The Nematoda phylum includes many helminth species parasitising humans and domestic and wild animals (23). Gastrointestinal nematodes (GIN) are globally distributed in small ruminants and negatively impact animal health and production. All grazing sheep are exposed to GIN infections, which are regarded as one of the most important ovine diseases worldwide and cause weight loss, diarrhoea, and death (7). Production losses result from the loss of appetite, tissue and organ damage, reduced feed conversion, and deaths of heavily infected animals. The amounts of weight gain and milk production in infected animals are 15% and 22% less than those of noninfected animals, respectively, and in addition, the cost of anthelmintic therapies indirectly impacts livestock productivity (19).
Different strategies are implemented for GIN control in domestic animals, but anthelmintic therapy is the most widespread method of helminth control globally (9). The most commonly used anthelmintics are benzimidazoles, macrocyclic lactones, or imidazothiazoles (1). These are broad-spectrum drugs with wide safety margins, but their extensive use has led to anthelmintic resistance (AR) development, which occurs when a drug’s recommended dosage cannot effectively treat the infected animal (8). The arising and spread of AR are accelerated by underdosing, repeated use of the same anthelmintic, and treatment of the whole flock (3).
Parasitic diseases and the emergence of AR are significant human health problems (25). This resistance is widespread in sheep-producing countries, is a persistent and growing issue in all parts of the world, and requires immediate attention (11). Several reports of AR development have been published in recent years, and it has become a global problem in sheep and goats in the last three decades (2, 22).
Successful helminth control strategies partially depend on accurately detecting AR in an area. However, several factors other than resistance may be responsible for anthelmintic therapy failure. For example, faulty flock drenching equipment may lead to livestock not being thoroughly dewormed. Anthelmintic underdosing due to inaccurate estimation of the animal’s weight or anthelmintic dose is another common cause of anthelmintic failure (24).
Various
One of the drawbacks of the FECRT is that it does not provide accurate information about the nematode species parasitising the tested animals and endowed with resistance. Some nematodes such as
In the Kurdistan Region, northern Iraq, the sheep population numbers more than three million head and is mainly raised on pasture grazing. Their farmers use anthelmintics to reduce the burden of GIN infection. The most commonly used dewormers are albendazole, ivermectin, and levamisole, administered
Arbat District is located 23 km southeast of the Sulaymaniyah Governorate, Kurdistan Region, northern Iraq. The district comprises 71 villages, and mainly sheep and cattle farming represents the primary income source. The sheep population is around 65,000, and the animals are kept in different size flocks and raised on free pasture throughout the year.
Farmers in Arbat District drench their sheep with anthelmintics once or twice a year to control GIN infections. Avermectins are the most commonly used, followed by imidazothiazoles and benzimidazoles. Veterinary practitioners reported the therapeutic inefficiency of antinematodal drugs in several herds after drenching the sheep with several anthelmintics. These incidences urged the carrying out of a standardised study to investigate the efficacy of the commonly used anthelmintics in this region.
Ten farms were visited to investigate their management practices. A questionnaire was distributed among the farmers, putting several questions about the number, age, and sex of the sheep on each farm, the date of previous treatment, and the type of anthelmintic used. Farms were included in the study by meeting qualifying criteria: keeping ≥ 40 sheep between the ages of six and eighteen months, grazing them on pasture, not having treated them with anthelmintics for the previous three months, and scoring a mean pre-treatment faecal egg count of ≥ 200 eggs per gram (EPG) for their animals.
Three farms met the selection criteria and were included in this study. The combined number of sheep raised on the studied farms was 3,623, accounting for about 5.6% of Arbat’s sheep population. The other farms were excluded for various reasons, such as carrying out treatment with anthelmintics in the previous 12 weeks, keeping too few animals of the required age, or the owners’ declining to participate in the study.
Forty sheep of both sexes, 6 to 18 months old, were selected from each farm, divided into four groups of 10, and tagged appropriately. The first group served as the control and was drenched with distilled water. Groups 2 to 4 were administered a single oral dose of albendazole, ivermectin, or levamisole, respectively.
