LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY
Tomas Kupčinskas
ANTHELMINTIC RESISTANCE AND
CONTROL OF GASTROINTESTINAL
NEMATODES ON SHEEP FARMS
IN LITHUANIA
Doctoral Dissertation Agricultural Sciences Veterinary (02A) Kaunas, 2017 1Dissertation has been prepared at the Department of Veterinary pathobiology (former Infectious Diseases) of the Veterinary Academy of the Lithuanian University of Health Sciences during the period of 2012-2016.
Scientific Supervisor
Prof. Habil. Dr. Saulius Petkevičius (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A)
Dissertation is defended at the Veterinary Research Council of the Lithuanian University of Health Sciences:
Chairperson
Prof. Dr. Jūratė Šiugždaitė (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A)
Members:
Assoc. Prof. Dr. Alvydas Malakauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A)
Prof. Dr. Vaidas Oberauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A)
Prof. Dr. Algimantas Paulauskas (Vytautas Magnus University, Biomedical Sciences, Biology – 01B)
Dr. Herve Hoste (French National Institute for Agricultural Research, Biomedical Sciences, Zoology – 05B)
Dissertation will be defended at the open session of the Lithuanian University of Health Sciences, Veterinary Academy, 2017 on 10 of February at 1:00 p.m. in the Dr. S. Jankauskas auditorium.
Address: Tilžės 18, LT-47181, Kaunas, Lithuania
LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA
Tomas Kupčinskas
AVIŲ VIRŠKINAMOJO TRAKTO
NEMATODŲ ANTIHELMINTINIS
ATSPARUMAS IR KONTROLĖ
LIETUVOJE
Daktaro disertacija Žemės ūkio mokslai
Veterinarija (02A)
Kaunas, 2017
Disertacija rengta 2012–2016 metais Lietuvos sveikatos mokslų universitete, Veterinarijos akademijoje, Veterinarinės patobiologijos katedroje (buvusi Užkrečiamųjų ligų), Parazitologijos laboratorijoje.
Mokslinis vadovas
Prof. habil. dr. Saulius Petkevičius (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A)
Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterinarijos mokslo krypties taryboje:
Pirmininkė
Prof. dr. Jūratė Šiugždaitė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A)
Nariai:
Doc. dr. Alvydas Malakauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A)
Prof. dr. Vaidas Oberauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A)
Prof. dr. Algimantas Paulauskas (Vytauto Didžiojo universitetas, biomedi-cinos mokslai, biologija – 01B)
Dr. Herve Hoste (Prancūzijos žemės ūkio nacionalinis mokslinių tyrimų institutas, biomedicinos mokslai, zoologija – 05B)
Disertacija ginama viešame Lietuvos sveikatos mokslų universiteto Veteri-narijos akademijos VeteriVeteri-narijos mokslo krypties tarybos posėdyje 2017 m. vasario 10 d. 13.00 val. Dr. S. Jankausko auditorijoje.
Adresas: Tilžės g. 18, LT-47181, Kaunas, Lietuva
TURINYS
ABBREVIATIONS ... 7
INTRODUCTION ... 8
1. LITERATURE REVIEW ... 10
1.1. The sheep parasites ... 10
1.2. The life cycle of sheep nematodes ... 11
1.3. Influence of temperature and moisture on larval development and survival ... 12
1.4. Pathophysiological aspects of trichostrongylid infections ... 13
1.5. Host immunity to trichostrongylid infections ... 14
1.6. Control of trichostrongylid infections in sheep ... 14
1.6.1. Anthelmintics... 14
1.6.2. Biological control ... 16
1.6.3. Anthelmintic resistance ... 17
1.6.4. Refugia and anthelmintic treatments regimens ... 18
1.7. Methods for detection of anthelmintic resistance ... 20
1.7.1. The faecal egg count reduction test ... 20
1.7.2. The egg hatch test ... 20
1.7.3. The larval development test ... 20
1.7.4. The molecular methods ... 21
2. MATERIAL AND METHODS ... 22
2.1. Study farms ... 22
2.1.1. Questionnaire survey ... 22
2.1.2. The anthelmintic resistance study assessed by in vivo FECRT ... 22
2.1.3. The anthelmintic resistance study assessed by in vitro methods ... 22
2.1.4. The experimental trial in two different treatment systems ... 23
2.2. Parasitological techniques ... 24
2.2.1. Modified McMaster method ... 24
2.2.2. Coprocultures... 25
2.2.3. Collection of larvae from grass ... 25
2.2.4. Baermann technique ... 25
2.2.5. Sedimentation method ... 26
2.2.6. Eggs preparing for in vitro methods ... 26
2.2.7. Yeast extract preparation for in vitro larval development test ... 26
2.2.8. Egg hatch discrimination dose test ... 27
2.2.9. Micro-agar larval development test ... 27
2.2.10. Faecal egg count reduction test (FECRT) ... 28
2.2.11. Pathophysiological analysis and performance indicators ... 28 5
2.3. Statistical analysis ... 29
3. RESULTS ... 30
3.1. A questionnaire survey on worm control practices used on Lithuanian sheep farms ... 30
3.2. Prevalence of anthelmintic resistance using in vivo FECRT ... on smallholder sheep farms in Lithuania ... 32
3.3. Prevalence of anthelmintic resistance to benzimidazoles and ivermectin aglycone on sheep farms in Lithuania assessed by in vitro methods ... 34
3.4. Anthelmintic resistance to benzimidazoles and levamisole on sheep farms in Lithuania detected by in vitro micro-agar larval development test ... 36
3.5. The efficacy of two different anthelmintic treatment regimens ... against natural gastrointestinal nematode infections in sheep ... 38
3.5.1. Eggs in the faeces ... 38
3.5.2. Pasture contamination ... 40
3.5.3. Meteorological and other observations ... 40
3.5.4. Mean weight gains in lambs ... 41
3.5.5. Body condition score ... 42
3.5.6. Dag score ... 43
4. DISCUSSION ... 44
4.1. Worm control practices based on questionnaire survey ... 44
4.2. The anthelmintic resistance assessed by in vivo FECRT ... 45
4.3. The prevalence of anthelmintic resistance assessed by in vitro methods ... 46
4.4. The efficacy of two different anthelmintic treatment regimens against natural gastrointestinal nematode infections in sheep ... 48
CONCLUSIONS ... 51 REFERENCES ... 52 PRACTICAL RECOMMENDATIONS ... 67 PUBLICATIONS ... 68 PUBLICATION I ... 70 PUBLICATION II ... 76 PUBLICATION III ... 80 SUMMARY IN LITHUANIAN ... 87 ACCESSORIES ... 103 CURRICULUM VITAE ... 107 ACKNOWLEDGEMENTS ... 108 6
ABBREVIATIONS
ABZ – AlbendazoleAR – Anthelmintic Resistance BZ – Benzimidazoles
CI 95% – Confidence limit of the mean DD – Discrimination Dose
DMSO – Dimethylsulfoxide ED 50 – Effective Dose, 50% ED 99 – Effective Dose, 99% EHT – Egg Hatch Test
EHDDT – Egg Hatch Discrimination Dose Test EPG – Eggs Per Gram faeces
FBZ – Fenbendazole FEC – Faecal Egg Count
FECR – Faecal Egg Count Reduction [%] FECRT – Faecal Egg Count Reduction Test GIN – Gastrointestinal Nematodes IVM – Ivermectin
IVM Ag – Ivermectin Aglycone L1 – First stage larvae L2 – Second stage larvae L3 – Third stage larvae L4 – Fourth stage larvae
LC 50 – Lethal Concentration, 50% LC 99 – Lethal Concentration, 99% LEV – Levamisole
MALDT – Micro-agar Larval Development Test MIC – Minimum Inhibitory Concentration ML – Macrocyclic Lactone
TBZ – Thiabendazole TT – Targeted Treatment
TST – Targeted Selective Treatment
WAAVP – World Association for the Advancement of Veterinary Parasitology
INTRODUCTION
Gastrointestinal nematode (GIN) infections remain the most prevalent and significant parasitic diseases in grazing sheep worldwide, with important economic losses to the sheep industry [55, 90, 144]. Infections caused by GIN leads to the production loss, morbidity and in certain cases mortality [78, 108, 120]. The most important genera are the trichostrongylids Teladorsagia,
Trichostrongylus and Haemonchus [113]. Haemonchus contortus is the most
dangerous blood sucking parasite in small ruminants which is more prevalent in southern regions of Europe. Due to the global warming the cases of
Haemonchus contortus in small ruminants has been reported in Nordic
countries and Lithuania [50, 97, 143]. Furthermore, Haemonchus contortus, was the first parasite ever to develop resistance to phenothiazine in the USA in 1957 [52]. In Lithuania, sheep farming has been in progress for the last 5-10 years and the number of sheep since 2005 (22,000) increased to 174,312 in 2016 in Lithuania [161].
