EVALUATION OF ANTHELMINTIC CONTROL OF INTESTINAL HELMINTH INFECTIONS IN DOGS AND IT’S LIMITING FACTORS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Žydrūnė Vienažindienė

EVALUATION OF ANTHELMINTIC

CONTROL OF INTESTINAL HELMINTH

INFECTIONS IN DOGS AND IT’S

LIMITING FACTORS

Doctoral Dissertation Agricultural Sciences,

Veterinary (A 002)

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Dissertation has been prepared at the Department of Veterinary Pathobiology, Veterinary Academy of the Lithuanian University of Health Sciences during the period of 2011–2019.

Scientific Supervisor

Prof. Dr. Mindaugas Šarkūnas (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – A 002).

Dissertation is defended at the Veterinary Research Council of the Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Albina Aniulienė (Lithuanian University of Health Sciences, Agri cultural Science, Veterinary, – A 002).

Members:

Prof. Dr. Jūratė Šiugždaitė (Lithuanian University of Health Sciences, Agri cultural Sciences, Veterinary – A 002);

Assoc. Prof. Dr. Dainius Zienius (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – A 002);

Dr. Petras Prakas (Nature Research Centre, Natural Sciences, Biology – N 010);

Prof. Dr. Manuela Schnyder (University of Zurich, Agricultural Sciences, Veterinary – A 002);

Dissertation will be defended at the open session of the Veterinary Research Council of Lithuanian University of Health Sciences, Veterinary Academy, on 30 of August, 2019 at 1:00 p.m. in the Dr. S. Jankauskas auditorium.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Žydrūnė Vienažindienė

PROFILAKTINĖS DEHELMINTIZACIJOS

PROGRAMŲ NUO ŠUNŲ VIRŠKINAMOJO

TRAKTO HELMINTOZIŲ VERTINIMAS

IR JĮ RIBOJANTYS VEIKSNIAI

Daktaro disertacija Žemės ūkio mokslai,

veterinarija (A 002)

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Disertacija rengta 2011–2019 metais Lietuvos sveikatos mokslų universitete, Veterinarijos akademijoje, Veterinarinės patobiologijos katedroje.

Mokslinis vadovas

prof. dr. Mindaugas Šarkūnas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterinarijos mokslo krypties taryboje:

Pirmininkė

prof. dr. Albina Aniulienė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002).

Nariai:

prof. dr. Jūratė Šiugždaitė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002);

doc. dr. Dainius Zienius (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002);

dr. Petras Prakas (Gamtos tyrimų centras, gamtos mokslai, biologija – N 010);

prof. dr. Manuela Schnyder (Ciuricho universitetas, veterinarinė parazi-tologija, veterinarija – A 002).

Disertacija bus ginama viešame Veterinarijos mokslo krypties tarybos po-sėdyje 2019 m. rugpjūčio 30 d. 13.00 val. Lietuvos sveikatos mokslų univer-siteto Veterinarijos akademijos Dr. S. Jankausko auditorijoje.

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ABBREVIATIONS

A. caninum – Ancylostoma caninum

C – Placebo – Control group

CAPC – Companion Animal Parasite Center CPEP – Canadian Parasite Expert Panel CI 95% – Confidence limit of the mean

ESCCAP – European Scientific Counsel for Companion Animal Parasites

D1 – treated monthly with Drontal®_Plus

D3 – treated every three months with Drontal®_Plus

D6 – treated every six months with Drontal®_Plus

DNA – deoxyribonucleic acid

D. caninum – Dipylidium caninum D. immitis – Dirofilaria immitis

E. multilocularis – Echinococcus multilocularis

E. intermedius – Echinococcus intermedius (syn. Echinococcus granulosus) E. granulosus s.l. – Echinococcus granulosus sensu lato

E. canadensis – Echinococcus canadensis

E. aerophila – Eucoleus aerophila (syn. Capillaria aerophila)

L₁ – First stage larvae

L₂ – Second stage larvae

L₃ – Third stage larvae

L₄ – Fourth stage larvae

M1 – treated monthly with Milbemax®

M3 – treated every three months with Milbemax®

M6 – treated every six months with Milbemax®

Mo – Month N – Number

P1 – treated monthly with Profender®

P3 – treated every three months with Profender®

P6 – treated every six months with Profender®

PCR – polymerase chain reaction SD – standard deviation

T. canis – Toxocara canis T. cati – Toxocara cati

T. leonine – Toxascaris leonine T. hydatigena – Taenia hydatigena T. pisiformis – Taenia pisiformis T. crassiceps – Taenia crassiceps T. ovis – Taenia ovis

T. taeniaeformis – Taenia taeniaeformis T. multiceps – Taenia multiceps

T. serialis – Taenia serialis T. solium – Taenia solium T. brauni – Taenia brauni

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INTRODUCTION

Dogs may be involved in transmission of many potentially zoonotic organ-isms of parasitic origin. Since the number of dogs bred in cities and in rural areas has recently been increasing worldwide, the risk for the transmission of parasite infections has been expected to increase accordingly [109]. The most commonly identified intestinal parasites in dogs are Toxocara canis, Taeniidae, Eucoleus aerophilus, Capillaria putorii and Trichuris vulpis [42, 73, 117]. Depending on the species and abundance of parasites, intestinal helminth infections in dogs may cause various clinical symptoms, such as vomiting, diarrhea, anemia, anorexia, dermatitis, loss of condition, intestinal disorders and severe cases could be fatal [6, 11] or may be a source for human infection [31, 80]. It is particularly important for zoonotic pathogens in dogs.

In previous study it was shown that 10.8% of village dogs in Lithuania were sheding Toxocara spp. and 14.2% – Taeniidae eggs in their faeces. Ad-ditionaly, in 3.3% of faecal samples hookworm and Capillaria spp. eggs, in 2.1% – Toxascaris leonina eggs were detected [13]. Toxocara canis is one of the most prevalent intestinal nematodes diagnosed in 3.1–34% of dogs reared in different environments in Europe [6, 34, 42, 51, 62, 82, 87, 117]. The most serious and concentrated source of Toxocara–infection is the bitch nursing a litter and the puppies aged between 3 weeks and 6 months [31].

As no practical measures are avilable to reduce the risks for human infec-tion, prevention of initial contamination of the environment is therefore the most important tool to prevent the transmission of these parasites [49]. This can be achieved by periodic anthelminthic treatments, or treatments based on the results of periodic diagnostic faecal examinations.

The European guidelines for worm control in dogs consider the individual risk situation for dogs: the recommendations on frequency of anthelmintic treatment may vary from a single annual up to monthly anthelmintic treat-ments [38]. However, in Swiss study it was shown that treatment of dogs with a combination of pyrantel embonate, praziquantel, and febantel every 3 months (Drontal®_{Plus, Bayer, Germany) for a period of 12 months had}

insuffiecient effect in reducing the detection rate of helminth eggs in their faeces. A more frequent treatment did not necessarily resulted in a further decrease of parasite detection. Despite the regular treatment, the yearly in-cidence of T. canis was 32%, while hookworms, T. vulpis., Capillaria spp., and Taeniidae eggs were detected in 11–22% of the dogs at least once a year [94]. Interestingly, parasitological findings 1–3 months after treatment did not resulted in significant differences for individual parasites such as Toxocara

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spp., Taeniidae and hookworms. While prepatent periods of investigated par-asites are longer than one month, the authors suggested that the limited effica-cy of treatment and/or coprophagia may have contributed to the egg findings after the treatment. However, in this study Toxocara eggs have not been de-termined to species level by morphometric analyses or PCR. Confirmatively, Fahrion et al. [39] and Nagy et al. [76] identified 31.5% and 7.3% of Toxocara eggs in faeces of Swiss dogs as T. cati. Therefore, especially coprophagia related to cat faeces could pose a major challenge for coprological analyses determining the prevalences of intestinal parasites in dogs. In an environment highly contaminated with embryonated T. canis eggs, frequent reinfection may occur leading to recurring patent infections even in pre–exposed adult dogs, as experimentally documented [40].

