Anthelmintic resistance against Helminth Parasites in domestic Livestock and its remedial measures
Jayanta Kumar Chamuah, Plabita Goswami, Trishita Banik, Angughali Aheto Sumi and Limasungla Imchem
ICAR-NRC on Mithun, Medziphema, Nagaland-797106
Abstract
The scope of application for addressing anthelmintic resistance encompasses a range of products and processes aimed at mitigating this pressing issue in veterinary medicine. Key components include the development and deployment of diagnostic tools to swiftly identify resistant parasites, facilitating targeted treatment strategies. Research and development efforts focus on understanding resistance mechanisms and exploring novel compounds to combat resistance effectively. Integrated parasite management (IPM) strategies, combining non-chemical methods with anthelmintic treatments, are integral in reducing reliance on drugs and slowing resistance development. Educational initiatives play a crucial role in disseminating best practices among veterinarians, farmers, and extension workers to optimize anthelmintic use and minimize resistance emergence. Regulatory measures, including guidelines for responsible drug use and surveillance programs, are essential for monitoring resistance levels and informing policy decisions. Collaborative efforts among researchers, industry stakeholders, and policymakers are paramount to addressing anthelmintic resistance comprehensively. Continuous evaluation and adaptation of intervention strategies ensure responsiveness to evolving resistance patterns and emerging challenges. Through the coordinated implementation of these products and processes, the scope of application for anthelmintic resistance management aims to safeguard livestock health while preserving the efficacy of anthelmintic treatments.
Introduction
Helminths are a various types of parasites causing a major health problem for animals in dif‐ ferent parts of the globe. Control of helminthiasis has largely relied on the use of pharmaceutical anthelmintics. Unfortunately, the exhaustive use of anthelmintic drugs has led to a serious and dramatic level of anthelmintic resistance. Anthelmintic resistance is a heritable loss of se sitivity of an anthelmintic in a parasite population that was in the past susceptible to the same anthelmintic. The development of anthelmintic resistance is evident to different helminths of almost every animal species and to different groups of anthelmintic in several continents. Frequent treatment, underdosing, genetics of the parasite, and targeting and timing of mass treatment are predisposing factors for anthelmintic resistance. Upregulation of cellular efflux mechanisms, an increase in drug metabolism, a change in drug receptor sites that reduces drug binding or the functional consequences of drug binding, and a decrease in drug receptor abun‐ dance through reduced expression within the parasite are the main mechanisms of anthelmintic resistance. In vivo method like fecal egg count reduction test and in vitro method such as egg hatch assays, larval motility test, larval development test and PCR can be used for the detection of anthelmintic resistance. Proper utilization of anthelmintic drugs, using combined anthelmintic and applying other alternatives are essential strategies to slow down the development of anthelmintic resistance. As anthelmintic resistance is a serious challenge throughout the world, proper utilization of the existing anthelmintics and reducing dependence on anthelmintics should be implemented to reduce its challenge.
The economic loss to the livestock industry that is occurred due to ectoparasitic infestation like tick injury, tick bite and tick paralysis in animals has a direct negative impact on production performance of animals that affects gross national income. Besides, the ectoparasites also play a role in transmitting various pathogenic microbes to domestic animals as well as human beings. The effective control of ectoparasites remains a challenging task due to cost involvement and increase development of acaricidal drug resistance. Keeping all these points in view the usage of anti-ectoparasitic vaccines appears to be a novel concept for limiting such loss from tick infestation. However, in the last few decades there has been some progress in the field of development of vaccines against ectoparasites that may be credited to handworks of few scientists from different parts of the world. Tremendous achievement in the said field has been reported especially on ticks.
