LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Faculty of Veterinary Medicine

Constantin Marc David Heinicke

A relative study on association between Sarcoptes scabiei and intestinal helminth infection in a population of hunted red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania.

Asociacijos tarp užsikrėtimo Sarcoptes scabiei ir helmintais vertinimas Lietuvoje sumedžiotų rudųjų lapių (Vulpes vulpes) ir usūrinių šunų (Nyctereutes procyonoides) populiacijose.

MASTER THESIS of Integrated Studies of Veterinary Medicine

Supervisor Prof. Dr. Mindaugas Šarkūnas

Kaunas, LT 2020

Annex 4

THE WORK WAS DONE IN THE DEPARTMENT OF VETERINARY PATHOBIOLOGY CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

I confirm that the presented Thesis “A relative study on association between Sarcoptes scabiei and intestinal helminth infection in a population of hunted red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania.”. 1. Has been done by me; 2. Has not been used by any other Lithuanian or foreign university; 3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.

Constantin Marc David Heinicke (date) (name, surname) (signature)

CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE ENGLISH LANGUAGE IN THE DONE WORK I confirm the correctness of the English language in the done work. Genė Pečiukevičienė (date) (editor’s name, surname) (signature)

CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE THESIS Prof. Dr. Mindaugas Šarkūnas (date) (name, surname of the head of (signature) department/clinic/institute)

Reviewer of the Master Thesis

(name, surname) (signature) Evaluation of the defense commission of the Thesis:

(date) (name, surname of the secretary of the (signature) defense commission)

2 | P a g e

1. TABLE OF CONTENT

1. TABLE OF CONTENT ______3

2. TABLE OF FIGURES AND IMAGES ______4

3. SUMMARY ______5

4. SANTRAUKA ______7

5. INTRODUCTION ______9

6. LITERATURE REVIEW ______12

6.1. Red fox (Vulpes vulpes) ______12 6.2. Raccoon dog (Nyctereutes procyonoides) ______13 6.3. Synergism or Mutualism of parasitic species ______13 6.4. Coprological analysis of endoparasites ______14 6.5. Red fox and raccoon dog endoparasites______14 6.5.1. Sarcoptes scabiei ______14 6.5.2. Nematodes ______15 6.5.3. Cestodes ______17 6.5.4. Trematodes ______17 6.5.5. Protozoa ______18 7. METHODS AND MATERIALS ______19

7.1. Biosecurity ______19 7.2. Collection of sample individuals ______19 7.3. Identification and tracking of samples ______20 7.4. Test group ______20 7.5. Coprological analyses ______22 7.5.1. Sample preparation ______22 7.5.2. Sample analysis ______22 7.6. Statistical analysis ______23 8. RESEARCH RESULTS ______25

9. DISCUSSION ______34

10. CONCLUSION ______37

11. PERSONAL SUGGESTION ______38

12. BIBLIOGRAPHY ______39

3 | P a g e

2. TABLE OF FIGURES AND IMAGES

Figure 1 – The distribution of hunted and collected red fox test subjects in different sex and age groups, the title and number indicate the individuals in each group...... 21 Figure 2 – The distribution of hunted and sampled raccoon dog test subjects in different sex and age groups, the number indicating the individuals in each group...... 22 Figure 3 – Prevalence of Sarcoptes sp. in hunted Raccoon dog (light grey) and red fox (dark grey). Numbers represent species prevalence in percentage...... 25 Figure 4 – Local distribution of Sarcoptic mange in percent of all gathered samples in Lithuanian provinces...... 26 Figure 5 – The detection frequency of excreted helminth eggs and coccidian oocysts in hunted Red fox and Raccoon dog sample population...... 27 Figure 6 – The frequency of Sarcoptes scabiei and a selected parasite with the calculated prevalence for each species in Red fox or Raccoon dog. The “*“showing statistical significance of (p<0, 05) and “**” showing high statistical significance of (p<0.005)...... 29 Figure 7 – Comparison of cross detection of red foxes which have Sarcoptes scabiei and a specific other parasite. The “*” shows a p value that is below 0, 05 and “**” shows a high statistically significant with (P < 0.005). The “^” suggest synergistic or mutualistic activity between parasites. The “˅“suggests that they have a negative effect on each other...... 31

4 | P a g e

3. SUMMARY

The presented thesis with the Title: “A relative study on association between Sarcoptes scabiei and intestinal helminth infection in a population of hunted red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania.” was completed by me, Constantin Marc David Heinicke. To date, there is a lack of published information available on Sarcoptes scabiei and their interaction with other parasite species. In present study the association between ectoparasite Sarcoptes scabiei and detected intestinal parasite infections in hunted red foxes and raccoon dogs was evaluated, which gives an estimative view on prevalence of parasite infections in Lithuania’s medium sized carnivore populations as well as the relation between parasite species found and their abundance is highlighted in the present study. All foxes and raccoon dogs were divided into two groups each. The foxes in the first group (n =27) were infected with Taeniidae spp; Toxocara canis, Toxascaris leonina, Uncinaria stenocephala, Eucoleus aerophila, Capillaria putorii, Trichuris vulpis, Alaria alata, Coccidia spp; while foxes in the second group (n= 13) were infected with Sarcoptes sp. and one or more of the following: Toxocara canis, Uncinaria stenocephala, Capillaria putorii, Trichuris vulpis, Alaria alata, Coccidia spp.. The raccoons in the third group (n=6) were non-infected with Sarcoptes scabiei but these parasites were found: Toxocara canis, Toxascaris leonina, Uncinaria stenocephala, Eucoleus aerophila, Capillaria putorii, Trichuris vulpis, Alaria alata. While in the fourth group (n=8) raccoon dogs were infected with Sarcoptes sp. and one or more of each of these intestinal parasites: Toxocara canis, Uncinaria stenocephala, Capillaria putorii, Trichuris vulpis, Alaria alata. All foxes and raccoon dogs analyzed in present study were feral and had been obtained from all, but three provinces (Alytaus, Marijampoles and Telšiai) of Lithuania. All age groups and both sexes were gathered all year round. In total 88 animals were coprologically examined. The study was carried out from December 2017 till May 2020, at the Department of Veterinary Pathobiology, Veterinary Academy, LUHS. The parasite eggs which were found in the excrements of the test groups show a correlation between Sarcoptes sp. and endoparasites in both populations. As a matter of fact, we have the data suggesting the mutualistic or synergistic behavior as well as the reverse effect in which, for instance Uncinaria stenocephala showed a 4.40% increase (Ρ = 0.020) in raccoon dog population and a 3.80% (Ρ = 0.013) increase in the red fox population, the prevalence observed with the combination of Sarcoptes scabiei in contrast when Uncinaria stenocephala appeared on its own. The mechanisms causing it are still not known. For more detailed analysis, the investigation on this topic should be continued and more factors, for

5 | P a g e example host immunity parameters, parasitic behavior regarding the hosts temperature change, the behavioral changes of the host due to Sarcoptosis and its effects on alternate parasite infection. Larger sample sizes of feces should be collected as well.

Keywords: Sarcoptes scabiei, coinfection – disease, endoparasites, Vulpes vulpes, Nyctereutes procyonoides, Lithuania;

