Abstract

Farming of wild ruminants in the United States, and particularly in Texas, has been on the increase over the past several years. Little is known of anthelmintic resistance in wild ruminants, with the majority of research being conducted with sheep and goats. The availability of an assay to detect resistant larvae to specific anthelmintics has been limited. DrenchRite®, a larval development assay, tests for resistance against benzimidazoles, levamisole, benzimidazole/levamisole combination, and avermectins/milbemycins, has been produced by CSIRO (Australian Commonwealth Scientific and Industrial Research Organization, McMaster Laboratory) through Horizon Technology in New South Wales, Australia. Using DrenchRite®, anthelmintic resistance has been diagnosed for two herds of wild ruminants, one of elk (Cervus elaphus) and one of Armenian red sheep (Ovis orientalis).

Introduction

In both Texas and Australia, anthelmintic resistance is a major problem, with Haemonchus sp. being the main contributor to this problem in Texas.^2-8,10 Having an anthelmintic resistance screen which evaluates different anthelmintics in one test would be beneficial not only to the veterinarian but to the producer who will have the results in about 1 wk. For positive results, the appropriate measures can be taken to curb resistant worms in the population by decreasing their numbers while improving the health of the herd.

Methods

The protocol that was followed was adapted from the DrenchRite® user manual.¹ Separate fecal samples were taken from the two populations, one rectally (sheep) and the other collected from the ground (elk). A trichostrongyle eggs/g of feces (epg) count was taken for both populations. Since the epg count was above 100, a sufficient amount of eggs were present for evaluation. A fecal slurry was prepared and rinsed through a 210 µm sieve. The filtrate was then washed free-flow through additional sieves of 70 and 37 µm. The eggs were recovered on the 37 µm sieve. A sugar gradient was prepared to remove any extra debris and recover the eggs. After centrifugation the eggs were pipetted from the gradient and recovered onto a 37 µm sieve and rinsed with water to remove any sugar solution. The eggs were recovered, counted and divided according to the manufacturer’s instructions¹ so that each of the 96 wells in the assay plate would contain 40 to 70 eggs. Nystatin (Fungizone) was added to combat any fungus that might have disrupted the parasite’s development.

The DrenchRite® plate contains 12 columns with 96 wells that are color-coded: a clear lane for the control wells, 4 green lanes for susceptible, 3 yellow lanes for weak to intermediate resistance, and 4 red lanes for highly resistant larvae. The 8 rows of drug-containing wells have increasing concentrations from each anthelmintic class. After dispensing the eggs, the plates were incubated for a period of 6.5 days at a range of 25 to 26°C. During this incubation time, a growth medium was added in order to provide nutrients to the developing larvae. Also the plates were checked periodically for loss of hydration within the wells. Larvae that had hatched and developed to the third stage were identified to genus and counted.^9,11

Results

No prior anthelmintic use was documented with this particular herd of elk. The predominant genera present from the elk were Trichostrongylus and Ostertagia. For Ostertagia, there was a 99% efficacy found with the benzimidazoles (Table 1). With levamisole, 4.17% of the population was seen as highly resistant, and 95.83% being moderately or not resistant. A 68% efficacy was found for the moderate/no resistance population while a 32% was found for the highly resistant population. An overall population efficacy was found to be 65.16%. The predominant genera found for this data was Trichostrongylus. For the benzimidazole/levamisole combination, Ostertagia was present along with Trichostrongylus in the control wells, but when the critical well (LD50) was reached, only one or the other species was identified. Ostertagia was the only genera seen with the efficacy being estimated at 100%. The genera of Trichostrongylus was eliminated before reaching the critical well (LD50) so an assumption of 100% efficacy was estimated for this genera with the benzimidazole/levamisole combination (Table 1).

Table 1. Efficacy seen with larvae from elk

SR: suspected resistance; S: susceptible

Trichostrongylus was the only genera that displayed a suspected resistance to the avermectins/milbemycins with Ostertagia having an assumed susceptibility. It should be noted that in the DrenchRite® manual, it states that resistance to the avermectin/milbemycins is rare in the field by comparison with the first broad spectrum anthelmintics (e.g., thiabendazole, levamisole) introduced. DrenchRite® can be used to detect the presence of resistance but not as yet, to quantify efficacy of the avermectins/milbemycins.¹

Anthelmintic treatment history in the Armenian red sheep was known for the last 6 yr. This group of red sheep consisted of eight females and two males that originated from west Texas. The females had been given pyrantel tartrate in the feed and also ivermectin. This regimen was alternated 4 times/yr. The males had also been given pyrantel tartrate in their feed but the last time ivermectin was given was in July 1994.

The predominant genus found from these red sheep was Haemonchus. Forty-six percent efficacy was seen with the benzimidazoles (Table 2). With levamisole, 14.6% of the population was considered highly resistant, while 85.4% were seen as moderately or not resistant. A 90% efficacy was estimated for the moderate/no resistance population while a 10% efficacy was found for the highly resistant population. An overall population efficacy was estimated at 76.9%. A 98% efficacy was seen with the benzimidazole/levamisole combination. For the avermectins/milbemycins, no resistance was suspected (Table 2).

Table 2. Efficacy seen with larvae from red sheep

NSR: no suspected resistance; NP: not present

Discussion

DrenchRite® has been tested mainly with sheep and goats. This research shows that it has application in wild sheep and cervid species. It can likely be applied to all exotic ruminant species because this test is independent of the host species and many exotic hoofstock are medicated with anthelmintics (e.g., zoos, game parks, deer farms, etc.). Note: This is in partial fulfillment of a master’s thesis.

Acknowledgments

Acknowledgments and thanks to the following individuals for their help and assistance with this research: Mr. Jeff Craven, Gabriella Foxworth, DVM, Donald Davis, PhD, and Mr. Rob McCook.

Literature Cited

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