Abundant variation in microsatellites of the parasitic nematode Trichostrongylus tenuis and linkage to a tandem repeat

An understanding of how genes move between and within populations of parasitic nematodes is important in combating the evolution and spread of anthelmintic resistance. Much has been learned by studying mitochondrial DNA markers, but autosomal markers such as microsatellites have been applied to only a few nematode species, despite their many advantages for studying gene flow in eukaryotes. Here, we describe the isolation of 307 microsatellites from Trichostrongylus tenuis, an intestinal nematode of red grouse. High levels of variation were revealed at sixteen microsatellite loci (including three sex-lined loci) in 111 male T. tenuis nematodes collected from four hosts at a single grouse estate in Scotland (average He = 0.708; mean number of alleles = 12.2). A population genetic analysis detected no deviation from panmixia either between (F(ST) = 0.00) or within hosts (F(IS) = 0.015). We discuss the feasibility of developing microsatellites in parasitic nematodes and the problem of null alleles. We also describe a novel 146-bp repeat element, TteREP1, which is linked to two-thirds of the microsatellites sequenced and is associated with marker development failure. The sequence of TteREP1 is related to the TcREP-class of repeats found in several other trichostrongyloid species including Trichostrongylus colubriformis and Haemonchus contortus

Profiling in wildlife crime : recovery of human DNA deposited outside

Incidents of bird of prey persecution receive a lot of media coverage in the UK, with investigations rarely recovering sufficient evidence to proceed to prosecution. One of the main challenges is to identify a suspect, as these offences are carried out in remote locations without witnesses, and crime scenes may not be found for days. However, traps, poisoned baits and bird of prey carcasses can be recovered from these crime scenes. This study aimed to determine whether reportable human DNA profiles could be recovered from any of these substrates after periods of time outside. Experiments depositing human touch DNA on duplicate substrates (traps, rabbit baits and corvid carcasses) set for 0, 1, 2, 4, 7 and 10 days outside were carried out, with DNA recovery and profiling following standard operating procedures for Scottish Police Authority Forensic Services. Weather conditions varied among experiments, including some heavy rainfall. Results demonstrated that it was possible to obtain reportable DNA profiles from all substrates after at least 1 day outside. Most promisingly, the traps showed no drop-off in DNA persistence over the experiments as complete DNA profiles were obtained after the full 10 days outside. A further experiment using 4 bird of prey carcasses confirmed that it is possible to obtain reportable human DNA profiles from them after 1 day outside (n = 2 reportable profiles). These results show that touch DNA can persist in an outdoor environment, and provide a tantalising avenue for inquiry in bird of prey persecution investigations

Validation of presumptive tests for non-human blood using Kastle Meyer and Hemastix reagents

Kastle Meyer and Hemastix reagents are presumptive tests commonly used in forensic casework for the detection of blood, and their suitability has been reviewed in numerous publications. However, studies to date have focused on the validation of these tests on human blood alone, and no published work has looked at the sensitivity, specificity and effect on DNA analysis when using these reagents to presumptively test for animal blood. The aim of this study was to validate the two reagents for use with animal blood, and compare their performance in order to choose the best test based on the circumstances in wildlife crime investigation. The sensitivity, specificity, stability and robustness of the methods were assessed by experiments with dilutions of animal blood (from 1:4 to 1:65536) using direct and indirect (rub) tests, potential interfering substances, blood sources from different species and aged blood. The effects of the two reagents on subsequent DNA analysis were also investigated. During the direct tests, Kastle Meyer showed a higher sensitivity, detecting blood down to a dilution of 1:16,384, one order of magnitude lower than Hemastix. However during the rub test, Hemastix showed a higher sensitivity, detecting blood down to a dilution of 1:64 on porous materials while Kastle Meyer was positive only down to a dilution of 1:16. Moreover, when using the same swab for presumptive testing and DNA extraction, Hemastix testing allowed amplification of a sufficient amount of DNA for species identification at its limit of sensitivity on porous materials (1:64) while Kastle Meyer inhibited most amplification of DNA at its less sensitive limit of 1:16 dilution. On the other hand, Hemastix showed a much lower specificity, producing false positive results when exposed to tomato, potato, rust, avian uric acid, bleach and sink rot, while Kastle Meyer only produced a faint positive reaction from potato. Both tests performed equally well detecting fresh blood of different animal species. The stability test gave comparable results among the tests except for aged fish blood stains, where the Kastle Meyer test performed poorly. Owing to its ease of use, higher sensitivity, and lack of interference with downstream DNA analysis, and despite its reduced specificity compared to Kastle Meyer, the Hemastix method is more appropriate for use in wildlife crime investigations. Positive results would always be confirmed with DNA analysis and the low interference of the reagent will allow the use of a single swab for presumptive testing and DNA sampling

Cross-species utility of microsatellite markers in trichostrongyloid nematodes

The development of microsatellite markers for parasitic nematodes has been hampered by technical difficulties in isolation and PCR amplification. We have investigated the potential for circumventing these problems using microsatellites from 3 trichostrongyloid species on a panel of 7 species. Ten of the 22 PCR primer pairs tested amplified in species other than the target species, usually in closely related species, and 2 new variable loci were discovered in the sheep parasite <i>Trichostrongylus vitrinus</i>. This study provides evidence that cross-species testing of microsatellite primers can be an effective alternative to isolation de novo

Macrogeographic population structure in a parasitic nematode with avian hosts

Much remains to be discovered about the population genetic structure of parasites, despite the importance of such knowledge to understanding the processes involved in the spread of drug resistance through populations. Here we present a study of population genetic diversity in <i>Trichostrongylus</i> tenuis, an avian parasitic nematode infecting both poultry and game birds, where anthelmintic use is common. We examined diversity of nicotinamide dehydrogenase subunit 4 (<i>nad4</i>) mtDNA sequences within and between seven locations: five in the UK (red grouse hosts), one in Iceland (domestic goose) and one in Norway (willow grouse). Within-UK comparisons showed high nucleotide diversity (π = 0.015, n = 23) but no structure between locations (ΦST = 0.022, P = 0.27), with over 97% of variation distributed within-hosts. The highest diversity was found in Iceland (π = 0.043, n = 4), and the lowest in Norway (π = 0.003, n = 4). Differentiation between countries was considerable (ΦCT = 0.44, P < 0.05), in spite of the potential mixing effects of gene flow via migrating wild hosts and the poultry trade. However, significant pairwise FST values were found only between Norway and the other locations. Phylogenetic analysis provided statistical support for a separate clade for Norwegian samples only, with unresolved diversity leading to a star-shaped relationship between Icelandic and UK haplotypes. These results suggest that Norwegian T. tenuis are isolated, but that there is some connectivity between UK and Icelandic populations. Although anthelmintic resistance has not yet been reported for <i>T. tenuis</i>, the population structure is such that emerging resistance has the potential to spread by gene flow over a large geographic scale