Research paperAn automated, multiplex-tandem PCR platform for the diagnosis of gastrointestinal nematode infections in cattle: An Australian-European validation study
Introduction
A rapidly increasing human population, combined with a warming climate and changes in the temporal and geographical distribution of major animal pathogens, represent major challenges for efficient and sustainable agricultural and livestock production. Underpinning the response to these challenges is the need for increasingly sensitive and rapid diagnostic methods that can detect subtle changes in the epidemiology of common livestock diseases (van Dijk et al., 2008, Skuce et al., 2013, Moustafa, 2015).
Gastrointestinal (GI) nematode infections (Order Strongylida) are common and widespread in domestic livestock, including sheep, goats and cattle, and are of major socioeconomic importance in both developing and developed economies (Charlier et al., 2015). In the developed world, infections with GI nematodes are associated predominantly with lost production, such as decreased weight gain, reduced feed consumption, impaired reproduction and ill-thrift (Hawkins, 1993, Perry and Randolph, 1999). Together, these can significantly reduce the productivity and profitability of farming enterprises (Charlier et al., 2014). Infections with GI nematodes are routinely treated with broad-spectrum anthelmintics, which until recently were derived from only three chemical classes (benzimidazole, levamisole and macrocyclic lactone) (Besier and Love, 2003). Two more classes have recently become available for use in sheep, monepantel (an amino-acetonitrile derivative) and derquantel (paraherquamide), but these are not yet marketed for use in cattle (Kaminsky et al., 2008). Resistance to anthelmintics in GI nematodes of cattle is not yet as severe, nor as prevalent, as it is in small ruminants (Kaplan, 2004, Kaplan and Vidyashankar, 2012), although reports are becoming increasingly common (Kaplan, 2004, Rendell, 2010, Felippelli et al., 2014, Geurden et al., 2015). The development of new anthelmintics is expensive, and can take decades, whereas resistance can develop more rapidly, often within a few years of the release of a new drug (Kaplan, 2004). Clearly there is a need to preserve the effectiveness of existing anthelmintics, and this can be supported by more frequent monitoring of worm burdens and adoption of management practices that delay the development of resistance (Besier et al., 2010, Larsen, 2014, Leathwick, 2014).
The routine diagnosis of GI nematode infections is traditionally based on counting their eggs in a microscopic chamber using a faecal flotation method (‘worm egg counts’, MAFF, 1986). However, this provides little information on the infecting species because, apart from Nematodirus spp., the eggs of most important GI nematodes are morphologically indistinguishable unless viewed individually at high magnification (Georgi and McCulloch, 1989). This is labour-intensive and expensive and potentially unreliable, since there is substantial overlap between the size of eggs from different species. Consequently, larval culture (LC) is needed to identify which genera of nematodes are predominant in any given sample. This requires incubation of faecal samples for 7–10 days to hatch infective third-stage larvae (L3), which are then identified using published information on size and morphology of the important nematode genera and species (Levine, 1968, van Wyk et al., 2004). This approach is both laborious and potentially inaccurate, as there is considerable overlap in the measurements of these genera (Roeber and Kahn, 2014). In addition, the temperature at which faeces are incubated can markedly affect the relative abundance of the infective larvae harvested and counted, and so the results of LC may not reflect the original population of nematode eggs that would ultimately go onto pasture (Berrie et al., 1988, Dobson et al., 1992, Roeber and Kahn, 2014). Another limitation of traditional diagnostic methods is that in cattle, worm egg counts are generally lower compared to small ruminants, and so flotation methods that are suitable for sheep and goats are not sensitive enough to accurately monitor worm burdens in adult cattle. This has implications for both the routine monitoring of worm egg counts and testing for anthelmintic resistance using the faecal egg count reduction test (Mes et al., 2001, Levecke et al., 2012).
A limited number of PCR or RT-PCR assays have been developed for the diagnosis of GI nematode infections in cattle, but these are either limited in the number of species or genera they detect, or involve time-consuming, manual reactions and visualisation of PCR amplicons during electrophoresis, increasing the potential for cross-contamination between samples. In addition, these do not allow a quantitative or even semi-quantitative estimate of the proportion of the genera or species present (Zarlenga et al., 2001, Höglund et al., 2013). More recently, a deep amplicon sequencing approach has been described that shows very promising results in quantifying the composition of gastrointestinal nematode communities in cattle (Avramenko et al., 2015). However, the high technical requirements of this approach might limit its usefulness to research applications but are less suitable for the routine diagnosis of GI nematode infections in veterinary service laboratories.