The intensive and incorrect use of anthelmintics, under-dosing and treatments with the same anthelmintics led to the development of anthelmintic resistance (AR) [16, 43]. The problem of AR is increasing and it is not uncommon to find sheep or goat farms where resistance exist not only to a single drug, but to all available anthelmintic drugs [26, 42, 74, 87, 131, 177]. The most widely used AR detection methods are in vivo faecal egg count reduction test (FECRT), and in vitro larval development test (LDT) and egg hatch test (EHT) [43, 79, 150, 178]. The AR in small ruminants has been reported worldwide including Australia [15, 146], New Zealand [179], North, Central and South America [54, 114, 156, 181], South Africa [169] and many European countries: Slovak Republic [39], Greece [121], Spain [2], Nether-lands [18], Italy [157], France [30] and United Kingdom [13, 151].
Management strategies based on refugia (unexposed to drug) methods, such as targeted or treatment at the most appropriate time of the whole flock (TT), targeted selective treatments (TST), based on treatments to only those animals that will most benefit, the dilution of resistant with susceptible parasites help to maintain the alleles for susceptibility within the population [1, 57, 89, 105]. It is now considered that the maintenance of a parasite population in refugia, novel non-chemical approaches, improved pasture management and husbandry practices are the most important factors to avoid a faster development of AR and should be included in any potential prophylactic control regime suggested for nematode parasites [87, 88].
Previous studies on Lithuanian small ruminants and cattle farms have shown the high prevalence of GIN infection [125, 143, 147]. There is a numerous
information regarding the anthelmintic resistance situation in different European countries. In addition, the occurence of resistance in Europe is increasing, multidrug resistance has also been reported, which makes the control of gastrointestinal nematodes in sheep very difficult. The number of sheep is increasing every year in Lithuania, but the data on anthelmintic resistance is limited. This information is important to understand how anthelmintic resistance is common and what treatment strategies we should use, to reduce the anthelmintic resistance in nearly future.
Aim and objectives Aim of the study:
The aim of the present study was therefore to examine the prevalence of anthelmintic resistance in gastrointestinal nematode species to ivermectin, benzimidazoles and levamisole in Lithuanian sheep flocks.
Objectives of the study:
1. To evaluate the management and treatment strategy on sheep farms based on questionnaire survey.
2. To identify the anthelmintic resistant species of parasitic nematodes on sheep farms in Lithuania.
3. To determine the prevalence of anthelmintic resistance assessed by in vivo faecal egg count reduction test.
4. To determine the prevalence of anthelmintic resistance assessed by in vitro micro agar larval development test and egg hatch discrimination dose test. 5. To compare the efficacy of two different treatment systems on sheep farms.
Scientific novelty
The first time in Lithuania the prevalence of anthelmintic resistance to ivermectin, benzimidazoles and levamisole assessed by in vivo and in vitro methods on sheep farms were performed. Also, the first time the efficacy of two different treatment systems in sheep farms: targeted treatment and our modified targeted treatment combined with TST systems using parasitological and pathophysiological indicators were prepared and compared.
Practical significance
The results greatly improved knowledge on the level of AR in Lithuanian sheep farms and contributed for preparing the strategic control programmes against sheep gastrointestinal nematodes in the future based on risk factors which leads to the development of AR.
1. LITERATURE REVIEW
1.1. The sheep parasitesSheep are infected with a wide range of gastrointestinal nematodes. The most common and important gastrointestinal nematodes in terms of disease and production-loss are the trichostrongylids Teladorsagia, Trichostrongylus,
Haemonchus and Nematodirus. Other parasites, such as Cooperia, Chabertia, Oesophagostomum and Marshallagia mostly important as a part of a mixed
burden [113]. The most common sheep gastrointestinal parasites and their localization are shown in Table 1.1.1.
Table 1.1.1. Sheep gastrointestinal parasites and their localization [63, 154]
Localization The parasite
Abomasum Haemonchus contortus
Abomasum Teladorsagia circumcincta
Abomasum Trichostrongylus axei
Small intestine Trichostrongylus colubriformis Small intestine Trichostrongylus vitrinus Small intestine Nematodirus battus Small intestine Cooperia curticei
Small intestine Bunostomum trigonocephalum Small intestine Strongyloides papilosus
Large intestine Oesophagostomum columbianum Large intestine Oesophagostomum venulosum Large intestine Chabertia ovina
Large intestine Trichuris ovis
Climate influences infective larval availability and therefore rates of infection, through direct effects on the development and survival of the free-living stages and translation of larvae onto pasture. Therefore, the distribution of different species and the typical faunal composition in any given area in Europe vary as a role of climate [10, 113, 118].
Haemonchus contortus is the most dangerous gastrointestinal parasite and
tend to be more common and more of a threat to sheep health and production in warmer, southern areas, but due to global warming it is spread and into northern regions of Europe [49, 50]. Teladorsagia are dominant parasites in northern and temperate regions. Nematodirus battus is a major cause of disease in lambs only in northern Europe. However, Trichostrongylus and Nematodirus spp. are ubiquitous and their importance varies at local scale [61, 163].
In addition, mixed infections are common in all regions. Thus, even where
H. contortus is common, it is likely to be found alongside Teladorsagia and Trichostrongylus spp. [45, 138, 162].
1.2. The life cycle of sheep nematodes
The life cycles of most Trichostrongyles, Oesophagostomum and
Bunosto-mum are comparable: the cycles are direct, that is these nematodes do not
require other animals to comlete their life cycles [63].
Adult nematodes inhabit the gastrointestinal tract (abomasum, small or large intestine). The pre-parasitic phase of larval development is entirely free living. After male and female worms mate, produced eggs are passed in the feces of infected hosts. Contained embryos will develop into first stage larvae (L1) if temperature and humidity are optimal (22-26˚C and 100% humidity). L1 larvae develop into L2 stage larvae. First (L1) and second (L2) larval stages feed on fecal and soil bacteria but the third stage(L3) cannot feed because it is enclosed by a protective, impermeable sheath (the retained L2 cuticle). These ensheathed L3 larvaes survive by utilizing nutrients stored by the actively feeding L1 and L2 stages [123]. When infective L3 stage larvae complete their development (about 4-6 days after hatching), they are spread onto surrounding grass, where they become available for ingestion by grazing animals. In cooler temperatures the process may be prolonged [143, 147].
The parasitic phase begins when trichostrongylid infective L3 stage larvae are ingested by grazing animals. Exsheathment is the next event in the parasitic phase of these life cycles. Exsheathment sites are species specific and are always proximal to the predilection site of each particular trichostrongylid species. The L3 of the trichostrongylid nematodes penetrate the mucous membrane (Haemonchus, Trichostrongylus) or enter the gastric glands (Teladorsagia). During the next few days the L3 moult to the fourth stage (L4) and remain in the mucous membrane or in the gastric glands for 10-14 days. The L4 larvae emerge and moult into a young adult stage (L5). Mature females lay eggs approximately 2-3 weeks after infection. The time from infection to egg-laying by adult females is specific for each nematode species (e.g.
