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Anthelmintic resistance in goat herds—In vivo versus in vitro detection methods
Accepted Manuscript Title: Anthelmintic resistance in goat herds – in vivo versus in vitro detection methods Authors: M. Babj´ak, A. K¨onigov´a, M. Urda Dolinsk´a, J. Vadlejch, M. V´arady PII: DOI: Reference:
S0304-4017(18)30086-4 https://doi.org/10.1016/j.vetpar.2018.02.036 VETPAR 8620
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
1-1-2018 21-2-2018 21-2-2018
Please cite this article as: Babj´ak M, K¨onigov´a A, Urda Dolinsk´a M, Vadlejch J, V´arady M, Anthelmintic resistance in goat herds – in vivo versus in vitro detection methods, Veterinary Parasitology (2010), https://doi.org/10.1016/j.vetpar.2018.02.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Original Article
Anthelmintic resistance in goat herds – in vivo versus in vitro detection methods
a
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M. Babják a, A. Königová a, M. Urda Dolinská a, J. Vadlejch b, M. Várady a,*
Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 040 01, Košice, Slovak
b
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Republic
Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural
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Resources, Czech University of Life Sciences Prague, Kamýcká 957, 165 21 Prague 6–
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Suchdol, Czech Republic
A. Königová ([email protected])
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M. Babják ([email protected])
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Urda-Dolinská ([email protected])
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J. Vadlejch ([email protected])
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*Corresponding author. Tel.: ++421 55 6334455.
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E-mail address: [email protected] (M. Várady).
Highlights:
The first survey of benzimidazole resistance on Slovak goat farms was conducted
High prevalence of resistant parasites were confirmed
Using double doses of treatment may underestimate the level of resistance 1
The in vitro tests provided reasonable identification of low levels of resistance
Abstract Anthelmintic resistance (AR) is a serious threat to animal health and has a major economic
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impact worldwide due to production and financial losses. The aim of this study was to
determine the occurrence of AR on 30 goat farms in Slovakia during the pasturing seasons
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and to compare three widely used in vitro and in vivo methods for detecting AR in field
conditions. A three-year survey was conducted during the pasturing seasons of 2014–2016.
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Goats on each farm were split into treated and control groups and were treated by
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recommended (5 mg/kg body weight) and double doses (10 mg/kg b.w.) of albendazole.
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Comparisons between percent reduction in a faecal egg count reduction test (FECRT) and an
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egg hatch test (EHT) and the presence of L3 larvae in a larval development test (LDT) using resistant concentrations of benzimidazole (BZ) were monitored after treatment. The FECRT
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indicated percent reductions of 69.2-86.2% for the single dose and of 36.3–45.4% for the double dose. The EHT indicated that all farms had BZ-resistant nematodes. Low (< 15%
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hatching) and high (> 15% hatching) levels of resistance were detected on 13 and 17 farms,
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respectively. The LDT failed to detect resistant larvae on seven farms but detected low and high levels of resistance on seven and 14 farms, respectively. The data indicate a moderate correlation between in vitro and in vivo tests for detecting BZ resistance among the 30 goat
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farms. The hatching detected by the EHT and the presence of L3 larvae by the LDT at resistant BZ concentrations provided reasonable identification of low levels of resistance in the parasite populations, but the use of a double dose for a treatment may underestimate the real occurrence of low levels of resistant parasites on goat farms.
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Keywords: Anthelmintics; Benzimidazole; Goats; Resistance; Detection method
1. Introduction Gastrointestinal (GI) nematodes cause the most prevalent and economically important
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diseases of small ruminants. The constant repetition of anthelmintic treatment on many farms is still the first option for prophylaxis rather than finding alternative methods to limit GI
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parasitism and finding solutions to improve control strategies, including mainly grazing management. Anthelmintic resistance (AR) is common throughout the world. The
development of AR is a genetic process where nematodes with resistance alleles survive
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treatment, thereby increasing the frequency of resistant alleles in worm populations after
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repeated application of an anthelmintic. Treatment thus serves as a selector of resistant alleles
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(Wolstenholme et al., 2004). Many factors such as underdosing, using the same group of
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anthelmintics for a long time, frequent treatment, or lack of quarantine are responsible for
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accelerating the development of AR (Torres-Acosta and Hoste, 2008). Benzimidazoles (BZs) and macrocyclic lactones (MLs) are the most frequently used groups of anthelmintics on
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Slovak farms with small ruminants (Čerňanská et al., 2008).
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Surveys of the occurrence of AR in Slovakia, however, have only been conducted on sheep farms. The occurrence of BZ resistance was studied in 1991-1993 (Várady and Praslička, 1993) and 2003-2004 (Čerňanská et al., 2006); ineffective treatment was first
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documented on six of 77 farms (7.8%) and a decade later on two of 29 farms (6.9%). The extent of ivermectin (IVM, an ML) resistance was also confirmed twice in 2003-2004 on eight of 26 sheep farms (30.8%) (Čerňanská et al., 2006) and in 2014 on 14 of 49 farms (28.5%) (Dolinská et al., 2014). These results indicated that the status of BZ and IVM resistance on sheep farms had not changed during the respective decades. The occurrence of
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AR on sheep farms is well known, but no data are available from goat farms, despite an annual increase in goat numbers in Slovakia and an increased demand for goat-milk products. Goats are often farmed together with sheep, so farmers usually use the same farming techniques for both species. This practice is the most common managerial flaw, especially using the same anthelmintic doses, which is a serious threat for the development of AR on a
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farm. Goats have a higher metabolic activity than sheep and thus require higher dose rates for effective treatment (Veneziano, 2004). Using the sheep dose for both species leads to the
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selection of resistant parasites in goats, so resistance can then be transferred to sheep (Charles et al., 1989).
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FECRT is recommended by the World Association for the Advancement of Veterinary
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Parasitology (WAAVP) as the test of choice, especially in surveys for resistance. The test,
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however, is time-consuming and expensive, inter-animal variation is high, and the
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pharmacokinetics of the drug in the host may lead to data of poor quality. Interest has therefore tended towards in vitro tests such as the egg hatch test (EHT) and the larval
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development test (LDT), which are faster and cheaper to perform and are therefore more
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suitable for large surveys.
The main goal of this study was to conduct the first survey of occurrence of BZ resistance
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on Slovak goat farms. A secondary goal was to compare the data obtained from in vivo and in vitro tests to identify the simplest and most effective tool for estimating susceptibility/resistance in vitro. Other goals were to examine the morphology of resistant
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larvae and to survey the practices used on the farms.
2. Materials and methods
2.1. Selection of farms and sampling 4
Animal use and study design were approved by the Ethics Committee of the Institute of Parasitology of the Slovak Academy of Sciences in accordance with the national legislation in Slovakia - Animal Welfare Act No. 23/2009. Permission to collect study samples was granted by participating small ruminant farms.
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Farms were selected from the central register of livestock in Slovakia. A minimum of 24 goats per farm was a necessary requirement in the survey. Thirty farms distributed throughout the country were selected. Faecal samples from the 30 herds of dairy goats were collected
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directly from the rectum from September 2014 to December 2016. The animals had not been treated for GI nematodes for eight weeks prior to sampling. Individual counts of eggs per
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gram (EPG) for each goat were determined by a modified McMaster technique with a
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sensitivity of 50 EPG (Coles et al., 1992).
