An Overview of Anthelmintic Resistance in Domestic Ruminants in Brazil

: Gastrointestinal nematodes (GIN) signiﬁcantly, negatively impact livestock worldwide, and their control depends on the use of chemotherapy drugs. However, this approach is unsustainable as anthelmintic resistance (AR) is growing widespread. This article provides a comprehensive overview of the historical and current data published on AR in domestic ruminants in Brazil. Alternative measures of GIN control have been discussed to provide helpful information to prevent the development of AR in the country. This review consisted of a search of technical and scientiﬁc publications between January 1960 to January 2023, using online sources such as PubMed, Scielo and Google Scholar. Eighty-three articles published over the last six decades reporting AR in sheep ( n = 43), goats ( n = 20) and cattle ( n = 20) were included. A total of 37.3%, 25.4% and 37.3% evaluated one, two and three or more molecule classes, respectively. Among all studies, 82.1% used fecal egg count reduction test as a method of AR diagnosis. In conclusion, AR is an urgent and emerging issue for ruminant production in Brazil. It is necessary to evaluate on a large scale the distribution and management of anthelmintic drugs and discuss strategies that delay this phenomenon’s development.


Introduction
Gastrointestinal nematodes (GIN) are important pathogens of grazing ruminants, responsible for economic losses in animal production worldwide [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].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].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 (salicy-benzimidazole + macrocyclic lactones + other combinations (37.3%; 31/83).Most of these studies (83.1%) used FECRT as a method of AR diagnosis.Currently, for the interpretation of FECRT, the recommendations of the World Association for the Advancement of Veterinary Parasitology (WAAVP) are based on classification criteria that outline how the observed data (values for the upper and lower 90% CI or results of separate hypothesis tests) are compared to the expected effectiveness and the lower limit of effectiveness (which serve as the values for the upper and lower limits of the gray zone), accepting a Type 1 error rate of 5% [41].
Eighty-three peer-reviewed articles reporting AR in domestic ruminants (sheep, goats and cattle) were published over the last six decades.Of all these studies, 51.8% (43/83) were reported in sheep, 24.1% (20/83) in goats and 24.1% (20/83) in cattle.Most of the studies were carried out with molecules from different classes.Of all studies, 37.3% (31/83) evaluated only one class, 25.4% (21/83) two drug classes and 37.3% (31/83) three or more drug classes.Figure 1 illustrates the map with a graphical representation of the distribution of the class of drugs used in the considered articles: macrocyclic lactones (24.1%; 20/83), combination of benzimidazole + macrocyclic lactones (14.5%; 12/83) and benzimidazole + macrocyclic lactones + other combinations (37.3%; 31/83).Most of these studies (83.1%) used FECRT as a method of AR diagnosis.Currently, for the interpretation of FECRT, the recommendations of the World Association for the Advancement of Veterinary Parasitology (WAAVP) are based on classification criteria that outline how the observed data (values for the upper and lower 90% CI or results of separate hypothesis tests) are compared to the expected effectiveness and the lower limit of effectiveness (which serve as the values for the upper and lower limits of the gray zone), accepting a Type 1 error rate of 5% [41].From 1960 to 1999, 18 articles (21.7%) reported AR in domestic ruminants, with one in cattle (5.6%; 1/18), three in goats (16.7%; 3/18) and fourteen (77.8%; 15/18) in sheep.From 2000 to 2023, the number of reports tripled, with 65 articles (78.3%) distributed across 14 states and 127 municipalities.Most of the studies were concentrated in the southern (39.8%; 33/83), northeastern (31.3%; 26/83), southeastern (22.9%; 19/83) and midwestern (6.0%; 5/83) regions.Despite lower reliability, some references [33,38, that do not include animal numbers or locations were considered in this review.Figure 2 illustrates the map with the graphical representation of the distribution of AR in domestic ruminants from Brazil.across 14 states and 127 municipalities.Most of the studies were concentrated in the southern (39.8%; 33/83), northeastern (31.3%; 26/83), southeastern (22.9%; 19/83) and midwestern (6.0%; 5/83) regions.Despite lower reliability, some references [33,38, that do not include animal numbers or locations were considered in this review.Figure 2 illustrates the map with the graphical representation of the distribution of AR in domestic ruminants from Brazil.