Coprophagia is an important factor that should not be neglected when per-forming and interpreting parasitological analyses. Several parasite stages that are only passing the intestinal tract may not be easily distinguished from dog specific parasites (e.g., Capillaria spp. or Taeniidae eggs from cat faeces). Furthermore, it may be possible that the prevalence of canine parasites is overestimated due to coprophagic ingestion of fox faeces, which is abundant in rural as well as urban areas [94].

Despite of few attempts the strategies for control of canine intestinal hel-minth infections still needs further detailed evaluation. Additionally, there is no experimentaly documented information on other possible factors that could limit the evaluation of control strategies in clinical field studies.

Aim and objectives

The aim of this study was to evaluate the control of intestinal helminth infections in dogs with different anthelmintics administered at three different intervals in field conditions and highlight its limiting factors.

Objectives of the study:

1. To evaluate the efficacy of febantel/pyrantel–embonate/praziquantel (Drontal®_{Plus, Bayer), emodepside/praziquantel (Profender}®_{, Bayer) or}

mil bemycin oxime/praziquantel (Milbemax®_{, Elanco) administered at 1-, 3-}

and 6-month intervals for control of intestinal helminth infections in dogs; 2. To examine the factors limiting the evaluation of control programs in field

conditions;

3. To evaluate the prevalence of E. intermedius (pig strain, G7), E. multilocularis and Taenia spp. in a population of village dogs after 6 years following long lasting regular praziquantel treatment during E. intermedius control program.

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Scientific novelty and practical significance

In this study the efficasy of three broad spectrum anthelmintics admin-istered at three different regimes against intestinal helminth infections was evaluated in a controled field study with naturally infected village dogs.

It was recorded, that exrection of T. canis eggs one month after the treat-ment of dogs with emodepside/praziquantel (Profender®_{, Bayer) and}

milbe-mycin oxime/praziquantel (Milbemax®_{, Elanco), has decreased significantly}

on few occasions in October, November, February and March.

Meanwhile, monthly treatment with febantel/pyrantel–embonate/prazi-quantel (Drontal®_{Plus, Bayer) was only significant in February as compared}

to those in the placebo-control group. The anthelmintic treatment of dogs every 3- and 6-months was insufficient in reducing the excretion of helminth eggs in dogs.

The factors that may limit the evaluation of anthelmintic control programs in dogs were evaluated. It was shown that excretion of T. canis eggs was higher (P>0.05) in chained dogs one month after the treatment as compared to those of dogs kept in pens. The regular removal of faeces from environment of restrained dogs has decreased (P>0.05) the excretion of T. canis, Taeni-idae, Trichuris spp. and Capillaria spp. eggs with faeces. Additionally, the morphometric differentiation of T. canis and T. cati eggs has improved the specificity of Toxocara spp. diagnosis by 34.5%. The established specificity of T. canis or T. cati egg morphometry was 100% if more than 3 eggs were measured. The sensitivity of the T.canis/T.cati PCR was highest (86.36%; P<0.05) if more than 3 eggs were present in the sample.

The evaluation of efficacy of E. intermedius control study by examination of village dogs after 6 years showed that transmission of E. intermedius was most probably supressed indirectly in treated group by reduced number of pigs and dogs in the villages while transmission of E. multilocularis – by fluctuations in red fox populations.

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1. LITERATURE REVIEW

1.1. The most frequent intestinal helminth infection in dogs 1.1.1. Nematodes

1.1.1.1. Toxocara spp.

Dogs are infected with T. canis by ingestion of embryonated eggs or hypo-biotic (arrested) L₃ in paratenic hosts; even older immune dogs may acquire new patent infections if exposed to low numbers of eggs [40]. Pups are infect-ed either prenatally in the last trimester of gestation or by larvae in milk from the bitch. Transplacental transmission accounts during more infections than the lactogenic route [17] and represents either recent infection of the pregnant bitch or reactivated hypobiotic larvae after somatic migration in the immune bitch [99].

The life cycle is typically migratory: after ingestion of eggs in a fully sus-ceptible host, hatched larvae migrate through the liver and lungs while moult-ing from L₃ to L₄, are coughed up through the trachea (L₄ to L₅) to finally develop into adults that reside in the small intestine of the definitive hosts. Eventually, eggs in large numbers (thousands per day) are excreted in the faeces. In the immune host, the larvae do not perform tracheal migration, but re–enter the circulation for somatic migration (i.e. L₃ relocate to skeletal muscles, kidneys, mammary gland, CNS and other organs) [99]. For T. canis, the prepatent period thus varies with the route of infection; eggs can be found in puppies 2–3 weeks of age after prenatal infection, while priepatency is 4–5 weeks after ingestion of eggs followed by tracheal migration [82]. Eggs are usually excreted for 4 months.

A dog infected with adult T. canis worms may shed thousands of eggs each day in the faeces. As this ascaride is a very common intestinal parasite in dogs, in urban areas where a relatively large number of dogs have an access to relatively little green space for their physiological needs, the contamination of soil with eggs in public areas of many cities is expected to be high [47].

Toxocara spp. eggs are unembryonated and therefore noninfective when

excreted in the faeces of dogs and cats. Eggs can develop to the infective larvated stage within a period of 3 weeks to several months, depending on soil type and environmental conditions such as temperature and humidity. Embryonated eggs have remained viable for at least 1 year under optimal circumstances [84].

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The routine deworming of pet dogs is aimed to reduce the risk for the pat-ent tapeworm and nematode infections in dogs and to prevpat-ent human infec-tions [87]. Humans are infected following ingestion of embryonated T. canis eggs, from which larvae hatch. The most frequent route of Toxocara spp. transmission to humans is via contaminated soil, the infection can also occur following ingestion of contaminated raw vegetables or fruits. Children have the highest risk of infection as they have an increased opportunity of exposure to contaminated soil or sandpits while playing outdoors [82]. People with a soil-related job (e.g. mechanics, gardeners, farmers, street cleaners) may be at the higher risk of infection with toxocarosis, as shown by their higher seroprevalence compared with values found in people with non-soil related occupations [88].

1.1.1.2. Trichuris vulpis

Trichuris vulpis, the dog whipworm, causes an intestinal parasitosis of

rel-evance in current canine veterinary practice. Its occurrence is well–known in pets, kennelled dogs and stray animals, and its eggs contaminate the ground in urban areas all over the world. Moreover, T. vulpis has been occasionally incriminated, though not convincingly substantiated, as a cause of zoonosis [122].

T. vulpis has a direct life cycle. Adult T. vulpis inhabit the large intestine

of domestic and wild canids, e.g. dogs and foxes. They are 4.5–7.5 cm long in which the thick, broad tail is about one quarter of the total length of the body. After mating, the females release trichuroid eggs containing a single cell which reach the environment via the faeces and, depending on moisture and temperature conditions, embryonate in the soil over a period of three– eight weeks to form an infectious larva inside the egg [56, 111].

Infective eggs may survive in soil for several years and can be a source of reinfection for dogs exposed to a contaminated environment. After ingestion, the larvae hatch in the small intestine and undergo a histotropic phase in the mucosa of the gastrointestinal tract before the adult stage can be found in the caecum or colon. T. vulpis is a blood–feeding nematode, with the anterior end of the body embedded in the mucosa of the caecum. Infection can lead to enteritis which – if massive – can become haemorrhagic [60]. The prepatent period of T. vulpis is reported to be 9–10 weeks, and adults live for up to 16 months [37].