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2.2. FECRT
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The FECRT was conducted following the WAAVP recommendation (Coles et al., 1992, 2006). Goats on each farm were split into treated and control groups. The goats were treated
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with the ovine recommended dose of albendazole (ABZ) (5 mg/kg body weight), and if
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sufficient animals were available another group was treated at twice the recommended dose rates of sheep (10 mg/kg body weight). The minimum number of goats in each group was 12. Animals in each group were resampled 7-10 days after treatment. The percent reduction in
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helminth eggs (%FECR) was calculated using three methods. First method (Dash et al., 1988): %FECR=100×(1−[T2/T1][C1/C2])
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where T1 and T2 are arithmetic means of the treated group before and after treatment, respectively, and C1 and C2 are arithmetic means of the control group sampled at the same time as the treated group, respectively. Second method (Kochapakdee, 1995):
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%FECR=100×(1−[T2/T1]) where T1 and T2 are arithmetic means of the treated group before and after treatment,
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respectively, with no control group. Cabaret and Berrag (2004):
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%FECR=(1/n)∑(100×(1−[Ti2/Ti1])
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where Ti1 and Ti2 are pre- and post-treatment EPGs, respectively, in host i from a total of
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n hosts. The parasite population was considered resistant if %FECR was < 95% (McKenna,
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1990).
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2.3. EHT
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The EHT was performed following Coles et al. (1992, 2006). Eggs were stored anaerobically as described by Hunt and Taylor (1989). Suspensions of helminth eggs were prepared from pooled samples from each farm. Five concentrations (0.05, 0.1, 0.3, 0.5 and 1
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µg/mL of thiabendazole (TBZ) in dimethyl sulfoxide (DMSO) were added to 24-well plates containing the egg suspensions (100 eggs/mL). One well in each plate row containing 10 µL of DMSO instead of TBZ was used as a control. Each concentration of TBZ was tested twice for each farm. The plates were incubated at 27 °C for 48 h, and 10 µL of an iodine solution (1g of iodine and 2 g of potassium iodide in 100 mL distilled water) was then added to each
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well. The proportion of hatched eggs was recorded in each well. The concentrations of TBZ required to inhibit 50 and 99% of the eggs from hatching (ED50/ED99) were determined.
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2.4. LDT
Helminth eggs recovered from faeces were incubated for seven days in 96-well microtitre
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plates with culture medium (yeast extract with Earle's Balanced Salt Solution and physiologic salt solution) in an aquatic solution of various concentrations (range from 0.0006 to 1.28 µg/mL) of TBZ (Coles et al., 2006). The proportions of eggs and L1, L2 and L3 larvae were
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determined for each well. The concentrations of TBZ that inhibited development to the L3
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stage by 50 and 99% (LD50/LD99) were determined for each farm.
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2.5. Morphological identification of resistant species in the LDT
Infectious third-stage larvae (L3) in the wells with high concentrations of TBZ were
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isolated, and the resistant species were identified as described by Van Wyk et al. (2013). Each
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L3 was examined for morphological features and its body length was measured.
2.6. Questionnaire survey
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All cooperating farmers were requested to provide data about their farms. Most of the
questions were about pastures (size and possibility of rotation), size and composition of goat herds, frequency of anthelmintic treatment, treatment strategy, choice of drugs, and knowledge about AR. Farmers answered by e-mail or by personal contact during visits to the farms. 7
2.7. Data analysis
The data were analysed by a statistical logistic regression model to determine ED50 and
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ED99 in the EHTs and LD50 and LD99 in the LDTs.
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3. Results
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3.1. Prevalence study
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A total of 996 samples from 30 farms were examined by a modified McMaster technique
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(Coles et al., 1992). The prevalence of strongyle eggs was 91.80%. Strongyloides papillosus
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(3.66%), and Moniezia spp. (2.44%).
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(12.90%) was the dominant species followed by Trichuris spp. (7.22%), Nematodirus spp.
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3.2. FECRT
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All three calculation methods were used for 26 of 29 farms (Table 1). We were able to apply single and double doses of ABZ on 22 of the farms. The remaining farms did not have enough animals to apply a double dose or to form a control group. The Dash et al. (1988)
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method identified BZ resistance on 18 of 26 farms (69.23%) using a single dose of ABZ and on seven farms (38.88 %) using a double dose. The Kochapakdee (1995) method identified BZ resistance on 21 of 29 farms (72.41%) using a single dose and on 8 of 22 farms (36.36%) using a double dose. The Cabaret and Berrag (2004) method identified BZ resistance on 25 of 29 farms (86.20%) using a single dose and on 10 of 22 farms (45.45%) using a double dose.
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Resistant parasites were thus detected by one or more methods on 26 of the 29 farms (89.65%).
3.3. EHT
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The threshold ED50 detected AR on 15 of the 30 farms (50.0%). ED50 ranged from 0.02 to 1.05 µg/mL. AR was also detected by FECRT on these 15 farms. The percent egg hatch at 0.1
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µg/mL TBZ ranged from 47 to 91% on farms with resistance. The percent egg hatch in the negative controls for all farms was > 95%. Comparison between %FECR and percent egg hatch at 0.3 µg/mL TBZ is shown in Table 2. Table 3 shows percent hatching at the higher
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concentrations of TBZ. The percent egg hatch at 0.1 µg/mL TBZ for the 15 farms where ED50
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was below the threshold of 0.1 µg/mL ranged from 5.5 to 38.5%. The percent egg hatch at 0.3
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µg/mL TBZ ranged between 0 and 27.50%. The percent egg hatch in 0.3 µg/mL TBZ for
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farms where ED50 was > 0.1 µg/mL ranged from 13 to 86%. These results indicate the
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presence of resistant parasites on these farms and the probability for developing more AR in the coming years. %FECR at a single dose (Cabaret and Berrag, 2004) and hatching at 0.3
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µg/mL TBZ in the EHT are compared in Table 3. Evidence of decreasing drug efficacy with
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more hatched eggs is clear.
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3.4. LDT
The proportions of eggs and L1, L2, and L3 larvae were determined for each concentration
of TBZ. A farm was considered as harbouring resistance if infectious L3 larvae were observed at ≥ 0.08 µg/mL TBZ. Infectious larvae were found at these concentrations on 20 of 28 farms (71.42%). LD50 and LD99 were determined for each farm where test has been performed. The
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thresholds of 0.01 µg/mL for LD50 and 0.02 µg/mL for LD99 identified resistant parasites on all farms. The numbers of L3 larvae at the higher concentrations of TBZ and LD50 and LD99 are presented in Table 4. %FECR at a single dose (Cabaret and Berrag, 2004) and the presence of L3 at ≥ 0.08 µg/mL TBZ in the LDT are compared in Table 5. Evidence of
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3.5. Morphological identification of resistant species in the LDT
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decreasing drug efficacy with more L3 larvae is clear.
Infectious L3 larvae were isolated from wells with concentrations of 0.08-1.28 µg/mL TBZ. L3 larvae were present at these concentrations on 18 farms. Haemonchus contortus was
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the dominant species (50.92%) followed by Trichostrongylus spp. (39.84%) and Ostertagia
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3.6. Questionnaire survey
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spp. (9.24%).
The size of the herds varied from 30 to 150 goats, with an average of 65 goats. Ninety
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percent of the farms were conventional, with milk production, meat production, or both. Two farms had hobby herds, and one farm was registered as organic. White and Brown short-
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haired goats were the most common breeds, followed by Anglo-Nubian goats and Alpine goats. Farmers treated their goats with anthelmintics twice a year on 24 farms (80%), and
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anthelmintics were never used on three farms. Two farms treated more than twice a year, and one farm used an anthelmintic treatment every time signs of disease (diarrhoea, weight loss) were noticed. All farmers who treated twice a year used BZ in spring and ML in autumn. Two farms treated only with BZ. Farmers on each farm where treatment was applied treated all animals in the herd. Twenty-four farms (80%) used pasture rotation; the other farms could not
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rotate pastures regularly. Goats on 25 farms (83.33%) grazed together with sheep. The farmers had no information about goat parasites or the presence of parasites on their farms. Three farmers (10%) suspected insufficient drug efficacy. Only half of farmers had knowledge about AR in gastrointestinal parasites of small ruminants. A double dose of anthelmintics recommended for goats was never used on any of the farms. Farmers were
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asked about the quarantine of introduced animals; new animals were treated before
introduction on most farms (25 farms, 83.33%), but the efficacy of drugs and the composition
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of parasite fauna had not been determined on any of the farms.
