Gastrointestinal nematodes (GIN) are important pathogens of grazing ruminants, responsible for economic losses in animal production worldwide ^[1][2][3][1,2,3]. The controlling of these parasites has been a challenge for producers and there is an emerging need to seek effective alternatives that do not cause animal toxicity [4]. Among Brazilian ruminant livestock, the most common GIN are those belong to the following genera: Haemonchus and Ostertagia (parasite of abomasum); Trichostrongylus (parasite of small intestine and abomasum); Cooperia (parasite of small intestine); and Oesophagostomum, also known as the nodular worm, which parasitizes the large intestine ^[5][6][7][5,6,7]. Infections by these parasites are characterized by lesions in the gastrointestinal mucosa, which impair the absorption of nutrients, reducing body weight gain and milk production. In addition, some species (e.g., Haemonchus contortus) are hematophagous [8]. Studies have already been conducted in Brazil to assess the economic impact of GIN infection in ruminants ^[9][10][9,10]. For instance, a reduction of 0.6 kg/cow/day of milk in dairy cattle is estimated, with a potential annual loss of up to USD 1870.48/animal [11]. In sheep, losses may reach approximately USD 400/animal/year [12].

GIN control has been largely achieved by using both broad (benzimidazoles, imidazothiazoles, hydropyrimidines and macrocyclic lactones) and narrow-spectrum (salicylanilides, nitrophenols and organophosphate) anthelmintics [13]. More recently, amino-acetonitrile derivatives have emerged as a new chemical class of synthetic anthelmintics, effective against GIN of sheep [14]. Nevertheless, the excessive dependence on these substances has led to the development of anthelmintic resistance (AR) in all species of domestic ruminants to all classes of anthelmintics [15]. In Europe ^[16][17][18][16,17,18], Africa ^[19][20][21][19,20,21], Asia ^[22][23][22,23], Oceania ^[24][25][26][24,25,26] and the Americas ^{[27][28][29][30]}[27,28,29,30], this phenomenon is associated with multiple drugs, threatening the viability of ruminant livestock production, especially small ruminants ^[18][31][32][18,31,32].

In Brazil, the first AR reports were on thiabendazole and ivermectin in sheep in the country’s southern region ^[33][34][33,34]. Similarly, benzimidazoles resistance was also detected in goats from the northeast region. In the same period, AR was also detected in goats treated with albendazole, parbendazole and levamisole [35], which raises the discussion about different dosages for small ruminants [36]. Soon after, the resistance of Haemonchus spp. to oxfendazole and albendazole was detected in cattle from the southern region [37]. With the recent increase in AR in Brazilian herds, the development of new compounds is pivotal, as well as the integration of rational GIN control ^[38][39][38,39].

2. Current Methods for Detection of AR

The primary method for detecting resistance is FECRT, which can be used with all anthelmintic groups. Nematode eggs are counted at pre- and post-treatment times defined according to the anthelmintic group used ^[40][131]. However, it is unsuitable for detecting resistance levels below 25% ^[41][132]. Several factors must be considered when planning an FECRT (i.e., study design, sample size considerations, choice of fecal egg count (FEC) method, statistical data analysis and interpretation) ^[42][133]. Other in vitro tests have been used less, such as the EHT, established to detect drug resistance in the benzimidazole class ^[43][134]. In addition, it is possible to use tests evaluating larval development and motility (LDT and LMIT) [15]. Particularly in cattle, most animals in a herd, even the young ones, have lower FEC, making diagnosing AR difficult [20]. For example, the McMaster technique is the most used method in studies of AR detection but it has a detection limit of 50 EPG ^[44][135]. The use of methods with higher detection, such as FLOTAC (one EPG) and Mini-FLOTAC (five EPG), might be encouraged in this kind of analysis ^[45][46][136,137]. The consensus is that there is a need for improvements in the AR detection methods, such as more reliable parasitological tests and an increase in the number of animals required for simultaneous testing on several drugs ^[47][119]. With the limitations of current in vivo and in vitro resistance tests, molecular tools can potentially improve drug resistance diagnosis ^[48][138]. The development of molecular diagnostics for anthelmintic resistance has been one of the leading research topics involving the molecular mechanisms of drug resistance ^[49][139]. Thus, developing molecular markers for diagnosing resistance can help develop new anthelmintic drugs ^[50][140]. The molecular mechanism of resistance is better understood for benzimidazoles; therefore, it offers a potential opportunity to expand molecular diagnostic tests for drugs of this class ^[51][141]. For example, in Brazil, some studies were conducted using the β-tubulin isotype gene, a marker to monitor resistance ^[51][52][53][141,142,143]. In addition, molecular characterization is an essential tool for the validation and phylogenetic analysis of nematodes, such as allele-specific polymerase chain reaction, endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC) and “Nemabiome” internal transcribed spacer 2 (ITS-2) amplicon sequencing ^[54][55][144,145]. A recent development in large-scale surveillance is the “Nemabiome” approach, which applies deep amplicon sequencing of barcoded PCR products ^[56][146]. Although initially developed for species identification and quantification, it has recently been adapted to assess the presence of resistance by benzimidazoles by deep sequencing of β-tubulin amplicons ^[57][147]. In general, molecular tests have greater sensitivity and specificity and can provide powerful tools to overcome many of the disadvantages of classical methods of AR. However, it requires further research to be used as a practical universal tool in the field.

