Jane E. Hodgkinson

The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution

BackgroundThe liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually.ResultsHere we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs.ConclusionsThe genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions

Graphical abstract

Anthelmintic efficacy against cyathostomins in horses in Southern England.

Cyathostomins are considered to be the most important group of helminths to affect equids due to their high prevalence, potential pathogenicity and ability to develop anthelmintic resistance. Their control relies almost exclusively on frequent anthelmintic use. Currently, fenbendazole (FBZ), pyrantel embonate (PYR), ivermectin (IVM) and moxidectin (MOX) are licensed for use in horses in the UK. With no new anthelmintics likely to be licensed in the near future, it is essential that investigations into the efficacy of current anthelmintics in different locations are performed to help inform control programmes. Here, efficacy of FBZ, PYR, IVM and MOX in horse populations in the South of England was investigated. Horses with a strongyle faecal egg count (FEC) of ≥50 eggs per gram (EPG) were enrolled onto a faecal egg count reduction test (FECRT) study. Efficacy was determined by calculating the percentage reduction in FEC between the group mean at Day 0 and 14 days post-treatment. Efficacy was indicated when a group arithmetic faecal egg count reduction (FECR) of ≥90% was recorded for FBZ and PYR, and ≥95% for IVM and MOX. Between March and December 2012, 404 FECRT were performed on 12 yards examining 101, 110, 93 and 100 equids for FBZ, PYR, IVM, and MOX, respectively. FBZ resistance was identified on all yards (mean FECR range 0-65.8%). On 10 of 12 yards, PYR efficacy was >90% (91.0-99.4%) and on two yards, PYR resistance was suspected (86.8-87.2%). IVM (96.4-100%) and MOX (99.9-100%) were >95% efficacious on all yards. As the prevalence of FBZ resistance was 100%, the future use of this anthelmintic for the control of strongyles should be questioned. PYR should be used strategically to reduce reliance on the macrocyclic lactone class products. Over-dispersion of FEC between horses was observed (average k=0.21) with 80% of the strongyle eggs counted measured in 15% of horses tested, strongly supporting the application of targeted helminth control programmes in this host species.

Helminth egg excretion with regard to age, gender and management practices on UK Thoroughbred studs

Few studies have described the combined effect of age, gender, management and control programmes on helminth prevalence and egg shedding in grazing equines. Here, fecal samples collected from 1221 Thoroughbred horses, residing at 22 studs in the UK, were analysed. The distribution of strongyle eggs amongst individuals in relation to age, gender and management practices was investigated. Fecal worm egg counts (FWECs), described as the number of eggs per gramme (epg) of feces, were determined using a modification of the salt flotation method. The FWEC prevalence (mean%) of strongyles, Parascaris equorum, tapeworm spp. and Strongyloides westeri was 56, 9, 4 and 8%, respectively. Strongyle, P. equorum, tapeworm spp. and S. westeri infections were detected on 22 (100%), 11 (50%), 9 (41%) and 8 (36%) of studs, respectively. Within all age and gender categories, strongyle FWECs were highly over-dispersed (arithmetic mean = 95 epg, aggregation parameter k=0·111) amongst horses. Animal age, last anthelmintic type administered and management practices (for example, group rotation on grazing) most strongly influenced strongyle prevalence and level of egg shedding (P < 0·05). Overall, 11% of equines (range: 234-2565 epg) were responsible for excreting 80% of the strongyle eggs detected on FWEC analysis. The results confirm that the judicious application of targeted treatments has potential to control equine strongyle populations by protecting individual horses from high burdens, whilst promoting refugia for anthelmintic susceptible genotypes.

Anthelmintic efficacy on UK Thoroughbred stud farms.

Anthelmintic drugs have been applied indiscriminately to control horse nematodes for over 40 years. We undertook a comprehensive study to investigate efficacy of the four available broad-spectrum anthelmintic drugs on 16 Thoroughbred stud farms using the faecal egg count reduction test. Efficacy against strongyles was determined by calculating the percentage of reduction in faecal egg count between the group mean at Day 0 and Days 14-17 post-treatment and the 95% lower confidence intervals estimated by non-parametric bootstrapping. Individual strongyle faecal egg count reduction tests (n=429) were performed in which 179, 131, 89 and 30 horses were administered ivermectin, moxidectin, pyrantel and fenbendazole, respectively. Moxidectin was efficacious in all tests (faecal egg count reduction range: 99.8-100%; 95% lower confidence intervals range: 96.8-100%) and reduced efficacy of ivermectin (faecal egg count reduction range: 85.7-100%; 95% lower confidence intervals range: 65-100%) was observed in one group of yearlings. Reduced pyrantel efficacy was observed in five groups of yearlings (faecal egg count reduction range: 0-73%; 95% lower confidence intervals range: 0-59.5%), but pyrantel was found to be efficacious when administered to mares (faecal egg count reduction range: 98-99.4%; 95% lower confidence intervals range: 91.8-99.3%). Low efficacy of fenbendazole was always observed (faecal egg count reduction range: 0.4-41%; 95% lower confidence intervals not calculable). Two further methods for estimating efficacy were applied and outputs obtained using all methodologies were in agreement. Efficacy against Parascaris equorum was assessed on four farms: fenbendazole had acceptable efficacy (faecal egg count reduction range: 97.5-99.9%; 95% lower confidence intervals range: 96.3-99.1%), but reduced efficacy of ivermectin was observed (faecal egg count reduction range: 25.5-91.2%; 95% lower confidence intervals range: 6.7-82.4%). Strongyle faecal egg count were analysed at approximately 2 week intervals for up to 12 weeks after anthelmintic drug administration to determine the egg reappearance period for moxidectin, ivermectin and pyrantel. The egg reappearance period for all three anthelmintic drugs was shorter than previously observed. Overall, our results indicate that ivermectin and moxidectin administration provided acceptable efficacy at 14 days; however, egg reappearance period results suggest that these products are working less effectively than measured previously. As shortened egg reappearance period is believed to be an early indicator of resistance, this highlights the issue of impending multi-drug resistance in strongyles on stud farms.