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4. Discussion
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AR is believed to be more frequent globally in parasites of goats than in parasites of
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sheep. The results of our study confirmed that BZ resistance was widespread on the farms that
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were examined. Our study represents the first national survey of the prevalence of BZ-
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resistant nematodes on goat farms in Slovakia. Such a high prevalence of BZ resistance was unexpected, particularly because previous studies of sheep reported a maximum BZ resistance
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of 10% (Praslička et al., 1994; Čerňanská et al., 2006), which may be due to two factors. Firstly, the majority of the goat farms had mixed grazing with sheep, which could accelerate
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the development of AR. Secondly, the farmers did not know about the higher dose requirement of BZ treatment for goats, and all had been applying the dose recommended for
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sheep. The results of our survey indicated a lack of knowledge about strategies of worm control and the problem of AR among Slovak goat farmers. The alternation of anthelmintic classes was adopted on 80% of farms where BZs were applied in spring and presumably MLs in autumn, which suggests that drenching frequency did not play an important role in the development of AR in the Slovak goat herds.
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The FECRT was assessed by three methods with different evaluation protocols. The results of the FECRT can be influenced by various factors such as environmental conditions, level of infection in the herd, and level of efficacy of the test anthelmintic (Cabaret and Berrag, 2004). %FECRs by single and double doses for all three methods were compared with the percent hatch for the EHT. The method based on individual evaluation of EPG (Cabaret
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and Berrag, 2004) was more accurate than the methods based on the arithmetic means of
treated and control groups (Dash et al., 1988) or the arithmetic mean of treated groups without
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a control (Kochapakdee, 1995). The interpretation of FECRT results can be difficult for farms with %FECRs near the threshold (Coles, 2005; Presidente, 1985). EPGs can be also influenced by compound infections with strongyles with high (H. contortus) or lower
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(Trichostrongylus spp., Teladorsagia circumcincta) fecundity and by the composition of
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resistant and susceptible parasites on a farm (Holm et al., 2014).
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Comparing the prevalence of BZ resistance using single and double doses of BZ identified notifiable differences. The prevalence decreased by 36.36 - 45.45% when the
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prevalence was calculated for a double dose of BZ. Orally administered drugs within the BZ, imidazothiazole/tetrahydropyrimidine, and ML (Chartier et al., 2001) classes are more rapidly
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cleared from the blood in goats than sheep (Bogan et al., 1987; Galtier et al., 1981; Gillham
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and Obendorf, 1985; Hennessy et al., 1993a,b; Sangster et al., 1991), so goats require higher dose rates. Our study, however, suggests that the real prevalence of resistance may be underestimated when using a double dose in FECRTs. In vitro hatching in the EHT at higher
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“resistant” concentrations has been previously reported as an excellent marker for diagnosing resistance in H. contortus (Várady et al., 2007). This parameter was able to provide early detection during the development of resistance in our study, especially if the alleles for resistance were rare in the parasite population (Čudeková et al., 2010).
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ED50 from the EHT identified the presence of AR on 15 of the 30 farms (50%) using a threshold of 0.1 µg/mL TBZ. Hatching on the other 15 farms ranged from 5.5 to 38.5% for an ED50 < 0.1 µg/mL and from 0 to 27.50% for an ED50 of 0.3 µg/mL TBZ. These results indicate the presence of almost 30% resistant parasites on some farms that had been identified as susceptible using ED50. Von Samson-Himmelstjerna et al. (2009) recommended decreasing
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the threshold to 0.05 µg/mL TBZ when H. contortus is present on a farm. A previous study
(Babják et al., 2017) reported that H. contortus was present on 23 of 30 farms (76.6%) before
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treatment. If we had decreased the threshold to 0.05 µg/mL, the EHT would have detected AR on 26 of the 30 farms, which would be in agreement with results obtained from the FECRT from the single-dose groups evaluated by the Cabaret and Berrag (2004) method. H. contortus
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represented 50.92% of the 549 L3 larvae in the wells of the LDT at the higher concentrations
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of TBZ. We detected resistance in this species on every farm where it occurred. The
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phenotype–genotype correlation for BZ resistance in the EHT among multispecies infection
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under field conditions have not been described, but our data indicated that using hatching
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criteria was probably more sensitive than the double dose in the FECRT for estimating the prevalence of resistant individuals in the parasite population.
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The majority of in vitro tests are not considered suitable for use in field screening
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surveys, so only the EHT and LDT performed well enough to identify widespread AR (Várady et al., 2006). We have previously discussed the advantages and disadvantages of both tests for surveying AR in sheep parasites (Várady et al., 2006; Dolinská et al., 2012). When
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the level of resistance is high, both tests can effectively detect resistance, but a delineation dose (DD) or hatching percentage in DD is more sensitive than ED50/LD50 for detecting smaller proportions of resistant worms in a population. The development of L3 larvae at “resistant” concentrations (≥ 0.08 µg/mL TBZ) could serve as a marker for the evaluation of the resistant part of the population, similar to the EHT. 13
5. Conclusion
In conclusion, the results of our study confirmed the high prevalence of BZ resistance on Slovak goat farms. The high incidence of resistant H. contortus is currently a serious problem,
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and the lack of knowledge of the mechanism of AR development, the epidemiology of
gastrointestinal parasites, and good farming management will pose a more serious threat in
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the future. BZ anthelmintics are the most commonly used drugs on Slovak farms, so drugs
from other classes might be more effective against GI parasites in goats. Further research is needed to detect resistance in goats to drugs from other classes, and continuing to monitor and
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control the spread of BZ resistance in Slovakia is necessary. Using double doses of BZs for
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treating goats is probably not optimal, because it cannot detect low levels of resistance. The
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evaluation of hatching percentage in the EHT and the presence of L3 larvae in the LDT at the
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resistance in a population.
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“resistant” concentrations is recommended to avoid misdiagnosing AR at low levels of BZ
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Conflict of interest statement
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The authors declare that they have no conflicts of interest.
Acknowledgements
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This study was supported by funds from the Slovak Research and Development Agency
APVV 14-0169 and Scientific grant agency VEGA 2/0120/16. We thank S. Spišáková and M. Krčmárik for their technical assistance. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 14
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Haemonchus contortus and Trichostrongylus colubriformis. J. Vet. Pharmacol. Ther. 16,
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Holm, S.A., Sörensen, C.R.L., Thamsborg, S.M., Enemark, H.L., 2014. Gastrointestinal nematodes and anthelmintic resistance in Danish goat herds. Parasite 21, 1–10. https://doi.org/10.1051/parasite/2014038
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Hunt, K.R., Taylor, M.A., 1989. Use of the egg hatch assay on sheep faecal samples for the
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Kochapakdee, S., Pandey, V.S., Pralomkarm, W., Choldumrongkul, S., Ngampongsai, W.N.,
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Lawpetchara, A., 1995. Anthelmintic resistance in goat in southern Thailand. Vet. Record 137, 124–125. https://dx.doi.org/10.1136/vr.137.5.124
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McKenna, P.B., 1990. The detection of anthelmintic resistance by the faecal egg count reduction test: an examination of some of the factors affecting performance and
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interpretation. N. Z. Vet. J. 38, 142–147. https://doi.org/10.1080/00480169.1990.35640
Praslička, J., Várady, M., Čorba, J., Veselý, L., 1994. A survey of anthelmintic resistance in
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Slovakia. Vet. Parasitol. 52, 169–171. https://doi.org/10.1016/0304-4017(94)90048-5
Presidente, P.J.A., 1985. Methods for Detection of Nematode Resistance to Anthelmintic. In: CSIRO Publishing, Melbourne, pp.13–28.
