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The distribution and anthelmintic resistance status of Trichostrongylus colubriformis, T. vitrinus and T. axei in lambs in New Zealand a
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TS Waghorn , JS Knight & DM Leathwick
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AgResearch Grasslands, Private Bag 11008, Palmerston North 4442, New Zealand Accepted author version posted online: 09 Dec 2013.Published online: 05 Feb 2014.
To cite this article: TS Waghorn, JS Knight & DM Leathwick (2014) The distribution and anthelmintic resistance status of Trichostrongylus colubriformis, T. vitrinus and T. axei in lambs in New Zealand, New Zealand Veterinary Journal, 62:3, 152-159, DOI: 10.1080/00480169.2013.871193 To link to this article: http://dx.doi.org/10.1080/00480169.2013.871193
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New Zealand Veterinary Journal 62(3), 152–159, 2014
Scientific Article
The distribution and anthelmintic resistance status of Trichostrongylus colubriformis, T. vitrinus and T. axei in lambs in New Zealand TS Waghorn*§, JS Knight* and DM Leathwick*
Abstract
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AIM: To determine the distribution of the three common Trichostrongylus spp. infecting sheep and their resistance status on farms throughout New Zealand, using PCR. METHODS: Cultures were prepared from faecal samples from 70 farms while conducting faecal egg count reduction tests (FECRT) in lambs between 2010 and 2012. Trichostrongylus-type infective stage larvae (L3) were recovered from cultures, derived from untreated control and albendazole-, levamisole- and ivermectintreated groups of lambs on each of the farms involved, and these were identified to species using PCR analysis of the second internal transcribed spacer region of ribosomal DNA. The species composition of the larvae present in cultures from the untreated control groups was examined across all farms to assess any potential differences in geographical distribution. In addition, the species composition of larvae cultured from the untreated and anthelmintic-treated lamb groups were compared to determine which species exhibited resistance to each of the anthelmintics used in the FECRT. RESULTS: Of 67 farms with Trichostrongylus spp. present, 42 (63%) cultures from the untreated control groups contained all three Trichostrongylus spp. and no significant geographical patterns in their distribution were detected. Seven samples contained only one species. Irrespective of the anthelmintic efficacy levels, Trichostrongylus colubriformis dominated cultures prepared from lambs following treatment with albendazole (99.1 (95%CI = 97−100)% of larvae) or levamisole (81.6 (95%CI = 75.3−87.9)% of larvae), indicating the presence of widespread resistance in this species. In cultures prepared from levamisoletreated lambs, small numbers of T. axei larvae were also frequently present (5.4 (95% CI = 1.3−12.4)% of larvae). Resistance to ivermectin was not found in any of the three Trichostrongylus spp. after PCR identification. Although larvae were identified, based on length, as being Trichostrongylus spp., for 24 of the 48 samples cultured following treatment with ivermectin, 100% of the larvae present were identified as Teladorsagia circumcincta. CONCLUSIONS:
As
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previous
surveys,
all
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* AgResearch Grasslands, Private Bag 11008, Palmerston North 4442, New Zealand § Author for correspondence. Email:
[email protected] http://dx.doi.org/10.1080/00480169.2013.871193 © 2014 New Zealand Veterinary Association
Trichostrongylus spp. were common throughout New Zealand and no geographical patterns were detected in the current study. On all farms where resistance to albendazole and/or levamisole was indicated (i.e. efficacy 95%, T. colubriformis still tended to dominate in post-treatment cultures. While this could reflect a lower susceptibility of T. colubriformis to these anthelmintics, it seems more likely to indicate the presence of resistant genotypes in these populations. Similarly, T. axei also tended to be present after treatment with levamisole, which likely reflects a known lower susceptibility of this species to these anthelmintics. KEY WORDS: PCR, Trichostrongylus spp., distribution, anthelmintic resistance, New Zealand
Introduction Nematode parasites are regarded by New Zealand sheep farmers as their most important animal health issue (Lawrence et al. 2007) and the impact of these parasites on productivity can be greatly exacerbated by the presence of anthelmintic resistance (Miller et al. 2012). The ability to manage both parasitic disease and anthelmintic resistance is dependent on an understanding of parasite epidemiology, which in turn requires the accurate identification of the parasite species involved (Coles et al. 2006; Sweeny et al. 2012). To date, identification of nematode parasite species has generally been based on the morphology and morphometry of adult male worms (Soulsby 1971). However for routine diagnostic purposes the cost of sacrificing animals to obtain adult worms cannot normally be justified, and so the most commonly used practice is the microscopic differentiation of infective stage larvae (L3) extracted from faecal cultures (Lancaster and Hong 1987; Van Wyk et al. 2004; Coles et al. 2006). This practice has proven useful in the identification of the worm genera involved in cases of clinical disease and has been particularly useful in the diagnosis of anthelmintic resistance (McKenna 1996, 1997). However for some nematodes differentiation of larval stages has limitations. In particular, the three species of the genus Trichostrongylus commonly infecting sheep in New Zealand, i.e.