Like other animals, mithun is prone to suffer from different ectoparasites and among parasitic agents, ticks are one of the contributory factors for their ticks injury, ticks pyaemia, ticks paralysis besides transmitting various microorganism including bacteria, virus, helminth and protozoal parasites to the host animals. Besides, huge ticks infestation leads million dollar loss of money in terms of reduce production in terms of meat and milk in world wide. A relative humidity of 80%, an environmental temperature of 15-35°C and varying soil pH of 6.5-8.5 are considered to be conducive for optimum development of ticks nymph and larvae and this climatic condition prevails in the north-eastern hilly region making an ideal atmosphere for perpetuation of life cycle of different ticks. But no systematic studies on the epidemiology and genetic characterization of the ticks from the north east region of the country. Sporadic studies have done on the ticks, prevalence, could not lead to the formulation of strategic control measures against ticks. . Ticks are one of the major obligate ectoparasite causing harm to livestock in different form as mechanical injury, tick paralysis and transmitting different micro-organism to host animals. Due to suitable environmental condition prevailing in the region, the tick population is at high level and causing severe health problem in free ranging animals including mithun. Due to difficult terrain and minimum trained manpower available in this part of the country the subject has not been addressed suitably and there is a huge gap in knowledge which is essential for the development of control measure. Livestock production in the North East is pre-dominantly the endeavor of small holders. Though there is vast potential for growth in this sector, production of meat and, milk has not achieved self-sufficiency level in this region. The livestock rearing is affected by disease condition which sometimes causes huge loss in terms of mortality and morbidity. Among different diseases, ectoparasitic infestations especially ticks is a common problem and most economically important pests of mithun and allied domesticated livestock. It damages the host both by direct and indirect ways. Direct damage leads to loss of blood, reduced growth of animals, reduction in milk production and loss in value of hide due to tick bite marks. The indirect damage is through transmission of lethal pathogens. The NEH region provides ideal condition mainly because of pastoral rearing of cattle for perpetuation of life cycle of different ticks. The control of ticks is solely focused on repeated application of synthetic chemicals leading to many problems. The problem is further accelerated by the fact that many tick populations have become resistant to one or other of the acaricide used to control ticks. The evaluation of resistance attempted against synthetic pyrethroids (deltamethrin and cypermethrin) in Rhipicephalus (Boophilus) microplus ticks collected from nine districts of three agro-climatic zones of north-western Himalayan region of India (Gadara et al., 2019). Determination of metabolic resistance mechanisms in pyrethroid-resistant and fipronil-tolerant brown dog ticks was studied by Eiden et al. (2017). Therefore, along with scientific community farmers of this region should be aware with condition and preventive measure should be taken in the farm of alteration of chemicals, use of herbal drugs, and genetic selection of individual breeds of domestic livestock with proper integrated tick control measure in near future. Therefore, effective botanical acaricides against leech populations will be developed in this project. So, this project is taken up to formulate effective Phyto formulation against leech as per Institute mandate.