The eggs may remain viable and infective in the environment for years, leading to high infection rates in dogs and consequent difficulties in

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trolling canine trichurosis. More specifically, T. vulpis eggs may survive from cold winter to hot summer, especially in wet and shady areas. Unless exposed to extreme conditions for long periods, desiccation and sunlight often do not affect egg viability. Eggs are a constant source of (re-) infection for dogs liv-ing in contaminated environments, thus the incidence of trichurosis is higher in adult dogs, which also have higher parasitic burdens than younger animals [6, 56, 111].The prevalence rates of intestinal trichurosis in adult dogs may also be due to an absence of a transplacental and/or transmammary transmis-sion of T. vulpis, due to its long pre-patent period, and due to a likely inability to elicit a protective immune response [43, 111].

1.1.1.3. Eucoleus aerophila (syn. Capillaria aerophila)

Eucoleus aerophila is a trichuroid parasitic nematode affecting the

respira-tory systems of domestic (i.e., dogs and cats) and wild (e.g., foxes and muste-lids) carnivores and occasionally of humans [111, 121]. The adult lungworms live embedded in the epithelia of the bronchioles, bronchi, and trachea of the definitive host. The females lay eggs that are coughed, swallowed, and released via faeces into the environment, where they undergo further devel-opment through the infectious stage. Animals become infected by ingesting embryonated eggs or earthworms from the environmental, which are con-sidered an intermediate or paratenic host [5, 18, 122]. The eggs reach the infective stage in about 30–45 days. After ingestion, the larvae mirgate to the lungs, where they evolve into adulthood and reach their sexual maturity after about 3–6 weeks post infection [5, 16 , 111].

The parasite is responsible for damage of lung parenchyma and the in-fection induces chronic bronchitis characterized by a wide range symptoms in canine and feline hosts. Even it is considered as subclinical infection, al-though the parasite may cause a chronic bronchitis [16, 53, 111].

Animals may display minimal respiratory signs (e.g., bronchovesicular sounds) or inflammation, sneezing, wheezing, and chronicss dry cough; when bacterial complications occur, the cough may become moist and productive, leading to bronchopneumonia and respiratory failure and additionally, heavy parasite burdens may lead to mortality [16, 53, 111].

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1.1.2. Cestodes 1.1.2.1. Taeniidae

The definitive hosts for Taenia spp. cestodes are usually carnivores. A de-finitive host becomes infected when it eats tissues from the intermediate host that contain metacestodes with larvae. The larvae attach to the small intestine and develop into adult tapeworms. T. hydatigena prepatent period in 7 to 9 weeks, and T. pisiformis is 6 weeks. The mature worm consists of a scolex, which is attached to the intestine, followed by a neck and immature, mature and gravid proglottids (segments). Gravid proglottids, containing the eggs, detach from the worm and are shed in the faeces. The proglottids of some species also crawl through the anal sphincter outside the intestine or into the environment. The eggs are immediately infective [29].

In carnivores, taeniasis is caused by T. crassiceps, T. ovis, T. taeniaeformis,

T. hydatigena, T. multiceps, T. serialis and T. brauni and is acquired by eating

tissues from a variety of intermediate hosts including ruminants, rabbits and rodents [45].

1.1.2.2. Echinococcus granulosus s.l.

The adult Echinococcus granulosus s.l. (3–6 mm long) resides in the small bowel of the definitive hosts, dogs or other canids. Gravid proglottids release the eggs that are passed in the faeces. After ingestion by a suitable intermedi-ate host (under natural conditions: sheep, goat, swine, cattle, horses, camel), the egg hatches in the small bowel and releases an oncosphere that penetrates the intestinal wall and migrates through the circulatory system into various organs, especially the liver and lungs. In these organs, the oncosphere devel-ops into a cyst that enlarges gradually, producing protoscolices and daughter cysts that fill the cyst interior. The definitive host becomes infected by ingest-ing the cyst-containingest-ing organs of the infected intermediate host. After ingest- inges-tion, the protoscolices evaginate, attach to the intestinal mucosa, and develop into adult stages in 32 to 80 days [29, 35, 113].

E. granulosus sensu lato represents a complex of different species which

are responsible for the disease cystic echinococcosis [115]. From this group of species, E. granulosus s.s. (including genotypes G1-3 and micro variants) is the most important cause for human infection worldwide; while the gen-otypes G6 and G7 of E. granulosus (also known as E. canadensis and E.

intermedius, respectively) is second [4]. E. intermedius G7 circulates mainly

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tribution is rather patchy being endemic in countries like Nepal [59], Poland [85], Lithuania [13], Argentina [106]; cases have been described in Austria, Germany, Czheck Republic, Hungary Spain, Slovakia, Romania and Ukraine [32, 92].

1.1.2.3. Echinococcus multilocularis

Echinococcus multilocularis, is one of the most pathogenic zoonotic

para-sites in Europe, leading to alveolar echinococcosis in humans [35]. In Europe, intermediate hosts of E. multilocularis include arvicolid rodents (Arvicola

terrestris) and the common vole (Microtus arvalis) although domestic dogs,

cats and other species are also sporadically infected. The red fox is referred to as the primary definitive host for E. multilocularis and responsible for the contamination of the intermediate host habitat, disseminating eggs with the faeces [35]. The adult stage of E. multilocularis that inhabits the small intes-tine of carnivores is a cestode of only 1.2–4.5 mm length. E. multilocularis affects the liver of humans as a slow growing, destructive tumor, with ab-dominal pain, biliary obstruction, and occasionally metastatic lesions into the lungs and brain and larval growth (in the liver) remains indefinitely in the pro-liferative stage, resulting in invasion of the surrounding tissues [36, 113, 114].

The highest endemicity levels occur in the northern prealpine regions, the high Tatra mountains between Poland [69] and Slovakia [33], and the moun-tainous areas stretching from southern Belgium [52] and northern [127] to central Germany.

1.2. The prevalence of intestinal helminths in dogs

Intestinal helminths of dogs are the major epidemiological challenge worldwide. In the absence of knowledge, uncontrolled and untreated dogs are excreting intestinal helminth eggs with faeces, which contaminates the environment.

The most commonly identified intestinal helminths in pet dogs in the United States were ascarids (2.2%), hookworms (2.5%), whipworms (1.2%),

Giardia (4.0%), and Cryptoisospora (4.4%) [63].

In European countries such as Poland the dogs were infected with U.

stenocephala (11%), T. canis (20.62%), T. leonina (2.91%), T. vulpis (0.27%), Taenia spp. (3.45%) [117]. In the eastern and northern regions of Hungary

– T. canis (24.3–30.1%), T. vulpis (20.4–23.3%), Capillaria spp. (0–7.3%),

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of examined dogs [42]. In Belgium, 18.1% of dogs were excreting T. canis, 7.2% T. leonina, 1.2% T. vulpis eggs [126]. In Germany the prevalence of T.

canis was 4.0%, hookworms – 0.9%, T. leonina – 0.4% [7]. 1.3. Control of helminth infections in dogs 1.3.1. Anthelmintics

Effective control of many parasitic zoonoses depends upon a detailed knowledge of life cycle patterns, particularly the role humans play in per-petuating transmission cycles like in case of E. granulosus in kangaroo-dog [116] or pig – dog situations [13]. For control of mixed infections including helminths from different classes, broad spectrum anthelmintics are used on the large scale.

Different parasitic classes are available for treatment and control of intes-tinal nematodes, being (pro-) benzimidazoles (e.g. febantel, fenbendazole), tetrahydropirimidines (e.g. pyrantel), cyclooctadepsipeptides (i.e. emode-pside) and macrocyclic lactones (e.g. ivermectin, selamectin, moxidectin, milbemycin oxime) the most used. Various anthelmintics such as pyrantel pamoate [20], praziquantel, pyrantel embonate and febantel [64], ivermectin and pyrantel pamoate [21], emodepside and praziquantel [2] have been used to treat gastrointestinal cestode and nematote infections in dogs over last de-cades. However some compounds may exhibit different efficacy against var-ious stages or different species of helminths and therefore may require more frequent treatment.