Teladorsagia – 17-21 days, Haemonchus – 2-3 weeks, Cooperia – 15-18 days.)
and is called the prepatent period [6, 140].
1.3. Influence of temperature and moisture on larval development and survival
Eggs of the ovine trichostrongylids develop to the infective L3 larval stage above a threshold of around 4°C, with some species, such as Haemonchus
contortus, having a higher threshold of around 8°C [85, 118]. Development of
larvae accelerates with increasing temperature. However, since mortality also increases within increasing temperature. The optimum temperature range for development varies by species and the highest among the trichostrongylid is for H. contortus (25-37°C) and the lowest for Teledorsagia circumcincta (16-30˚C) [118]. Optimal temperatures for L3 survival are low, but not freezing, with survival for several months in water at 3°C [81]. Lower temperatures are less favourable and Haemonchus contortus L3 in particular are poorly resistant to freezing. L3 of most species survive well over winter on pasture and infect susceptible hosts, especially newborn lambs, in the spring [5, 139]. The temperature response of Nematodirus battus must be considered separately, because of its peculiar life cycle [61]. Development of N. battus is slow and takes about 7 weeks at 20°C or lower temperature. Larvae do not hatch, but require a cold stimulus over several weeks, fallowed by temperatures within the hatching range of around 11-17°C [163, 165].
Compared with temperature, the effects of moisture on the free-living stages of the trichostrongyloid nematodes have received less attention [118]. However, moisture is needed for the development of trichostrongylid larvae to the L3 stage, but unless evaporation greatly exceeds precipitation, this is adequate within faecal pellets [116, 117]. Free water is needed for L3 to migrate out of faeces, but is not necessary for onward migration onto grass [164]. In more arid regions, lack of rainfall limits larval development, migration out of faeces and survival on the herbage [113].
Other climatic variables, such as desiccation and ultraviolet irradiation may increase mortality, with differences between species [162]. High temperatures often occur alongside dry and sunny conditions, so desiccation and ultraviolet irradiation should be considered alongside temperature in estimating loss of L3 from pasture [113].
1.4. Pathophysiological aspects of trichostrongylid infections
Infections with GIN usually involve several different species of parasites, which may have an additive pathogenic effect on the host. The pathogenic effect of GIN may be clinical or subclinical. Young animals are most susceptible. The effect of GIN infections depend on GIN specie and the number of parasites, host immunity system, nutritional status and environ-mental conditions [63]. Subclinical form mostly is related with loss of appetite and reduced production. However, to diagnose a sublinical form is still difficult [176].
Clinical signs of parasitism depend on different GIN species. In
Tricho-strongylus infection, the pepsinogen is activated with high concentration in
blood. High concentration of pepsinogen resulting into blood protein reducing, peeling of epithelium of gastrointestinal tract and increased secretion of mucin glycoprotein. Heavy infections of Trichostrongylus cause inappetence, diarrhoea, rapid weight loss and death [160].
Haemonchus contortus is the most dangerous parasite in sheep. The
symptom most commonly associated with Haemonchus infection is blood loss, white mucous membranes and anemia. After the sheep has ingested L3 larvae, the worm burrow into the mucosal of the stomach, nourishing on the red blood cells, which can be life-threatening to the sheep. An infected sheep can bleed to death within hours. Infections with Haemonchus mostly result into dehydratation, diarrhea, rough hair coat, depression, uncoordination, reduced growth. Another obvious sign is a „bottle jaw“ (fluid accumulation in sub-mandubular tissues). In some of cases the high level of Haemonchus contortus larvae resulting into animals death [175].
In Teladorsagia spp. infection, an elevation of abomasal pH and rise in blood pepsinogen concentration are observed. This cause loss of differentiation and stretching of the abomasal mucousa. In relation to acid secretion in the abomasum the levels of gastrin in the blood are greatly elevated [143, 147]. The clinical signs as weight loss, reduced feed intake, diarrhoea, gastritis may be seen [137].
Trichuris spp., Chabertia spp. and Oesophagostomum spp. can parasitaze in
the large intestine. Heavy infections with Trichuris spp. is not very common and mostly may be seen in very young lambs. In Chabertia spp. the immunity develops quickly, and outbreaks are seen only under conditions of severe stress. Congestion and edema of the cecal mucosa, ulceration and small hemorrhages accompanied by diarrhea and unthriftiness, are seen. Diarrhea caused by Oesophagostomum spp. usually develops during the second week of infection. As the diarrhea progresses, sheep become emaciated and weak. Continuing presence of numerous adult worms may result in a chronic
infection. The sheep become weak, lose weight despite a good appetite, and show intermittent diarrhea and constipation [154].
1.5. Host immunity to trichostrongylid infections
Sheep and goat are infected with the same principal GIN species, which provoke similar pathological changes and economic consequences [72]. However, immune responses against GIN are less efficient in goats than in sheep. The immunity of the host responds to GIN infection with production of specific IgA, IgE, IgG antibodies, eosinophilia ans mucosal mastocytosis, dependent on the activation of T helper 2 cells [143]. The acquisition of a fully expressed immune response appears delayed in goats. There are four different consequences, associated with the development of immune response in sheep: L3 establishment, worm development and growth, female fertility and egg production and persistency of adult worms. After the first contacts with GIN, the ability of goats to control challenge infections is much lower than that of sheep and that the „immune memory“ after anthelmintic treatment does not last as long [73, 76].
Another important reason associated with the GIN infections is that goat metabolize anthelmintics faster than do sheep and resulting in a lower short lived plasma levels of the active drug in other words a much lower bio-availability [64].
In addition, the sheep breed such as Red Maasai, the Barbados Blackbelly and St. Croix are more resistant to helminth infections, specifically
Haemonchus contortus, than other breed [63].
1.6. Control of trichostrongylid infections in sheep 1.6.1. Anthelmintics
The most used and common control and prophylaxis measure against sheep GIN is anthelmintics. The three broad-spectrum anthelmintic drug classes are most commonly used in sheep: macrocyclic lactones (ML), benzimidazoles (BZ) and imidazothiazoles. These anthelmintics are different by their chemical structure, range activity and spectrum (Table 1.6.1.1.).
Table 1.6.1.1. The anthelmintic spectrum of efficacy against sheep parasites
[107]
Anthelmintic class Nematode Fluke Cestode Ectoparasites
Benzimidazoles + +/– +/– –
Imidazothiazoles + – – –
Macrocyclic lactones + – – +
Ivermectin is the most popular anthelmintic from ML against sheep GIN. Ivermectin also shows insecticidal and acaricidal activities. Ivermectin was discovered in 1975 by identifyingfrom the bacterium Streptomyces avermitilis [24].
Ivermectin kills by interfering with nervous system and muscle function, in particular by enhancing inhibitory neurotransmission. Ivermectin binds to invertebrate-specific members glutamate-gated chloride channels in the membranes of invertebrate nerve and muscle cells, causing increased permeability to chloride ions, resulting in cellular hyper-polarization, followed by paralysis and death [38, 84]. Ivermectin mostly is administering subcutaneously at 0.2 mg/kg of sheep body weight in some of cases (goat GIN) the doses must be twice or at least 1.5 times higher, compared to sheep [65, 115].
Benzimidazoles (BZ) are broad-spectrum anthelmintics. FBZ and ABZ are the most used anthelmintics from benzimidazoles against sheep and goat nematodes, cestodes and in higher doses fluke [158, 187]. Thiabendazole (TBZ) was the first BZ, which was discovered in 1950 [107]. FBZ and ABZ inhibits the polymerisation of tubulin to microtubules. This interferes with essential structural and functional properties of the cells of helminths, such as formation of the cytoskeleton, formation of the mitotic spindle and the uptake and intracellular transport of nutrients and metabolic products [93]. The toxity of FBZ and ABZ is low, but it is not suggested to use in early gestation, because of its teratogenic effect [11, 188]. FBZ and ABZ are administering orally at 7.5-15.0 mg/kg of sheep body weight, depending on anthelmintic and parasites [28, 35].