Review on Anthelmintic resistance
One of the first cases of resistance to the imidazothiazole LEV emerged in the early 1980s roughly 10-20 years after its initial release. Given that by that time multiple BZ compounds, such as TBZ, fenbendazole, and oxfendazole had started showing signs of reduced efficacy, it was understood that to delay the emergence of widespread LEV-resistance action needed to be taken. Early studies investigated LEV in order to determine its precise mode of action as well as the possible mechanisms of resistance. Although some evidence of inhibition of metabolic enzymes was shown, it was soon established that LEV acted primarily by stimulating nerve cells through cholinergic (acetylcholine dependent) muscle synapses in susceptible nematodes .It was concluded that the LEV-resistant H. contortus was most likely similar to the pseudo-wild, mildly resistant C. elegans isolates, belonging to complementation groups unc-29, 38, 63, 74, and/or lev-8, 9. More than half a decade later, both Sangster et al. (1998) and Moreno-Guzmán et al. (1998) performed binding experiments with radioactively labeled LEV which demonstrated that, unlike for the free-living C. elegans where resistance was associated with strains deficient in receptors, resistance to LEV in different H. contortus isolates appeared to be governed by changes in the normal function of the receptor – i.e., increased or decreased binding affinity and/or binding sites for the drug. Both studies concluded that LEV was binding to a distinct population of AchRs and that the most likely route of resistance development is through the modifications of these receptors leading to altered affinities for the ligand. Since AchRs were found to not only be the direct targets of LEV but also associated with resistance, a lot of effort was put into understanding their structure and interactions with other cellular components. In free-living nematodes, LEV-sensitive AchRs (L-AchRs) (Lewis et al., 1987) were found to be heteropentamers, composed of three α and two non-α subunits. In addition, at least three ancillary proteins have been indicated to be crucial for the expression and assembly of L-AchRs (Boulin et al., 2008). In parasitic nematodes, such as H. contortus, on the other hand, at least two different LAchRs have been functionally reconstituted by assembling different sets of receptor subunits – Hco-L-AChR1 (composed of subunits Hco-unc-29.1, Hco-unc-38, Hco-unc-63a, and Hco-acr-8) and Hco-L-AChR2 (sharing thesame subunit composition but lacking Hco-acr-8) . Furthermore, Boulin et al. (2011) demonstrated that the presence (or absence) of a single subunit, Hco-acr-8, plays the main role in determining the sensitivity of the receptor to LEV. This observation was further supported by the findings of Fauvin et al. (2010) and later by Barrère et al. (2014), who uncovered and described a truncated hco-acr-8 mRNA transcript (referred to as Hco-acr-8b) at the time only found in LEV resistant isolates of H. contortus and occurring due to a 63 bp deletion in the second intron of hco-acr-8. Although it is tempting to speculate, so far, no data is available on whether this truncated form of the Hco-acr-8 subunit (Hco-acr-8b) results in the dominance of the receptor Hco-L-AChR2 (lacking a full-length Hco-acr-8 subunit) over Hco-L-AChR1 in the parasite or if an altogether new type of receptor (including Hco-acr-8b) is assembled described an alternative transcript of the hcounc- 63, Hco-unc-63b, only found to be expressed in the resistant isolates of trichostrongylids, suggesting that LEV-resistance in parasitic nematodes could potentially be established by the parasite acquiring mutations in one (or more) of the L-AChR subunits, leading to the translation of truncated LAChRs. Despite being a rather intuitive and sensible theory behind LEVresistance in parasitic nematodes, especially in the case of Hco-acr-8b, it has been shown to not hold true in numerous cases as the 63 bp deletion, thought to give rise to Hco-acr-8b, was shown to be present at relatively high frequencies in both susceptible and resistant isolates of H. contortus. In fact, Doyle et al. (2022) suggested that rather than the 63 bp deletion in intron 2, a non-synonymous point mutation in exon 4 (leading to the substitution Ser à Thr at P168) in hco-acr-8 seemed to be a better predictor of the LEV-resistant phenotype. However, this finding requires further confirmation. Several research groups attempted to investigate LEV resistance from a different angle – by identifying changes or patterns of change in gene expression upon exposing different roundworm parasite isolates to LEV. One of the first studies of this kind was carried out by Williamson et al. (2011), who found increased expression of both hco-acr-8 and hco-acr-8b, whilst the expression of genes coding for the subunits Hco-unc-29.3 and Hco-unc- 63 were down-regulated. A great body of work on expression level changes of AChR subunit, ancillary protein, and P-gp genes was produced by Sarai et al. In their 2013 study, Sarai et al. (2013) compared changes in gene expression levels in one susceptible and three LEV-resistant field isolates of H. contortus. Apart from a consistent down-regulation of hco-unc-63a in adult stages of the parasite, no reliable change in expression level patterns was observed for either AChR, ancillary protein, or P-gp gene groups. A year later, the authors compared