1.3.1.1. Febantel

The febantel is transformed into fenbendazole in the stomach and the in-testine of the host, shortly after ingestion. Once transformed its behavior is comparable to the one of fenbendazole [129]. Febantel is broad–spectrum effective against cestodes and nematodes, including T. vulpis, Taenia spp. and

D. caninum [1]. However, its effectivenes against somatic nematode larvae

was possible only by administration of increased doses of febantel [41, 65]. Therefore, under therapeutic doses the somatic larvae remains unaffected in host tissues and may be a sourse of reinfection. It is also more effective against ancylostomids than ascarids [25].

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1.3.1.2. Emodepside

Emodepside is highly effective against mature adult, immature adult and L₄ stages of nematodes and >94% effective against L₃ larval stages of T. canis [3]. As demonstrated by laboratory studies [3, 97, 98, 101], emodepside plus praziquantel tablets (like Profender®_{) demonstrate a broad spectrum of}

activi-ty against gastrointestinal helminths including immature stages of nematodes and cestodes.

1.3.1.3. Pyrantel pamoate

Pyrantel pamoate is poorly absorbed from the gastrointestinal tract [73] it is therefore effective against mature or premature nematodes in the intestine only [67]. Meanwhile its efficacy against larval T. canis is significantly re-duced [89], thus increasing the risk for reinfection.

1.3.1.4. Milbemycin oxime

The milbemycin oxime has high efficacy in removal of roundworms and hookworms from naturally infected dogs and cats with patent infections [9, 95]. Milbemycin oxime is available in associations either with lufenuron or praziquantel. In dogs, the oral formulation of milbemycin oxime with lufenuron has shown 91.5% efficacy against natural ascarid infections [10]. In the multicentre field study mentioned earlier the formulation containing milbemycin oxime and praziquantel has achieved geometric mean of egg counts reduced by 99.4%–99.8% in dogs infected by roundworms and A.

caninum [2]. Milbemycin oxime has been previously shown to have activity

in dogs against L₃ and L₄ stages of D. immitis as a monthly heartworm pre-ventive and efficacy against the adult intestinal nematodes, T. canis, T.

leoni-na, A. caninum and T. vulpis [2, 72, 102, 100].

Milbemycin oxime has been recently marketed in a monthly chewable tab-let for dogs, also containing the insecticide spinosad. This formulation has a 99.3–100% efficacy in treating and controlling intestinal nematodes in natu-rally and experimentally infected dogs [100, 105].

Milbemycin oxime and praziquantel [91] have been used to treat intesti-nal cestode and nematode infections in naturally and experimentally infected dogs.

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1.3.1.5. Praziquantel

The praziquantel is highly effective against mature and immature E.

gran-ulosus and E. multilocularis, mature Taenia spp. and D. caninum [101]

ces-todes. Its efficacy against different Taenia species has also received some attention. Oral doses of 1 mg praziquantel per kg of body weight have been reported to be 100% effective against T. ovis [46] and T. pisiformis [28], while this dose was described to have variable efficacy against T. hydatigena [28].

1.3.2. The strategies for control of helminth infections

1.3.2.1. Control strategy proposed by European Scientific Counsel for Companion Animal Parasites (ESCCAP)

For adult dogs, ESCCAP recommends an individual risk assessment for each animal to determine whether anthelmintic treatment is necessary, and how often. There is little information about the impact of re-treatment inter-vals on parasite burdens and environmental contamination on which to base a maximum re-treatment interval under different epidemiological conditions. Therefore, a treatment frequency of at least 4 times per year with no more than 3 months between each treatment is a general recommendation [38].

As the pre-patent period for Toxocara spp. after ingestion of larvae via predation of paratenic hosts (rodents) or infective eggs from the environment is over four weeks, monthly treatment will minimise the risk of patent in-fections. As an alternative to repeated treatments, faecal examinations can be performed at suitable intervals, followed by anthelmintic treatment where positive results are found [38].

1.3.2.2. Control strategy proposed by Companion Animal Parasite Center (CAPC)

To prevent environmental contamination, all pups should be routinely treated with pyrantel pamoate at 2, 4, 6, and 8 weeks of age and then placed on a monthly preventive with efficacy against Toxocara spp. Nursing dams should be treated for ascarids at the same time as their litters. Pregnant bitch-es may be treated during pregnancy with daily fenbendazole or 2 to 4 timbitch-es with a high dose of ivermectin to prevent transplacental and transmammary transmission of T. canis larvae to the pups [19].

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Treatment of tapeworms in dogs must be combined with appropriate man-agement, such as flea control and prevention of ingestion of prey species; in the absence of these changes, re-infection is likely to occur. In dogs that are allowed outside or that are known to have predatory behavior, a heartworm preventive containing praziquantel will routinely treat infections with Taenia spp. and other cyclophyllidean cestodes [19].

1.3.2.3. Control strategy proposed by Canadian Parasite Expert Panel (CPEP)

With their approach, in low-risk households (both pets at low risk for par-asite exposure and people at low risk of infection), treatment is based on fecal examination results or, if fecal testing is not performed, once or twice–yearly treatment is recommended. In high risk households, fecal testing is recom-mended 3–4 times per year, with treatment based on results, or administration of routine preventive treatment at least 2, and preferably 3–4 times per year [26].

1.3.2.4. Implementation of control strategies in field conditions

Pet dogs in rural areas are often infected with a variety of gastrointes-tinal helminths such as tapeworms, ascarids, hookworms and whipworms. Zoonotic species may also be developing in the canine‘s intestines, playing an important role in the transmission of helminthic zoonoses such as echi-nococcosis [31] and toxocarosis to humans [48]. Monthly treatments for the prevention of the establishment of patent intestinal T. canis in dogs older than 4–8 weeks have been recommended by CAPC [19]. The European guidelines for worm control in dogs consider the individual risk situation for dogs: the recommendations on frequency of anthelmintic treatment may vary from a single annual up to monthly anthelmintic treatments [38]. However, the sci-entific basis for a widly accepted recommendation, that animals should be treated at least 4 times a year, with no more than 3 months between each treatment is rather vague and further evidence for such a generalized strategy is needed.

As some classes of anthelmintics exhibit different efficacy against vari-ous stages or different species of helminths, the frequency of application for dogs permanently exposed to infection pressure can be determined in field studies. However, the field experimental strategy with naturally infected dogs has not been validated so far in detail. In a study with privately owned Swiss dogs it was shown that anthelmintic treatments with a combination of

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py-rantel embonate, praziquantel and febantel (Drontal®_{Plus, Bayer) in three}

months intervals had a questionable impact on preventing excretion of canine helminths eggs [94]. Regular anthelmintic treatment in this study did not re-sulted in significant decrease in Toxocara spp., Trichuris spp. and hookworm egg excretion with dog faeces. As prepatent period for all of these helminths is shorter than 3-month period, reinfection of dogs and excretion of helminth eggs in their feces could have occurred. However, accidental ingestion and intestinal passage of unembryonated T. canis, T. cati, and Trichuris spp. eggs, resulted from coprophagic behavior of dogs, presumably could have limited to some extend the evaluation of anthelmintic control strategy in this study.