6 | P a g e

4. SANTRAUKA

Aš, Constantin Marc David Heinicke, parašiau šį magistro darbą pavadinimu “Asociacijos tarp užsikrėtimo Sarcoptes scabiei ir helmintais vertinimas Lietuvoje sumedžiotų rudųjų lapių (Vulpes vulpes) ir usūrinių šunų (Nyctereutes procyonoides) populiacijose”. Šiandien turime labai mažai informacijos apie Sarcoptes scabiei ir jų sąsajas su kitomis parazitų rūšimis. Šiame tyrime buvo ištirta asociacija tarp ektoparazito Sarcoptes scabiei ir žarnyno parazitų sukeltų infekcijų rudosioms lapėms bei usūriniams šunims, kas padeda susidaryti apytikrį vaizdą apie tiriamų parazitų paplitimą Lietuvos vidutinio dydžio mėsėdžių populiacijose. Tyrime tirta rastų žarnyno parazitų gausa ir taip išsiaiškintos bei aprašytos sąsajos tarp parazitų sąveikos. Tiek lapės, tiek usūriniai šunys buvo padalinti po dvi grupes. Pirmoji lapių grupė (n=27) buvo užsikrėtusi Taeniidae spp; Toxocara canis, Toxascaris leonina, Uncinaria stenocephala, Eucoleus aerophila, Capillaria putorii, Trichuris vulpis, Alaria alata, Coccidia spp.; o antroji lapių grupė (n=13) buvo užsikrėtusi Sarcoptes sp. ir vienu ar daugiau iš toliau paminėtų: Toxocara canis, Uncinaria stenocephala, Capillaria putorii, Trichuris vulpis, Alaria alata, Coccidia spp.. Trečiosios grupės usūriniai šunys (n=6) nebuvo užsikrėtę Sarcoptes scabiei, tačiau juose rasti šie parazitai: Toxocara canis, Toxascaris leonina, Uncinaria stenocephala, Eucoleus aerophila, Capillaria putorii, Trichuris vulpis, Alaria alata, o ketvirtosios grupės usūriniai šunys (n=8) buvo infekuoti Sarcoptes sp. bei vienu ar daugiau iš toliau paminėtų žarnyno parazitų: Toxocara canis, Uncinaria stenocephala, Capillaria putorii, Trichuris vulpis, Alaria alata. Visos lapės bei usūriniai šunys panaudoti tyrime buvo laukiniai ir medžioti visoje Lietuvos teritorijoje, išskyrus Alytaus, Marijampolės ir Telšių apskritis. Tirti gyvūnai buvo medžiojami apvalius metus, jie buvo visų amžiaus grupių ir abiejų lyčių. Koprologinė analizė buvo atlikta 88 sumedžiotiems individams. Tyrimas buvo vykdomas nuo 2017 metų gruodžio iki 2020 metų gegužės mėnesio LSMU Veterinarijos akademijoje, Veterinarinės patobiologijos katedroje. Tiriamųjų išmatose rasti parazitų kiaušinėliai rodo koreliaciją tarp Sarcoptes sp. ir endoparazitų abejose gyvūnų populiacijose - turime duomenų, kurie rodo, jog yra mutualistinių ar sinergistinių veiksnių, taip pat ir atvirkštinis efektas, kurie daro įtaką parazitų paplitimui, kaip, pavyzdžiui, Uncinaria stenocephala rodė 4.40% paplitimo padidėjimą (Ρ = 0.020) usūrinių šunų populiacijoje bei 3.80% paplitimo padidėjimą (Ρ = 0.013) rudųjų lapių populiacijoje būnant kartu su Sarcoptes scabiei, nei lyginant su paplitimu būnant vienintele parazitų populiacija. Mechanizmai, kurie už tai atsakingi, vis dar nežinomi. Išsamesnei analizei tyrimai šia tema turėtų būti tęsiami ir daugiau faktorių, tokių kaip imuniteto parametrai, parazitų reakcija į šeimininko temperatūros pokyčius, šeimininko elgesio pokyčiai

7 | P a g e susirgus sarkoptoze ir infekcijos įtaka antrinei parazitinei infekcijai turėtų būti tyrinėjami. Taip pat turėtų būti surinkti didesni išmatų mėginių vienetai.

Gairės: Sarcoptes scabiei, koinfekcija - liga, endoparazitai, Vulpes vulpes, Nyctereutes procyonoides, Lietuva;

8 | P a g e

5. INTRODUCTION

The red fox (Vulpes vulpes) and the raccoon dog (Nyctereutes procyonoides) are widely distributed throughout Europe and they carry a multitude of diseases and parasites, some of these are zoonotic and pose high risk not only to humans but also to their pets and livestock. Apart from the eminent danger of rabies, which is held at bay with the oral vaccination (1), there are some dangerous parasites which are also zoonotic. Some other parasites are Echinococcus multilocularis, Trichinella spp., Toxocara canis and Sarcoptes scabiei(2). This is where the present study has its point of interest due to its novelty of the following comparison since the medical field at this point mostly concentrates on zoonotic diseases. However, they should also focus on other parasites because they can cause severe diseases and infections for livestock, pets and human beings(3).The red fox is found throughout the northern hemisphere (4). With its main habitation in Europe, it is the most abundant predator here in northern Europe. The raccoon dog is an alien predator and it originally derived from Eurasia. It is an omnivore making it versatile, while the fox is a carne-omnivore (5,6). Nevertheless, this does not show that red foxes are less adaptable to their surroundings. The red fox and raccoon dog have a huge impact on the ecosystem due to their number and wide range of prey. The raccoon dog counts as the invasive species, the one which poses a direct threat to biodiversity and native species and is, as stated before, alien here in Europe (4,5). They both are prone to infect themselves with Sarcoptes scabiei. This ectoparasite is hard to control in the wild and does also cause diseases for livestock, pets as well as humans. The burrowing mite is one of the most widely spread ectoparasites and it is very potent as a parasite that has the ability to cause high mortality (7) or even to diminish a species in a certain region (8). Sarcoptes sp. infection results from the direct contact with an infected animal. Most predators are burrowing animals that live in burrows in their social structure with their pack sharing a confined space, which makes it quite easy for the mite to be transmitted. Sarcoptic mange causes a severe dermatitis resulting in the loss of hair and proper skin functions; in the cutis it is active, thus, leaves its hosts furless and without protection against the weather. In a cold or wet climate this leads to a “hair to hide” quick death(7,9). Furthermore, as mentioned earlier, the fox and raccoon dog are vectors as hosts or intermediate hosts for many other parasites, some of them zoonotic. They, as Sarcoptes sp; have a direct impact on the population and bring harm to our pets(10). Most are shared over the fecal oral route, mainly due to the behavior of most meso-predators and apex-predators like the wolf (Canis lupus) that perform Coprophagia (the eating of excrements)(11,12). Infections are not only rapidly

9 | P a g e transmitted and spread throughout the population, but they are also transmitted to other species. This also means a high prevalence is reached swiftly and is easily maintained in most species. Needless to say, it makes it equally hard to control or eradicate them (13,14). Their correlation or coinfection with Sarcoptes sp. could have a huge impact on epidemiological factors as well as clinical signs and population statues. So far, I have found only one Journal article talking about the coinfection and mutualism between Sarcoptes sp. and bacteria (15). I also found an article talking about parasite-parasite interaction which highlights its presence and importance(15). Of course, there is a high number of articles about Sarcoptosis and any sort of intestinal helminth, just to name some(3,7–9,16–21), but none highlights or focuses on their interaction between the above mentioned parasites. The goal was to try to analyze this through their prevalence in combined detection of Sarcoptes sp. and one parasite species named above at once and compare the results to calculated reference prevalence. This was done in a similar way in the study comparing bacterial infection(22). Also, it is very important to see the differences between the two species of predators, there are lots of articles found on this matter (2,8,10,20,21,23–25) and to add or update the data in Lithuania regarding the differences of detection and prevalence. Also, it is important how they are balanced in between the predatory species and to establish the hotspots of the disease in the country. The most recent studies of the two predator species that were conducted in Lithuania and had a similar focus were about infective pathogens of the raccoon dog in 2014(10) and about helminth infection in both populations in 2012(2).The aim of the present study is to estimate the prevalence of Sarcoptes scabiei, Taeniidae spp; Toxocara canis, Toxascaris leonina, Uncinaria stenocephala, Eucoleus aerophila, Capillaria putorii, Trichuris vulpis, Alaria alata, Coccidia spp. in Lithuania’s red fox and raccoon dog populations. Furthermore, it aims at highlighting the association between Sarcoptes scabiei and fecal found parasites, such as Taeniidae spp; Toxocara canis, Toxascaris leonina, Uncinaria stenocephala, Eucoleus aerophila, Capillaria putorii, Trichuris vulpis, Alaria alata, Coccidia spp.

The tasks of the research are:

1. Evaluation of the prevalence of Sarcoptes scabiei based on external examination of corpses and analysis continuing with grouping of the species and their collection district. 2. Evaluation of prevalence of Taeniidae spp; Toxocara canis, Toxascaris leonina, Uncinaria stenocephala, Eucoleus aerophila, Capillaria putorii, Trichuris vulpis, Alaria alata, Coccidia spp. in target species true adjusted McMaster fecal flotation technique.

10 | P a g e

3. Evaluation of the combined prevalence of Sarcoptes scabiei and each found parasite and a calculated reference prevalence to establish a relation between ectoparasites and endoparasites.