expression level changes upon exposing highly resistant fractions of the H. contortus population to varying LEV concentrations (Sarai et al., 2014). As a consequence, a distinct pattern was observed,wherein at low LEV concentrations, expression levels of some AChR genes increased (e.g., hco-unc-38, hco-unc-63b, and hco-unc-16) together with non-specific P-gps (such as hco-pgp-3, hco-pgp-4, hco-pgp-10, and hco-pgp-14), whereas at intermediate and higher concentrations of the drug, downregulated expression of primarily AChR subunits and ancillary proteins occurred. Interestingly, in the same study, the expression of hco-acr-8b was not detected. In 2015, Sarai et al. (2015) conducted selection (with LEV) experiments on a naïve isolate of H. contortus for nine generations. Throughout the generations and across the different parasite life stages, the authors found differing levels of expression for many of the previously mentioned AChR subunit as well as ancillary protein genes, which appeared to be mostly down-regulated in L1 but up-regulated in L3 and adult stages. Taken together, it appears that the mechanisms of resistance that ensure worm survival could not only be LEV concentration, but also parasite life-stage dependent. The complicating issues with most of these studies were that the isolates used for comparison were either multi-resistant (thus making it difficult to delineate the expression pattern changes occurri occurring specifically due to LEV39 resistance) and/or of different geographic or historic origin, which could result in considerable changes in expression or allele frequency patterns, completely unrelated to resistance. Overall, it appears that LEV-resistance is a truly polygenic trait (Sangster et al., 1998), determined by multiple mutations and/or transcriptional changes associated with, among others, AChR, ancillary protein, and perhaps even P-gp genes. Further innovative, perhaps even genomic, approaches will thus be required to untangle LEV resistance in parasitic nematodes and identify the major contributing factors.
Types of Anthelmintic resistance:
There are three primary types of AR: Single-Drug Resistance, Multiple-Drug Resistance, and Cross-Resistance.
- Single-Drug Resistance: This occurs when parasites develop resistance to a single class of anthelmintic drugs. For example, parasites that resist treatment with benzimidazoles alone would fall under this category. Single-drug resistance is often the first stage of AR and can quickly expand to other drugs if not managed.
- Multiple-Drug Resistance (MDR): In MDR, parasites are resistant to two or more classes of anthelmintics, even if the drugs work through different mechanisms. MDR is particularly alarming because it limits treatment options, as no single class of anthelmintics is effective. For instance, parasites may develop resistance to both benzimidazoles and macrocyclic lactones, which have distinct mechanisms of action.
- Cross-Resistance: Cross-resistance arises when resistance to one drug class confers resistance to another, often closely related, class. This can happen if the drugs share a similar mechanism, making it easier for parasites to adapt to both. For example, resistance to one type of macrocyclic lactone can confer resistance to others within the same group, even if they have slight structural differences.
Detection of Anthelmintic Resistance:
For Anthelmintic Resistance test: In vivo Methods
- Faecal Egg Count Reduction Test (FECRT) :
The anthelmintic efficacy of a chemical is determined by comparing worm egg counts from the animal before and after treatment. This test has been thoroughly standardized, allowing it to be widely used. Resistance is evident when two requirements are met, according to FECRT: the percent reduction in egg count is less than 95%, and the lower limit of its 95% confidence range is equal to or less than 90%. A post-treatment egg count for benzimidazoles should be performed 10–14 days after the anthelmintic has been delivered. Because anthelmintic treatment can temporarily stop eggs from being laid without killing adult nematodes, itis a good idea to use it. Egg production may be decreased if the time between treatments is less than 10 days, resulting in an overestimation of anthelmintic efficacy with the benzimidazoles anthelmintic. As a result, collecting feces samples 10–14 days following treatment is recommended. Fecal samples should be taken less than 7 days after treatment if levamisole resistance is suspected. As a result, depending on the anthelmintic group, the duration between treatment and the second egg count varies: 7–10 days for benzimidazoles; 3–7 days for tetrahydro pyrimidines and imidazothiazoles; 14–17 days for macrocyclic lactones. The Formula for faecal egg count reduction test are as follows:
FECR = 100 x (1-[T2/T1][C1/C2]), where T1 and T2 represented the mean pre- and post-treatment faecal nematode egg counts (FECs) of a treated group, and C1 and C2 represented the mean pre- and post-treatment FECs of an untreated
Ii.Polymerase Chain Reaction (PCR) for Benzimidazole resistance:
The genotyping of resistant (rr) or susceptible (rS and SS) adult worms or larvae is possible with this technology, which is based on the use of PCR. Worms can be genotyped for the mutation on β-tubulin residue 200 (phenylalanine to tyrosine), which is implicated in BZ resistance, by employing four primers in the same reaction mixture.
