By Melissa George, BS, MS

BVSc Student at The University of Queensland, PhD Candidate at The University of Georgia
Research Assistant at Bovine Dynamics Pty Ltd.
Email: melissa@bovinedynamics.com.au, Mobile: 0448 770 900, Address: PO Box 740, Kenmore, QLD 4069

January 2017

 

The challenge of drench resistance is a frustrating and constant issue for sheep producers in many parts of the world. While drench treatments are an important aspect of internal parasite control, Integrated Parasite Management programs (http://www.wormboss.com.au/tests-tools/management-tools.php) which combine non-chemical and chemical strategies are key to sustainable control of internal parasites.

Although drench resistance is a severe problem today, few long-time small ruminant producers may remember an era prior to the development of drench resistance—a time when virtually 100% efficacy was status quo. For example, ivermectin was introduced to Australia in 1988 and a national survey conducted from 1991 to 1992 found no evidence of ivermectin resistance (Overend et al., 1994). Ivermectin was considered the magic bullet in worm control in this period. However, resistance eventually developed and a national survey conducted from 2009 to 2012 identified ivermectin resistance on 87% of properties tested (Playford, 2013).

Worm Replacement—Background

What if you could go back in time before the development of drench resistance and implement sustainable parasite control strategies to help slow the development of resistance? Would you try a new strategy of worm control to reclaim your drenches and have effective chemicals to control internal parasites?

A study conducted in Dr. Ray Kaplan’s laboratory at The University of Georgia, USA asked the question: Can we replace resistant worms with susceptible worms?

The technique of worm replacement was previously tested in five studies across the world with several different replacement strategies and many seemed promising with successful improvements in susceptibility (Bird et al., 2001; Moussavou-Boussougou et al., 2007; Sissay et al., 2006; Wyk and Schalkwyk, 1990).  However, all prior studies evaluated worm replacement in the short term—a year or less from the intervention, which only represents one or two grazing seasons. The study conducted at The University of Georgia was the first to evaluate the strategy long-term, monitoring resistance in the population of parasites for 3.5 years following replacement.

Haemonchus contortus in the southeast United States—an evil worm

Haemonchus contortus or Barber’s pole worm, is a highly pathogenic, blood-feeding gastrointestinal parasite that can cause anaemia and death (Fig. 1). This is the primary internal parasite of concern in small ruminants in the southeast United States as it thrives in subtropical climates. Commonly referred to as ‘the evil worm’, H. contortus has a remarkably high propensity to develop drench resistance due to its large effective population size and high mutation rate (Gilleard, 2013). In the southeast US, drench resistance to multiple classes of drenches is a widespread problem, providing a significant challenge to effective parasite control (Howell et al., 2008). Novel approaches to internal parasite control such as ‘worm replacement’ are desperately needed.

 

Methods—how does one replace resistant worms with susceptible worms?

In spring of 2011, the University of Georgia’s sheep flock was experiencing severe anaemia and weight loss due to infection with a multiple-resistant population of H. contortus. This study aimed to intervene in this situation through replacement with a susceptible laboratory isolate of H. contortus. Luckily, the University acquired a new property that was confirmed clean of worms through grass washings and soil samples (research methodologies to assess the level of worm larvae on pasture). If the farm manager had simply used the old and unsustainable ‘Treat and Move’ strategy where all animals would be treated with the most effective drench combination possible (probably not 100% effective) and moved to the new pasture, the pasture would have been immediately contaminated with only resistant worms, even though there may have been only a few worms surviving this treatment. This would have amplified the current resistance problem and further perpetuated the clinical symptoms associated with barber’s pole worm. Thus, the research team tested a novel approach to parasite control to replace the resistant worms with susceptible worms founded around the dilution principle to increase the ratio of susceptible to resistant worms and therefore slow the development of resistance. 

In this study, sheep were removed from the contaminated pasture and housed on concrete floors, treated with a multiple day regiment of Levamisole and Albendazole, reducing worm egg counts by 99.0 % (98.8, 99.2). Next, sheep were orally infected with 5,000 susceptible* third-stage larvae (L3) of H. contortus and moved to the clean pasture previously described (Fig. 2). Sheep were left to seed the pastures with the susceptible larvae for 1.5 months following replacement. A subset of ewes was drenched with albendazole and a worm egg count reduction test (WECRT) yielded 98.5% (98.1, 98.8) reduction in worm egg count, confirming successful replacement of the resistant ‘evil worm’ with the susceptible laboratory isolate (Fig. 3).

*The susceptible strain was confirmed as being susceptible to all drench groups.

While replacement of resistant parasites with susceptible parasites appeared to be successful, the research team wanted to go further and evaluate the long-term sustainability of this practice. An Integrated Parasite Management program was implemented including the use of FAMACHA^©, a diagnostic tool that matches the colour of the mucous membranes of the lower eyelid to anaemia level, allowing selective treatment of individuals that are suffering and leaving resilient animals untreated to maintain refugia. In other words, the only sheep that are drenched are those showing signs of suffering from infection, leaving many animals, and their resident worms, without exposure to the drench. Albendazole was selected as the treatment of choice to allow the research team to further study the development of resistance to the benzimidazole class of drenches also known as the white wormers. 

Is worm replacement sustainable?

Three diagnostic tests were used to monitor the development of drench resistance including the WECRT, DrenchRite® Larval Development Assay (LDA), and pyrosequence genotyping to measure the frequency of genetic changes associated with benzimidazole resistance. Additional work regarding the population genetics of the population was conducted, however, is outside of the scope of this article.