11 | P a g e

6. LITERATURE REVIEW

6.1. Red fox (Vulpes vulpes) The red fox is (Vulpes vulpes) Europe’s most abundant medium sized predator. It is also found throughout the globe with high adaptability skills showing a remarkable variety of habitats and food sources. There are more than 40 red fox sub-species found globally (26) of which one is found in Lithuania. Before the oral vaccine control of rabies the red fox accounted for 46% of cases in the wild life population as the study from 2004 (27) shows. The exact population size of Red foxes in Lithuania is unknown; however, it can be speculated. The latest population count was done in 2001 ranging at around 22.900 individuals in area of around 65.300 km³. Due to the oral vaccine in years from 1995 till 2002 it was estimated that the population since then has increased threefold (27). Unfortunately, there is no other data available to us, at the current time, we can only conclude from the annual hunting registry last published in 2013 that the fox population has been steady since 2002 (28). To determine more accurate numbers of population size, different methods should be used. For example, surveying habitats by a trained field ecologist would be most accurate but would only be viable in small areas and cannot be extrapolated to a country scale. Also, effective and widely used is the night counting index as well as road kills and line transection (1) as the red fox is a predominant vector for other diseases owing to its high adaptability and a huge variety of food sources and its high mobility range from 2-20km each day in an area of around 52 ha(6). The individuals found in Lithuania show a high prevalence for any number of diseases such as parasites. According to the performed in 2019, foxes have shown Babesia spp., Borrellia spp., Mycoplasma spp., Dirofilaria sp. and many more putting the detected vector-borne pathogens at an 83, 9% rate. From the 31 individuals tested, 26 had one or multiple vector borne diseases (23). In Lithuania, the foxes also carry a high rate of intestinal parasites displayed in a study published in the year 2012 (2). It was done by the Lithuanian University of Health Sciences in collaboration with the University of Zürich. They analyzed a large sample of 310 red foxes and 99 raccoon dogs, and found that both species were highly infected with a multitude of Nematodes (Uncinaria stenocephala, Toxocara canis, Capillaria plica, Eucoleus aerophilus, Capillaria putorii, Crenosoma vulpis, Trichinella, Mastophorus muris, Syphacia obvelata, Heligmosomum costellatum ), Cestodes (Echinococcus multilocularis, Mesocestoides, Taenia polyacantha, Taenia crassiceps, Taenia taeniaeformis) and Trematodes (Alaria alata, Alaria alata metacercaria, Echinostomatidae, Opistorchis felineus) . The focus of the above mentioned study was on the transmission of E. multilocularis.(2) 12 | P a g e

6.2. Raccoon dog (Nyctereutes procyonoides) The raccoon dog which is also named as an adaption specialist from Asia, the only canid that performs hibernation and it has five subspecies (29). It was introduced as a fur farm animal to western Russia and spread quickly throughout northern and central Europe. Their original habitat are the Mongolian plains, northern Indochina and the southeastern corner of Russia (5). Their introduction was performed by the former Soviet Union and their controlled states in Siberia as well as North-Eastern Europe first in the year 1929 and continued till 1955(5). Raccoon dogs are omnivorous canids that prefer to inhabit the areas with fresh water sources such as rivers and streams(30). Moreover, they choose open forest habitats with a diverse food resource and where they can find or create shelter, but they have also been observed in meadows or farm land (5). (Nyctereutes procyonoides) are night active as the name suggests. They are great scavengers but also succeed in hunting. They are a direct threat and rival to foxes in many ways. The habitat and food sources are broadly similar, and the raccoon dogs even have the advantage of being not so selective when it comes to burrow or foodstuffs. Their adaption is almost flawless. The fox and the raccoon dog also compete in the diseases they carry. Both are known to carry rabies Trichinella spp. and the mange. Rabies is a deadly virus form that causes encephalitis that is caused by neurotropic virus of the genus Lyssavirus(31). A recent experimental study indicates the main epidemiological cycle of rabies in the European wildlife is maintained by the red fox and the raccoon dog (32). They also analyzed the epidemiology of rabies in Lithuania. In the past ten years, rabies in Lithuania has been successfully suppressed by oral vaccination, which has effectively reduced the prevalence of rabies among predators (27). Thus, the other diseases are now sliding more into appearance and are next for permanent monitoring and control programs. These diseases include not only sarcoptic mange and ectoparasite but also endoparasites like Toxocara canis or Trichuris vulpis. Sarcoptic mange is a global infectious and parasitic skin disease of mammals. The cause is Sarcoptes scabiei, it is a zoonoses. The raccoon dog as well as many other wild species develop extensive skin lesions and eventually die as a consequence, because of secondary skin infection or lack of protection against the weather due to the baldness (9).

6.3. Synergism or Mutualism of parasitic species Scarce data can be found on the parasitic interaction between the named parasite especially the focused ectoparasite Sarcoptes sp. and endoparasites. In a study done in 2014 the link is drawn, between the Sarcoptes sp. produced inhibitory chemicals and the favorable outcome of this for bacteria. That later resulted in a secondary bacterial dermatitis (22). Another study was conducted

13 | P a g e on the pathogenic impacts on the bats (Vombatus ursinus) infected with the Sarcoptes mite. This study did not highlight any secondary parasites, but there is a clear evidence that it eases the hurdles for other parasitic infestations. It produced the findings about the severe heat loss and failure of thermoregulation that the host experiences as well as lowered metabolic rates and the huge impact on the infected individual and their foraging, resting and social behaviors. All these factors led to the immune compromise which occurred due to Sarcoptes sp. and gave rise to possibilities of further secondary infections (16).

6.4. Coprological analysis of endoparasites In the study performed in central Italy they focused on the accuracy of the coprological egg analysis in comparison to detecting the adult parasites in the intestine. Both methods then were compared by the prevalence of endoparasites in foxes. It was found that the accuracy varies between species from Dipylidium caninum at 43% to 95% in Ascarides. The accuracy was tested with the help of coprological egg flotation technique, whereas for reference technique necropsy and count of the adult parasites was used that had been collected from the intestines and then morphological differentiation and pcr determination of adult helminths was performed (20).

6.5. Red fox and raccoon dog endoparasites 6.5.1. Sarcoptes scabiei Sarcoptic scabiei also referred to as mange or scabies is caused by the Sarcoptes scabiei mite, which is a highly contagious mammalian ectoparasite. It is an infectious agent for wildlife, domestic animals, and humans worldwide(3). It belongs to the suborder Sarcoptiformes, family of Sarcoptidae(33). Sarcoptes scabiei has a characteristic oval shape, a tortoise-shaped body with a flat ventral surface and a convex back. The surface of the mites body is covered with fine stripes; the dorsal specific surface has areas with several thick bristles, and adult females have areas with many cornified spines, which is an important taxonomic feature (33). Sarcoptes scabiei have been detected in more than 100 mammals and marsupials (34). This infection is different from many other mites, with that they inhabit the epidermis of the skin, digging tunnels in the outer layer of the epidermis. They can penetrate the skin surface at all stages of life (17). Penetration is achieved by chewing movement of the chelicerae. The mites penetrate the stratum corneum into the stratum granulosum and stratum spinosum, where they consume living cells or penetrate further to create a cave. Due to the continuous growth of the epidermis, many caves including the eggs and feces are closed in, in the epidermis. The fertilized female lays eggs in these tunnels and has a life expectancy of 4-6 weeks. The eggs are ovoid in shape, and they are laid at a rate of 3-4 per day. They hatch

14 | P a g e within three days, and larvae with three pairs of legs appear. Some larvae migrate from the female breeding channel and move into the skin. It is found that other individuals migrate throughout the skin or stay in the original channels or their extensions, sometimes called molting pockets. There, in 3 to 4 days they develop into the first nymph stage (protonymphs), and into the second nymph stage (tritonymph) after about 3 more days. These nymph stages remain in the tunnels and molting pockets, stay on the skin surface, or form new tunnels and pockets. The nymph stage has four pairs of legs. It takes 2 to 4 days for the tritonymphs to metamorphose into adults. Therefore, it takes about 2 weeks to develop from an egg to an adult (3). The infection of scabies in isolated populations can have a serious and drastic outcome. For example, it is believed that scabies was the one leading causes of the extinction of red foxes on Bornholm, a Danish island (8). Also, a mortality rate from ~70% was found in the study carried out by Pence and Windberg in 1994 with the population of north American coyotes infected with Sarcoptes sp.(7). This is a clear illustration of the severe effect sarcoptic mange can have on a population. 6.5.2. Nematodes 6.5.2.1. Toxocara canis Toxocara canis is mostly seen in dogs and red foxes but can also be found in other canids. The life cycle of this species is complicated; there are four possible infection modes. The most common rout of an infection is through an ascaridae egg that contains a L31 being infected. The temperature must be optimal then it takes 4 weeks after ingestion till they hatch in the small intestine. The larvae are spread through the bloodstream. They pass through the liver to the lungs, where the development to the second stage occurs. Then, the larvae return to the intestine through the trachea where it goes through the two final stages. This process occurs in dogs usually not more than 2-3 months of age (14). 6.5.2.2. Toxascaris leonina Toxascaris leonina mainly infects carnivores or omni-carnivores such as cats, foxes, dogs but it has also been found in many other wild canids or felids. The infection stages of Toxascaris sp. are as follows: the first one is the eggs; the second stage is larvae or the third-stage larvae which is present in the intermediate host of mice. This means, all the previously mentioned and many more canids, that have mice as food source, have the possibility to get infected. The parasite eggs rapidly progress to the infection stage (about 1 week) much faster than the relative species which take about four weeks. Toxascaris sp., also known as Toxascaris limbata, then hatches and the larvae enter the wall of the small intestine; they remain there for about two weeks. This parasite does not have larval migration unlike other similar species. The third stage larvae appear after about 11 days and change