Levamisole (LEV) is a broad-spectrum antihelmintic, which belongs to the class of synthetic imidazothiazole derivatives and was discovered in 1966. Levamisole works as a nicotinic acetylcholine receptor agonist that causes continued stimulation of the parasitic worm muscles, leading to paralysis [7]. LEV is administering orally or by injection subcutaneously or musculary at 5.0-7.5 mg/kg of sheep body weight [159]. LEV has a narrow therapeutic safety index and overdosing can lead to a poisoning and death, however there is no teratogenic and embryotoxic effect [53].
In addition, two new broad-spectrum anthelmintic classes are available now on sheep GIN control programmes: the amino-acetonitrile derivatives and the spiroindoles [83, 129]. However, a novel anthelmintic drugs are create in a slower rate than the anthelmintic resistance develop, for example case of resistance to monepantel, which first became available in New Zealand in 2009 [55].
1.6.2. Biological control
The aim of biological control is to help for increasing problem of anthelmintic resistance, which resulting in economical damage and animal welfare [67].
There are some reports on plants with anthelmintic properties and another suitable organism for the biological control of the free-living stages of nematode parasites of livestock – nematophagous fungi Duddingtonia flagrans [68, 148, 182].
The seeds of Azadirachta indica, Caesalpinia crista, Vernonia
anthel-mintica, Fumaria parviflora, leaves of Ananas comosus, Actinidia chinensis
and the fruits of Embelia ribes have been reported to cause effective reductions of >80% in FEC of trichostrongylids in sheep, goats and calves [9, 68, 82, 145].
Other examples are tannin containing plants which have known anthelmintic activity against GIN of ruminants, particularly sheep and goats [71, 130].
Duddingtonia flagrans produces thick walled chlamydospores in abudance
and has a superior ability to survive passage through the gastrointestinal tract and harsh environmental conditions in livesctock [94]. This fungus has the potential to break the life cycle of nematode parasites by capturing infective larval stages before they migrate from dung to pasture, where they would otherwise be acquired by grazing animals [95]. Various cereal grains provide an ideal substrate for growth and production of spores of nematophagous fungi.
Biological control is one of the more promising non-chemotherapeutic approaches to parasite control, but different factors need to be considered, such as sufficient doses of fungi, a proper equipment installation into feeding systems, stocking rate and level of overwintering pasture larval infectivity [183].
1.6.3. Anthelmintic resistance
Anthelmintic resistance is the heritable ability of the worm to tolerate a normally effective dose of the anthelmintic. The rate of emergence of resistant strains has generally been lower in temperate zones in the northern hemisphere compared to other regions due to different climate, parasite species and treatment frequency. However, occurence of resistance in Europe is increasing and anthelmintic resistance not only to the single drug, but also multidrug has also been reported, which makes the control of gastrointestinal nematodes in small ruminants very difficult [26, 87, 177].
Haemonchus contortus was the first parasite ever to develop resistance.
Resistance to phenothiazine was reported in the USA in 1957 [52]. Resistance has developed mainly in Haemonchus contortus, Teladorsagia circumcincta,
Trichostrongylus colubriformis and Cooperia spp., affecting Australia, New
Zealand, South Africa, many European, Asian countries and both American continents [34].
In most European countries, the recent reports of anthelmintic resistance mainly refer to cases of benzimidazole (BZ) and/or levamisole (LEV) resistance and with increasing numbers of cases of resistance to macrocyclic lactones (ML), mostly for ivermectin [122]. There are some reports of anthelmintic resistance to doramectin (15% efficacy) in the Netherlands and moxidectin (44% efficacy) in Switzerland and Southern Germany [18, 132]. Resistance to triclabendazole has also been reported in Netherlands and Ireland [111, 112].Anthelmintic resistance to benzimidazoles was found on 83% of the sheep farms examined in western France [29], 11.0% in Norway [49], 13.6% in Spain [106], 100% in Slovak Republic [46]. In Italy, levamisole resistance was found in all the farms and ivermectin resistance in two out of the three farms examined [157]. In Greece, Teladorsagia spp. remains the dominant nematode infecting small ruminants and over 16% strains studied were found to be benzimidazole-resistant strains [121].
The mechanisms of resistance can broadly be divided into two main processes: firstly, into a change in the target molecule and secondly, into a mechanism which inactivates or removes the drug from the environment of the target molecule [143].
The most popular methods to detect anthelmintic resistance are in vivo faecal egg count reduction test and in vitro egg hatch test and larval development test.
1.6.4. Refugia and anthelmintic treatments regimens
Anthelmintics are still the most common measure in sheep GIN control strategies. Anthelmintics can be used strategicaly or therapeuticaly combined with grazing management and/or biological control measures [186]. However, anthelmintics should be used properly using in treatment strategies or refugia based methods – strategies.
One of the most popular anthelmintic treatment strategie in Europe is to treat animals before the grazing season and after the grazing season (turn out, turn in). Using this treatment regimen farmers avoid a pasture contamination in spring and health problems caused by GIN during the winter [89].
Another suggestion is to treat animals every three weeks during the grazing period started from the beginnig for a minimum of three to four times by the system „0-3-6-9“ helps to decrease the pasture contamination by GIN eggs [17].
However, the most promising treatment strategies, which could help to avoid a faster development of AR, are refugia based.
The rate of development of AR is related with the proportion of the parasite population which is exposed to anthelmintic and it could be slowed by maintaining this proportion of population in refugia (unexposed to drug) [105, 170]. The refugia was first described by ecologists as a local environment that has escaped regional ecological change and therefore provides a habitat for endangered species. However, the refugia in parasitic communities cannot be directly applied where anthelmintic susceptible population exists alongside the anthelmintic resistant population and can interbreed freely. The request is that parasites in refugia must complete their life cycles and pass on susceptible alleles to the next parasitic generation [88]. The concept of refugia is that worms left unexposed to drugs are able to help to maintain the alleles for susceptibility within the population. These worms in refugia can dilute the resistant genotypes on pasture thus reducing the proportion of resistant parasitic adult worms that are likely to mate with other resistant adults [88, 168].
Two methods were considered to optimise treatments: targeted treatment (TT) – treatments given to the whole group of animals at the most appropriate time (e.g. before the grazing season to avoid a pasture contamination) and targeted selective treatment (TST), where treatments are direct to only animals showing clinical symptoms or reduced productivity [127, 170]. The TST strategie requires the ability to identify animals within a flock, which need a treatment. For this purpose, there are a variety parasitological (FEC), pathophysiological (body condition score, diarrhoea score, dag score, anaemia)
or production (weight gain, milk production) markers were evaluated as indicators for treatment [89].
Parasitological indicator for treatment is based on the level of FEC. The threshold of FEC varies between the countries and different studies beginning from 200 EPG to 500 EPG. The threshold mostly is related with the point between subclinical and clinical form. As an indicator the FEC has a great potential, but there are still some limitations to use it in practice, especially in farms with high number of animals [88].
Body condition score as an indicator was tested in several countries using a five point scale method. For example, in Greece ewes were treated if they had a body condition score of 2 or less [59].
Diarrhea score and dag score also have a correlation with a high number of GIN. Diarrhea score as an indicator was evaluated in Morocco and showed a correlation with FEC, but it should be used under the farming conditions [119]. At mid to late summer there is a positive correlation between increased dag score and high FEC in lambs, due to other parasites such as protozoa or
Moniezia in early spring [19]. Dag scores are estimated on a 5-point scale: 1-
no dags, 5- extensive dags [25].
Some of studies in Greese and Italy have shown that milk production could be an appropriate marker to identify those animals requiring treatment, whlist maintaining production and have indicated the importance of the timings of treatments as these needs to be given at times when milk withdrawal is not a problem [36, 127].