A PCR–ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis

We report the use of six oligoprobes designed from intergenic spacer region sequences to identify fourth-stage larvae (L4) of the tribe Cyathostominae. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed as a positive control to identify all these members of the Cyathostominae. The intergenic spacer region was amplified by PCR using conserved primers. Initially, three oligoprobes were used in Southern blot analysis. To facilitate high-throughput identification, these and a further four oligoprobes were developed for use in a PCR-ELISA. All probes were validated for their ability to detect cyathostomin PCR products in the PCR-ELISA, using DNA from morphologically identified adult parasites. Initially, 712 L4 were isolated from the diarrhoeic faeces from horses (n=17) with clinical larval cyathostominosis. PCR products from 522 of these L4 were subjected to analysis, with 413 L4 being identified as one of the aforementioned species. With reference to individual species analysis, 28.5% of the 522 L4 were identified as C. longibursatus, 25.7% as C. nassatus, 15.9% as C. ashworthi, 7.3% as C. goldi and 1.7% as C. catinatum. No L4 were identified as being C. insigne species. When L4 within faeces from individual horses were compared, no sample was found to comprise parasites of one species. The least number of species identified in a single sample was two. This study suggests that clinical larval cyathostominosis is predominantly caused by mixed-species infections.

PCR-based differentiation of Fasciola species (Trematoda: Fasciolidae), using primers based on RAPD-derived sequences

Abstract The zoonotic liver flukes Fasciola hepatica and F. gigantica co-exist in parts of Africa and Asia. The two species have similar life-cycles but different transmission characteristics. Although the identification of adult Fasciola to species level is traditionally based on differences in size and shape, recent studies have demonstrated this method to be unreliable. Species of Fasciola can be distinguished by staining and comparing the morpho-anatomy of the gut and ovaries or by iso-enzyme analysis but such approaches are time-consuming and require specialist skills. Two primer sets, based on RAPD-derived sequences from English F. hepatica and Ghanaian F. gigantica, can now be used, in two separate PCR, to distinguish F. hepatica from F. gigantica. When the PCR were used to investigate 10 flukes (five from the U.K. and five from Peru) morpho-anatomically identified as F. hepatica and 10 (five from Ghana and five from Sudan) morpho-anatomically identified as F. gigantica, all 20 flukes were correctly identified to species level. The PCR were validated using 175 flukes collected, over a 12-year period, from different countries and both cattle and sheep.

Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses

Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-specific. The fifth probe recognised all 16 cyathostomin species but did not bind to members of the genus Strongylus. Furthermore, these probes were used to identify individual infective L3, eggs and L4 indicating that they will be invaluable to furthering the study of the epidemiology and pathogenesis of these important equine nematodes.

Identification of putative markers of triclabendazole resistance by a genome-wide analysis of genetically recombinant Fasciola hepatica

Despite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

Evolution of acaricide resistance: phenotypic and genotypic changes in field populations of Rhipicephalus (Boophilus) microplus in response to pyrethroid selection pressure.

There have been few, if any, studies of arthropod field populations quantifying the kinetics of evolution of phenotypic and genotypic resistance to chemicals in response to the presence or absence of selection pressure. A prospective intervention study was undertaken over 2 years in Mexico to measure changes in resistance phenotype and genotype in the presence or absence of pyrethroid selection pressure on field populations of Rhipicephalus (Boophilus) microplus ticks on 11 farms with varying degrees of pyrethroid resistance. The resistance phenotype was evaluated by bioassay in a larval packet test expressed as the resistance factor (RF) derived from probit analysis of dose mortality regressions, and resistance genotype by an allele-specific PCR (AS-PCR) to determine the frequency of a sodium channel mutation (F1550I) associated with pyrethroid resistance. To validate the AS-PCR, a Pyrosequencing™ method was developed to detect the F1550I mutation. There was good concordance with the genotypes identified by both Pyrosequencing™ and AS-PCR (Kappa: 0.85). On five farms cypermethrin (CY) was exclusively used at intervals and on six farms amitraz was used. On two of the five CY-treated farms, the experiment was prematurely terminated due to unacceptably high levels of tick resistance. For all five farms, after 8-24 months of continued selection pressure with CY, the RF had increased 2-125-fold. The frequency of the resistance allele increased on all five farms from a starting range of 5-46% to a range of 66-95% after 8-24 months. On six farms treated with amitraz neither the RF nor the frequency of the resistance allele changed. A clear correlation between the phenotype and genotype was found in three of four treated farms confirming that the F1550I mutation is a major cause of synthetic pyrethroid resistance in Mexico. These results show that the pyrethroid resistance trait is stable (> 2 years) and that resistance is acquired much faster than it is lost. Hence, alternation of pyrethroid acaricide with other chemicals is likely to lead to the stepwise acquisition of synthetic pyrethroid resistance but not additional prolongation of its efficacious lifespan.