Recently, the use of multiplexed-tandem PCR (‘MT-PCR’) (Stanley and Szewczuk, 2005) has been described for the specific diagnosis of GI nematode infections in small ruminants (Roeber et al., 2012). MT-PCR consists of two amplification phases: (i) a primary ‘target enrichment’ phase, through a small number of PCR cycles, using multiplexed primer sets, and (ii) a subsequent analytical amplification phase (using a diluted product from the primary amplification as a template), consisting of the targeted amplification, in tandem rather than by multiplex, of each genetic locus using specific, nested primers. Using this method, the initial amplification phase is limited to 10–15 cycles and so interactions between or among multiplexed primer sets are minimized. This reduces competition or the generation of artefactual products, hence limiting amplification bias which would otherwise prevent downstream quantification (Stanley and Szewczuk, 2005). The primary amplicons are diluted prior to their use as templates in the secondary phase, and so primer carry-over and PCR inhibition are also reduced. Conducting the secondary (analytical) amplification phase in tandem means that this method can be coupled to a single-channel RT-PCR thermocycler, allowing rapid screening of multiple samples in parallel and quantification employing one fluorogenic dye (e.g., SYTO-9) at a reduced cost.
In the present paper we adapt techniques previously developed for small ruminants to the diagnosis of key species and genera of GI nematodes in cattle. The aim was to critically evaluate the performance of this new test using known positive control genomic DNA samples from target nematode species, and compare it with the current routine diagnostic tests, namely faecal egg counts and LC. Further, to examine the utility of MT-PCR as a routine diagnostic test for GI nematode infections in cattle, and its suitability for screening bovine faecal samples in different regions, we deployed this test in three different laboratories in Australia, Belgium and Scotland. Each laboratory analysed faecal samples from naturally infected cattle in their respective region, comparing the results of MT-PCR with their routine diagnostic methods.
Section snippets
Faecal sample procurement and worm egg counts
Three laboratories participated in this study. Collection of faecal samples from cattle and subsequent microscopic and molecular testing were carried out by the Mackinnon group at the University of Melbourne (Werribee, Australia), the Laboratory of Parasitology at Ghent University (Merelbeke, Belgium) and Moredun Research Institute (Edinburgh, Scotland). In Australia, between November 2014 and January 2016 a total of 144 fresh faecal samples were collected from cattle from different properties
Assay performance—analytical sensitivity, specificity, repeatability and reproducibility
Positive MT-PCR results were obtained from every sample and each replicate containing 5–1000 H. placei eggs for assays of Haemonchus spp. and pan-nematode (Table 1), and there was no inhibition evident in any of the samples as indicated by the amplification of the spike control. Amplification in the pan-nematode assay was more efficient resulting in higher gene copy numbers recorded for every sample and replicate. An almost perfect linear correlation between egg numbers and observed MT-PCR gene
Discussion
This study demonstrated the MT-PCR platform, together with the developed cattle parasites kits, to be an advanced method for the specific diagnosis of GI nematode infections in cattle. The testing of samples spiked with known quantities of monospecific H. placei eggs showed sensitive and linear amplification of samples ranging between 0.125 eggs to a 1000 eggs. The Haemonchus spp. assay was slightly less sensitive in the range of 0.125–5 eggs, which resulted in some of the replicates not being
Conclusion
In conclusion, MT-PCR together with the developed cattle parasites kits has been demonstrated to be an effective and advanced tool for the specific diagnosis of GI nematode infections in cattle. MT-PCR was far superior to the traditional LC, both in terms of sensitivity and specificity as well as the time, labour and expertise it requires. The assay panels used performed well when tested on a diverse range of samples from different countries and/or regions, and also with different parasite
Conflict of interest statement
All authors declare that there is no conflict of interest. FR is an employee of AusDiagnostics and DH is an employee of Merial.
Acknowledgments
This study was funded by Merial Animal Health. The authors would like to acknowledge the financial support of the Scottish Government’s Rural and Environment Science and Analytical Services (RESAS) to PS & AAM, Moredun Research Institute. SMF is supported by an Australian Research Council Discovery Early Career Researcher Award. Aspects of this research were conducted on the Victorian Life Sciences Computation Initiative’s Peak Computing Facility at the University of Melbourne, an initiative of
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