1 L3 third-stage larvae 15 | P a g e their form and shape to L4 within 3 to 5 weeks after infection. Adult stage appears from about 6 weeks post infection; they lie dormant in the intestinal lumen. The preparation period of Toxascaris sp. is 10-11 weeks. They are common to be found in foxes (21) and they are endemic in many mammals around the globe (35). 6.5.2.3. Uncinaria stenocephala Uncinaria s. is also called the “Northern hookworm” (36) because of its local spread throughout the northern hemisphere. The adults can usually be found in the small intestines, resulting in the following symptoms: anemia, diarrhea, anorexia and even interdigital dermatitis. The adult itself is rather small about 1.0 cm long and has a sexual dimorphism resulting in the females being larger than the males which grow around 2-4 mm in body length. A sharp rise of infection was observed in July usually as a result of a longer period of high temperatures, reaching its peak in and around September(37). The typical hosts are dogs, cats and foxes as well as other wild canids, various other mammals act as paratenic hosts(38). 6.5.2.4. Eucoleus aerophila Also known as Capillaria aerophila, as the name suggests, is mostly found in the respiratory tract ( bronchi and trachea) of foxes, coyotes, mustelids, occasionally in dogs and cats, and it can also be found in humans(39). Males are around 24mm in length; females are around 10mm longer. The males spicule sheath is covered with spines and it only has one spicule (18). 6.5.2.5. Capillaria putorii A filamentous worm, around 10 mm long; males are 5-8 mm in length and the females’ measure 9-15 mm. They are also referred to by the name of Aonchotheca putorii and are found in Europe, northern America as well as New Zealand where they infect the small intestines and stomach of usually wild mammals like foxes, raccoons, mink but they also infect domesticated cats (24). It is known to cause severe gastritis, the parasites causes gastric ulceration and with that causes a secondary anemia in its hosts (40). In 2014 a study was done in Lithuania proving Capillaria sp. in all surveillance species pine marten (Martes martes), stone marten (Martes foina), American mink (Neovison vison) and European polecat (Mustela putorius). A prevalence from 20% in European polecat to 50% in the pine martens was established in the statistical analysis (41). 6.5.2.6. Trichuris vulpis Trichuris vulpis is a large intestine parasite infecting not only dogs, cats but also foxes and other wild canids. They are around 4-8 mm cm in length. Trichuris vulpis has a direct life cycle, with eggs passing in feces. The penetration of adult worms is into the intestinal mucosa and is associated with inflammation usually causing diarrhea. The factors affecting the development of clinical symptoms are in direct correlation of the number with the location of adult whipworms. The

16 | P a g e severe inflammation and anemia can have a high impact on the host. The whipworms egg morphology is the tool for diagnosis; it is barrel-shaped, thick-walled and possesses bipolar mucus plugs. The eggs can be detected during fecal flotation. Carve adult worms release eggs intermittently. Therefore, a negative result does not rule out infection (42). A study in Thailand proves that dogs are used as vectors of the parasite spread throughout a rural schoolchildren community (43). 6.5.3. Cestodes 6.5.3.1. Taeniidae sp. Most prone to cause danger for humans is Echinococcus multilocularis due to its latency and complicated diagnostics/treatment. All the Echinococcus species have been detected in foxes and they can survive very long outside a host making the control of the parasite very challenging. In the study of Echinococcus sp that was conducted in north western Poland they surveyed an astonishing 61% prevalence rate in the local (Vulpes vulpes) population. They analyzed 620 foxes (44). Echinococcus multilocularis is common in the whole North America and Asia, Europe and Russia. The records of E. multilocularis increased in 2008 and the Ukraine reported first cases in their red fox population. They examined 145 red foxes 4 of which were infected. They also observed that they were surrounded by countries endemic of E. multilocularis, and suggested that the red fox might be the vector due to its adaptability and long distance of travel in relation to the intermediate hosts, rodents (45). 6.5.4. Trematodes 6.5.4.1. Alaria alata Alaria a. is found in the small intestines of dogs, cats, foxes, minks and other wild canids. The front of the worm is spoon shaped. The hind part has a conical shape and inherits the reproduction organs. The life cycle is quite interesting, freshwater snails are involved as the first intermediate host; the second intermediate host involve either amphibians or reptiles. Where it enters tadpole true their ingestion, if mice then eat the infected tadpole, it makes them the paratenic host. As soon as a fox eats a mouse, the mesocercariae start excessively migrating throughout the host including its diaphragm and lungs. Here they develop into the metacercariae. Then they return to the intestines to mature into adult flukes. The entire prepatent period takes around 2 to 4 weeks. In France a study was done on the meat digestion analysis to detect trichinella in wild boar (Sus scrofa). They study shows that it can also be used to find Alaria sp. in wild boar meat (19). Research carried out in the Netherlands reported that the local fox population there had a prevalence of 10.9% of Alaria sp. in the population. They didn’t establish any relation between the parasites count, in regard to sex, time of year and place of origin(46).

17 | P a g e

6.5.5. Protozoa 6.5.5.1. Coccidia spp. McMaster method is the simplest technique for detecting the presence and estimating the number of coccidial oocysts in feces. The technique is the same as the one described for helminthological diagnosis, although the small size of the oocysts makes the microscopic examination more prolonged. If the animal has acute clinical signs of coccidiosis, such as blood‐ stained feces, and many thousands of oocysts are present, one may consider the diagnosis confirmed. Unfortunately, with the more pathogenic species of Coccidia spp; clinical signs may appear during the merogonous phase or when oocyst production has just started, so the negative or low oocyst count does not necessarily indicate that the clinical diagnosis is negative. The oocyst count is also of little value in a prolonged coccidial infection. In a case report on fur farmed foxes, coccidiosis was found in six silver foxes (Vulpes vulpes) and four blue foxes (Alopex lagopus) they found two species in the fecal samples. Isospora canivelocis and Isospora canis were present in the sick animals. They were diagnosed by morphological factors, particularly the size. After being treated with oral sulfadiazine-trimethoprim the foxes recovered and gained back their usual weight (47). In a field study done in Denmark a prevalence of 2.9% of coccidia oocysts was found in the fecal samples of 68 red foxes from the metropolitan surroundings of Copenhagen. The standard McMaster fecal flotation technique (25) was used.

18 | P a g e

7. METHODS AND MATERIALS

The work was fully carried out at the Department of Veterinary Pathobiology of the Lithuanian University of Health Sciences in the period of 2017 till 2020.

7.1. Biosecurity Due to the high danger and risk working with foxes and other predators, as well as biohazardous materials, that have a high number of different zoonotic parasites and diseases, biosecurity should be discussed and kept at highest standards. It was started with the mandatory four day freezing at -80°C of the bagged cadavers before handling them, although the literature suggests -70°C for at least four days(48). After that they were brought to the Pathology department, were they were only unpacked in the area of a special necropsy room that was closed for others and could only be entered dressed in our specific clothing. This clothing included a hair net, a mouth-nose mask, an apron, shoulder length gloves, a second pair of gloves that where put over the shoulder length gloves and overshoes. Rubber boots were worn under the lab coat. After sample collection, all samples were again frozen at -80°C and all surfaces were wiped down with bleach, the protective clothing was discarded, and hands were scrubbed and disinfected. When at work with samples, gloves and lab coats were worn. After preparing the microscopical slides the laboratory fume cupboard was left and the microscopy analysis began. When the work was completed and all samples were counted, the surfaces were sanitized with bleach, all biochemical materials were disposed of in the biohazard trash and hands were scrubbed and disinfected.

7.2. Collection of sample individuals The samples were provided by the Institute for Veterinary and Food Risk Assessment (Veterinarijos ir maisto Rizikos vertinimo institutas). They were relayed to the Lithuanian University of Health Sciences. At the pathology department of our university we performed the necropsy of the individuals. We started by unfreezing the individuals for around 24 to 48 hours, after that our investigation was begun. This is where I examined the individuals for Sarcoptes scabiei and obtained the needed fecal samples from the colon for my study. The feces were dropped into a sample bag which was labeled with the local identification number of the individual plus “FECES” on the outside and I put a label inside with the same local ID. After the procedure the bag was sealed and collected in a container with my other samples. In addition to the previously

19 | P a g e described process, the bag was frozen for a minimum of four days at -80°C to preserve it in case of a necessity for further investigation.