3. How to Prevent AR Development?

It has already been proved that the excessive and incorrect use of anthelmintics to control GIN infections has resulted in AR. However, concerns about the use of these products are more comprehensive than studies of AR itself. Recently, with the improvement in awareness about the consumption of organic products, there has been a rise in concern with the potential residual effect of these products in meat and milk, derived products from ruminants that are widely consumed worldwide ^[58][148]. Despite reducing the withdrawal period, the risks associated with residues in milk intended for human consumption and dairy products may be present and should be considered ^[59][149]. For example, a study with moxidectin demonstrated that this molecule may be present in goat milk for up to 21 days ^[60][150]. Additionally, to the direct consequence of using anthelmintics, the excretion of these by-products may also be considered an essential threat from an environmental perspective ^[61][151]. The access of anthelmintic residues into the environment resulting from the direct excretion of the original drugs and metabolites in pastures during grazing, as well as through the dispersion of the manure and slurry containing anthelmintic residues, represents a potential risk for the environment ^[62][152]. Hence, studies focusing on controlling GIN but with a rational use of these chemical molecules might be encouraged. Investigating the antiparasitic activity of natural bioproducts can contribute to the development of alternative treatments and a reduction in dependence on conventional chemotherapy ^[63][153]. The antiparasitic activity of plants derives mainly from biologically active compounds known as secondary metabolites, which could lead to the detection of new antiparasitic molecules ^[64][154]. For example, flavonoids and condensed tannins may have anthelmintic effects, as demonstrated in a study inhibiting in vitro sheathing of larvae (L3) of H. contortus ^[65][155]. In addition, using nanoparticles can provide good results in the treatment of parasitic infections because they increase the bioavailability and biodistribution of drugs. However, the safety of using nanoparticles from a broader perspective needs to be better investigated ^[66][67][156,157]. So far, most of the studies have been conducted in lab conditions, as they have low cost, repeatability and allow the use of different stages (i.e., eggs and larvae) ^[62][152]. Although these plant alternatives can be cheap and accessible, they have limitations. These molecules’ potential adverse toxicity effects in vivo are generally controversial or completely unknown ^[67][68][157,158]. In vivo studies consist of oral administration of the leaves (fresh, hay and flour), aqueous or ethanolic extracts and oil of plants to ruminants infected naturally or experimentally with GIN ^[61][69][151,159]. Therefore, the association of standardized in vivo and in vitro methods is paramount for evaluating the effectiveness of plant products, especially for the determination of EC₅₀ and EC₉₀ (50% and 90% maximal effective concentration, respectively), which allows comparing the activities of different plants ^[70][160]. In order to postpone the development of AR, it is necessary to integrate GIN control measures. Therefore, some factors are essential to be considered: (i) good management has a direct effect on the health of animals with feeders and drinkers that avoid waste and contamination ^[71][161]; (ii) strategies such as grazing rotation, co-grazing with other appropriate species and manure management are alternatives to reduce the use of anthelmintics ^[72][162]; (iii) the improvement of animal resistance through genetic selection to reduce the use of chemoprophylaxis ^[73][163]; and (iv) to optimize the effectiveness of anthelmintics in populations of multiresistant nematodes, drug combinations can be used ^[74][114]. It is worth emphasizing the importance of carrying out anthelmintic efficacy tests for choosing the chemical groups to be used. The need to develop new anthelmintics for the management of AR is evident; however, it is a slow and expensive process ^[75][164]. Furthermore, it is crucial to use existing anthelmintics in a way that minimizes the impact of AR ^[76][165].