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Sangster, N.C., Rickard, J.M., Hennessy, D.R., Steel, J.W., Collins, G.H., 1991. Disposition of oxfendazole in goats and efficacy compared with sheep. Res. Vet. Sci. 51, 258–263. https://doi.org/10.1016/0034-5288(91)90074-X Torres-Acosta, J.F.J., Hoste, H., 2008. Alternative or improved methods to limit gastrointestinal parasitism in grazing sheep and goats. Small Rum. Res. 77, 159–173.
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https://doi.org/10.1016/j.smallrumres.2008.03.009
Urda Dolinská, M., Königová, A., Babják, M., Várady, M., 2016. Comparison of two in vitro
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methods for the detection of ivermectin resistance in Haemonchus contortus in sheep. Helminthologia 53, 120–125. https://doi.org/ 10.1515/helmin-2015-0002
Van Wyk, J.A., Mayhew, E., 2013. Morphological identification of parasitic nematode
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infective larvae of small ruminants and cattle: a practical lab guide. Onderstepoort J Vet
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in Slovakia. Veterinářství 4, 142.
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Várady, M., Praslička, J., 1993. The occurrence of gastrointestinal nematodes in sheep farms
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Várady, M., Čerňanská, D., Čorba, J., 2006. Use of two in vitro methods for the detection of anthelmintic resistant nematode parasites on Slovak sheep farms. Vet. Parasitol. 135,
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325–331. https://doi.org/10.1016/j.vetpar.2005.10.006 Várady, M., Čudeková, P., Čorba, J., 2007. In vitro detection of benzimidazole resistance in
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Haemonchus contortus: Egg hatch test versus larval development test. Vet. Parasitol. 149, 104–110. https://doi.org/10.1016/j.vetpar.2007.07.011
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Veneziano, V., 2004. Control of gastrointestinal strongyles in goats. Il controllo delle strongilosi gastro-intestinali dei caprini. Parassitologia 46, 245–250.
Von Samson-Himmelstjerna, G., Walsh, T.K., Donnan, A.A., Carrière, S., Jackson, F., Skuce, P.J., Rohn, K., Wolstenholme, A.J., 2009. Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and
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pyrosequencing. Parasitology 136, 349–358. https://doi.org/10.1017/S003118200800543X Wolstenholme, A., Fairweather, I., Prichard, R., Von Samson-Himmelstjerna, G., Sangster, N.C., 2004. Drug resistance in veterinary helminths. Trends Parasitol. 20, 469–476.
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PT
ED
M
A
N
U
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https://doi.org/10.1016/j.pt.2004.07.010
19
Table 1 %FECR using the three calculation methods. Farm
Cabaret and Berrag
number
(2004)
Kochapakdee (1995)
Dash et al. (1988)
Double
Single
Double
Single
Double
dose
dose
dose
dose
dose
dose
1
67.5
-----
62.3
-----
76.2
-----
2
84.9
-----
85.7
-----
88.8
-----
3
-----
-----
-----
-----
-----
4
70.9
-----
76.2
-----
83.6
5
75.0
-----
73.9
-----
-----
6
93.1
-----
92.2
-----
7
91.5
-----
87.2
-----
8
93.6
98.4
95.4
97.8
9
53.2
66.1
45.0
10
66.3
65.0
55.9
11
67.4
82.7
74.6
12
100
100
13
91.9
98.9
14
100
100
15
97.5
16
89.6
---------
86.7
-----
94.5
97.3
54.0
69.6
76.9
77.9
88.4
82.4
50.0
62.3
100
100
100
100
94.0
99.0
94.0
99.0
100
100
100
100
98.2
97.1
98.5
97.5
97.7
98.2
88.0
98.1
88.1
98.1
U
-----
M
SC R
-----
90.8
N
63.6
A
ED
PT
17
IP T
Single
94.8
99.2
92.7
99.1
88.4
98.6
92.4
100
98.1
100
97.7
100
90.8
90.9
90.7
91.7
90.5
91.1
20
89.9
98.5
91.6
98.2
89.9
98.0
21
92.1
93.9
92.8
96.6
92.5
96.7
22
93.9
-----
97.2
-----
97.5
-----
23
93.0
93.8
93.5
95.2
95.6
97.0
24
82.4
86.8
85.7
91.5
83.5
88.1
25
92.3
98.0
91.6
98.0
-----
-----
26
65.4
70.8
63.7
69.5
63.5
69.1
27
91.5
97.0
94.0
97.3
98.2
99.2
18
A
CC E
19
20
41.5
66.3
13.8
62.4
49.6
65.3
29
90.4
99.6
92.2
97.5
-----
-----
30
81.3
93.8
89.8
92.4
-----
-----
A
CC E
PT
ED
M
A
N
U
SC R
IP T
28
21
Table 2 Comparison between % FECR at a single dose (5 mg/kg) and % hatching at 0.3
n
% Hatching (mean ± SD)
95-100
3
1.8 ± 2.0
90-95
13
14.6 ± 12.4
80-90
5
25.5 ± 20.4
40-80
8
70.6 ± 17.2
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%FECR
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µg/mL TBZ in the EHT.
A
CC E
PT
ED
M
A
N
U
SD, standard deviation.
22
Table 3 Comparison between ED50, ED99, and hatching (%) at 0.1 and 0.3 µg/mL TBZ in the EHT. Farm
ED50(µg/mL)
ED99 (µg/mL)
Hatching at 0.1
Hatching at 0.3
(µg/mL)
(µg/mL)
0.64
3.06
88.50
86.00
2
0.04
0.34
5.50
4.50
3
0.07
0.29
28.50
4
0.60
2.65
76.50
5
0.76
3.06
88.00
80.50
6
0.05
0.54
13.50
5.50
7
0.04
0.27
8
0.11
1.25
9
0.70
7.24
10
1.05
11
0.30
12
0.04
12.00
U
SC R
75.50
2.50
51.00
17.50
70.00
73.00
87.00
84.50
2.15
85.50
49.50
0.23
10.50
1.50
0.24
17.00
2.00
A
N
7.00
M ED
27.70
PT 0.06
CC E
13
IP T
1
0.05
0.21
12.00
0.00
15
0.06
0.32
9.00
4.00
16
0.05
0.31
17.50
5.50
17
0.05
0.37
15.00
6.50
18
0.06
0.61
21.00
11.00
19
0.22
11.00
69.00
57.00
A
14
23
0.10
3.20
48.00
36.00
21
0.10
4.06
47.00
38.00
22
0.20
2.20
75.00
35.00
23
0.08
9.10
38.50
31.50
24
0.17
4.60
56.50
53.00
25
0.10
2.68
55.50
13.00
26
0.45
2.10
91.00
27
0.02
1.50
12.00
11.00
28
0.20
11.00
55.50
38.50
29
0.05
4.76
11.50
11.00
30
0.05
12.40
32.00
27.50
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20
A
CC E
PT
ED
M
A
N
U
SC R
77.00
24
Table 4 Results of the LDT for 28 goat farms in Slovakia. Number of L3 larvae at ≥
Farm
LD50 (µg/mL)
LD99 (µg/mL)
0.08 µg/mL TBZ 53
0.396
8.854
2
4
0.018
0.377
3
0
0.018
0.022
4
33
0.209
1.490
5
56
0.448
6
1
0.042
7
0
0.015
8
0
0.010
0.064
9
69
0.380
17.020
10
62
0.300
4.020
11
75
12
0
13
0
14
0
15 16
25.660 0.104
U
SC R
0.029
6.640
0.043
0.073
0.013
0.030
0.023
0.033
1
0.011
0.062
8
0.020
0.122
2
0.021
0.742
-----
-----
-----
-----
-----
-----
10
0.054
6.340
35
0.045
1.600
22
30
0.125
1.200
23
6
0.018
1.510
24
13
0.022
3.660
25
13
0.029
3.490
26
27
0.529
4.050
27
1
0.018
1.624
28
21
0.160
10.250
29
0
0.025
0.260
20
CC E
21
PT
18 19
A
N
0.064
M
ED
17
A
IP T
1
25
A ED
PT
CC E
IP T
SC R
U
N
A
M
30 29 0.084 6.920
26
Table 5 Comparison between %FECR at a single dose (5 mg/kg) and the presence of L3 at ≥
n
Number of L3 (mean ± SD)
95-100
3
0.33 ± 0.57
90-95
11
8.0 ± 12.77
80-90
5
12.8 ± 9.62
40-80
8
49.5 ± 20.11
SC R
%FECR
IP T
0.08 µg/mL TBZ in the LDT.