The FAMACHA© system developed in South Africa is using as an indicator of anaemia caused by haematophagous parasites, such as
Hea-monchus contortus. The FAMACHA© has been successfuly used in many
countries and has a great potential identify animals, which requires a treatment [20, 100]. The FAMACHA© categories range from 1 – red (nonanaemic) to 5 – white (severely anaemic) [103]. Using this system, only sheep scored 4 and 5, have to be treated. The FAMACHA© helps to reduce the number of treatments required and slow down the development of AR by increasing refugia [86]. Furthermore, FAMACHA© is not a suitable indicator for selective treatment in regions, where H. contortus is not a dominant parasite [127].
1.7. Methods for detection of anthelmintic resistance 1.7.1. The faecal egg count reduction test
The most widely used test to assess anthelmintic efficacy is the in vivo faecal egg count reduction test (FECRT), recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP) [31]. The test is based on a comparison of the number of eggs per gram (EPG) of faeces on the day of application to the number of eggs 10-17 days later, depends on anthelmintics use. Flocks with FECRs <95% and lower limits <90% were considered as harbouring GINs resistant to an anthelmintic. If only one of these conditions is met, resistance is suspected. Generally, in vivo techniques are rather time and money consuming and are often characterized by low reproducibility of results that may be caused by differences in drug pharmacodynamics in treated animals [174]. However, still this method is one of the most popular because of its simplicity.
1.7.2. The egg hatch test
Egg hatch test is one of the most widely used in vitro method for the detection of benzimidazole resistance, especially in field diagnostics. This method is based on the ovicidal properties of benzimidazoles and the ability of eggs of resistant populations to embryonate and hatch in a higher concentration of benzimidazole than can eggs from sensitive populations.
The original test was described by Le Jambre (1976). A modified version recommended by the WAAVP is that according to Coles et al. [31, 32]. The egg hatch test can also be used to detect resistance in parasites of goats [51, 126].
1.7.3. The larval development test
The larval development test is another common in vitro method to detect anthelmintic resistance to benzimidazoles (BZ), macrocyclic lactones (ML) and levamisole (LEV). This method is based on larval ability to survive and develop in environments of various concentrations of anthelmintics. The first time it was described by Coles et al., 1988. The test has the great advantage of the simultaneous detection of efficacy/inefficacy of the two broad-spectrum anthelmintics [174]. To increase the ability of the test to differentiate between ivermectin-resistant and susceptible isolates, instead of ivermectin, an avermectin analogues, such as ivermectin aglycone, eprinomectin are used
[92]. The larval development test is the most sensitive test for detection ML resistant nematodes of sheep and goats.
1.7.4. The molecular methods
Molecular technology demonstrate the presence of parasites based on their antigenic components or DNA segments and there are not influenced by environmental factors that usually can interfere with the results of a stool test [134, 152]. For the identification of resistant parasites include the diagnostic methods such as polymerase chain reaction, real-time polymerase chain reaction, random amplified polymorphic DNA, microsatellite marker method [152, 153]. The molecular methods provide high levels of specificity and sensitivity, but because of high costs is still used only in limited and special cases [4, 136, 185].
Anthelmintic resistance in trichostrongyloid parasites of sheep has been described throughout the world and has become an important factor in limiting efficient production in many countries. Another important factor - multidrug resistance has also been reported, which makes the control of gastrointestinal nematodes in sheep very difficult. The number of sheep in Lithuania is increasing, but the data on anthelmintic resistance is limited. This information is important to understand how anthelmintic resistance is common and what treatment strategies we should use, to reduce the anthelmintic resistance in sheep nematodes.
2. MATERIAL AND METHODS
The work was carried out at the Department of Veterinary Pathobiology (former Infectious Diseases) of the Lithuanian University of Health Sciences within the period 2012-2016. The study was performed in compliance with Lithuanian animal welfare regulations (No. B1-866, 2012; No. XI-2271, 2012) and was approved by the Lithuanian Committee of Veterinary Medicine and Zootechnic Sciences (Protocol No.07/2010).
2.1. Study farms 2.1.1. Questionnaire survey
A questionnaire surveyed 71 sheep farmers. Twenty-nine farms were visited, and the farmers were interviewed personally. Forty-two sheep farmers from the list of the Lithuanian sheep breeders association were interviewed by telephone. All farmers were asked about their practices of farm management: number of animals, sheep breeds, size of pastures, and worm-control practices: treatment times and frequency, products, and dosages of anthelmintic drugs (Accesory 1).
2.1.2. The anthelmintic resistance study assessed by in vivo FECRT A total of 25 sheep farms, mainly in central and southern Lithuania, were visited between April 2014 and November 2014 (Publication III). Eighteen of these farms were included in the study based on EPG ≥140. Sheep older than 18 months in each flock were divided into two groups of 15 randomly selected animals, marked with different coloured sprays, and treated with two different anthelmintics (fenbendazole at 7.5 mg/kg body weight and ivermectin at 0.2 mg/kg body weight). Farms were visited twice. The selected flocks had been regularly treated with anthelmintics. All flocks had grazing animals at the time of the study. The last anthelmintic treatment had been given at least eight weeks before the beginning of the study. The selected flocks consisted of approximately 40-500 animals.
2.1.3. The anthelmintic resistance study assessed by in vitro methods The survey was conducted from August 2013 to November 2014. A total of 33 and 23 farms, respectively, in two different investigations mainly from central and southern Lithuania with previous history of the use of
fenbendazole, levamisole and/or ivermectin for at least of 3-5 years were enrolled in the study for AR detection. Only the farms where the last anthelmintic treatment was carried out for at least 10 weeks before the onset of the study were included. The farms where only BZ were used – egg hatch discrimination dose test (EHDDT) was conducted for detection of AR to BZ. The farms where only ML and LEV were used – micro agar larval development test (MALDT) was conducted for detection of AR to ivermectin aglycone (IVM-Ag) and LEV. On farms, where two or three classes of drugs were used - both tests EHDDT and MALDT were conducted to test for multidrug resistance.
The size of sheep flocks was 40-1000 animals per farm. On majority of farms Lithuanian black-headed sheep and crossbreed sheep were reared. On all farms the animals were grazed on pastures from 01-10 of April till 15-31 of October.
2.1.4. The experimental trial in two different treatment systems
The survey was conducted during the grazing period from 15 of April to 31 of October 2014. Three sheep farms in district of Prienai (54.72509, 24.049087 (WGS)) Southern part of Lithuania were enrolled in the survey. On targeted treatment (TT) and combined targeted selective treatment (combined TST) farms different treatment regimens were applied, where treatments were based on FEC while no treatments were administered on control farm during the study. The combined TST treatment was consisted of two different treatment methods, targeted treatment of the whole flock (before turn out in spring) and targeted selective treatment (the animals with EPG ≥300 were treated in beginning of July). The size of sheep farms were 24, 57 and 28 animals on TT, combined TST and control farms, respectively. The farms were comparable by grazing pressure (9 animals per hectare, including lambs), sheep breeds on the farm (Lithuanian blackheaded, Romanov and crossbreeds), location (the distance between the farms was max. 15 km) and management system (no pasture rotation). On all three farms the lambs were grazing together with ewes starting from 15-25 of April until the end of October on the same pasture without moving.
On Farm TT 24 mix-breeds of Romanov and Lithuanian blackheaded (10 adult and 14 young individs) were grazing on the pasture of 2.3 hectares (339 kg/ha). During the last 4 years the animals were regularly treated with anthelmintics twice a year (spring and autumn) before turn out and turn in.
On Farm with combined TST Farm 57 (21 adult and 36 young individs) Lithuanian blackheaded, Romanov, and crosbreeded with Berichon du Cher
animals were grazing on the pasture of 5.7 hectares (371 kg/ha). During the last 8 years the animals were regularly treated with anthelmintics twice a year, before turn out and turn in.