Anthelmintic Resistance in Small Ruminants
Sixty-three (75.9%) peer-reviewed articles have been published on small ruminant AR, including 51.8% (43/83) in sheep and 24.1% (20/83) in goats.Anthelmintic resistance was reported in 92 municipalities from four regions of the country: (i) south-states of Paraná, Rio Grande do Sul and Santa Catarina; (ii) southwest-states of Minas Gerais, Rio de Janeiro and São Paulo; (iii) midwest-state of Mato Grosso do Sul; and (iv) northeaststates of Ceará, Paraíba, Pernambuco and Rio Grande do Norte (Tables 1 and 2).The lack of information in the north of the country does not mean the absence of AR.However, it is worth mentioning that this region of the country concentrates the smallest number of small ruminants (2.3% of total Brazilian herd-IBGE, 2021), while the northeastern and southern regions possess the vast majority of goats and sheep (91.9%).Additionally, most of the research has been conducted in these regions.

Anthelmintic Resistance in Small Ruminants
Sixty-three (75.9%) peer-reviewed articles have been published on small ruminant AR, including 51.8% (43/83) in sheep and 24.1% (20/83) in goats.Anthelmintic resistance was reported in 92 municipalities from four regions of the country: (i) south-states of Paraná, Rio Grande do Sul and Santa Catarina; (ii) southwest-states of Minas Gerais, Rio de Janeiro and São Paulo; (iii) midwest-state of Mato Grosso do Sul; and (iv) northeast-states of Ceará, Paraíba, Pernambuco and Rio Grande do Norte (Tables 1 and 2).The lack of information in the north of the country does not mean the absence of AR.However, it is worth mentioning that this region of the country concentrates the smallest number of small ruminants (2.3% of total Brazilian herd-IBGE, 2021), while the northeastern and southern regions possess the vast majority of goats and sheep (91.9%).Additionally, most of the research has been conducted in these regions.Overall, 24,449 animals were assessed primarily through FECRT (85.7%; 54/63).These studies evaluated different classes of drugs but mainly molecules belonging to the class of benzimidazole (i.e., albendazole, thiabendazole, oxfendazole, mebendazole and febendazole), macrocyclic lactones (i.e., ivermectin, doramectin, abamectin and moxidectin) and imidazothiazole (i.e., levamisole and tetramisole).
The AR is more serious than has been documented so far.The gradual growth from its somewhat sporadic occurrence in the early 1960s to the current situation threatens the sustainability of production systems.The primary tool adopted for controlling GIN parasites is the use of anthelmintics, which generally positively impact the well-being and health of domestic and production animals [3].It is known that AR is an evolutionary process that is unpredictable if anthelmintics are used intensively in a herd [102].Nonetheless, it is possible to reduce the rate of resistance development by modifying anthelmintic use strategies [15].Particularly in sheep, resistance of H. contortus is associated with economic losses and mortality [3,103].In addition, there is more research and wider availability of drugs for this animal species (51.8%), reflecting more AR reports compared with goats and cattle.For example, the nematodes of sheep treated intensively with monepantel may show resistance to this drug in three months [104].Similarly, lambs treated with levamisole every 42 days may present resistant nematode populations after the third treatment [7].
With the decreasing effectiveness of anthelmintics, the prophylaxis of GIN infections in small ruminants has become more challenging.Hence, the rational and integrated use of these compounds with sustainable measures of control is necessary to prevent AR [105].Unfortunately, GIN of sheep developed resistance to a more recent molecule available commercially, the amino-acetonitrile derivatives [14,[106][107][108].