A
CC E
PT
ED
M
A
N
U
SD, standard deviation.
27
S0304-4017(18)30086-4 https://doi.org/10.1016/j.vetpar.2018.02.036 VETPAR 8620
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
1-1-2018 21-2-2018 21-2-2018
Please cite this article as: Babj´ak M, K¨onigov´a A, Urda Dolinsk´a M, Vadlejch J, V´arady M, Anthelmintic resistance in goat herds – in vivo versus in vitro detection methods, Veterinary Parasitology (2010), https://doi.org/10.1016/j.vetpar.2018.02.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Original Article
Anthelmintic resistance in goat herds – in vivo versus in vitro detection methods
a
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M. Babják a, A. Königová a, M. Urda Dolinská a, J. Vadlejch b, M. Várady a,*
Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 040 01, Košice, Slovak
b
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Republic
Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural
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Resources, Czech University of Life Sciences Prague, Kamýcká 957, 165 21 Prague 6–
A
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Suchdol, Czech Republic
A. Königová ([email protected])
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M. Babják ([email protected])
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Urda-Dolinská ([email protected])
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J. Vadlejch ([email protected])
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*Corresponding author. Tel.: ++421 55 6334455.
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E-mail address: [email protected] (M. Várady).
Highlights:
The first survey of benzimidazole resistance on Slovak goat farms was conducted
High prevalence of resistant parasites were confirmed
Using double doses of treatment may underestimate the level of resistance 1
The in vitro tests provided reasonable identification of low levels of resistance
Abstract Anthelmintic resistance (AR) is a serious threat to animal health and has a major economic
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impact worldwide due to production and financial losses. The aim of this study was to
determine the occurrence of AR on 30 goat farms in Slovakia during the pasturing seasons
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and to compare three widely used in vitro and in vivo methods for detecting AR in field
conditions. A three-year survey was conducted during the pasturing seasons of 2014–2016.
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Goats on each farm were split into treated and control groups and were treated by
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recommended (5 mg/kg body weight) and double doses (10 mg/kg b.w.) of albendazole.
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Comparisons between percent reduction in a faecal egg count reduction test (FECRT) and an
M
egg hatch test (EHT) and the presence of L3 larvae in a larval development test (LDT) using resistant concentrations of benzimidazole (BZ) were monitored after treatment. The FECRT
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indicated percent reductions of 69.2-86.2% for the single dose and of 36.3–45.4% for the double dose. The EHT indicated that all farms had BZ-resistant nematodes. Low (< 15%
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hatching) and high (> 15% hatching) levels of resistance were detected on 13 and 17 farms,
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respectively. The LDT failed to detect resistant larvae on seven farms but detected low and high levels of resistance on seven and 14 farms, respectively. The data indicate a moderate correlation between in vitro and in vivo tests for detecting BZ resistance among the 30 goat
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farms. The hatching detected by the EHT and the presence of L3 larvae by the LDT at resistant BZ concentrations provided reasonable identification of low levels of resistance in the parasite populations, but the use of a double dose for a treatment may underestimate the real occurrence of low levels of resistant parasites on goat farms.
2
Keywords: Anthelmintics; Benzimidazole; Goats; Resistance; Detection method
1. Introduction Gastrointestinal (GI) nematodes cause the most prevalent and economically important
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diseases of small ruminants. The constant repetition of anthelmintic treatment on many farms is still the first option for prophylaxis rather than finding alternative methods to limit GI
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parasitism and finding solutions to improve control strategies, including mainly grazing management. Anthelmintic resistance (AR) is common throughout the world. The
development of AR is a genetic process where nematodes with resistance alleles survive
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treatment, thereby increasing the frequency of resistant alleles in worm populations after
N
repeated application of an anthelmintic. Treatment thus serves as a selector of resistant alleles
A
(Wolstenholme et al., 2004). Many factors such as underdosing, using the same group of
M
anthelmintics for a long time, frequent treatment, or lack of quarantine are responsible for
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accelerating the development of AR (Torres-Acosta and Hoste, 2008). Benzimidazoles (BZs) and macrocyclic lactones (MLs) are the most frequently used groups of anthelmintics on
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Slovak farms with small ruminants (Čerňanská et al., 2008).
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Surveys of the occurrence of AR in Slovakia, however, have only been conducted on sheep farms. The occurrence of BZ resistance was studied in 1991-1993 (Várady and Praslička, 1993) and 2003-2004 (Čerňanská et al., 2006); ineffective treatment was first
A
documented on six of 77 farms (7.8%) and a decade later on two of 29 farms (6.9%). The extent of ivermectin (IVM, an ML) resistance was also confirmed twice in 2003-2004 on eight of 26 sheep farms (30.8%) (Čerňanská et al., 2006) and in 2014 on 14 of 49 farms (28.5%) (Dolinská et al., 2014). These results indicated that the status of BZ and IVM resistance on sheep farms had not changed during the respective decades. The occurrence of
3
AR on sheep farms is well known, but no data are available from goat farms, despite an annual increase in goat numbers in Slovakia and an increased demand for goat-milk products. Goats are often farmed together with sheep, so farmers usually use the same farming techniques for both species. This practice is the most common managerial flaw, especially using the same anthelmintic doses, which is a serious threat for the development of AR on a
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farm. Goats have a higher metabolic activity than sheep and thus require higher dose rates for effective treatment (Veneziano, 2004). Using the sheep dose for both species leads to the
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selection of resistant parasites in goats, so resistance can then be transferred to sheep (Charles et al., 1989).
U
FECRT is recommended by the World Association for the Advancement of Veterinary
N
Parasitology (WAAVP) as the test of choice, especially in surveys for resistance. The test,
A
however, is time-consuming and expensive, inter-animal variation is high, and the
M
pharmacokinetics of the drug in the host may lead to data of poor quality. Interest has therefore tended towards in vitro tests such as the egg hatch test (EHT) and the larval
ED
development test (LDT), which are faster and cheaper to perform and are therefore more
PT
suitable for large surveys.
The main goal of this study was to conduct the first survey of occurrence of BZ resistance
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on Slovak goat farms. A secondary goal was to compare the data obtained from in vivo and in vitro tests to identify the simplest and most effective tool for estimating susceptibility/resistance in vitro. Other goals were to examine the morphology of resistant
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larvae and to survey the practices used on the farms.