7.3. Identification and tracking of samples The foxes had a 4-digit identification number from the “Veterinarijos ir maisto Rizikos vertinimo institutas”. With the help of it we could follow up on some important details like date of culling, location of culling, sender of carcass, and some other details. This date was very important for later analyses of the samples. A local identification number was given to every single individual for 2 tracking every sample and individual. The local identification number consists of two letters describing the species, for example “La” for the red fox in Lithuanian “Lape”, likewise, “Us” for the raccoon dog “usūrinis šuo” (LT3). After two letters a two-digit number of the year when the sample was taken followed, for example “19” for the year 2019 and “20” for 2020, then followed the numeration of samples starting with “1”. Here are some examples: La/19/26, Us/20/14, La/20/101; Thus, every sample had its individual code to prevent the loss or confusion of samples. The data of the collected individuals, test samples, their physical values and individual identification were gathered in one two-sided document. They were collected in our laboratory journal in which we kept track of all samples and values taken as well as some notes. All the sample bags were also marked twice once with a permanent marker with our local identification number and the contents of the given sample bag. Furthermore, a handwritten sticking label was inserted into the bag and glued to the inside. All this was done to prevent the loss of samples due to mislabeling or unreadable labels.

7.4. Test group My test group was diverse regarding age, sex and species; it is interesting to see the difference in between the two species. Although there was a sample size difference between the groups, clear differences which can be shown in the presented study in the first two tables (figure 1, figure 2). I displayed the setup of my trial in different distinct groups.

The total number of individuals in the shown population of Figure 1 is 40 individuals of hunted and collected red foxes. To divide this by sex, you can find a sum of 30 males and 10 females are shown on the right. The lower bar in the named figure shows the diversity of the sexes.

2 Identification 3 Lithuanian 20 | P a g e

Moreover 25 % are female and 75% are male individuals. The rest of Figure 1 is used to display the differences in age groups. The upper bar of the chart depicts the total number of 40 individuals which can be divided into young and adult individuals. The bar chart shows 82.5% adult individuals and 17.5% of young individuals respectively. That is to say, 33 adults were found in comparison to 7 young individuals. Each bar makes up 100% and represents all the individuals of the red fox available here that were used in this study.

Red fox sample population age/sex distribiution

Age 7, Young 33, Adult

Sex 30, Male 10, Female

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Figure 1 – The distribution of hunted and collected red fox test subjects in different sex and age groups, the title and number indicate the individuals in each group.

The following Figure number 2 is also presented as a bar chart. Here the constellation of the hunted and sampled raccoon dog population is illustrated. Raccoon dog population is represented by 8 female and 6 male individuals, of those 13 were adults and 1 was under 1 year of age and was considered a youngster.

Figure 2 gives data on a total of 14 individuals which are divided by age depicted in the top bar. The sample numbers expressed as a percentage show 7, 14% young individuals and 92.86% adult individuals respectively. The subdivision of the raccoon dog samples by sex is illustrated in the same figure. The figure 2 displays a percentage of 57.14% female individuals compared to a total of 42.85% of male individuals. This bar chart represents the same subdivision as the one mentioned before.

21 | P a g e

Raccoon dog sample population age/sex distribiution

Age 1, Young 13, Adult

Sex 8, Female 6, Male

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Figure 2 – The distribution of hunted and sampled raccoon dog test subjects in different sex and age groups, the number indicating the individuals in each group.

7.5. Coprological analyses 7.5.1. Sample preparation To ensure the full homogenization of the flotation liquid, it had to be prepared at least a day before. Flotation liquid the zinc chloride suspension with a specific density of 1.45g/cm³ was prepared(49). Also, the fecal samples had to be taken out of the freezer around two hours prior to testing, for the purpose of defrosting. Individual fecal samples were homogenized with a spatula and a sample of 2.5 g was placed into a 15 ml tube, diluted with water (1:4) and centrifuged at 1600g for 10 min. The sediment (about 2 ml) was resuspended in 6 ml of ZnCl2 solution (density: 1.45g/cm³) and vortexed vigorously. Tubes were filled with the same ZnCl2 solution and centrifuged for 30 min at 400g. The floated particles from the meniscus of the sample were collected four times from each sample by means of the wire loop and transferred onto the slide for further microscopy. All tubes and slides were marked with the local ID and the inspection of the sample started. 7.5.2. Sample analysis I focused the microscope at 40x magnification and started to scan the samples always from top right to left bottom, drawing parallel viewing lines. By counting every egg, I saw and determined the species and noted it into my laboratory book under the local ID of the sample. During my sample analyses I found and recorded the following species, all species have been determined by morphological differentiation method: Taeniidae spp., Toxocara canis, Toxoascaris leonina, Uncinaria stenocephala, Eucoleus aerophilus, Capillaria putorii, Trichuris vulpis, Alaria alata, in addition to some Coccidia spp. and even Sarcoptes sp. as adult. Furthermore, I found an

22 | P a g e abundance of two kinds of broken eggshells. This is the suspected impact of the multiple deep- freezing procedures and the stress it causes on the eggs and the water found in their cells. Through investigative morphological analyses I determined that one of those shells belonged to Toxocara sp. and the other one to Alaria alata. I determined this by closer inspection of the eggshells and ruling out other parasites due to the eggshell’s characteristics, using characteristics, such as size, shape, the surface of the shell as well the thickness and the general appearance. The values in my result stand combined with the determined species. So, the suspected A. alata where added to A. alata and suspected T. canis was added to T. canis. In the supplied figures 5, 6 and 7 a statistically calculated prevalence next to the detected prevalence can be observed. This value was calculated to prove synergism of a combined parasite set. Meaning that if two different parasite species are found in a single individual the detected prevalence is combined and statistically compared resulting in a calculated prevalence of the combined parasites. Now if the detected prevalence happens to be higher than the calculated prevalence value, it suggests that the parasites are catalyzed by each other’s presence. This synergistic relation can happen due to one or many reasons. For example, one parasite lowering the immune system, weakening the host in general, initiates a chemical cascade, sets free or produces nutrients for the second parasite. This in the end results in a higher prevalence of both combined than each one separately in a host population (15).

7.6. Statistical analysis For the statistical analysis SPSS and Excel from Microsoft were used, throughout my sample collection and data collected Excel was used. I used the descriptive statistics frequency, one sample T test and the bivariant correlate tool from SPSS to cross-reference my existing data package against test values and to find links and connections. The data were analyzed in a multitude of directions by starting with a general overview of the sample group, then separating them into different groups by sex, age and species. It was followed by a juxtaposition of both referenced species and their mange prevalence. Furthermore, the prevalence of each detected egg from the feces was done. Later all the collected data were added up together and analyzed with the help of the above-mentioned tools to find mutualism or synergism of the parasites. Lastly, the whole data was tested against the Confidence index of 95% and the Chi-square test was done to categorize in statistically significant for comparisons. In this work (p4 < 0.05) was deemed statistically significant

4 P value (R. A. Fisher) 23 | P a g e and (p < 0.005) was deemed highly significant. The general data was displayed as 5% (x6/ y7, 95% CI “lower CI” – “upper CI”) and the P value was displayed in this manner (p = 0.05).

5 Prevalence 6 Detected animals 7 Number of individuals in group 24 | P a g e

8. RESEARCH RESULTS

It was found that mange infection was more prevalent (Figure 3) in raccoon dogs 46.15% (12/26, CI 95% 26.59 ─ 66.63) as compared to those of red foxes 32.14% (27/84, CI 95% 22.36 ─ 43.22), (Ρ = 0.04). The difference between them was statistically significant.

0.5 46.15% 0.45

0.4

0.35 32.14%

0.3

0.25 Red fox Raccoon dogs Prevalence, % Prevalence, 0.2

0.15

0.1

0.05

0 Sample species

Figure 3 – Prevalence of Sarcoptes sp. in hunted Raccoon dog (light grey) and red fox (dark grey). Numbers represent species prevalence in percentage.

In Figure 4 the distribution of mange infected red foxes and raccoon dogs combined can be seen, showing both populations together. They are organized in local areas by Lithuanian districts. In the legend below you will see the percentage of infected animals gathered from that region. Shown in grey are the provinces which lack data since we didn’t receive any individual from these regions

25 | P a g e

Figure 4 – Local distribution of Sarcoptic mange in percent of all gathered samples in Lithuanian provinces.

