Molecular Study of Benzimidazole Resistance in Teladorsagia circumcincta Isolated from Sheep in North of Iran.

Background: Resistance to benzimidazole (BZ) compounds is common in Teladorsagia circumcincta populations in sheep and goats worldwide. Given the importance of anthelmintic resistance and shortage of information on single nucleotide polymorphisms (SNPs) in this prevalent nematode in Iran, this study was conducted. Methods: From June to September 2016, abomasa of 139 sheep of different sexes and ages in Amol City slaughterhouse, northern Iran were examined for isolation of nematodes. Totally 45 male T. circumcincta confirmed by both microscopical and nested-PCR-RFLP methods were included in this study. Susceptibility or resistance of each single T. circumcincta worm to benzimidazoles was assessed using allele-specific PCR. Results: Frequency of genotypes in the present study were 33.33% heterozygote BZ and 66.67% BZ homozygote sensitive. No homozygote resistant worm was found. Conclusion: Resistance against BZs in T. circumcincta of sheep has occurred at a low prevalence in the north of Iran. However, mutated genes might get dominant under drug selection in future. Hence, periodic investigations for early detection of mutated alleles in nematode populations using accurate and sensitive molecular methods such as PCR-RFLP is recommended.


Introduction
astrointestinal parasitism is one of the most important causes of production losses in small ruminants (1).
Although anthelmintic therapy used to prevent and control such infections, this may lead to the development of resistance in parasitic helminths which is becoming a serious problem for the livestock industry worldwide (2). In fact, development of resistance in nematodes of veterinary importance is influenced by a number of host-related physiological and environmental factors but importantly can result from operational factors such as frequent treatment with the same group of anthelmintics or single-drug regimens, anthelmintics-underdosing and mass treatment (3). Moreover, global warming has been also associated with development of resistance in certain nematodes (4).
Other than morphological and physiological differences such as size and fertility in sensitive vs resistant T. circumcincta worms (13), genetic basis of resistance has attracted a lot of attention. BZ resistance in T. circumcincta has been associated with several independent single nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene at codons F167Y (14) and F200Y (15). However, resistance in other helminths such as Haemonchus contortus and Trichuris trichiura is correlated to one extra SNPs polymorphisms at codon E198A (16). Recently an additional polymorphism in substitution of glutamate for leucine (E198L) was found in T. circumcincta in Ireland albeit with a low frequency (17). This finding gives weight to the existence of additional determinants of resistance other than known mutations.
Benzimidazoles are used for more than 40 years as treatment or preventive chemotherapy of sheep nematodes in Iran. However, PCR-based studies performed on the status of BZ resistance in the nematode populations of sheep are limited to few reports on worms with unknown origin and/or unknown year of sampling (18)(19)(20)(21)(22).
Given the importance of anthelmintic resistance and shortage of information on SNP polymorphisms of T. circumcincta, the present study was conducted to evaluate benzimidazole resistance in this nematode isolated from sheep in north of Iran. Furthermore, a historical mini-review on the anthelmintic efficacy studies in Iran is presented in the discussion section and some key points for better planning of next studies are suggested.

Study area, isolation of worms and microscopic analysis
From June to September 2016, 139 sheep of both sexes (48 males and 91 females) and different ages (<6 months, n=7; 6 months-1 year, n=42; 1 year<, n=90) slaughtered at Amol abattoir, Mazandaran Province, north of Iran were randomly chosen. Abomasa of individual sheep were removed, put in plastic bags and transferred to the laboratory of Veterinary Office of Amol city under cold chain within hours. Recovered parasite specimens were collected in separate microtubes containing PBS and transferred to the Laboratory of Parasitology, Faculty of Veterinary Science, Bu-Ali Sina University of Hamedan, Iran. Adult male T. circumcincta worms were then specified according to morphological characters under light microscope (23) and stored in ethanol in separate microtubes at -20 °C until further analysis.

Molecular identification of nematodes
DNA extraction from 45 isolated male T. circumcincta confirmed by light microscopy was carried out using a commercial DNA extraction kit (Yekta Tajhiz Azma, Iran) according to the manufacturer's instruction. To confirm the identification of the nematodes, a previously described nested-PCR-RFLP method targeting isotype 1 β-tubulin gene was used (24). A negative control contained no DNA was used in all PCR runs. The nested-PCR products were digested using RsaI restriction enzyme to confirm the identities of isolated nematodes (24).

Allele-specific PCR
To determine the susceptibility or resistance of isolated T. circumcincta to benzimidazoles, a previously introduced allele-specific PCR was performed (25).

Results
The results of the nested-PCR reactions and RFLP analysis confirmed the identity of all nematodes as T. circumcincta.
Allele-specific PCRs showed that the BZ resistance allele was present in 15 (33.33%) out of 45 examined T. circumcincta. No homozygous resistant (BZ RR ) genotype was found. The other 30 nematodes were homozygous susceptible (BZ SS =66.67%). Table 1 shows resistance allele frequencies according to age and sex of the examined sheep.