2. Materials and methods
2.1. Selection of farms and sampling 4
Animal use and study design were approved by the Ethics Committee of the Institute of Parasitology of the Slovak Academy of Sciences in accordance with the national legislation in Slovakia - Animal Welfare Act No. 23/2009. Permission to collect study samples was granted by participating small ruminant farms.
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Farms were selected from the central register of livestock in Slovakia. A minimum of 24 goats per farm was a necessary requirement in the survey. Thirty farms distributed throughout the country were selected. Faecal samples from the 30 herds of dairy goats were collected
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directly from the rectum from September 2014 to December 2016. The animals had not been treated for GI nematodes for eight weeks prior to sampling. Individual counts of eggs per
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gram (EPG) for each goat were determined by a modified McMaster technique with a
A
N
sensitivity of 50 EPG (Coles et al., 1992).
M
2.2. FECRT
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The FECRT was conducted following the WAAVP recommendation (Coles et al., 1992, 2006). Goats on each farm were split into treated and control groups. The goats were treated
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with the ovine recommended dose of albendazole (ABZ) (5 mg/kg body weight), and if
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sufficient animals were available another group was treated at twice the recommended dose rates of sheep (10 mg/kg body weight). The minimum number of goats in each group was 12. Animals in each group were resampled 7-10 days after treatment. The percent reduction in
A
helminth eggs (%FECR) was calculated using three methods. First method (Dash et al., 1988): %FECR=100×(1−[T2/T1][C1/C2])
5
where T1 and T2 are arithmetic means of the treated group before and after treatment, respectively, and C1 and C2 are arithmetic means of the control group sampled at the same time as the treated group, respectively. Second method (Kochapakdee, 1995):
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%FECR=100×(1−[T2/T1]) where T1 and T2 are arithmetic means of the treated group before and after treatment,
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respectively, with no control group. Cabaret and Berrag (2004):
N
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%FECR=(1/n)∑(100×(1−[Ti2/Ti1])
A
where Ti1 and Ti2 are pre- and post-treatment EPGs, respectively, in host i from a total of
M
n hosts. The parasite population was considered resistant if %FECR was < 95% (McKenna,
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1990).
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2.3. EHT
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The EHT was performed following Coles et al. (1992, 2006). Eggs were stored anaerobically as described by Hunt and Taylor (1989). Suspensions of helminth eggs were prepared from pooled samples from each farm. Five concentrations (0.05, 0.1, 0.3, 0.5 and 1
A
µg/mL of thiabendazole (TBZ) in dimethyl sulfoxide (DMSO) were added to 24-well plates containing the egg suspensions (100 eggs/mL). One well in each plate row containing 10 µL of DMSO instead of TBZ was used as a control. Each concentration of TBZ was tested twice for each farm. The plates were incubated at 27 °C for 48 h, and 10 µL of an iodine solution (1g of iodine and 2 g of potassium iodide in 100 mL distilled water) was then added to each
6
well. The proportion of hatched eggs was recorded in each well. The concentrations of TBZ required to inhibit 50 and 99% of the eggs from hatching (ED50/ED99) were determined.
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2.4. LDT
Helminth eggs recovered from faeces were incubated for seven days in 96-well microtitre
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plates with culture medium (yeast extract with Earle's Balanced Salt Solution and physiologic salt solution) in an aquatic solution of various concentrations (range from 0.0006 to 1.28 µg/mL) of TBZ (Coles et al., 2006). The proportions of eggs and L1, L2 and L3 larvae were
U
determined for each well. The concentrations of TBZ that inhibited development to the L3
A
N
stage by 50 and 99% (LD50/LD99) were determined for each farm.
ED
M
2.5. Morphological identification of resistant species in the LDT
Infectious third-stage larvae (L3) in the wells with high concentrations of TBZ were
PT
isolated, and the resistant species were identified as described by Van Wyk et al. (2013). Each
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L3 was examined for morphological features and its body length was measured.
2.6. Questionnaire survey
A
All cooperating farmers were requested to provide data about their farms. Most of the
questions were about pastures (size and possibility of rotation), size and composition of goat herds, frequency of anthelmintic treatment, treatment strategy, choice of drugs, and knowledge about AR. Farmers answered by e-mail or by personal contact during visits to the farms. 7
2.7. Data analysis
The data were analysed by a statistical logistic regression model to determine ED50 and
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ED99 in the EHTs and LD50 and LD99 in the LDTs.
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3. Results
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3.1. Prevalence study
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A total of 996 samples from 30 farms were examined by a modified McMaster technique
A
(Coles et al., 1992). The prevalence of strongyle eggs was 91.80%. Strongyloides papillosus
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(3.66%), and Moniezia spp. (2.44%).
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(12.90%) was the dominant species followed by Trichuris spp. (7.22%), Nematodirus spp.
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3.2. FECRT
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All three calculation methods were used for 26 of 29 farms (Table 1). We were able to apply single and double doses of ABZ on 22 of the farms. The remaining farms did not have enough animals to apply a double dose or to form a control group. The Dash et al. (1988)
A
method identified BZ resistance on 18 of 26 farms (69.23%) using a single dose of ABZ and on seven farms (38.88 %) using a double dose. The Kochapakdee (1995) method identified BZ resistance on 21 of 29 farms (72.41%) using a single dose and on 8 of 22 farms (36.36%) using a double dose. The Cabaret and Berrag (2004) method identified BZ resistance on 25 of 29 farms (86.20%) using a single dose and on 10 of 22 farms (45.45%) using a double dose.
8
Resistant parasites were thus detected by one or more methods on 26 of the 29 farms (89.65%).
3.3. EHT
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The threshold ED50 detected AR on 15 of the 30 farms (50.0%). ED50 ranged from 0.02 to 1.05 µg/mL. AR was also detected by FECRT on these 15 farms. The percent egg hatch at 0.1
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µg/mL TBZ ranged from 47 to 91% on farms with resistance. The percent egg hatch in the negative controls for all farms was > 95%. Comparison between %FECR and percent egg hatch at 0.3 µg/mL TBZ is shown in Table 2. Table 3 shows percent hatching at the higher
U
concentrations of TBZ. The percent egg hatch at 0.1 µg/mL TBZ for the 15 farms where ED50
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was below the threshold of 0.1 µg/mL ranged from 5.5 to 38.5%. The percent egg hatch at 0.3
A
µg/mL TBZ ranged between 0 and 27.50%. The percent egg hatch in 0.3 µg/mL TBZ for
M
farms where ED50 was > 0.1 µg/mL ranged from 13 to 86%. These results indicate the
ED
presence of resistant parasites on these farms and the probability for developing more AR in the coming years. %FECR at a single dose (Cabaret and Berrag, 2004) and hatching at 0.3
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µg/mL TBZ in the EHT are compared in Table 3. Evidence of decreasing drug efficacy with
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more hatched eggs is clear.
A
3.4. LDT
The proportions of eggs and L1, L2, and L3 larvae were determined for each concentration
of TBZ. A farm was considered as harbouring resistance if infectious L3 larvae were observed at ≥ 0.08 µg/mL TBZ. Infectious larvae were found at these concentrations on 20 of 28 farms (71.42%). LD50 and LD99 were determined for each farm where test has been performed. The
9
thresholds of 0.01 µg/mL for LD50 and 0.02 µg/mL for LD99 identified resistant parasites on all farms. The numbers of L3 larvae at the higher concentrations of TBZ and LD50 and LD99 are presented in Table 4. %FECR at a single dose (Cabaret and Berrag, 2004) and the presence of L3 at ≥ 0.08 µg/mL TBZ in the LDT are compared in Table 5. Evidence of
SC R
3.5. Morphological identification of resistant species in the LDT
IP T
decreasing drug efficacy with more L3 larvae is clear.