On control farm 28 animals (13 adult and 15 young individs), mostly Lithuanian blackheaded, Romanov and crossbreeds were grazing on the pasture of 3 hectares (273 kg/ha). The animals were not treated with anthelmintics during the last 6 years.
The age of lambs was 4-8, 2-12 and 4-8 weeks on TT, combined TST and control farms in the begining of study. Some of lambs weighing 35-50 kg were withdrawn from the study for slaughter starting from the begining of August.
On Farms TT and with combined TST the animals were previously treated mostly with injectable ivermectin. On all three farms the tupping season started in the end of August or October. The animals on all three farms were examined for the presence of Fasciola hepatica and lungworm infections on two occasions (April and September). In addition, before the study, all farms were examined for anthelmintic resistance to ivermectin (IVM-Ag) and benzimidazoles (TBZ) using in vitro tests. No anthelmintic resistance was detected on any farm.
The data on monthly precipitation and average temperature were obtained from the Lithuanian Hydrometeorological Service from the meteorological station situated 5-15 km from the examined farms.
2.2. Parasitological techniques 2.2.1. Modified McMaster method
The numbers of nematode eggs (EPG) and oocysts per gram of faeces were determined using a modified MacMaster technique with a sensitivity of 20 eggs per gram of faeces [66, 128]. The faecal samples from each animal were taken from the rectum. Weighted 4 g of faeces, mixed with 56 ml of water and left for 30 min. Then the faecal suspension through a tea strainer or a double-layer of cheesecloth were filtered into a plastic container. 10 ml of filtered faecal suspension was pipetted into a centrifuge tube and centrifuged for 7 min. at 1200 RPM (revolutions per minute). After the centrifugation the supernatant was removed with a pipette and a flotation fluid (zinc chloride solution with density 1.4) was added to the 4ml mark. The sediment was mixed with flotation fluid and the both sides of McMaster chamber werre filled with this suspension. After 3 min. the eggs and oocysts were counted in both sides of chambers. The number of eggs per gram of faeces were calculated as fallows:
the egg counts of the two chambers were added together and multiplied the total by 20.
2.2.2. Coprocultures
Post-treatment larval cultures were prepared from pooled faecal samples for the flocks with FECRT efficacies <100%. The pooled samples were composed of the faeces collected from each animal of the group. Ten grammes of faeces were mixed with 4 g of vermiculite and incubated for 7 days at 27°C (water was added to maintain an adequate moisture level). Third-stage larvae (L3) were then recovered from the coprocultures by a Baermann technique [31]. The L3 were morphologically differentiated and identified according to MAFF [98] and Van Wyk et al. [167]. The first 100 L3, or all L3 when <100 developed, were identified.
2.2.3. Collection of larvae from grass
Three replicate herbage samples of approximately 400g of weight were collected from the pasture grazed by sheep from each farm for the deter-mination of the numbers of nematode larvae, every two weeks. Herbage samples were collected by walking across the pasture in a W-shaped pattern every 10-20 steps. Grass within 20 cm of faecal pellets was avoided. Larvae were isolated, counted and results were expressed as the number of L3 per kg of dried grass [56].
2.2.4. Baermann technique
The Baermann technique was used to isolate lungworm larvae from faecal samples. The method is based on the active migration of larvae from faeces suspended in water and their subsequent collection and identification. 5-10g of fresh faeces were weighted and placed on a piece of double-layer cheesecloth. The cheessecloth was formed around the faeces as a „pouch“ and closed with a rubber band. This pouch was clipped with the stick and placed in the conical glass with lukewarm water for 24 hours at a room temperature. After that, a few ml of fluid with the larvae from the bottom of glass were taken and sedimented or left to sediment for another 24h. By using Pasteur pipette, 3 drops of the sediment fluid were transferred on microscope slide and fixed with Lugol‘s iodide [63]. The L3 were morphologically differentiated and identified according to MAFF [98] and Van Wyk et al. [167].
2.2.5. Sedimentation method
The sedimentation method was used to detect Fasciola hepatica eggs in the faeces. 10g of fresh faeces were mixed with 250 ml of water. The faecal suspension was filtered through a tea strainer into a conical glass. After 30 min. the supernatant was removed and the same procedure repeated. After that, 50 ml of tape water were poured on the sediment. After 5 min. the supernatant was removed and a few drops of 1% of methylene blue were added. The sediment was transferred into Petri dish and examined by microscope at 10×4 magnification [63].
2.2.6. Eggs preparing for in vitro methods
Faecal samples were taken rectally or from the ground (max. 1 hour old). Samples were taken from 10-15 animals from the farm (10-15 different faecal deposit in the ground) of 6-10 grams of faeces from each individ. Samples were concentrated in the plastic bag (100-120 grams) and mixed. Later put approximately 1/3 of mixed faeces in to the plastic container (250 ml) and filled with water 2/3 of container up to the top and closed [171].
Nematode eggs were isolated by sequential sieving of the faeces through three stacked sieves with mesh size 250, 100 and 20 µm. The material collected on the 20 µm sieve was washed with water into glass and left for sedimentation. After 15-20 min. the supernatant was removed and the sediment transferred into tubes with cover. The tubes were centrifuged for 2 min at 1500 RPM. The supernatant was removed and saturated sodium chloride solution until the a meniscus is formed was added and centrifuged again. After the centrifugation, the material (with eggs) were washed from the cover slips with distilled water into a glass. The water-egg suspension were washed with dionised water to remove salt. Finally, the number of eggs were counted by removing aliquots with micropipette. Adjusted to have the concentration about 100 eggs per 10µl [31].
2.2.7. Yeast extract preparation for in vitro larval development test 1 g yeast extract + 90 ml 0.85% NaCl. Autoclaved at 121°C 1.0 atmospheric pressure for 20 minutes. Added 3 ml of concentrated Earles solution per 27 ml yeast extract. Aliquot and freezed for several months [75]. 10× Earles in g/100ml is KCl 0.04, MgSO4 0.2, NaCl 6.68, NaHCO3 2.2, NaH2PO4 0.14, CaCl2 0.02.
2.2.8. Egg hatch discrimination dose test
To examine for the AR to BZ, the EHDDT was performed as described by Coles et al. [31]. A stock solution of thiabendazole (TBZ) (Sigma-Aldrich, Germany) was prepared by dissolving the pure compoud in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Germany). The final concentration was prepared by adding 10 µl of the TBZ solution to 1.99 ml of an aqueous suspension with approximately 150 eggs/ml-1 [46]. For the purpose of this study EHDDT was only used at the single working concentration of 0.1 µg/ml–1 (Accesory 3). As the control, 0.5% DMSO solution without anthelmintic was also included in the test. The egg suspensions were dispensed into 24-well plates (Nuncleon, Denmark) and incubated at 27°C for 48 h. The test was stopped by adding 10 µl of Lugol‘s iodine and first 100 eggs and/or larvae were subsequently counted in each well. The test was performed with two replicates.
2.2.9. Micro-agar larval development test
Tests were performed in 96-well microtiter plates as described by Coles et al. [32]. For MALDT thiabendazole (TBZ), levamisole (LEV) and IVM aglycone (IVM-Ag) was chosen because of the higher ability to differentiate between the IVM-resistant and susceptible isolates. To produce 12 final concentrations ranging from 0.0006 to 1.28 µg/ml–1 for thiabendazole, from 0.084 to 173.6 ng/ml–1 for IVM-Ag and from 0.0156 to 32 µg/ml–1 for levamisole stock drug solutions of TBZ and IVM-Ag were serially diluted 1:2 with dimethyl sulfoxide (DMSO) and of levamisole with deionised water (Accesory 2). Subsequently, 12 μl of each stock solution with different final concentrations were mixed with 150 μl of 2% Bacto agar (Difco, USA) and stored at +4°C for 5 min. To inhibit the fungal growth 10 μl of eggs (final number of eggs per well was 50) in a 0.3 mg/ml–1 solution of amphotericin B (Sigma-Aldrich, Germany) were mixed with 10 μl of yeast extract and then added to the agar [172, 46]. Yeast extract was prepared as described by Hubert and Kerboeuf [75]. Only DMSO (1.3%) was used in the control wells. The plates were incubated for seven days at 27°C. Incubation was terminated by adding Lugol's iodine solution into each well. After incubation, the proportion of unhatched eggs, L1-L2 and L3 stage larvae at each concentration was determined under an inverted stereomicroscope. For IVM-Ag resistance the threshold discriminating concentration of 21.6 ng.ml–1 was chosen, 0.04 µg.ml -1 for thiabendazole and 2 µg.ml–1 for levamisole.