L3 FEC FECRT ITS-2
Infective stage larvae Faecal nematode egg count Faecal egg count reduction test (s) Second internal transcribed spacer region
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T. colubriformis, T. axei and T. vitrinus, are problematic in that their larval stages are not easily differentiated. This is especially true when using morphometry as not only are there overlaps in larval and sheath tail lengths between the species (Gordon 1933), but culture conditions can also influence the size of the resulting L3 (Rossanigo and Gruner 1996; McMurtry et al. 2000). In an attempt to counter this problem McMurtry et al. (2000) used the number of tubercles on exsheathed L3 to differentiate between these three species. Although considerably increasing the cost, this technique proved useful except on some occasions when dealing with mixed nematode infections. Because T. axei and Teladorsagia circumcincta L3 both lack tubercles, and there is often an overlap in the lengths of the L3 of these species, they cannot always be differentiated using this method. Because of these issues the diagnosis of infection levels and the presence of anthelmintic resistance has, in general, not differentiated between species for this genus (Waghorn et al. 2006; McKenna 2010). Trichostrongylus spp. are, however, extremely prevalent in nematode infections of sheep in New Zealand (Brunsdon 1960; Vlassoff 1973; Hervé et al. 2003) and they frequently make up a majority of the L3 recovered from faecal cultures (T.S. Waghorn, unpublished data). Also, the genus is often represented amongst those surviving anthelmintic treatment and is therefore classified as being resistant (McKenna 1995, 2010; Waghorn et al. 2006). Given that there are differences in the biology of the three species (Brunsdon 1963; Beveridge et al. 1989; O’Connor et al. 2006), the ability to easily differentiate these species is likely to aid in their management. T. axei is an abomasal worm which is found in many ruminant and other hosts (Soulsby 1971) and is the cause of parasite problems in older cattle (Bisset 1994). T. colubriformis and T. vitrinus are small intestinal worms with preferred hosts of sheep and goats (McKenna 2009), although T. colubriformis can be found in most ruminants (Soulsby 1971). Cross infection between hosts with T. axei and T. colubriformis is common but is not seen with T. vitrinus which is often the dominant species especially in cooler/wetter environments (Beveridge et al. 1989). Recent advances in molecular biology have now made it possible to rapidly identify Trichostrongylus L3 to species using PCR techniques (Bisset et al. 2014). Previously, such procedures involved multiple rounds of PCR and enzymatic digests (Silvestre and Humbert 2000; Bott et al. 2009), however continuing advances have refined the techniques and allowed savings in both time and cost (Roeber et al. 2011). Here we use the latest refinements in the PCR method to demonstrate the utility of a molecular tool that can accurately differentiate between L3 of the three commonly found Trichostrongylus spp. and Te. circumcincta. The aim of the current study was to update our understanding of the distribution of the three Trichostrongylus spp. throughout New Zealand, and to determine which Trichostrongylus spp. exhibited resistance to the different anthelmintic groups using this tool.
Materials and methods Faecal samples from commercial farms throughout New Zealand were collected as part of faecal egg count reduction tests (FECRT) associated with other projects. Between 2010 and 2012 FECRT were completed on 70 farms. These farms were generally
Figure 1. The approximate location of the 70 sheep farms sampled for Trichostrongylus spp. around New Zealand. The North and South Islands show the different geographical regions used in the cluster and correspondence analysis. Stars indicate the locations of the six farms in the most distinctive branch from a cluster analysis dendrogram.
classed as breeding/finishing units running a mixture of sheep and beef cattle, of which there are approximately 3,500 in the South Island and 6,500 in the North Island of New Zealand (Anonymous 2013). All farms were allocated to one of nine geographical regions based on their location; the farm locations and regions are shown in Figure 1. On each of the farms involved, faecal samples were collected from eight to 12 lambs from each of an untreated control group and three anthelmintic-treated groups (albendazole, levamisole and ivermectin). Samples were collected from all animals on the day of treatment and again 7−10 days later. The number of nematode eggs per gram of faeces (faecal nematode egg count; FEC) was assessed using a modified McMaster method where each egg counted equated to 50 epg. Equivalent amounts (approximately 5 grams) of additional faeces from each individual sample were pooled within each treatment group to create faecal cultures for each farm. The pooled cultures were mixed with vermiculite and water to achieve a moist but not wet consistency, and then incubated at 22°C for 14 days before undergoing baermannisation to extract the L3. In each case 100 randomly selected L3 were identified to genus on the basis of the morphological characteristics of larval and sheath tail lengths, as well as assessment of head shape to differentiate Teladorsagia from Trichostrongylus genera (Soulsby 1971; Lancaster and Hong 1987). Treatment efficacies were calculated as described by Waghorn et al. (2006) using the equation below.
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1−
mean epg post-treatment × (n treatment larvae/100) mean epg pre-control × × 100 mean epg pre-treatment × (n control larvae/100) mean epg post-control
The proportion of visually identified L3 cultured from each sample was used to apportion egg counts to the dominant nematode genera (McKenna 1996). Anthelmintic resistance was considered to be present when the calculated efficacy for the genus was