The recommended anthelmintic dosages of 5.0 mg/kg of albendazole (Albenol-100), 0.2 mg/kg of ivermectin (Intermectin Drench), and 10.0 mg/kg of levamisole (Leva-200) were used. All drugs were produced by Interchemie (Venray, the Netherlands). Each animal’s dose was calculated according to its weight and administered using an automatic drenching gun. The study protocol was submitted to and approved by the Animal Care and Use Committee (ACUC) at the College of Veterinary Medicine, University of Sulaimani.
The FECRT determines an anthelmintic’s efficacy
Faecal egg count reduction percentages below 95% and/or lower 95% CI limits equal to or lower than 90% indicated resistance. If only one condition were met, resistance was suspected, and the herd was considered resistant if the FECR was < 95% and the lower CI was < 90% (4, 10).
The efficacies of albendazole, ivermectin and levamisole in the study region were compared statistically using a one-way analysis of variance (ANOVA), followed by Duncan’s
After incubation for one week, the third-stage larvae (L3s) were collected by the larvoscopic Baermann technique, previously described by Coles
Tests were performed in 96-well microtitre plates. Nematode eggs were recovered from the faeces by passage through sieves with 250 μm and 75 μm aperture sizes, followed by centrifugation on a two-step sucrose gradient (10% and 25% sucrose). The eggs were recovered from the top layer of the 25% sucrose and washed over a 25 μm sieve with water to remove residual sucrose.
The eggs were agitated gently in an 8.4 mg/L sodium hypochlorite solution for 12 min and subsequently washed with a large volume of water. The eggs had distilled water added in order that after the addition of amphotericin B (at a final concentration of 25.0 mg/mL) and tylosin tartare (at a final concentration of 800 μg/mL), 30 μL contained 50–60 eggs, and the solution was used immediately for larval development assays (6). Assay plates were prepared by adding 150 μL of nutrient broth to the wells of a 96-well plate. Egg solution in a 30 μL volume was added to each well.
The microtitre plates were put in a humid chamber to prevent drying and incubated overnight at 26°C. The next day, 2.0 μL of a growth medium was added to each well (14). The medium constituents were Earle’s salt solution (10% v/v), yeast extract (1% w/v), sodium bicarbonate (1 mM), and saline solution (0.9% sodium chloride w/v). The LDA used was a modified version of the method described initially by Taylor
The plates were incubated at 26°C for additional five days. After that, 10 μL of Lugol’s iodine solution was added to each well, and the content was put on a glass slide. The number of fully developed infective-stage larvae (L3) present in each well was counted. The percentage of L3 in the treatment wells was calculated by the formula 100 × (number of L3 in T/number of L3 in C), where T and C are the wells containing the test drugs and control wells, respectively. The IC50 of each anthelmintic was calculated using the Microsoft Excel 2016 program, and all tests were run in triplicate.
Percentage of third-stage larvae in the first 100 larvae isolated from a pooled post-treatment faecal sample of the control and treated groups
Farm | Pre-treatment FEC* (eggs/g) | Percentage of larvae by genus |
||
---|---|---|---|---|
1 | 764.1 ± 45.2 | 82.0 | 12.0 | 6.0 |
2 | 760.0 ± 48.1 | 76.0 | 15.0 | 9.0 |
3 | 604.2 ± 37.8 | 86.0 | 8.0 | 6.0 |
* Values represent mean pre-treatment FECs in 40 sheep ± SEM
Larval cultures of the post-treatment faecal samples revealed that
The ivermectin-resistant GINs were
Median inhibitory concentration (IC50) of anthelmintics preventive of nematode egg hatchability
Farm | IC50 |
||
---|---|---|---|
Albendazole (μg/mL) | Ivermectin (ng/mL) | Levamisole (μg/mL) | |
1 | 0.076 ± 0.004 | 8.030 ± 0.692 | 1.470 ± 0.090 |
2 | 0.071 ± 0.004 | 7.670 ± 0.622 | 1.450 ± 0.140 |
3 | 0.072 ± 0.007 | 8.200 ± 0.615 | 1.390 ± 0.120 |
Values represent the mean of three tests ± SEM
The median inhibitory concentrations for albendazole on the three farms were 0.076 ± 0.004 μg/mL, 0.071 ± 0.004 μg/mL, and 0.072 ± 0.007 μg/mL, respectively. This outcome indicated that albendazole effectively inhibited larval nematode development at the recommended concentrations.