1.4. The evaluation of limiting factors for the efficacy studies in field conditions

1.4.1. Contamination of environment with helminth eggs excreted with dog feaces

Toxocariosis may be a problem for both urban and rural dewellers. The study in Spain was comparing a rural area with an urban one. Similar T. canis infection rates of about 30% in dogs and a soil contamination with Toxocara eggs in the rural (9%) and in the urban (3.7%) zone [23] was found. However in a study conducted in the former East Germany on infection rates in dogs, the prevalence of T. canis was higher in rural (25.5%) than in urban (15.2%) dogs [61]. The study in Italy [51] has shown that the presence of nematode eggs in the environment was higher in rural areas (48.4%) as compared to the urban (26.2%) ones. The nematode eggs were detected in faeces of over 40% of the fenced dogs and 24% of urban and 47% of rural dogs who defecate in the area around the house.

Studies on examnination of soil contamination with helminth eggs in Eu-ropean countries shown that 16% of soil samples in Spain [27], 4% in Estonia [112], 12% in Czech Republic [34], 7% in Slovakia [83], 63.3% in Portugal [79] and 38–53% in Poland [74] were contaminated with helminth eggs.

It is known that infective Toxocara spp. eggs can survive for several years in the soil. Their tough outer shell reduces the effects of varying environmen-tal conditions [44, 66, 75]. Therefore considerable contamination of environ-ment with Toxocara eggs may constitute sites of zoonotic risks.

In Belgium [125] Toxocara spp. eggs were detection in 14% of the sand samples collected from public playgrounds and in 2% of samples collect-ed from kindergardens. Several developmental stages of Toxocara spp. eggs were observed during this study. However, microscopical differentiation

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based on differences in egg size was not considered reliable, since the study based on morphometric identification of embryonated eggs reported overlap-ping measurements for T. canis and T. cati [123]. Therefore no differentiation between T. canis and T. cati eggs was made.

According to Maizels and Meghji [68], some adult dogs to be susceptible to T. canis infection, even when repeatedly exposed to the parasite and despite of the production of specific antibodies. Therefore, not only the pups but also the adult dogs may significantly contribute to the contamination of the envi-ronment with eggs of Toxocara spp. during their lifetime.

In earlier study it was reported that most owners (56%) collected and re-moved their dog’s faeces from their yard at least four to five times per week. Such regular disposal of faeces would help reducing the risk of Toxocara spp. and other helminth eggs being maintained in the household environment [15]. According to Overgaauw [82] the education, along with an appropriate anthelmintic treatment regime and consequent reduction in environmental contamination with helminth eggs, would lead to a reduction in the incidence of most parasitic zoonoses transmited by dogs.

1.4.2. Coprophagia

Prevalence estimates of enteric helminth infections are usually based on cross-sectional studies, in which finding of helminth eggs in dog faeces at one time point is considered as proof of infection. However, some studies have suggested, that coprophagy in dogs may be responsible for finding of dog–typical [94, 130] as well as dog-atypical [39, 120] helminth parasite eggs in faecal samples in the absence of an actual infection. Generally speaking, coprophagy is likely to lead to an over estimation of the occurrence of patent helminth infections. The chance that eggs found during coproscopical exam-ination originate from eating contaminated faeces, rather than from an actual infection, depends on the parasite species in question, as not all parasites pro-duce eggs that pass through the gastrointestinal tract without being digested or at least morphologically affected [77].

Ascarids, for example, produce robust eggs that have been shown to pass through the gastrointestinal tract seemingly unaffected [31, 119]. These eggs need to mature for a longer time in the environment to become infective (e.g.

Toxocara spp.), and if ingested before reaching their infective stage, they can

passively pass through the gastrointestinal tract.

Coprophagy is a common behaviour among dogs. Dogs may consume their own faeces, faeces of other dogs and/or faeces of other animal species. Dogs consuming their own faeces are unlikely to affect prevalence estimates

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of patent infections. However, consuming faeces from other dogs may influ-ence such estimates, especially as it may concern faeces from dogs at risk of harbouring patent parasitic infections. As the result of consuming faeces from other species, eggs of non-dog parasites that are hard to distinguish morphologically from eggs of dog parasites can also affect the results of co-proscopical examinations. It is unclear however, how frequently coprophagic behaviour occurs among dogs and to what extent coprophagy may influence prevalence estimates of cross–sectional studies on dog parasites based on sin-gle faecal examinations [77].

1.4.3. The importance of intestinal passage of ingested eggs

Eggs may be excreted in the dogs faeces while the dog is not infected. To achieve a false positive diagnosis for patent infection, the stages must be recognizable after passing the gastrointestinal tract of a dog. In an environ-ment highly contaminated with embryonated T. canis eggs, frequent reinfec-tion may occur leading to recurring patent infecreinfec-tions even in pre-exposed adult dogs, as experimentally documented [40]. Nijsse et al., [77] assumed that Toxocara spp., Trichuris spp., Capillaria spp. and strongyle-type eggs are able to pass the digestive tract of a dog that were ingested due to coprophagia. Fahrion et al. [39] reported that following ingestion of felid faeces, dogs may shed T. cati eggs in their faeces after intestinal passage.

The intestinal passage of taeniid eggs after ingestion by dogs however seems to be doubtful. The results of investigations on hatching and activation of taeniid eggs showed, that this may be achieved by exposure of eggs in pancreatin (T. pisiformis; [103]), bicarbonate and pancreatin (T. pisiformis; [8]) or artificial gastric juice (T. solium; [90]). This suggests that the stimuli involved are not specific for particular intermedieate hosts and that the eggs may hach and activate in the gut of the definitive host as it was recorded by Vogue and Berntzen [128] or Greeve and Tyler [50]. In the study on intestinal passage of T. pisiformis, T. ovis and T. hydatigena eggs in dogs it was shown that majority of eggs hatched in the anterior half of small intestine of dogs and therefore were not available after faecal passage [22]. Furthermore, the pronounced anti-oncospheral antibody responses in dogs infected with ither

T. hydatigena or T. pisiformis eggs suggested that taeniid oncospheres are

able to penetrate the intestinal mucosa of canine definitive host and become active [58]. The eggs of E. multilocularis are also able to hatch in the gastro– intestinal tract of dogs as they were reported to cause metacestode infection in dogs [30]. This suggests that intestinal passage of taeniid eggs after ingestion is of minor concern as compared with nematode eggs.

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2. MATERIALS AND METHODS

2.1. The efficacy of three different dosing regimens

of febantel/pyrantel–embonate/praziquantel (Drontal®_{Plus, Bayer)} and emodepside/praziquantel (Profender®_{, Bayer) against}

gastrointestinal helminth infections in dogs 2.1.1. Study animals and sampling

A total of 894 village dogs with the opportunity for individual sampling of faeces from the ground (i.e. dogs on chain or in pens) living in a 50 km radius around Kaunas city, from Jonava, Kaišiadorys, Kaunas, Kėdainiai, Prienai and Šakiai districts, Lithuania, were included in this study. The dogs were randomly selected with the owners’ consent and subdivided in 7 groups as described below (Table 2.1.1.1). The sample size per group was calculated using StatTools (www.stattools.net) with expected prevalences of 1% for the treated and based on previous studies (T. canis 14.2% and Taeniidae 10.8%; [13]) of 10% for the control groups for these key parasites. Based on these assumptions the calculated minimal sample size was 100 dogs per group.

Every dog received an individual ID number and a general examination of the health status was carried out at day of study start (month 0) and repeated during the monthly visits (total 12 months). The fresh individual faecal sam-ples were collected monthly from the ground within the area of dogs (n=894) (i.e. dogs on chain (n=713) or in pens (n=181); dogs in regularly removed faeces (n=505) and non-removed faeces (n=389).