In Figure 5 the prevalence of intestinal parasites is displayed, with a statistically significant difference between the two species and the red fox population shows a higher detection rate. The red fox population had a higher prevalence of intestinal helminths than the tested raccoon dog population. The prevalence of both populations ranged from 35.71% (5/14, CI 95% 12.76 ─ 64.86) of prevalence in raccoon dog, with Toxocara canis compared to the prevalence of 52.50% (21/40, CI 95% 36.13 ─ 68.49) (p = 0.09) in red foxes. Although Toxoascaris leonina which wasn’t found in raccoons but incidence of 7.50% (3/40, CI 95% 1.57 ─ 20.39) (p = 0.027) was found in red foxes. The lower spectrum was also displayed by the 10.00% (4/40, CI 95% 2.79 ─ 23.66) of Coccidia spp. which was found only in red foxes (p =0.008). Alaria alata was detected at 28.57% (4/14, CI 95% 8.39 ─ 58.10) in raccoon dogs and 45.00% (18/40, CI 95% 29.26 ─ 61.51) (p = 0.014) in red foxes. Alaria sp. was the parasite that had a medium level of detection in both species. The percentage of incidence of Capillaria putorii was similar in the raccoon dog population with 21.43% (3/14, CI 95% 4.66 ─ 50.80) whereas Capillaria putorii had a total of 40.00% occurrences (16/40, CI 95% 24.86 ─ 56.67) (p = 0.003) in red foxes. Eucoleus aerophilus was not represented in raccoon dogs. Whereas this endoparasite showed 7.50% (3/40, CI 95% 1.57 ─ 20.39) (p = 0.027) in red foxes. Uncinaria stenocephala showed 21.43% (3/14, CI 95% 4.66 ─ 50.80) in raccoons and 35.00% (14/40, CI 95% 20.63 ─ 51.68) (p = 0.034) in red foxes. Taeniidae spp. was not be found in 26 | P a g e racoons but red foxes show an incidence of 10.00% (4/40, CI 95% 2.79 ─ 23.66) (p = 0.008) of this endoparasite.

detected prevalence of selected parasite in raccoon dog detected prevalence of selected parasite in red fox

0.00% Coccidia spp. 10.00%

28.57% Alaria alata 45.00%

21.43% Trichuris vulpis 30.00%

21.43% Capillaria putorii 40.00%

0.00% Eucoleus aerophila 7.50%

21.43% Uncinaria stenocephala 35.00%

0.00% Toxascaris leonina 7.50%

35.71% Toxocara canis 52.50%

0.00% Taeniidae spp. 10.00%

0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% Prevalence, %

Figure 5 – The detection frequency of excreted helminth eggs and coccidian oocysts in hunted Red fox and Raccoon dog sample population.

The detected mixed prevalence of Taeniidae spp. and Sarcoptes scabiei in the same red fox individuals was found to be at 2.50% (1/40, CI 95% 0.06 ─ 13.16) with a calculated value of 3.21% (1/40, CI 95% 0.61 ─ 13.16), (Ρ = 0.002) of combined prevalence, the p value here is under the 27 | P a g e defined (p < 0.05) defining it as statistically significant. From the raccoon dogs no individuals where found with combined detection of the named parasites. Foxes had a measured 15.00% (6/40, CI 95% 5.71 ─ 29.84) prevalence for Toxocara canis and Sarcoptes. The calculated combined value is 16.88% (7/40, CI 95% 7.34 ─ 29.84), (Ρ = 0.002), both values are marked with a star because their combined p value deems it scientifically highly significant. The raccoon dog with these two parasites is represented with a measured prevalence of 14.29% (2/14, CI 95% 1.78 ─ 42.81) and a calculated value of 16.48% (2/14, CI 95% 4.66 ─ 42.81), (Ρ = 0.003) being statistically significant. Toxoascaris leonina is again only represented by red foxes detected 2.50% (1/40, CI 95% 0.06 ─ 13.16) and an analytic calculated value of 2.41% (1/40, CI 95% 0.06 ─ 8.81), (Ρ = 0.0003), here the p value is also highly significant. In Uncinaria stenocephala a combined prevalence of 15.00% (6/40, CI 95% 5.71 ─ 29.84) was detected in foxes and a calculated value of 11.25% (5/40, CI 95% 4.19 ─ 23.66), (Ρ = 0.013). In the raccoon dog populations combined detection of Uncinaria sp. and Sarcoptes sp. has 14.29% (2/14, CI 95% 1.78 ─ 42.81) prevalence, the calculated value is lower at 9.89% (1/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.020). In the fox population no, detected individual was infected with Sarcoptes and Eucoleus aerophilus at the same time, but the calculated prevalence was 2.41% (1/40, CI 95% 0.06 ─ 8.81), (Ρ = 0.024) being statistically significant. Raccoon dogs did not show any prevalence neither with this parasite detected nor calculated. Capillaria putorii and Sarcoptes sp. were found with a detected prevalence in foxes of 15.00% (6/40, CI 95% 5.71 ─ 29.84) and a calculated value of 12.86% (5/40, CI 95% 5.71 ─ 26.80), (Ρ = 0.004) being statistically highly significant. The raccoon dog population had a combined prevalence of 21.43% (3/14, CI 95% 4.66 ─ 50.80) and a calculated prevalence of 9.89% (1/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.135) here the p value was insignificant with p=0.09. The combination of Sarcoptes sp. and Trichuris vulpis had a detection rate of 7.50% (3/40, CI 95% 1.57 ─ 20.39) in foxes and a calculated value of 9.64% (4/40, CI 95% 2.79 ─ 20.39), (Ρ = 0.005) again statistically highly significant. The raccoon dog population on the other hand had a detected prevalence of 21.43% (3/14, CI 95% 4.66 ─ 50.80) of the combination and a calculated prevalence of 9.89% (1/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.135) being statistically insignificant. In the parasite combination of Alaria alata and Sarcoptes sp. the measured prevalence in foxes was 15.00% (6/40, CI 95% 5.71 ─ 29.84) and calculated value of 14.46% (6/40, CI 95% 5.71 ─ 26.80), (Ρ = 0.0002) being highly significant. In raccoon dogs the measured value was 21.43% (3/14, CI 95% 4.66 ─ 50.80) and the calculated prevalence was 13.19% (2/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.052) being statistically insignificant. The last column is Coccidia spp. here the combined prevalence for the foxes was 2.50% (1/40, CI 95% 0.06 ─ 13.16) and the calculated prevalence was 3.21% (1/40, CI 95% 0.61 ─ 13.16), (Ρ = 0.002). The raccoon dogs didn’t have a present or calculated prevalence of the above stated combined parasites.

28 | P a g e

2.50% ** 3.21% Coccidia spp. 0.00% 0.00%

15.00% ** 14.46% Alaria alata 21.43% 13.19%

7.50% ** 9.64% Trichuris vulpis 21.43% 9.89%

15.00% ** 12.86% Capillaria putorii 21.43% 9.89%

0.00% * 2.41% Eucoleus aerophila 0.00% 0.00%

15.00% * 11.25% Uncinaria stenocephala 14.29% * 9.89%

2.50% ** 2.41% Toxascaris leonina 0.00% 0.00%

15.00% ** 16.88% Toxocara canis 14.29% ** 16.48%

2.50% ** 3.21% Taeniidae spp. 0.00% 0.00%

0.00% 5.00% 10.00% 15.00% 20.00% 25.00% Prevalence, %

detected prevalence Sarcoptes s. + additional detection of selected parasite red fox calculated prevalence Sarcoptes s. + additional selected parasite red fox detected prevalence Sarcoptes s. + additional detection of selected parasite raccoon dog calculated prevalence Sarcoptes s. + additional selected parasite raccoon dog

Figure 6 – The frequency of Sarcoptes scabiei and a selected parasite with the calculated prevalence for each species in Red fox or Raccoon dog. The “*“showing statistical significance of (p<0, 05) and “**” showing high statistical significance of (p<0.005).