Levamisole was also effective in inhibiting larval development, as a 3.0 μg/mL concentration inhibited approximately 82% of the larvae in all three farms. The IC50 of levamisole were 1.47 ± 0.09 μg/mL, 1.45 ± 0.14 μg/mL, and 1.39 ± 0.12 μg/mL for the three farms. These results showed that the nematode larvae were susceptible to levamisole’s inhibitory effects at the standard concentrations.
Ivermectin did not effectively inhibit larval developments at the recommended concentration of 3.5 ng/mL, and only at a concentration as high as 31.5 ng/mL did it inhibit larvae, doing so for about 79%. Ivermectin’s IC50 values were 8.03 ± 0.69 ng/mL, 7.67 ± 0.62 ng/mL, and 8.20 ± 0.62 ng/mL, respectively.
The LDA results showed that albendazole was the most effective anthelmintic drug at inhibiting nematode larval development at the prescribed concentration. Levamisole inhibited larval progress to L3 at concentrations slightly higher than the recommendations. Ivermectin, in contrast, was ineffective in inhibiting larvae at the recommended concentration, and the IC50 was about 2.3 times as high as that value.
Anthelmintic resistance is an increasing problem of small ruminant production worldwide that may render currently available drugs inefficient in the future, and accordingly, the development of control strategies has become a necessity (13). Implementing a suitable AR control program starts with identifying the common GINs and determining the extent of resistance in an area (15). In the north of Iraq, where the livestock industry is the key agricultural sector, GIN control is highly important, and investigating how AR is distributed among GIN species is required to improve the control strategy.
In the study area, GIN control is primarily dependent on anthelmintic treatment. This study investigated AR in Arbat District after reports of anthelmintic failure on several farms in the area. FECRT, considered the most reliable method of testing an anthelmintic drug’s utility under field conditions (18), was used to elucidate the efficacy of albendazole, ivermectin, and levamisole
The FECRT showed that ivermectin was the least efficacious anthelmintic in this study, while albendazole was the most effective. The questionnaire answers revealed that the drugs most commonly used by farmers in the study area were ivermectin, levamisole and albendazole, in order of frequency of implementation. Frequent use of an anthelmintic over an extended period usually provokes AR development (21). Ivermectin is an endectocide often used to control external arthropods and internal nematode parasites, which may justify its frequent use by sheep farmers. The drug’s frequent use might be the reason why it was the least effective anthelmintic.
One of the FECRT disadvantages is that the results’ accuracy is affected by such factors as underdosing. The tested drugs were administered to each animal to overcome this problem using recommended dosage rates based on each sheep’s weight. Therefore, the AR reported by the FECRT cannot be a result of deficient dose administration and must only be due to the emergence of resistant GINs. Furthermore, the FECRT results were confirmed using the
The LDA was conducted to determine the drugs’ IC50 values, and the results indicated that AR against ivermectin had developed on all three farms. The drug’s IC50 ranged between 7.67 and 8.20 ng/mL, while previous studies reported IC50 of 0.23 ng/mL (16, 20), meaning that ivermectin was only 3% as potent in the present study. In a previous study, the IC50 of levamisole on
In conclusion, this study is the first report which reveals AR occurrence in sheep in Arbat District, northern Iraq, confirmed by FECRT and LDA. Resistance to ivermectin was evident and was principally due to the emergence of resistant