and genotypes of the A. caninum infecting greyhounds. Fecal samples from recently retired greyhounds originating from geographically diverse areas of the USA were acquired from two greyhound adoption kennels, one active greyhound racing kennel, and three veterinary practices that work with adoption kennels. Fecal egg counts (FECs) were performed on fecal samples from 219 greyhounds, and despite almost all the dogs having been treated with one or more anthelmintics in the previous two to four weeks, the mean FEC was 822.4 eggs per gram (EPG). Resistance to benzimidazoles and macrocyclic lactones were measured using the egg hatch assay (EHA) and the larval development assay (LDA) respectively. We performed 23 EHA and 22 LDA on either individual or pooled feces, representing 81 animals. Mean and median IC50 and IC95 values for the EHA were 5.3 uM, 3.6 uM, and 24.5 uM, 23.4 uM respectively. For the LDA, mean and median IC50 values were 749.8 nM, >1000 nM respectively. These values range from 62 to 68 times higher than those we measured in our susceptible laboratory isolates. Pre-treatment fecal samples could not be obtained, however, post-treatment samples representing 219 greyhounds were collected. For samples collected <10 days post-treatment with albendazole, moxidectin, or a combination of febantel-pyrantel-moxidectin, the mean FEC were 349, 333, and 835 EPG, respectively. Samples collected 10-21 days post-treatment with albendazole, moxidectin, or pyrantel, yielded mean FEC of 1874, 335, and 600 EPG, respectively. Samples collected >21 days post-treatment with albendazole or moxidectin yielded mean FEC of 1819 and 1117 EPG, respectively. We obtained DNA from hookworm eggs isolated from 70 fecal samples, comprised of 60 individual dogs and 10 pools from multiple dogs. Deep sequencing of the isotype 1 {beta}-tubulin gene revealed the presence of the F167Y (TTC>TAC) resistance polymorphism in 99% of these samples, with 69% having [≥]75% resistant allele frequency. No resistance-associated polymorphisms were seen at any of the other {beta}-tubulin codons previously reported as associated with benzimidazole resistance in Strongylid nematodes. These clinical, in vitro, and genetic data provide strong evidence that racing and recently retired greyhound dogs in the USA are infected with MDR A. caninum at very high levels in terms of both prevalence and infection intensity.

molecular biology↗

bioRxiv2019

Evaluation of DNA extraction methods on individual helminth egg and larval stages for whole genome sequencing

Whole genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative, however, these samples are typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole genome sequencing of individual immature helminth samples. These approaches do not use whole genome amplification, a common but costly approach to increase the yield of low input samples. We first tested individual parasites from two species spotted onto FTA cards - egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni - before further testing on an additional six species - Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Dracunculus medinensis, Strongyloides stercoralis, and Trichuris muris - with an optimal protocol. Whole genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation between species, and within species but between life stages, described. These results demonstrate the feasibility of whole genome sequencing of individual parasites, and highlight a new avenue towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

genomics↗