2.1.2. Anthelmintic treatment of dogs

In this placebo-controlled and blinded field trial, dogs were divided into three groups and orally treated with febantel/pyrantel–embonate/praziquantel (Drontal®_{Plus, Bayer) (D1, D3, D6), emodepside/praziquantel (Profender}®_,

Bayer) (P1, P3, P6), or a placebo (control group, termed as C) at different time points during the study (Table 2.1.1.1). All dogs in the control group were administered with placebo on months 0 and 6. All dogs were weighted at the beginning of the study (and again after 2 months for young growing dogs) and the anthelminthic drug dosage was adjusted accordingly to the manufac-turer’s instructions. For ethical reasons, dogs in the control group were treat-ed with anthelminthic after the last sampling at the end of the study.

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Table 2.1.1.1. Study design of a controlled anthelminthic treatment study in

Lithuanian village dogs orally treated monthly (1), every three months (3) or every six months (6) with febantel/pyrantel–embonate/praziquantel

(Dron-tal®_{Plus, Bayer) (D1, D3, D6), emodepside/praziquantel (Profender}®_{, Bayer)}

(P1, P3, P6) or placebo (C) Dogs groups C D1 D3 D6 P1 P3 P6 No. of dogs at study start 202 114 113 117 119 115 114 No. of dogs at study end 177 95 100 95 102 101 89 No. of dogs lost

during study (12.3%)25 (16.6%)19 (11.5%)13 (18.8%)22 (14.2%)17 (12.1%)14 (21.9%)25 Treatment

frequency 6-month intervals Monthly 3-month intervals 6-month intervals Monthly 3-month intervals 6-month intervals No. of

treatments 2 13 5 3 13 5 3

2.1.3. Coprological analyses

Faecal samples were stored at 4°C until examination. Ten grams of faeces were used for sedimentation in 400 ml of water. Of those, two milliliters of fecal sediment (around 1/3 of the total sediment) were examined microscop-ically after flotation in zinc chloride solution (density 1.45 g/cm³). Toxocara eggs were morphologically differentiated according to the measurements as described [39]. If available, 10 Toxocara eggs per sample were microscopi-cally measured by using a 10x objective and ocular with integrated microme-ter scale. Eggs measuring 62.3 µm (range 61.8–62.7 µm) by 72.7 µm (range 72.2–73.3 µm) were considered as T. cati, while eggs measuring 74.8 µm (range 74.0–75.7 µm) by 86.0 µm (range 85.1–86.8 µm) were classified to

T. canis [39]. The samples with morphologically questionable Toxocara spp.

eggs (n=45) were further tested by PCR discriminating T. canis and T. cati [57]. All T. canis – PCR positive, but T. cati – PCR negative samples were considered as T. canis. Samples that were tested PCR – positive for T. cati, but negative for T. canis were considered as T. cati. PCR negative samples were further identified morphometrically according to remaining eggs with defined measurements best fitting to T. cati or T. canis [39]. Additionally, samples with varying numbers of measured Toxocara spp. eggs (from 1 to 10 per sample; n=156) were randomly selected for confirmation by PCR.

If taeniid eggs were detected by microscopy, DNA isolation and further molecular identification was performed (see next paragraph). Capillaria-like

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eggs were not differentiated to species level. The findings of hookworm as well as T. leonina eggs were not included in this evaluation.

2.1.4. Molecular analyses

Supernatant of positive samples as well as the wash fluid of the microscop-ic slide of the corresponding sample were sieved through a 40 µm mesh filter membrane for Toxocara eggs as described [71]. DNA extraction from

Toxo-cara eggs isolated from the filter was performed as described by Stefanić et

al. [107] and further molecular analysis was performed by PCR as described by Jacobs et al. [57].

Taeniidae positive faecal samples were further processed by sequential sieving through nylon nets followed by taeniid egg isolation and identifica-tion using an inverted microscope (egg F/Si method) [71]. DNA extracidentifica-tion from isolated taeniid eggs was performed as described by Stefanić et al. [107] followed by multiplex PCR according to Trachsel et al. [118] for the simul-taneous detection of E. granulosus (all strains), E. multilocularis and Taenia spp.

2.1.5. Statystical analyses

The minimal yearly incidences (one positive result during the year) for excretion of Toxocara spp., T. canis, T. cati, taeniid- and Capillaria-like eggs were calculated by including all dogs within the group that had positive de-tection of at least one egg during the study. Subsequent positive coproscopi-cal records in the same dog during the study were not included in this coproscopi- calcu-lation due to the long patency of possible helminth infections or reinfections. The exact binomial confidence intervals for the monthly excretion of T. canis,

T. cati, taeniid- and Capillaria-like eggs in each group were calculated using

the StatPages.net [86]. If the prevalence in treated group was lower than the lower limit of 95% CI in the control group and vice versa, the difference was considered as statistically significant. The difference for the minimal year-ly incidences for excretion of Toxocara, taeniid- and Capillaria-like eggs as well as the cumulative results for these parasites during November to March between chained dogs and dogs kept in pens was tested using Fisher’s Exact test (Graph Pad Prism, version 4.0). A P-value ≤0.05 was considered statisti-cally significant.

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2.2. The efficacy of three different dosing regimens of milbemycin oxime/praziquantel (Milbemax®_{, Elanco) against gastrointestinal}

helminth infections in dogs 2.2.1. Study animals and sampling

A total of 552 village dogs with the opportunity for individual sampling of faeces from the ground (i.e. dogs on chain or in pens) living in a 50 km radius around Kaunas city, from Jonava, Kaišiadorys, Kaunas, Kėdainiai, Prienai and Šakiai districts, Lithuania, were included in this study. The dogs were randomly selected with the owners’ consent and subdivided in 4 groups as described below (Table 2.2.1.1). The sample size per group was calculated using StatTools (www.stattools.net) with expected prevalences of 1% for the treated and based on previous studies (T. canis 14.2% and Taeniidae 10.8%; [13]) of 10% for the control groups for these key parasites. Based on these assumptions the calculated minimal sample size was 100 dogs per group.

Every dog received an individual ID number and a general examination of the health status was carried out at day of study start (month 0) and repeated during the monthly visits (total 12 months). The fresh individual faecal sam-ples were collected monthly from the ground within the area of dogs (n=552) (i.e. dogs on chain (n=462) or in pens (n=90)).

2.2.2. Anthelmintic treatment of dogs

In this placebo-controlled and blinded field trial, dogs were divided into three groups and orally treated with milbemycin oxime/praziquantel (Milbemax®_{, Elanco) (M1, M3, M6) or a placebo (control group, termed}

as C) at different time points during the study (Table 2.2.1.1). All dogs in the control group were administered with placebo on months 0 and 6. All dogs were weighted at the beginning of the study (and again after 2 months for young growing dogs) and the anthelminthic drug dosage was adjusted accordingly to the manufacturer’s instructions. For ethical reasons, dogs in the control group were treated with anthelminthic after the last sampling at the end of the study.

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Table 2.2.1.1.Study design of a controlled anthelminthic treatment study in Lithuanian village dogs orally treated monthly (1), every three months (3) or

every six months (6) with milbemycin oxime/praziquantel (Milbemax®_,

Elan-co) (M1, M3, M6) or placebo (C)

Dogs groups C M1 M3 M6

No. of dogs at study start 202 116 117 117 No. of dogs at study end 177 98 95 94 No. of dogs lost during study 25 (12.3%) 18 (15.5%) 22 (18.8%) 23 (19.7%) Treatment frequency 6-month

intervals Monthly 3-month intervals 6-month intervals No. of treatments 2 13 5 3

2.2.3. Other analyses

For coprological analyses see in 2.1.3. For molecular analyses see in 2.1.4. For statystical analyses see in 2.1.5.

2.3. Evaluation of the efficacy of E. intermedius (pig strain, G7) control program, by examination of dogs in treatment

and control areas after 6 years. 2.3.1. Study animals and sampling

In 2017 the study included a population of 334 dogs from the treatment and control areas of the same villages, where the long–term dog treatment was carried out four times a year, every two months, during the most inten-sive pig slaughtering period (October–April) in 2006–2010.