Infectious L3 larvae were isolated from wells with concentrations of 0.08-1.28 µg/mL TBZ. L3 larvae were present at these concentrations on 18 farms. Haemonchus contortus was
U
the dominant species (50.92%) followed by Trichostrongylus spp. (39.84%) and Ostertagia
M
ED
3.6. Questionnaire survey
A
N
spp. (9.24%).
The size of the herds varied from 30 to 150 goats, with an average of 65 goats. Ninety
PT
percent of the farms were conventional, with milk production, meat production, or both. Two farms had hobby herds, and one farm was registered as organic. White and Brown short-
CC E
haired goats were the most common breeds, followed by Anglo-Nubian goats and Alpine goats. Farmers treated their goats with anthelmintics twice a year on 24 farms (80%), and
A
anthelmintics were never used on three farms. Two farms treated more than twice a year, and one farm used an anthelmintic treatment every time signs of disease (diarrhoea, weight loss) were noticed. All farmers who treated twice a year used BZ in spring and ML in autumn. Two farms treated only with BZ. Farmers on each farm where treatment was applied treated all animals in the herd. Twenty-four farms (80%) used pasture rotation; the other farms could not
10
rotate pastures regularly. Goats on 25 farms (83.33%) grazed together with sheep. The farmers had no information about goat parasites or the presence of parasites on their farms. Three farmers (10%) suspected insufficient drug efficacy. Only half of farmers had knowledge about AR in gastrointestinal parasites of small ruminants. A double dose of anthelmintics recommended for goats was never used on any of the farms. Farmers were
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asked about the quarantine of introduced animals; new animals were treated before
introduction on most farms (25 farms, 83.33%), but the efficacy of drugs and the composition
SC R
of parasite fauna had not been determined on any of the farms.
U
4. Discussion
N
AR is believed to be more frequent globally in parasites of goats than in parasites of
A
sheep. The results of our study confirmed that BZ resistance was widespread on the farms that
M
were examined. Our study represents the first national survey of the prevalence of BZ-
ED
resistant nematodes on goat farms in Slovakia. Such a high prevalence of BZ resistance was unexpected, particularly because previous studies of sheep reported a maximum BZ resistance
PT
of 10% (Praslička et al., 1994; Čerňanská et al., 2006), which may be due to two factors. Firstly, the majority of the goat farms had mixed grazing with sheep, which could accelerate
CC E
the development of AR. Secondly, the farmers did not know about the higher dose requirement of BZ treatment for goats, and all had been applying the dose recommended for
A
sheep. The results of our survey indicated a lack of knowledge about strategies of worm control and the problem of AR among Slovak goat farmers. The alternation of anthelmintic classes was adopted on 80% of farms where BZs were applied in spring and presumably MLs in autumn, which suggests that drenching frequency did not play an important role in the development of AR in the Slovak goat herds.
11
The FECRT was assessed by three methods with different evaluation protocols. The results of the FECRT can be influenced by various factors such as environmental conditions, level of infection in the herd, and level of efficacy of the test anthelmintic (Cabaret and Berrag, 2004). %FECRs by single and double doses for all three methods were compared with the percent hatch for the EHT. The method based on individual evaluation of EPG (Cabaret
IP T
and Berrag, 2004) was more accurate than the methods based on the arithmetic means of
treated and control groups (Dash et al., 1988) or the arithmetic mean of treated groups without
SC R
a control (Kochapakdee, 1995). The interpretation of FECRT results can be difficult for farms with %FECRs near the threshold (Coles, 2005; Presidente, 1985). EPGs can be also influenced by compound infections with strongyles with high (H. contortus) or lower
U
(Trichostrongylus spp., Teladorsagia circumcincta) fecundity and by the composition of
A
N
resistant and susceptible parasites on a farm (Holm et al., 2014).
M
Comparing the prevalence of BZ resistance using single and double doses of BZ identified notifiable differences. The prevalence decreased by 36.36 - 45.45% when the
ED
prevalence was calculated for a double dose of BZ. Orally administered drugs within the BZ, imidazothiazole/tetrahydropyrimidine, and ML (Chartier et al., 2001) classes are more rapidly
PT
cleared from the blood in goats than sheep (Bogan et al., 1987; Galtier et al., 1981; Gillham
CC E
and Obendorf, 1985; Hennessy et al., 1993a,b; Sangster et al., 1991), so goats require higher dose rates. Our study, however, suggests that the real prevalence of resistance may be underestimated when using a double dose in FECRTs. In vitro hatching in the EHT at higher
A
“resistant” concentrations has been previously reported as an excellent marker for diagnosing resistance in H. contortus (Várady et al., 2007). This parameter was able to provide early detection during the development of resistance in our study, especially if the alleles for resistance were rare in the parasite population (Čudeková et al., 2010).
12
ED50 from the EHT identified the presence of AR on 15 of the 30 farms (50%) using a threshold of 0.1 µg/mL TBZ. Hatching on the other 15 farms ranged from 5.5 to 38.5% for an ED50 < 0.1 µg/mL and from 0 to 27.50% for an ED50 of 0.3 µg/mL TBZ. These results indicate the presence of almost 30% resistant parasites on some farms that had been identified as susceptible using ED50. Von Samson-Himmelstjerna et al. (2009) recommended decreasing
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the threshold to 0.05 µg/mL TBZ when H. contortus is present on a farm. A previous study
(Babják et al., 2017) reported that H. contortus was present on 23 of 30 farms (76.6%) before
SC R
treatment. If we had decreased the threshold to 0.05 µg/mL, the EHT would have detected AR on 26 of the 30 farms, which would be in agreement with results obtained from the FECRT from the single-dose groups evaluated by the Cabaret and Berrag (2004) method. H. contortus
U
represented 50.92% of the 549 L3 larvae in the wells of the LDT at the higher concentrations
N
of TBZ. We detected resistance in this species on every farm where it occurred. The
A
phenotype–genotype correlation for BZ resistance in the EHT among multispecies infection
M
under field conditions have not been described, but our data indicated that using hatching
ED
criteria was probably more sensitive than the double dose in the FECRT for estimating the prevalence of resistant individuals in the parasite population.
PT
The majority of in vitro tests are not considered suitable for use in field screening
CC E
surveys, so only the EHT and LDT performed well enough to identify widespread AR (Várady et al., 2006). We have previously discussed the advantages and disadvantages of both tests for surveying AR in sheep parasites (Várady et al., 2006; Dolinská et al., 2012). When
A
the level of resistance is high, both tests can effectively detect resistance, but a delineation dose (DD) or hatching percentage in DD is more sensitive than ED50/LD50 for detecting smaller proportions of resistant worms in a population. The development of L3 larvae at “resistant” concentrations (≥ 0.08 µg/mL TBZ) could serve as a marker for the evaluation of the resistant part of the population, similar to the EHT. 13
5. Conclusion
In conclusion, the results of our study confirmed the high prevalence of BZ resistance on Slovak goat farms. The high incidence of resistant H. contortus is currently a serious problem,
IP T
and the lack of knowledge of the mechanism of AR development, the epidemiology of
gastrointestinal parasites, and good farming management will pose a more serious threat in
SC R
the future. BZ anthelmintics are the most commonly used drugs on Slovak farms, so drugs
from other classes might be more effective against GI parasites in goats. Further research is needed to detect resistance in goats to drugs from other classes, and continuing to monitor and
U
control the spread of BZ resistance in Slovakia is necessary. Using double doses of BZs for
N
treating goats is probably not optimal, because it cannot detect low levels of resistance. The
A
evaluation of hatching percentage in the EHT and the presence of L3 larvae in the LDT at the
ED
resistance in a population.
