Anthelmintic Resistance in Livestock
Hardeep Kalkal, Amitava Paul and Shikha Tamta
International Institute of Veterinary Education and Research, Rohtak, Haryana, India- 124001
Introduction
Antiparasitic drug resistance is the genetic ability of parasites to survive treatment with a drug that was generally effective against those parasites in the past. It is due to the selection of a specific heritable trait in a population of parasites that results into significant increase in survivability of that parasite population to standard recommended dose of the drug. After an animal is treated with an antiparasitic drug, the susceptible parasites die and the resistant parasites survive to pass on resistance genes to their offspring. Antiparasitic resistance poses a significant threat to animal health and can result into substantial production losses. Antiparasitic resistance has been documented in livestock, both globally and within India.
Many factors contribute to antiparasitic resistance, including the biology of the parasite; the immune status of the host animal; treatment practices; drug properties; and certain livestock management practices. The situation has recently become alarming in India particularly anthelmintic resistance in the nematode parasites of grazing animals and acaricide resistance in livestock ticks. To help combat this emerging problem, there is a need to develop an antiparasitic resistance management strategy that promotes sustainable use of approved antiparasitic drugs by slowing the development of antiparasitic resistance in livestock parasites in India.
Anthelmintic resistance
Anthelmintics are a group of antiparasitic drugs that kill or expel parasitic helminth worms from the body of the host. Since 1940s anthelmintics remain mainstay of worm control andtreatment of parasitic infections in farm animals. However, resistant parasitic population to majority of the launched anthelmintics are recorded within two decades of their commercialisation, for instances, phenothiazine launched in 1940s as first anthelmintic and its resistance report came into record by 1957 in USA. Amongst livestock animals, sheep has witnessed maximum number of cases of anthelmintic resistance. In India, first report of anthelmintic resistance was published in 1976 from organised sheep farms of Uttarakhand region against phenothiazine and benzimidazole (thiabendazole) drugs. Hitherto, the importance of anthelmintic resistance remained in dim light until 1990 when the condition become alarming and started reporting throughout India.
Earlier work evaluated the knowledge that defined resistance in the year 1980, and from their study, they predicted the spread and future impact of resistance and also set goals for future research. The earliest report of AR was in 1964 for H. contortus resistance to benzimidazole in treated sheep and was also the first for a modern drug in production animals. Within 10 years of the first report of AR, resistance was found regularly in sheep parasites, followed by reports of resistance in horse and cattle nematodes. Although anthelmintics have been efficient and work quickly, nematodes have developed resistance in a number of sheep-producing countries such as Australia, South Africa, New Zealand, Switzerland and Italy. To this end the highest resistance has been observed with ivermectin (Ivomec®) and albendazole (Valbazen®) or fenbendazole (Safeguard® or Panacur®), and low to moderate resistance has been observed with levamisole (Levasole®, Tramisol®). Resistance to moxidectin (Cydectin®) is also prevalent and on the rise on many livestock farms. In Africa, anthelmintic resistance has been reported in both the commercial and resource-poor farming sectors in at least 13 countries, and, among the commercial farms in South Africa, the situation is considered the worst in the world, with high levels of Haemonchus contortus resistance to all classes of anthelmintics.
- Cross resistance
Ability of the parasite strains to survive the recommended therapeutic doses of chemically unrelated drugs having different modes of action.
- Side resistance
Parasites demonstrate resistance to a drug as a result of selection by another drug having similar mode of action.
- Multiple resistance
Parasites are resistant to two or more anthelmintic groups because of either selection by each group independently or by side resistance.
Risk factors for development of anthelmintic resistance
The control of gastrointestinal parasitism for small ruminants has long been under threat from the development of anthelmintic resistance by parasite populations. However, in recent years it has become evident that this is also an emerging problem for cattle. Resistance against drugs belonging to the same anthelmintic drug class is called side resistance, whereas cross and multidrug resistance refer to resistance against two or multiple drugs belonging to different anthelmintic drug classes. Development of AR can be limited by ensuring that the parasites are exposed to an effective drug dose and to consider the timing andfrequency of anthelmintic drug treatments so that only a small proportion of the population is exposed to the anthelmintic. The main factors for the selection for anthelmintic resistance are high-treatment frequency, underdosing and the use of the same anthelmintic class over several years. These factors, individually or in combination, together with the risk of underdosing and continued use of one class of anthelmintics, irrespective of efficacy status are frequently encountered factors enhancing development of anthelmintic resistance.
Anthelmintic resistance monitoring
Controlled Test
This test is the most reliable method of assessing anthelmintic efficacy but also costly in terms of labour requirements and animal usage and is now rarely used. In an attempt to reduce the costs and time taken, laboratory animal models have been used. To characterise the sensitivity of a field isolate, groups of worm free animals should be inoculated with infective larvae and the anthelmintic tested at 0.5, 1 and 2 times the recommended dose rate. Inclusion in the test of a known susceptible strain has been recommended. Resistance is generally confirmed when the reduction in geometric mean worm counts is less than 90%.