In Figure 7 the highest combined prevalence in the red fox population detected was that of Capillaria sp. in combination with Sarcoptes sp; together the measured prevalence was at 15.00% (6/40, CI 95% 5.71 ─ 29.84) that is 3.63% lower than the calculated reference value at 12.86% 29 | P a g e

(5/40, CI 95% 5.71 ─ 26.80), (Ρ = 0.004) Capillaria sp. measured alone had a prevalence parasite of 40.00% (16/40, CI 95% 24.86 ─ 56.67). Ranking second highest prevalence is suspected Alaria sp. with 15.00% (6/40, CI 95% 5.71 ─ 29.84) when combined measured 14.46% (6/40, CI 95% 5.71 ─ 26.80), (Ρ = 0.000) calculated and a single parasite prevalence of 45.00% (18/40, CI 95% 29.26 ─ 61.51). Then follows Uncinaria sp. with 15.00% (6/40, CI 95% 5.71 ─ 29.84) combined measured prevalence and 11.25% (5/40, CI 95% 4.19 ─ 23.66), (Ρ = 0.013). The parasite as a single prevalence is laying at 35.00% (14/40, CI 95% 20.63 ─ 51.68). Then follows Toxocara canis with 15.00% (6/40, CI 95% 5.71 ─ 29.84) combined prevalence, Sarcoptes sp. and Toxocara sp. has a calculated value of 16.88% (7/40, CI 95% 7.34 ─ 29.84), (Ρ = 0.002) Toxocara sp. was deemed highly statistically significant with a p value (p < 0.005) and had single prevalence in the fox population of 52.50% (21/40, CI 95% 36.13 ─ 68.49). Trichuris vulpis had a detected prevalence of 7.50% (3/40, CI 95% 1.57 ─ 20.39) and a calculated one of 9.64% (4/40, CI 95% 2.79 ─ 20.39), (Ρ = 0.005), whereas when alone, a prevalence of 30.00% (12/40, CI 95% 16.56 ─ 46.53) was detected. The lowest prevalence shown by Taeniidae spp., Toxoascaris Leonina, Eucoleus aerophilus, Alaria alata and Coccidia spp. Taeniidae spp. had a measured prevalence combined with Sarcoptes sp. of 2.50% (1/40, CI 95% 0.06 ─ 13.16) a calculated value of 3.21% (1/40, CI 95% 0.61 ─ 13.16), (Ρ = 0.002) and a single prevalence without Sarcoptic mange of 10.00% (4/40, CI 95% 2.79 ─ 23.66). Toxocara leonina was detected with a combined prevalence of 2.50% (1/40, CI 95% 0.06 ─ 13.16) and a calculated one of 2.41% (1/40, CI 95% 0.06 ─ 8.81), (Ρ = 0.00003) it was deemed highly statistically significant due to (p < 0.005). Eucoleus aerophilus and the mange mite combined weren’t detected in any fox together, the calculated value was 2.41% (1/40, CI 95% 0.06 ─ 8.81), (Ρ = 0.024) and the parasite alone was detected 7.50% (3/40, CI 95% 1.57 ─ 20.39). Alaria alata was found with a detection rate of 15.00% (6/40, CI 95% 5.71 ─ 29.84) with Sarcoptes sp. and was forecasted 14.46% (6/40, CI 95% 5.71 ─ 26.80), (Ρ = 0.0002) prevalence, thus, it also achieved high significance. The prevalence of it without the Sarcoptes sp. comparison was at 45.00% (18/40, CI 95% 29.26 ─ 61.51). Coccidia spp. was detected to be present in 2.50% (1/40, CI 95% 0.06 ─ 13.16) with the sarcotes mite and was projected to have a prevalence of 3.21% (1/40, CI 95% 0.61 ─ 13.16), (Ρ = 0.002) the relation between them is also statistically higly significant. The parasite could be found in 10.00% (4/40, CI 95% 2.79 ─ 23.66) of all analized red foxes.

30 | P a g e

60.00% 52.50%

50.00%

45.00% 40.00%

40.00%

35.00% 30.00%

30.00% Prevalence, % Prevalence,

20.00%

16.88%

**

14.46%

15.00% 15.00% 15.00% 15.00%

< > > >

**

12.86%

**

11.25%

*

9.64%

10.00% 10.00%

10.00% **

7.50%

<

7.50% 7.50%

3.21% 3.21%

2.41%

2.50% 2.50% 2.50%

2.41%

** **

> < <

**

*

0.00% < 0.00%

detected prevalence Sarcoptes s. + additional detection of selected parasite red fox calculated prevalence Sarcoptes s. + additional selected parasite red fox

detected prevalence of selected parasite in red fox

Figure 7 – Comparison of cross detection of red foxes which have Sarcoptes scabiei and a specific other parasite. The “*” shows a p value that is below 0, 05 and “**” shows a high statistically significant with (P < 0.005). The “^” suggest synergistic or mutualistic activity between parasites. The “˅“suggests that they have a negative effect on each other.

31 | P a g e

Toxocara canis had a combined prevalence of 14.29% (2/14, CI 95% 1.78 ─ 42.81), the calculated one was at 16.48% (2/14, CI 95% 4.66 ─ 42.81), (Ρ = 0.003) and the single detected prevalence with the specific parasite value was at 35.71% (5/14, CI 95% 12.76 ─ 64.86). Uncinaria sp. and Sarcoptes sp. were detected with prevalence of 14.29% (2/14, CI 95% 1.78 ─ 42.81) and a calculated one of 9.89% (1/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.02) which was deemed statisticaly significant (P < 0.05). The single parastites detection rate measured in at 21.43% (3/14, CI 95% 4.66 ─ 50.80). Capilaria sp. Trichuris sp. and Alaria sp. show all the same values with the combined detected prevalence of 21.43% (3/14, CI 95% 4.66 ─ 50.80), the calculated prevalence was different though. Capilaria sp. had a calculated mixed prevalence of 9.89% (1/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.135), Trichuris sp. of 9.89% (1/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.135) and Alaria sp. had a combined statisticaly calcualted value of 13.19% (2/14, CI 95% 1.78 ─ 33.87), (Ρ = 0.052) beeing statisticaly significant.The single measured prevalence of of Alaria sp. was at 28.57% (4/14, CI 95% 8.39 ─ 58.10), Capilaria putori was at 21.43% (3/14, CI 95% 4.66 ─ 50.80) and Trichuris sp. was at the same value of 21.43% (3/14, CI 95% 4.66 ─ 50.80).

32 | P a g e

40.00% 35.71%

35.00%

30.00% 28.57%

25.00%

21.43% 21.43% 21.43%

21.43% 21.43% 21.43%

> > >

Prevalence, % Prevalence, 20.00%

16.48%

**

14.29% 14.29% <

15.00% >

13.19%

9.89%

9.89% 9.89% * 10.00%

5.00%

0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00%

0.00% 0.00% 0.00% 0.00% 0.00%

detected prevalence Sarcoptes s. + additional detection of selected parasite raccoon dog

calculated prevalence Sarcoptes s. + additional selected parasite raccoon dog

detected prevalence of selected parasite in raccoon dog

Figure 8 – Comparison of cross detection raccoon dogs which have Sarcoptes scabiei and a specific other parasite. The “*” shows a p value that is below 0, 05 and “**” shows a high statistically significant with (P < 0.005). The “^” suggest synergistic or mutualistic activity between parasites. The “˅“suggests a negative effect on each other.