Discussion
In the present study, resistance to benizimidazole compounds was examined in 45 T. circumcincta nematodes isolated from 139 sheep in north of Iran by the detection of alleles responsible for resistance.
The emergence of resistance against anthelmintic drugs is an important issue, several studies have been carried out in Iran. Anthelminthics efficacy examinations started as early as 1959 with testing lead arsenate in the treatment of ovine monieziosis (26). After that, efficacy of different compounds such as diethylcarbamazine citrate, emetine hydrochloride, thiabendazole, tetramisole, Lugol's Iodin, ivermectin, levamisole and albendazole against nematodes in naturally infected ruminants in Iran was evaluated (10)(11)(12)(27)(28)(29). In a chain of field studies, resistance of Teladorsagia, Tri-chostrongylus and Haemonchus to levamisole and albendazole was examined in sheep of Khuzestan Province, southwestern Iran. These studies included sheep flocks from all parts of the province including mountainous, hilly and plain areas. Resistance of T. circumcincta and T. vitrinus to levamisole was found in 66.67% of studied flocks (10). Resistance of T. circumcincta and Marshallagia marshalli to albendazole in 27% of studied flocks was also observed (11). Methods of aforementioned works were copromicroscopical.
PCR-based detection of resistance alleles in sheep nematodes against commonly used benzimidazoles in Iran started in 2007. However, the researchers mainly focused on methodology other than field studies (21,22), in some articles authors did not state year or area of sampling (18,19,21,22) and in one study researchers pooled worms from each study area and proceeded the PCR-RFLP on extracted DNA from the pools (20). The relationship between treatment background and β-tubulin gene polymorphism of H. contortus was studied in seven regions of Khuzestan Province and researchers found that restriction profiles of β-tubulin gene in H. contortus varied in sheep with different treatment backgrounds (20). BZ resistance of adult H. contortus isolated from abomasa of sheep and goats was investigated in Khuzestan, Isfahan and Mazandaran provinces using a PCR-RFLP method which targeted point mutations at codons 167, 198 and 200 (18,19). None of the tested specimens were found resistant.
In the present study, frequency of genotypes were 33.33% BZ RS and 66.67% BZ SS in T. circumcincta of north of Iran. Sensitive diagnostic approaches such as PCR-RFLP are crucial as if the BZ-resistant nematodes reach to medium level in natural population, no reversion is possible even if another anthelmintic agent without any cross-resistance is used (30). In the only previous PCR-based study on the BZ resistance in field isolates in Iran, adult T. circumcincta from 35 untreated and 40 albendazole-treated sheep were examined (22). BZ RR genotype was found in 5 worms (6.66%) collected from BZ-treated sheep. In the latter study, the year and area of isolation of worms were not stated in the article so comparison of the results of that work with ours is not possible. However, presence of mutated genes in BZ RR isolates even at a low frequency and heterozygote BZ RS isolates in this study and a previous study from Iran (31.11% and 82.66%) (22), can lead to dominancy under drug selection in future.
In this study, it was not possible to collect accurate information about history of anthelminthic therapy in the studied sheep brought to slaughterhouse. Although in a previous study one group of sheep was treated before, but the time interval of therapy-to-study was not stated clearly (22). Therefore, for future studies detailed information about treatment background of sample population might help in better interpretation of results.
In this study breeds and geographical origin of sheep that worms were isolated from were not recorded. Generally, the emergence rate of anthelminthic resistance varies geographically in accordance with climatic and environmental factors, parasite species and treatment regimens adopted in various regions (31). This is why fecal egg count reduction test (FECRT) is recommended for selecting the anthelmintic of choice in each region or even flock of livestock. Recently, polymorphism in DRB1 and DBR2 loci of the major histocompatibility complex, which plays a key role in immune responses, has been found to be associated with various gastrointestinal nematodes' fecal egg count including Teladorsagia in Ghezel sheep breed in Iran (32,33). Therefore, potential value of markers such as Ovar-DRB as indicator of parasite resistant sheep breeds should be taken into account in applied animal breeding programs. For future studies, studying correlations between developed resistance and breed of animals is suggested.
In Iran, T. circumcincta has been reported from various herbivorous species (6-9, 34). Cross-species transmission of gastrointestinal nematodes between domestic and wild animals occurs normally (35). Therefore, transmission of worms with resistance genes from/to susceptible livestock species is likely. Hence, mapping of resistant population of worms will be useful for quarantine legislation as animal movement is an important factor in distribution of drug resistant helminths (1).
Reports on resistance to major classes of anthelmintics such as the benzimidazoles, macrocyclic lactones and imidazothiazoles/tetrahydropyrimidines from all parts of the world are numerous. Discovery of a new class of anthelmintics, the amino-acetonitrile derivatives, in 2008 (36) gave a lot of hope but did not last long as there are growing evidence on failure of monepantel against T. circumcincta (37). However, advancement of diagnostic tools, development of vaccines, sustainable use of existing anthelminthics with application of tests like FAMACHA for selective therapy and improvement of control practices will help for better control of helminth infections by 2030 (38).

Conclusion
Resistance against BZs in T. circumcincta of sheep has occurred at a low prevalence in the north of Iran. However, periodic investigations for early detection of mutated alleles in nematode populations using accurate and sensitive molecular methods such as PCR-RFLP will be of great importance to maintain the efficacy of currently available anthelmintics and prevent emergence of resistance in Iran. Furthermore, molecular-based investigations on resistance to other major classes of antihelminthics in Iran are suggested.