For E. intermedius control experiment 11 villages were selected accord-ing to slaughterhouse records for CE in the endemic area in the southwest of Lithuania, located between 36.7 and 47.6 km from the border with Poland. Infected pigs were traced back to the backyard farm where they were reared, according to their ear tag. The villages with the highest numbers of CE cases in pigs were assigned randomly to 2 areas: “treatment areas” with anthelmint-ic intervention in the dog populations and control areas without interventions. Six villages (Sasnava, Bitikai, Kantališkiai, Surgučiai, Purviniškės and Be-bruliškė) were assigned to the treatment area, while six other villages (Aukš-tosios, Baraginė, Dženčialauka, Puskelniai, Taukaičiai and Smilgiai) from the same endemic area were assigned to the control area. A highway separated the treatment and control areas. The shortest distance between control and

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treat-ment areas was 1.65 km. The fresh individual fecal samples were collected from the ground within the area accessible for dogs.

The total number of dogs in selected villages was estimated based on the average number of dogs per household. For this purpose, the number of dogs was counted in 436 households visited in 2006 and the average was 1.46 dogs per household. Based on this number, the total number of dogs was estimated to be 953 (540 in the control and 413 in the treatment areas) from 653 house-holds in all selected villages (370 in control and 283 in treated areas). From the total dog population estimated in both groups, between 62.5 and 72% were sampled and examined for the presence of intestinal helminths once a year in December during the period 2006–2010.The majority of dogs were tested each year; however, some dogs had to be withdrawn from the study (animals died or were given away) or additional new dogs that were brought by the same owner were introduced into the study. Most of the dogs were lo-cal crossbreeds, between 1 to 10 years of age with an average weight of 16 kg (8–50 kg). All dogs were included in the study with the consent of the owners. The owners of control and treated dogs were not blinded during the study.

In 2017, 6 years after the end of the intervention, faeces of dogs from the same control and treatment areas were sampled to document the situation and assess the sustainability of the control efforts after 6 years. The average num-ber of dogs/household was 0.6, yielding a calculated numnum-ber of 222 dogs in the control and 170 in the treatment areas. From these dog populations, 83.8% of dogs from the control areas and 87.2% from the treatment areas were sam-pled and examined in 2017.

2.3.2. Coprological analyses

Faecal samples were frozen at –80°C for 5 days to inactivate taeniid eggs and subsequently stored at –20°C until further use. The efficacy of the treatment strategy in dogs was measured by estimating the prevalence of patent taeniid infections, followed by DNA analyses for genus/species determination. Individual faecal samples were homogenised with a spatula and a subsample of 2.5 g was placed into a 12 ml tube, diluted with water (1:4) and centrifuged at 1600 g for 10 minutes. The sediment (about 2 ml) was resuspended with 6 ml of ZnCl₂ solution (density: 1.45) and vortexed vigorously. Tubes were filled with the same ZnCl₂ solution and centrifuged for 30 min at 400 g. The supernatant containing the eggs was passed through sieves of different sizes (100, 50 and 21 µm); the taeniid eggs (if present) were retained in the 21 µm mesh [71].

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2.3.3. Other analyses

For molecular analyses see in 2.1.4. For statystical analyses see in 2.1.5.

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3. RESULTS

3.1. The efficacy of three different dosing regimens

of febantel/pyrantel–embonate/praziquantel (Drontal®_{Plus, Bayer)} and emodepside/praziquantel (Profender®_{, Bayer) against}

gastrointestinal helminth infections in dogs 3.1.1. The dynamic of parasite egg excretion in non-treated control dogs

At the beginning of study (month 0; May), 4.02% of dogs in all groups (36/894) excreted T. canis, 0.78% (7/894) Taeniidae, 5.03% (45/894)

Capil-laria–like and 0.34% (3/894) T. vulpis eggs. T. cati eggs were found in 0.44%

(4/894) of samples at this time point. Out of 11 sequenced taeniid-egg posi-tive fecal samples, 6 were confirmed as T. pisiformis, one as E. intermedius., one as E. multilocularis, one as T. hydatigena, one as Taenia spp. and from one sample no sequence could be determined. No significant differences were found in the prevalence of T. canis between all groups at beginning of the study (month 0) (Table 3.1.1.1). The excretion of T. canis eggs in the place-bo-control group C (Fig. 3.1.1.1 A) was low in summer (2.68% in June, 2.29% in July and 2.34% in August), slightly elevated in autumn and increased in winter, reaching a significantly higher value in February (7.37%; P<0.05) as compared to those in June, July and August. No significant difference in egg excretion was observed for taeniid– or Capillaria–like eggs during the year.

3.1.2. The dynamics of parasite egg excretion in treated dogs

Dogs treated with febantel/pyrantel–embonate/praziquantel (Dron-tal®_{Plus, Bayer). In the febantel/pyrantel–embonate/praziquantel}

(Dron-tal®_{Plus) D1, D3 and D6 groups, 0.00–3.81% of dogs shed T. canis eggs}

one month after the treatment, while dogs in the placebo-control group (C) were monthly excreting T. canis eggs ranging between 1.13–7.37% (Table 3.1.1.1). A significant decrease in excretion of T. canis eggs one month after the treatment was found in group D1 in February (2.02%; 95% CI 0.25–7.11) as compared to the placebo-control group C (7.41%; 95% CI 4.11–12.12) in the same month (Table 3.1.1.1). In the febantel/pyrantel–embonate/prazi-quantel (Drontal®_{Plus) D1, D3 and D6 groups, none of the dogs were}

ex-creting taeniid eggs one month after the treatments (except 0.89% in August (group D1) and 0.87% in June (group D3)), while 0.95–3.80% of dogs in the placebo-control group C excreted taeniid eggs monthly.

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Table 3.1.1.1.