Worm Egg Count Reduction Tests

WECRT were completed 1.5 and 2.5 years following replacement. Surprisingly, within 1.5 years post-replacement, resistance to albendazole, ivermectin, and moxidectin was confirmed via WECRT (-56%, -200%, and 59.2% reduction in WEC, respectively); note that a negative value indicates that the worm egg count of animals following treatment were higher than untreated controls, which can happen due to normal variation in the animals used in each group (Fig. 5). It is important to note that resistance to the macrocyclic lactone class of drenches developed even though albendazole was the only drench used. This led researchers to hypothesize that the susceptible laboratory isolate possessed a fitness deficit—they were not fit to survive in the field—allowing the small number of residual resistant worms present post-replacement (i.e. that survived the initial treatment) to outcompete the susceptible worms.  The residual resistant worms were highly benzimidazole and macrocyclic lactone resistant and borderline resistant to levamisole. Resistance to levamisole developed within 2.5 years of replacement under minimal drug selection pressure.

DrenchRite® Larval Development Assay

The DrenchRite® is a diagnostic test to detect resistance to multiple classes of drenches in H. contortus (http://www.wormx.info/storeyhowell2012). This assay is extremely useful in the southeast US where H. contortus is the most common and most clinically important internal parasite of small ruminants. This assay was developed in Australia and the plates are still made in Australia and shipped to the US. Faeces is collected from a mob of sheep and eggs are isolated. Eggs are placed in a 96 well plate with doubling concentrations of three classes of drenches and control wells. Eggs are incubated for 7 days to allow hatching and development to third-stage larvae. Larval development is then assessed by a trained technician and the level of resistance to the benzimidazoles, macrocyclic lactones, and levamisole is reported. The graphs below express the results of this assay (Fig. 6). A shift to the right indicates a greater level of drug resistance. The susceptible laboratory isolate is displayed in blue and the residual resistant worms prior to replacement are displayed in red. A clear reversion to susceptibility at 1.5 months post-replacement is displayed by the leftward shift of the green line. Importantly, resistance to the benzimidazole and macrocylic lactone classes developed within 1.5 years following replacement. These results support those of the WECRT. 

The most striking result was the emergence of genetic changes associated with benzimidazole resistance. A mutation at position 200 of the beta tubulin isotype 1 gene is associated with resistance to benzimidazoles. The blue TTC indicates susceptibility and the red TAC indicates a single nucleotide polymorphism associated with resistance. Fig. 7 depicts the reversion in susceptibility following replacement and the gradual development of resistance over 1.5 years until fixation 2.5 years following replacement.

Phenotypic and genotypic measurements confirmed that a multiple-drug resistant field population of H. contortus was successfully replaced with a susceptible laboratory isolate, but the population rapidly reverted to resistance even under minimal drug selection pressure. These findings may be associated with differences in fitness between field and laboratory isolates.

Final Thoughts

This study confirmed that it is possible to replace a resistant population of H. contortus with a susceptible laboratory isolate. However, resistance can re-emerge rapidly and presents further challenges to this approach. It seems that the susceptible laboratory isolate was unable to thrive in a field setting and future replacement experiments should consider use of a field-adapted susceptible isolate and seeder lambs. Additionally, drenching with a fully efficacious drench to remove a greater proportion of the residual resistant worms may improve the sustainability of this approach. Although these results indicate that worm replacement remains a possible strategy in the future for sheep producers, it is important to note that we do have tools to manage drench resistance including non-chemical strategies for parasite control (http://www.wormboss.com.au/tests-tools/management-tools.php). 

References:

Bird, J., Shulaw, W.P., Pope, W., Bremer, C., 2001. Control of anthelmintic resistant endoparasites in a commercial sheep flock through parasite community replacement. Veterinary Parasitology 97, 219-225.

Gilleard, J.S., 2013. Haemonchus contortus as a paradigm and model to study anthelmintic drug resistance. Parasitology 140, 1506-1522.

Howell, S.B., Kaplan, R.M., Williamson, L.H., Burke, J.M., Miller, J.E., Terrill, T.H., Valencia, E., Williams, M.J., Zajac, A.M. 2008. Prevalence of anthelmintic resistance on sheep and goat farms in the southeastern United States. JAVMA 233, 1913-1919.

Moussavou-Boussougou, M.N., Silvestre, A., Cortet, J., Sauve, C., Cabaret, J., 2007. Substitution of benzimidazole-resistant nematodes for susceptible nematodes in grazing lambs. Parasitology 134, 553-560.

Miller, M., Howell, S., Vatta, A., Redman, E., Storey, B., Gilleard, J., Kaplan, R., 2015. Evaluation of worm replacement as a means to reverse the impact of multiple-anthelmintic resistant Haemonchus contortus on a sheep farm. In:  World Association for the Advancement of Veterinary Parasitology, Liverpool, England.

Overend, D.J., Phillips, M.L., Poulton, A.L., Foster, C.E., 1994. Anthelmintic resistance in Australian sheep nematode populations. Australian Veterinary Journal 71, 117-121.

Playford, M. et al, 2013. The prevalence of anthelmintic resistance on Australian sheep farms (2009-2012). In:  World Association for the Advancement of Veterinary Parasitology, Perth.

Sissay, M.M., Asefa, A., Uggla, A., Waller, P.J., 2006. Anthelmintic resistance of nematode parasites of small ruminants in eastern Ethiopia: Exploitation of refugia to restore anthelmintic efficacy. Veterinary Parasitology 135, 337-346.

Van Wyk, J.A., Van Schalkwyk, P.C., 1990. A novel approach to the control of anthelmintic-resistant Haemonchus contortus in sheep.Veterinary Parasitology 35, 61-69.