2.2.10. Faecal egg count reduction test (FECRT)
We selected the BZ and ML anthelmintic classes for evaluation in this survey, based on the information obtained from the questionnaire. One group in each flock was treated with the fenbendazole (FBZ) at 7.5 mg/kg body weight (Panacur® granules, Intervet International B.V., Boxmeer, Netherlands), and the other group was treated with IVM at 0.2 mg/kg body weight (Biomectin 1%, Vetoquinol Biowet Sp.zo.o., Gorzow Wlkp., Poland). The doses were based on the heaviest animal in each flock. FBZ was administrated orally over the back of the tongue, and IVM was administrated subcutaneously behind the scapula. Five of the 18 flocks received only one anthelmintic (IVM on three farms and FBZ on two farms), because of the difficulties of management. Individual faecal samples were collected on the day of treatment (T1). Animals with <140 EPG on day zero (T1) were removed from the trial. Eggs of Nematodirus spp. were not included in the counts. Individual post-treatment faecal samples (T2) were collected after 14 days from both groups in each flock treated with both anthelmintics, after 10 days from the flocks treated with only FBZ, and after 14 days from the flocks treated with only IVM. The FECR was calculated as:
FECR (%)=100×(1-(T2/T1))
where T2 is the arithmetic mean FEC post-treatment and T1 is the arithmetic mean FEC pre-treatment [21, 91].
2.2.11. Pathophysiological analysis and performance indicators
At each sampling occasion, young individs weights and body condition scores (on scale of 1-5 with 0.5 unit intervals) were recorded and estimated as described by Campbell et al. [25]. Dag scores were assessed according to guidelines by Australian Wool Innovations on a 5-point scale: 1- no dags, 5- extensive dags [8] (Fig. 2.2.11.1.).
Fig. 2.2.11.1. Visual sheep dag scores [8]
2.3. Statistical analysis
The lower and higher limits for 95% confidence interval were calculated following the WAAVP recommendations [31]. Descriptive statistics were calculated using Microsoft® Excel 2007 and IBM SPSS Statistics (Version 21.0). The differences in the level of resistance was calculated using Fisher‘s exact test performed by GraphPad Prism version 4.00.
The value P<0.05 was considered statistically significant.
3. RESULTS
3.1. A questionnaire survey on worm control practices used on Lithuanian sheep farms
A questionnaire surveyed 71 sheep farmers (Publication III). All farms were mainly specialized for meat production and practiced grazing, and 19.7% of the farms had an ecological status. Rotational grazing was used on 78.3% of the farms, and 21.7% of respondents kept sheep on the same pasture with a shelter during the grazing period. Pastures had a mean area of 19.53 ha (1-120 ha). The average number of sheep per farm was 149.9 (1-1700). The dominant sheep breeds showed in Fig. 3.1.1.
Fig. 3.1.1. The dominant sheep breeds in Lithuania, %
Lithuanian black-headed sheep was the dominant breed (46.5%). The remaining 11.9% consisted of breeds such as Merinofleischschaf, Ile de France, Texel, and Lacaune and crossbreeds. Sheep were usually pastured from March/April to October/November, and all sheep were housed during the winter.
An estimated 71.8% of sheep farmers used anthelmintics against GIN, but 9.5% of farmers declared that they treated their sheep only with the appearance of clinical symptoms such as diarrhoea, apathy and/or weight loss. The most commonly used classes of anthelmintics were ML (68.6%), and BZ (27.5%).
From the BZ group 68.7% albendazole and 31.3% FBZ were used. Levamisole was used very sporadically (3.9%) (Table 3.1.2).
Table 3.1.2. Worm-control practices on the sheep farms
Worm control factors Number (%)
Anthelmintic class used on sheep farms
Macrocyclic lactones 29 (56.9)
Benzimidazoles 8 (15.7)
Imidazothiazoles 2 (3.9)
Macrocyclic lactones and benzimidazoles 12 (23.5)
Treatment frequency (yearlings/ewes)
None 20 (28.2)
Once 7 (9.8)
One to two times 9 (12.7)
Twice 32 (45.1)
Two to three times 1 (1.4)
Three to four times 2 (2.8)
Treatment frequency (lambs)
None 20 (28.2)
Once 16 (22.5)
One to two times 3 (4.2)
Twice 30 (42.3)
Two to three times 2 (2.8)
Spring before turn out and autumn before turn in were the most common seasons to treat ewes. Yearlings and adults were usually treated together, with a mean annual drenching rate of 1.39. Most of the respondents declared that they treated lambs at the same time as the yearlings and adults to save time, with a mean number of treatments of 1.24 (Table 3.1.2). Of the respondents that used anthelmintics, 62.7% declared that they treated their animals twice every year. All respondents that used anthelmintics once per year treated their ewes in spring. A few farms added a treatment in summer (7.1%). Anthelmintics were rotated on 4.8% of the farms. Only one respondent reported four treatments per year. Annual treatments were usually performed without any parasitological analyses, and 11.9% of the respondents reported only a single coprological analysis during the entire period when sheep were kept.
Animal weights were visually appraised on 92.9% of the farms, and only 7.1% of farmers weighed their animals. Veterinarians treated the animals on 54.8% of the farms, and owners or farm workers treated the animals on 45.2% of the farms. Problems with sheep GINs were declared by 39.2% of the respondents.
3.2. Prevalence of anthelmintic resistance using in vivo FECRT on smallholder sheep farms in Lithuania
The arithmetic mean FEC, percentages of the FECR and 95% confidence intervals are presented in Table 3.2.2. The FECRs indicated the presence of FBZ resistance on three of the 15 farms where FBZ was used FECR (ranged from 44.9 to 85%), and FBZ resistance was suspected on one farm (94.3%, CI 93-99). Resistance to IVM was present on two of the 16 farms that used IVM (FECR of 74.9 and 77.2%). On one farm (Farm No.7) resistance was detected to both classes of anthelmintics (Publication III). Mean pre-treatment EPG counts varied from 216 to 3114.
The main genus of resistant GIN identified after treatment were
Tela-dorsagia spp. On all 6 positive farms, with distribution varying from 42 to
100%. Trichostrongylus spp. was found on five farms, with distribution varying from 4 to 100%, and 6-56% of the GINs on seven farms were
Cooperia spp. Chabertia ovina was found on two farms (4-18%), and Haemonchus contortus was found on only one farm (10%) (Table 3.2.1).
Table 3.2.1. Third-stage larvae (L3) identified in post-treatment coprocultures
on 6 sheep farms with AR in Lithuania treated with fenbendazole (7.5 mg/kg) and ivermectin (0.2 mg/kg)
Farm
no. Anthelmintic class (drug)
Larval identification (% L3) post–treatment
Telad. Trich. Chab. Haem. Coop.