Excr

etion of T

. canis eggs in dogs tr

eated orally with febantel/pyrantel–embonate/praziquantel

(Dr

ontal

®

Plus, Bayer) (D1, D3, D6), emodepside/praziquantel (Pr

ofender ® , Bayer) (P1, P3, P6) or Placebo (C) Mo of study Mo Excr etion of T . canis eggs in Placebo-contr ol dogs Excr etion of T. canis eggs in Dr ontal ®Plus tr eated gr oup Excr etion of T. canis eggs in Pr ofender ® tr eated gr oup C D1 D3 D6 P1 P3 P6 N pos./N % (95% CI) N pos./N % (95% CI) N pos./N % (95% CI) N pos./N % (95% CI) N pos./N % (95% CI) N pos./N % (95% CI) N pos./N % (95% CI) 0 May 9/202 4.46 (2.06–8.29) 3/1 14 2.63 (0.55–7.50) 3/1 13 2.65 (0.55–7.56) 6/1 17 5.13 (1.19–10.38) 6/1 19 5.04 (1.87–10.65) 7/1 15 6.09 (2.48–12.14) 2/1 14 1.75 (0.21–6.19) 1 Jun 6/224 2.68 (0.99–5.74) 1/1 12 0.89 (0.02–4,87) 0/1 15 0.00 (0.00–3.16) 1/1 15 0.87 (0.02–4.75) 0/1 18 0.00 (0.00–3.08) 1/1 17 0.85 (0.02–4.67) 3/1 14 2.63 (0.55–7.50) 2 Jul 6/218 2.29 (0.75– 5.27) 1/1 12 0.89 (0.02–4.87) 0/1 13 0.00 (0.00–3.21 2/1 16 1.74 (0.21–6.14) 0/1 14 0.00 (0.00–3.18) 1/1 16 0.86 (0.02–4.71) 1/ 111 0.90 (0.02–4.92) 3 Aug 5/214 2.34 (0.76 –5.37) 1/1 12 0.89 (0.02–4.87) 1/1 13 0.88 (0.02–4.83) 0/1 14 0.00 (0.00–3.18) 0/1 12 0.00 (0.00–3.24) 2/1 14 1.75 (0.21–6.19) 3/ 111 2.70 (0.56–7.70) 4 Sep 6/204 2.94 (1.09–6.29) 2/109 1.83 (0.22–6.47) 2/ 111 1.80 (0.22–6.36) 4/1 13 3.54 (0.97–8.82) 1/1 10 0.91 (0.02–4.96) 0/1 12 0.00 (0.00–3.24) 2/109 1.83 (0.22–6.47) 5 Oct 9/210 4.29 (1.98–7.98) 1/106 0.94 (0.02–5.14) 1/ 111 0.90 (0.02–4.92) 5/1 10 4.55 (1.59–10.29) 0/1 10 0.00 (0.00–3.30)* 0/1 12 0.00 (0.00–3.24) * 3/107 2.80 (0.58 –7.98) 6 Nov 7/199 3.52 (1.43–7.1 1) 2/109 1.83 (0.22–6.47) 2/108 1.85 (0.22–6.53) 5/1 10 4.55 (1.59–10.29) 0/109 0.00 (0.00–3.33)* 2/109 1.83 (0.22–6.47) 4/102 3.92 (1.08–9.74) 7 Dec 7/194 3.61 (1.46 –7.29) 2/107 1.87 (0.23–6.59) 2/106 1.89 (0.23–6.65) 1/106 0.94 (0.02–5.14) 1/108 0.93 (0.02–5.05) 0/108 0.00 (0.00–3.36) * 1/101 0.99 (0.02–5.39) 8 Jan 7/189 3.70 (1.50–7.48) 4/105 3.81 (1.05–9.47) 4/103 3.88 (1.07–9.65) 4/101 3.96 (1.09–9.83) 1/105 0.95 (0.02–5.19) 1/105 0.95 (0.02–5.19) 0/101 0.00 (0.00–3.59) * 9 Feb 14/189 7.41 (4.1 1–12.12) 2/99 2.02 (0.25–7.1 1)* 2/101 1.98 (0.24–6.97) * 8/98 8.16 (3.59–15.45) 2/105 1.90 (0.23–6.71)* 4/104 3.85 (1.06–9.56) 2/98 2.04 (0.25–7.18) * 10 Mar 9/188 4.79 (2.21–8.89) 1/98 1.02 (0.03–5.55) 3/100 3.00 (0.62–8.52) 10/96 10.42 (5.1 1–18.32) * 0/102 0.00 (0.00–3.55)* 0/102 0.00 (0.00–3.55) * 6/95 6.32 (2.35–13.24) 11 Apr 6/185 3.24 (1.20–6. 92) 2/97 2.06 (0.25–7.25) 2/100 2.00 (0.24–7.04) 11/97 11.34 (5.80–19.39) * 0/103 0.00 (0.00–3.52) 3/103 2.91 (0.60–8.28) 4/95 4.21 (1.16–10.43) 12 May 2/177 1.13 (0.14–4.02) 0/95 0.00 (0.00–3.91) 4/100 4.00 (1.10–9.93) 4/95 4.21 (1.16–10.43) 0/102 0.00 (0.00–3.55) 3/101 2.97 (0.62–8.44) 1/89 1.12 (0.03–6.10)

*P<0.05 as compared with the Placebo group in respective month.

Bold

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A

B

Fig. 3.1.1.1. Monthly detection of T. canis eggs

(A) in all dogs of placebo-control group C (n=202) (black triangles), placebo-control dogs in pens (13.9%) (red circles) or chained placebo dogs (86.1%) (green squares); (B) T. canis egg excretion 1 month after treatments with febantel/pyrantel–embonate/

praziquantel (Drontal®_{Plus, Bayer) or emodepside/praziquantel (Profender}®_{, Bayer)}

(each data point represents combined data from dogs treated at 1, 3, and 6-month intervals, in treated dogs (black triangles;

n_min=182, n_max=695), treated dogs in pens (20.8%) (red circles; n_min=52, n_max=157) or chained treated dogs (79.2%) (green squares; n_min=130, n_max=538).

Vertical bars represent 95% confidence interval.

Dogs treated with emodepside/praziquantel (Profender®_{, Bayer). In}

emodepside/praziquantel (Profender®_{, Bayer) P1, P3 and P6 groups, 0.00–}

2.63% of dogs were excreting T. canis eggs one month after the treatments (Table 3.1.1.1). Significant reduction of egg excretion one month after the treatment was recorded in the P1 group in October, November, February and

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34

March and in the P3 group in December and March (P<0.05). Additionally, two months after treatment, groups P3 (October) and P6 (January) and three months after treatment group P6 (February) revealed significant reductions in excretion of T. canis eggs (P<0.05). The excretion of Taeniidae eggs one month after the treatments with emodepside/praziquantel (Profender®_{, Bayer)}

(groups P1, P3 and P6) was recorded in 0.84% of dogs in June (month 1) in group P1 (Table 3.1.2.1). The effect of treatment was significant in groups D1 and P1 in April, when excretion of taeniid eggs in placebo-control dogs has peaked up to 3.80% (95% CI 1.54–7.68) (P<0.05).

3.1.3. The minimal yearly incidence of egg excretion in dogs treated with febantel/pyrantel–embonate/praziquantel (Drontal®_Plus, Bayer) or emodepside/praziquantel (Profender®_{, Bayer)}

As shown in Fig. 3.1.3.1, the monthly treatments with febantel/pyrantel– embonate/praziquantel (Drontal®_{Plus, Bayer) and emodepside/praziquantel}

(Profender®_{, Bayer) significantly decreased the minimal yearly incidence for}

excretion of Toxocara spp. (11.86%; 95%CI 6.64–19.10 in P1) and taeniid eggs (0.00%; 95% CI 0.00–3.24 in D1 and 0.00%; 95% CI 0.00–3.08 in P1, 1.75%; 95% CI 0.21–6.19 in P6) (P<0.01), while excretion of T. canis eggs was significantly reduced by monthly administration of emodepside/prazi-quantel (Profender®_{, Bayer) (4.24%; 95% CI 1.39–9.61 in P1) compared to}

placebo-control group C (eggs of Toxocara sp. in 26.34%; 95% CI 20.70– 32.62; of T. canis in 16.96%; 95% CI 12.29–22.53; and of taeniid eggs in 6.70%; 95% CI 3.80–10.80 of dogs) (P<0.01) (Fig. 3.1.3.1).

At the beginning of the study in May (month 0), 3.51% (D1), 1.77% (D3), 9.40% (D6) and 8.40% (P1), 6.96% (P3), 4.39% (P6) of dogs were excreting

Capillaria–like eggs in the febantel/pyrantel–embonate/praziquantel

(Dron-tal®_{Plus, Bayer) or emodepside/praziquantel (Profender}®_{, Bayer)}_groups,

re-spectively. One month after the treatment with febantel/pyrantel–embonate/ praziquantel (Drontal®_{Plus, Bayer) or emodepside/praziquantel (Profender}®_,

Bayer), 1.79% (D1), 1.74% (D3), 5.22% (D6) and 1.69% (P1), 3.42% (P3) 1.75% (P6) of dogs, respectively, were still excreting Capillaria-like eggs. The excretion of Capillaria-like eggs in the faeces was not significantly af-fected by any of the treatment regimes.

EVALUATION OF ANTHELMINTIC CONTROL OF INTESTINAL HELMINTH INFECTIONS IN DOGS AND IT’S LIMITING FACTORS

Žydrūnė Vienažindienė