Anthelmintic Resistance in Cattle
Twenty articles were found reporting AR in cattle.These reports originated from 51 municipalities, distributed in the southern (Rio Grande do Sul and Santa Catarina), southeastern (Minas Gerais and São Paulo), midwestern (Mato Grosso do Sul) and northeastern (Paraíba) regions of Brazil (Table 3).In total, 6729 individual animals were assessed for AR.Similarly, to small ruminants, most studies used FECRT (70.0%; 14/20) as a method for resistance detection.These studies evaluated different classes of drugs but mainly evaluated drugs from the class of macrocyclic lactones (avermectins and milbemycins).
In cattle, using chemical compounds to control infections by GIN is commonly conducted with broad-spectrum molecules (macrocyclic lactones, benzimidazoles, imidazothiazoles and salicylanilides).Mainly, macrocyclic lactones are used worldwide in ruminant livestock.They are available in different formulations, concentrations and associations, with ivermectin being the predominant chemical compound [31].Frequently, these drugs are administered without any technical criteria for drug selection, and this empirical and indiscriminate use has favored AR development.This incorrect use has additional implications for the effectiveness of anthelmintic treatments, as it causes the emergence and spread of parasite resistance [56].Unlike the development of resistance in small ruminants, in cattle, this phenomenon occurred slower; however, in recent decades, there has been a rapid increase in reports of AR in GIN infection of cattle worldwide [29,56,[109][110][111].It is imperative to address the AR issue in cattle, with the view of a significant threat to cattle productivity [102].The most frequent helminths in Brazilian cattle herds are Cooperia spp.and Haemonchus placei [122], which were identified in several reports of AR [67,71,116].Resistance of the genus Cooperia is also common in countries such as Argentina [73], United Kingdom [123], Mexico [124], Sweden, Belgium, Germany [125], United States [126,127] and Australia [128,129].Most of these reports are related to resistance to ivermectin.Infection by Cooperia punctata can significantly impact productivity by reducing weight gain and decreasing feed intake [127].In addition, it influences phosphorus kinetics, reducing food intake and altering phosphorus absorption and retention [130].

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 [131].However, it is unsuitable for detecting resistance levels below 25% [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) [133].Other in vitro tests have been used less, such as the EHT, established to detect drug resistance in the benzimidazole class [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 [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 [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 [119].
With the limitations of current in vivo and in vitro resistance tests, molecular tools can potentially improve drug resistance diagnosis [138].The development of molecular diagnostics for anthelmintic resistance has been one of the leading research topics involving the molecular mechanisms of drug resistance [139].Thus, developing molecular markers for diagnosing resistance can help develop new anthelmintic drugs [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 [141].For example, in Brazil, some studies were conducted using the β-tubulin isotype gene, a marker to monitor resistance [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 [144,145].
A recent development in large-scale surveillance is the "Nemabiome" approach, which applies deep amplicon sequencing of barcoded PCR products [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 [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.

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 [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 [149].For example, a study with moxidectin demonstrated that this molecule may be present in goat milk for up to 21 days [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 [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 [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 [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 [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 [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 [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) [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 [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 [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 EC 50 and EC 90 (50% and 90% maximal effective concentration, respectively), which allows comparing the activities of different plants [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 [161]; (ii) strategies such as grazing rotation, co-grazing with other appropriate species and manure management are alternatives to reduce the use of anthelmintics [162]; (iii) the improvement of animal resistance through genetic selection to reduce the use of chemoprophylaxis [163]; and (iv) to optimize the effectiveness of anthelmintics in populations of multiresistant nematodes, drug combinations can be used [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 [164].Furthermore, it is crucial to use existing anthelmintics in a way that minimizes the impact of AR [165].

Conclusion Remarks and Perspective for Future Research
The present review demonstrates that AR is an urgent and emerging issue for ruminant production in Brazil, especially in the southern and northeastern regions, where most of the data discussed herein were produced.It is necessary to evaluate on a large scale the distribution and management of anthelmintic drugs currently available and discuss strategies to prevent the development of this phenomenon.Technological advances in diagnostic tools associated with individual management of animals with continuous monitoring are fundamental issues to better guide the control of GIN infections.Several challenges remain, and we hope to enter a new anthelmintic era, including innovative, integrated control approaches and more sensitive and cost-effective diagnostic tools.

Figure 1 .Figure 1 .
Figure 1.Geographic distribution of anthelmintic resistance according of the class of drugs used in ruminants from Brazil.From 1960 to 1999, 18 articles (21.7%) reported AR in domestic ruminants, with one in cattle (5.6%; 1/18), three in goats (16.7%; 3/18) and fourteen (77.8%; 15/18) in sheep.From 2000 to 2023, the number of reports tripled, with 65 articles (78.3%) distributed Figure 1.Geographic distribution of anthelmintic resistance according of the class of drugs used in ruminants from Brazil.

Figure 2 .
Figure 2. Geographic distribution of anthelmintic resistance according to published records in cattle, goats and sheep from Brazil.

Figure 2 .
Figure 2. Geographic distribution of anthelmintic resistance according to published records in cattle, goats and sheep from Brazil.

Table 1 .
Distribution of anthelmintic resistance in sheep from Brazil.

Table 2 .
Distribution of anthelmintic resistance in goats from Brazil.

Table 3 .
Distribution of anthelmintic resistance in cattle from Brazil.