Faecal Egg Count Reduction Test
The faecal egg count reduction test (FECRT) is the main method of detection of anthelmintic resistance in nematodes of veterinary importance. In the FECRT, populations of gastrointestinal nematodes of sheep are considered susceptible when drug efficacy exceeds 95% (reduction in FECRT). Conversely, resistance is present when efficacy is < 95%. The equivalent efficiency benchmark for resistance is 90% for other host species. However, reductions in efficacy require interpretation in the light of different situations, where, for instance, the 95% cutoff is more complex than it seems because some drugs have very high efficacy (99.9%) against some parasite species but lower (say, 95%) for others in the same host. FECRT is an in vivo method that involves the nematodes in the sheep as the experimental unit. An advantage of FECRT is that it can be used with all groups of anthelmintics that are available today. The disadvantage is that the faecal egg count (FEC) levels do not always correspond to the number of adult worms inside the animals. However, FECs in young sheep correlate fairly well to the burden of adult worms, at least compared to the situation in adult sheep.
Egg Hatch Assay
Egg hatch test (EHT) is an in vitro test that can be used to measure AR. EHT can only measure BZ resistance. In practice, fresh eggs are either diluted in increasing concentrations of thiabendazole (TBZ) or diluted in a predetermined concentration (discriminating dose) and incubated for 48 hours. The eggs hatched are then counted under an inverted microscope. Discriminating doses have been established in nematode species such as H. contortus. A discriminating dose is the dose required to prevent hatching of 99% of susceptible eggs. The EHT can detect resistance if there are at least 2-3% resistant eggs. Egg hatch test and other in vitro tests generate dose-response lines. This allows the calculation of parameters, such as the concentration that kills 95% of eggs (the EC95), a single parameter used to compare isolates. Resistant worms will have a higher EC95 because a higher drug concentration is required to kill them. Such assays are underutilised tools for measuring resistant phenotypes. However, they have been fundamental tools for studying the results of experimental genetic crosses.
Larval Feeding Inhibition Assay
A larval feeding assay devised for detection of macrocyclic lactones and imidazothiazoles resistance in gastrointestinal nematodes. It is based upon the principle that the concentration of anthelmintic required to inhibit larval feeding in 50% of L1 juvenile of nematode is higher in parasites resistant to either macrocyclic lactones or imidazothiazoles than those of susceptible isolates indicating development of resistance against these anthelmintics.
Larva Development Test
Most anthelmintics affect the metabolism of a parasite in some way that affects parasite growth. In LDT, L1 are cultured to L3 in the presence of heat treated lyophilised Escherichia coli, as a food source, and the anthelmintic under test. Suitable controls are also run without the presence of anthelmintic. Further a dose response curve is generated to determine LD50 values.
Larval Paralysis Test
A larval paralysis test has been developed for the detection of levamisole and morantel resistance. In the assay, infective third stage larvae are incubated for 24h in serial dilutions of the anthelmintic. After this time the percentage of paralysed larvae is determined at each concentration and a dose-response line plotted and compared to known reference strains.
Larva Migration Inhibition Assay
L3 obtained from larval cultures are isolated and stored in ventilated cell culture flasks at 6–10 °C in a fridge for a maximum of 3 months prior to use. LMIAs were performed before and (in case of a positive EPG) after treatment. L3 are subjected to different concentrations of ivermectin and then migrated and non-migrated larvae are counted under a stereo microscope and the percentage of non-migrated larvae to the total amount of larvae are calculated.
Use of molecular techniques for AR monitoring
Nowadays, the traditional parasitological diagnostic techniques involving mainly microscopy have been complemented by a variety of new techniques and tools, mostly molecular in nature. To date, traditional methods are still routinely used despite the fact that they can be labour and time intense to perform. PCR-based procedures have been proven to have greater sensitivity and specificity than ‘conventional’ diagnostic approaches reliant on microscopy and/or immune detection.
Perspective (future control and prevention methods, necessary research)
Many of the approaches that are available for prevention of AR are still being researched and evaluated, and most of them are at present not suitable for thecommunal grazing systems of many resource-poor farmers; therefore, further research must still be conducted to ensure adaptability to both commercial and resource-poor farming operations.
In order to win the battle against the emergence of AR, correct use of anthelmintics and on-farm training about gastrointestinal helminths infecting livestock must be provided. Such training should be ongoing and provided by extension officers together with animal health technicians. Training initiatives should incorporate practical demonstrations and focus on aspects such as the importance of correct dosage, when to alternate anthelmintic classes and treatment frequency