1 IVM FBZ 88 – 100 – – – – – 12 – 4 IVM FBZ 100 58 42 – – – – – – – 5 IVM FBZ 40 – – – – – 10 – 50 – 7 IVM FBZ 44 42 – 4 – 4 – – 56 50 13 IVM FBZ n.d. 52 n.d. 28 n.d. – n.d. – n.d. 20 15 IVM FBZ 54 – 22 – 18 – – – – 6
Telad.: Teladorsagia; Trich.: Trichostrongylus; Chab.: Chabertia; Haem.: Haemonchus; Coop.: Cooperia
n.d.: not done
Tavle 3.2.2. Mean eggs per gram of faeces (EPG), faecal egg count reduction
percentages (FECR%) and 95% confidence intervals (CI) on 18 sheep farms in Lithuania treated with fenbendazole (7.5 mg/kg) and ivermectin (0.2 mg/kg)
Farm
no. Anthelmintic class (drug) pre-treatment EPG (range) post-treatment EPG (range) FECR% CI
1 IVM FBZ 596 (160-1340) 300 (140-600) 34 (0-140) 10 (0-100) 96.7 (94-100) 94.3 (93-99) 2 IVM FBZ 1346 (160-3540) 446 (160-1040) 26 (0-100) 0 98.1 (97-100) 100 3 IVM FBZ 378 (140-1240) 415 (140-1080) 0 0 100 100 4 IVM FBZ 544 (140-2120) 332 (140-1000) 82 (0-220) 2 (0-20) 99.4 (99-100) 85 (73-98) 5 IVM FBZ 3114 (140-11720) 2843 (140-19760) 120 (0-940) 0 96.2 (95-100) 100 6 IVM FBZ 264 (140-640) 267 (140-420) 0 0 100 100 7 IVM FBZ 428 (160-1080) 278 (140-620) 68 (0-140) 98 (0-520) 75.6 (66-85) 77.2 (68-89) 8 IVM FBZ 736 (140-3120) n.d. n.d. 0 n.d. 100 9 IVM FBZ 1366 (160-6680) 788 (140-2660) 0 0 100 100 10 IVM FBZ 352 (140-1060) 216 (140-400) 6 (0-40) 4 (0-40) 98.3 (96-100) 98.2 (98-100) 11 IVM FBZ 287 (140-620) n.d. n.d. 0 100 n.d. 12 IVM FBZ 388 (140-680) n.d. n.d. 0 n.d. 100 13 IVM FBZ 783 (140-2960) n.d. 432 (60-1360) n.d. 44.9 (31-56) n.d. 14 IVM FBZ 414 (140-1100) 246 (140-560) 0 0 100 100 15 FBZ 2724 (140-15120) 0 100 IVM 1080 (160-3600) 272 (0-2140) 74.9 (65-84) 16 IVM FBZ 1044 (140-6840) 223 (140-680) 0 0 100 100 17 IVM FBZ 238 (140-760) n.d. n.d. 0 n.d. 100 18 IVM FBZ 725 (140-2680) 868 (140-3240) 0 0 100 100 33
3.3. Prevalence of anthelmintic resistance to benzimidazoles and ivermectin aglycone on sheep farms in Lithuania assessed by in vitro
methods
The survey was conducted from August 2013 to November 2014. A total of 33 farms, mainly in central and southern Lithuania, were enrolled in the study. On 12 of the 33 farms, where only BZ were used, an EHDDT was conducted for detection of AR to BZ. On eight other farms where only ML were used, MALDT was conducted for detection of AR to IVM. On the remaining 13 farms, where both classes of drugs were used, both tests were applied to test for multidrug resistance to BZ and IVM (Publication I).
Instead of using the conventional threshold values (ED50 or ED99), the number of hatched eggs at a discrimination dose (DD) concentration of 0.1 µg/ml-1 was used, because DD prevents 99% of the susceptible eggs from hatching. The percentage of hatched eggs was categorised into low, medium or high, based on farm status (susceptible/resistance) determined by hatching in the EHDDT. BZ-resistant GIN were found in all 25 farms investigated. On 36% of these farms (9/25; 95% CI 18.0-57.5), a high level of resistance (>40% of hatching) was recorded.
A medium level of resistance in GIN nematodes was recorded on 24% of farms (6/25; 95% CI 9.4-45.1), while a low level of resistance (<20% of hatching) was recorded on 40% of farms (10/25; 95% CI 21.1-61.3) (Fig. 3.3.1). However, the differences in incidence of different levels of resistance were not significant (P>0.05)
Fig. 3.3.1. Number of sheep farms with different percentage levels of hatched
eggs at a threshold DD of 0.1 µg/ml–1 TBZ in EHDDT on 25 farms
The results for IVM resistance among GIN determined using MALDT in
vitro on 21 sheep farms are presented in Figure 3.3.2. On 61.9% of these farms
(13/21; 95% CI 38.4-81.9), L3 larvae had developed at the threshold concentration of 21.6 ng/ml-1. No resistance to IVM was detected on 38.1% of farms (8/21; 95% CI 18.1-61.6), all of which were in the group of farms where both classes of anthelmintics were used.The percentage of developed L3 larvae at the threshold concentration was categorised as low (<30% development of larvae) or high (>30%). The differences between groups where AR to IVM was detected and no resistance to IVM was found were not significant (P>0.05). A low level of resistance was detected on 84.6% of farms (11/13; 95% CI 54.6-98.1) (P<0.05), while a high level of resistance was recorded on 15.4% of farms (2/13; 95% CI 1.9-45.4). The differentiation of L3 larvae at the discri-mination concentration in the MALDT revealed the presence of
Telador-sagia/Trichostrongylus spp. in all tests.
Fig. 3.3.2. Number of sheep farms with the development of larvae
at a threshold of 21.6 µg/ml–1 IVM aglycone in LDT on 21 farms
Low levels of multidrug resistance were detected on five (38.5%; 95% CI 13.9-68.4) out of 13 sheep farms. From this group where both classes of anthelmintics were used, AR to BZ was found on all farms, while AR to IVM-Ag was found on only five out of 13 farms.
3.4. Anthelmintic resistance to benzimidazoles and levamisole on sheep farms in Lithuania detected by in vitro micro-agar larval development test
The survey was conducted from May 2014 to November 2014. The in vitro method MALDT was used for the detection of benzimidazole and levamisole resistance. Twenty-three sheep farms with regullar use of anthelmintic treatments were selected for the study. Seventeen sheep farms were tested by MALDT for anthelmintic resistance (AR) to benzimidazoles and 6 sheep farms were tested for AR to levamisole (Publication II).
A total 23 sheep farms were investigated. No resistance to thiabendazole (TBZ) were found on 5 farms (29.4%; 95% CI 10.3-56.0). Benzimidazoles resistant gastrointestinal nematodes were found on 12 farms (70.6%; 95% CI 44.0-89.7). Three (25%; 95% CI 5.5-57.2) farms had high level of resistance, 3 farms had a medium level of resistance (25%; 95% CI 5.5-57.2) and 6 (50%; 95% CI 21.1-78.9) farms had a low level of resistance. In addition, on one farm the level of developed L3 larvae on the concentration 0.04 µg.ml–1 for thiabendazole was very high – 79.5% (Fig. 3.4.1).
Fig. 3.4.1. Number of sheep farms with the development of larvae at a
threshold of 0.04 µg/ml-1 thiabendazole in MALDT on 17 farms
No resistance to levamisole were found on 4 farms (66.6%; 95% CI 22.3-95.7). Resistance to levamisole with low percentages (<3.6%) of developed L3 larvae found on 2 farms (33.4%; 95% CI 4.3-77.7).
Fig. 3.4.2 and Fig. 3.4.3 summarizes the results of minimum inhibitory concentration (MIC) values, obtained by MALDT. On the basis of MIC values
in thiabendazole resistance is advanced on 9 (52.9%; 95% CI 27.8-77.0) farms (MIC ≥0.08 µg.ml–1), in levamisole on 2 (33.4%; 95% CI 4.3-77.7) farms (MIC≥4 µg.ml–1).
Fig. 3.4.2. Comparison of thiabendazole MIC values in 17 sheep farms
Fig. 3.4.3. Comparison of levamisole MIC values in 6 sheep farms