M
“resistant” concentrations is recommended to avoid misdiagnosing AR at low levels of BZ
PT
Conflict of interest statement
CC E
The authors declare that they have no conflicts of interest.
Acknowledgements
A
This study was supported by funds from the Slovak Research and Development Agency
APVV 14-0169 and Scientific grant agency VEGA 2/0120/16. We thank S. Spišáková and M. Krčmárik for their technical assistance. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 14
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Sangster, N.C., Rickard, J.M., Hennessy, D.R., Steel, J.W., Collins, G.H., 1991. Disposition of oxfendazole in goats and efficacy compared with sheep. Res. Vet. Sci. 51, 258–263. https://doi.org/10.1016/0034-5288(91)90074-X Torres-Acosta, J.F.J., Hoste, H., 2008. Alternative or improved methods to limit gastrointestinal parasitism in grazing sheep and goats. Small Rum. Res. 77, 159–173.
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Várady, M., Praslička, J., 1993. The occurrence of gastrointestinal nematodes in sheep farms
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Veneziano, V., 2004. Control of gastrointestinal strongyles in goats. Il controllo delle strongilosi gastro-intestinali dei caprini. Parassitologia 46, 245–250.
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pyrosequencing. Parasitology 136, 349–358. https://doi.org/10.1017/S003118200800543X Wolstenholme, A., Fairweather, I., Prichard, R., Von Samson-Himmelstjerna, G., Sangster, N.C., 2004. Drug resistance in veterinary helminths. Trends Parasitol. 20, 469–476.
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CC E
PT
ED
M
A
N
U
SC R
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https://doi.org/10.1016/j.pt.2004.07.010
19
Table 1 %FECR using the three calculation methods. Farm
Cabaret and Berrag
number
(2004)
Kochapakdee (1995)
Dash et al. (1988)
Double
Single
Double
Single
Double
dose
dose
dose
dose
dose
dose
1
67.5
-----
62.3
-----
76.2
-----
2
84.9
-----
85.7
-----
88.8
-----
3
-----
-----
-----
-----
-----
4
70.9
-----
76.2
-----
83.6
5
75.0
-----
73.9
-----
-----
6
93.1
-----
92.2
-----
7
91.5
-----
87.2
-----
8
93.6
98.4
95.4
97.8
9
53.2
66.1
45.0
10
66.3
65.0
55.9
11
67.4
82.7
74.6
12
100
100
13
91.9
98.9
14
100
100
15
97.5
16
89.6
---------
86.7
-----
94.5
97.3
54.0
69.6
76.9
77.9
88.4
82.4
50.0
62.3
100
100
100
100
94.0
99.0
94.0
99.0
100
100
100
100
98.2
97.1
98.5
97.5
97.7
98.2
88.0
98.1
88.1
98.1
U
-----
M
SC R
-----
90.8
N
63.6
A
ED
PT
17
IP T
Single
94.8
99.2
92.7
99.1
88.4
98.6
92.4
100
98.1
100
97.7
100
90.8
90.9
90.7
91.7
90.5
91.1
20
89.9
98.5
91.6
98.2
89.9
98.0
21
92.1
93.9
92.8
96.6
92.5
96.7
22
93.9
-----
97.2
-----
97.5
-----
23
93.0
93.8
93.5
95.2
95.6
97.0
24
82.4
86.8
85.7
91.5
83.5
88.1
25
92.3
98.0
91.6
98.0
-----
-----
26
65.4
70.8
63.7
69.5
63.5
69.1
27
91.5
97.0
94.0
97.3
98.2
99.2
18
A
CC E
19
20
41.5
66.3
13.8
62.4
49.6
65.3
29
90.4
99.6
92.2
97.5
-----
-----
30
81.3
93.8
89.8
92.4
-----
-----
A
CC E
PT
ED
M
A
N
U
SC R
IP T
28
21
Table 2 Comparison between % FECR at a single dose (5 mg/kg) and % hatching at 0.3
n
% Hatching (mean ± SD)
95-100
3
1.8 ± 2.0
90-95
13
14.6 ± 12.4
80-90
5
25.5 ± 20.4
40-80
8
70.6 ± 17.2
SC R
%FECR
IP T
µg/mL TBZ in the EHT.
A
CC E
PT
ED
M
A
N
U
SD, standard deviation.
22
Table 3 Comparison between ED50, ED99, and hatching (%) at 0.1 and 0.3 µg/mL TBZ in the EHT. Farm
ED50(µg/mL)
ED99 (µg/mL)
Hatching at 0.1
Hatching at 0.3
(µg/mL)
(µg/mL)
0.64
3.06
88.50
86.00
2
0.04
0.34
5.50
4.50
3
0.07
0.29
28.50
4
0.60
2.65
76.50
5
0.76
3.06
88.00
80.50
6
0.05
0.54
13.50
5.50
7
0.04
0.27
8
0.11
1.25
9
0.70
7.24
10
1.05
11
0.30
12
0.04
12.00
U
SC R
75.50
2.50
51.00
17.50
70.00
73.00
87.00
84.50
2.15
85.50
49.50
0.23
10.50
1.50
0.24
17.00
2.00
A
N
7.00
M ED
27.70
PT 0.06
CC E
13
IP T
1
0.05
0.21
12.00
0.00
15
0.06
0.32
9.00
4.00
16
0.05
0.31
17.50
5.50
17
0.05
0.37
15.00
6.50
18
0.06
0.61
21.00
11.00
19
0.22
11.00
69.00
57.00
A
14
23
0.10
3.20
48.00
36.00
21
0.10
4.06
47.00
38.00
22
0.20
2.20
75.00
35.00
23
0.08
9.10
38.50
31.50
24
0.17
4.60
56.50
53.00
25
0.10
2.68
55.50
13.00
26
0.45
2.10
91.00
27
0.02
1.50
12.00
11.00
28
0.20
11.00
55.50
38.50
29
0.05
4.76
11.50
11.00
30
0.05
12.40
32.00
27.50
IP T
20
A
CC E
PT
ED
M
A
N
U
SC R
77.00
24
Table 4 Results of the LDT for 28 goat farms in Slovakia. Number of L3 larvae at ≥
Farm
LD50 (µg/mL)
LD99 (µg/mL)
0.08 µg/mL TBZ 53
0.396
8.854
2
4
0.018
0.377
3
0
0.018
0.022
4
33
0.209
1.490
5
56
0.448
6
1
0.042
7
0
0.015
8
0
0.010
0.064
9
69
0.380
17.020
10
62
0.300
4.020
11
75
12
0
13
0
14
0
15 16
25.660 0.104
U
SC R
0.029
6.640
0.043
0.073
0.013
0.030
0.023
0.033
1
0.011
0.062
8
0.020
0.122
2
0.021
0.742
-----
-----
-----
-----
-----
-----
10
0.054
6.340
35
0.045
1.600
22
30
0.125
1.200
23
6
0.018
1.510
24
13
0.022
3.660
25
13
0.029
3.490
26
27
0.529
4.050
27
1
0.018
1.624
28
21
0.160
10.250
29
0
0.025
0.260
20
CC E
21
PT
18 19
A
N
0.064
M
ED
17
A
IP T
1
25
A ED
PT
CC E
IP T
SC R
U
N
A
M
30 29 0.084 6.920
26
Table 5 Comparison between %FECR at a single dose (5 mg/kg) and the presence of L3 at ≥
n
Number of L3 (mean ± SD)
95-100
3
0.33 ± 0.57
90-95
11
8.0 ± 12.77
80-90
5
12.8 ± 9.62
40-80
8
49.5 ± 20.11
SC R
%FECR
IP T
0.08 µg/mL TBZ in the LDT.
A
CC E
PT
ED
M
A
N
U
SD, standard deviation.
27