Management of Anthelmintic resistance:
Managing anthelmintic resistance (AR) in domestic livestock is essential to maintaining animal health, productivity, and profitability. AR is a significant problem worldwide, as resistance to commonly used dewormers can lead to ineffective treatments and increased disease prevalence. Effective management strategies rely on a multifaceted approach, combining pharmacological and non-pharmacological methods to control parasite populations and slow resistance development.
- Strategic and Targeted Treatment: Instead of routinely deworming all animals, targeted treatment (treating only those animals with high parasite loads) and targeted selective treatment (treating only the animals most affected by parasitic infections, such as young, sick, or highly productive animals) can help reduce drug exposure. This strategy prevents unnecessary treatment of animals with low or manageable parasite burdens, slowing down the development of resistance by reducing selective pressure.
- Rotational Use of Drug Classes: Parasites develop resistance more rapidly when exposed to a single class of drugs. Rotational deworming involves alternating between different classes of anthelmintics, such as benzimidazoles, macrocyclic lactones, and imidazothiazoles. By changing drug classes periodically, livestock managers can reduce the likelihood of parasites developing cross-resistance. However, it is important to base drug rotation on fecal egg count reduction tests (FECRT) to determine the effectiveness of each class before rotation.
- Refugia-Based Strategies: Refugia refers to the proportion of the parasite population not exposed to anthelmintics, such as larvae in the environment or untreated animals. Maintaining a population of susceptible parasites in refugia reduces the overall level of resistance in a herd by diluting the genetic pool of resistant parasites. This approach helps maintain drug efficacy by ensuring that susceptible genes remain within the parasite population.
- Non-Chemical Control Methods: Integrating pasture management techniques can significantly help in managing AR. Practices such as rotational grazing, pasture resting, and mixed-species grazing can reduce parasite loads on pastures by disrupting the life cycle of worms. Additionally, biological control agents like nematophagous fungi, which feed on parasite larvae in the environment, can be used to reduce worm burden in pastures.
- Monitoring and Surveillance: Routine monitoring of parasite loads through fecal egg count (FEC) tests and evaluating drug efficacy through FECRT is crucial. This allows early detection of resistance and informs decisions about drug use. By tracking parasite burdens, livestock managers can make evidence-based decisions on whether deworming is necessary and which drugs are still effective.
- Selective Breeding for Resistance: Some animals have natural genetic resistance to parasites. Selecting and breeding livestock with higher natural resistance to parasitic infections can reduce overall herd susceptibility, decreasing reliance on anthelmintics over time. This approach has shown success in certain breeds of sheep and cattle and can contribute to sustainable parasite management.
- Education and Awareness: Educating livestock owners, farmers, and veterinarians on best practices for deworming, understanding AR, and adopting integrated parasite management (IPM) strategies is crucial. Well-informed stakeholders are more likely to implement sustainable practices that reduce AR development.
In conclusion, managing AR in domestic livestock requires a holistic approach that combines selective drug use, alternative parasite control methods, genetic selection, and regular monitoring. Implementing these practices effectively can help sustain anthelmintic efficacy, maintain animal health, and support long-term productivity in the livestock industry.