33 | P a g e

9. DISCUSSION

It is very interesting to observe as displayed in Figure 3 of my study, that the prevalence of mange in raccoon dogs is much higher than in foxes. This can have many reasons, it is fascinating that in a study 16 years ago, the prevalence of Sarcoptes sp in raccoon dogs was at 7% and the red foxes at around 4% (10), compared with 46.15% in raccoon dogs and 32.14% in red foxes. This is a 650% increase in the raccoon population and an 800 % proliferation/increment in the fox population over the last 16 years. It is an upsetting increase but it is equally important to admit that we have an estimated threefold population since then due to the oral rabies vaccine established around 2001(50). Figure 4 establishes that the highest percentage 90% to 100% of the foxes and raccoon dogs that had mange was delivered from the north west. From 45% to 60% of all animals that we received from Kaunas and Vilnius regions had Sarcoptes scabiei, they are marked depicted in red. And in the north east from 15% to 30% of individuals were infected with sarcoptic mange. In Figure 5 also a clear relation is seen. Here the red foxes succumb to the raccoon dogs. The Red fox population had a much higher detection rate of the intestinal parasites than the raccoon dogs but Toxocara canis and Trichuris vulpis numbers were statistically significantly lower, for example, Capillaria sp. with 40% in foxes and 21.1% in raccoon dogs and there was not a single endoparasite found that was more abundant in raccoon dogs. I am inclined to believe that the both predatory species have a different combination of parasites: the raccoon dog is heavier infested with ectoparasites and the red fox is more prone to intestinal parasites. All in all, nothing raises the prevalence of most diseases as much as overpopulation. A study done in Britain also suggests that the prevalence of Sarcoptes sp. is higher in lower population areas of red fox due to the bad habitation and improper food sources. In the present study this can be underlined as you can see it in Figure 3. It is due to the hostility of their habitat that the foxes are weak and are more prone to infection (51). In addition, for all I know, in recent years the hunt of predators has decreased. Due to the lack of reported data, I can only speculate that the same trend that we have in Germany also might apply to Lithuania. In Germany the hunted foxes steadily decline, most hunters in Germany hunt for meat and due to negative press, fur is not worn so often it makes the fox a very unpopular animal to hunt. Some might even argue that to kill just for selection is not ethical. What is interesting from the German hunting office statistics is that although the hunted foxes number decreases the number of “Fall wild” (animals that are shot due to disease or injury) has increased over the recent years (52). In Figure 4 you can see the spread of Sarcoptes throughout Lithuania. It is unfortunate that we don’t 34 | P a g e have a complete data set; nonetheless, you can see a high infection rate in many provinces. It would be very interesting to analyze the factors in different provinces to figure out what exactly has an impact on these variances. You can see a distinct grouping of the states which gives me confidence that the data is not random. As I mentioned, unfortunately, my data set is too small to conclude at reasonable confidence level for this purpose. In Figure 5 you can see that the raccoon dog in general had a lesser abundance and prevalence of intestinal parasites. Why didn’t I find any Coccidia, Eucoleus aerophilus, Toxoascaris leonina or Taeniids? As cited before, the study done in Lithuania in 2012 detected each of those parasites in the raccoon dog population too. It could have occurred due to my small sample size of raccoon dogs or the epidemiological study might have changed in Lithuania in regard to those parasites (2). For certain a larger sample size of the raccoon dog would have improved the understanding of this specific matter. Although regarding these parasites a trend can also be observed in the fox population. They are at the lower spectrum of the prevalence regarding the other parasites. In general, in both populations Alaria alata, Toxocara canis, Trichuris vulpis and Capillaria putorii were the most prevalent. These are also mostly spread thru Coprophagia and are also found in our pets, mainly dogs. A study carried out by my professor also highlights the wild carnivores as important vector among village dogs. These parasites were also found in the domestic village dogs (53). In Figure 6 the correlation between calculated and measured prevalence is highly fascinating. There is statistically significant suggestive data that we have mutualistic or synergistic behavior and likewise the data that shows that if an individual is carrying Sarcoptes sp. it is less prone to have other parasites. For example, with Uncinaria sp. we have an increase in detection in relevance to the reference percentage in both species if the individual carries the Sarcoptes mite. In foxes a 3.75% increase and in raccoon dogs a 4.39% were detected, both being statistically significant. On the other hand, we have Toxocara sp. which dwindled in both species with its prevalence if infected with Sarcoptes sp. In the red fox test population, it diminished by 1.88% and in the raccoon dog test group a 2.20% decrease was detected. Regretfully, at this stage I cannot give any direct answers as to why this happened. I can only speculate that it might have something to do with the immunosuppression and the weakening of the host, also the hypothermia caused by Sarcoptosis could influence the helminths and their prevalence. Finding the mechanism that influences either the parasites or the host that lead to the prevalence will be a serious challenge because a large number of factors come into play and it might be a multifactorial mechanism. It would be interesting to investigate this further to see if Sarcoptes sp. or the intestinal parasites have a direct effect on one another, or if the effect is indirect. It may be that even the behavior of the host changes with Sarcoptosis. In Figure 8 it is clear that there is a lack of sample size, but due to the small group size it is a very careful projection. I am inclined to believe that in order to get a more determined evaluation a larger sample pool is needed, 35 | P a g e and further analysis of the hosts is necessary too. We can already see a trend though there was a significant difference in parasitic species when they carried two or more parasites at a time. As discussed, a trait or interaction has not been proven with the hosts yet, but it is particularly interesting and it might be deemed most plausible due to the previously mentioned immune suppression and many chemical and hormone interactions the parasites cause as well as the hypothermia and the altered behavior. One might even trigger a cascade that makes a certain food source more likable or make the host choose a more prone habitat for other parasites. It is most intriguing to do more tests and run another set with a more balanced focus. In general, the accomplishments are very interesting and intrigue to undertake more extensive research. I would have wished for more raccoon dog samples to get a more equal basis and to get a more stable and less biased result. Also, it was very hard to obtain the 4 grams of fecal sample for the analyses; many animals fell short or didn’t have any feces. It is reasonable to assume that when animals are dying and going through agony, most of them lose the feces right there and then. It would be a good idea to ask hunters to collect the fecal sample if dropped at the place of death.

36 | P a g e

10. CONCLUSION

All in all, we can sum up that the presented study was a success and has pointed us in a new direction. We should focus more on parasitic infections as a whole and see how parasites interact with each other, which will give us more information in general and also about the named parasites and their abundance in relation to other parasites. Regarding my data, a clear relationship of Sarcoptes sp. and the intestinal found parasites has been proven. It works in both ways; some parasites are Synergetic with Sarcoptes sp. showing a higher prevalence in Sarcoptosis and others show a clear regression when Sarcoptes sp. is present. Nevertheless, further investigations and studies must be done to find out more about the parasitic infestation of the given animals and what an infection of multiple parasites means for each of those and for the host itself. After all, they could be considered roommates and, in my view, it is merely impossible not to affect each other while they are being shared by one host. The literature, sources and statistics that have been read, make me conclude that the red fox due to its volume and the raccoon dog because it shares the habitat and food source with the fox both are much infested populations. Its sick status is due to either unfriendly habitat or lack of proper diet sources or due to overpopulation caused by the factors, such as low predation, high food sources, positive habitats and falling away of main diseases. The overpopulation of the red fox is caused specifically by the oral rabies vaccine. Low hunted numbers and low number of larger predators or rivals, the high food sources due to our culture and its comfort around civilization and the ever more friendly habitats also added to this. With human’s agriculture, we are leaving less and less protection and shelter for the fox’s prey. It is specifically seen that the raccoon dog suffers more from Sarcoptic mange while the red fox population must deal with more endoparasites. Sarcoptosis is proven to be a locality prevalent disease, shaping Lithuania’s incidences at highest in northwestern and second highest in southeastern provinces. Both species are highly prevalent for both endo and ectoparasites and there is evidence suggesting that endo and ectoparasites do have a synergistic effect on each other in the raccoon dog population. It should be further investigated with a higher sample size.

37 | P a g e

11. PERSONAL SUGGESTION

I suggest reconsidering the nowadays hunting strategy in Lithuania and funneling more resources into the proper count of species. In order to do a proficient and ecologically accurate hunting plan, exact numbers are needed. But it takes no specialist to see that the predator species are suffering, on the one hand, from alien species like the raccoon dog, being a direct rival and contributing to the pressure on the predator population and vectoring diseases in-between them, and on the other hand, from the high prevalence of infectious diseases and parasites. This can only result from two factors as mentioned above. On the one hand, overpopulation of predators and, on the other hand, the poor habitat choices they must make because of overpopulation and overall human intervention into their original habitat with agriculture, urban sprawl and human activity. Thus, on the one hand, there is overpopulation but, on the other hand, there are not enough resources to keep a nourished healthy fox population. Due to the harsh intervention of humanity into the red fox’s habitat, I also deem us humans accountable and responsible for keeping it in a healthy balance. This is now where a proper population count is considered necessary and where an effective and balanced hunting plan comes into play. At this point it is clear that too few foxes are hunted and by far too few raccoon dogs too due to the lack of pressure on hunters in selecting as well as on the governments for not seeing their obligation to rule harder against alien species. The government must make the hunt on pest species as easy as possible and even oblige hunters to do it, for example, with the assistance of the introduction of new laws they could legalize night vision and suppressors to ease the pest hunt. In history, rewards were paid to eradicate wild species effectively. I feel strongly that this would be a good start to help against the raccoon dog population and with it gone, I am sure the red fox population would recover. Another point of interest would be to make hunting more popular again. Making younger people interested in hunting and giving more education to the population. These days, this would also be an opportunity to continue regaining interest in hunting again. As regards running a similar or improved test series, more samples are needed especially from the raccoon dog population. Furthermore, biosecurity should be enhanced with fully enclosed disposable suits. In addition, hunters should be asked to obtain fecal samples at the place of animal’s death because, to my way of thinking, lots of samples are lost this way.

38 | P a g e

12. BIBLIOGRAPHY

1. European Commission, Directorate-General for Health and Food Safety. Rabies eradication in the EU. 2017.

2. Bružinskaitė-Schmidhalter R, Šarkūnas M, Malakauskas A, Mathis A, Torgerson PR, Deplazes P. Helminths of red foxes ( Vulpes vulpes ) and raccoon dogs ( Nyctereutes procyonoides ) in Lithuania. Parasitology. 2012 Jan;139(1):120–7.

3. Bucko M, Samantha G. SARCOPTES SCABIEI Aetiology Epidemiology Diagnosis Prevention and Control Potential Impacts of Disease Agent Beyond Clinical Illness References [Internet]. OIE; 2019. Available from: https://www.oie.int/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/Disease_car ds/newcards/Sarcoptes_scabiei_(Infection_with).pdf

4. Carter A, Luck GW, McDonald SP. Ecology of the red fox (Vulpes vulpes) in an agricultural landscape. 2. Home range and movements. Aust Mammalogy. 2012;34(2):175.

5. Kauhala K, Kowalczyk R. Invasion of the raccoon dog Nyctereutes procyonoides in Europe: History of colonization, features behind its success, and threats to native fauna. Current Zoology. 2011 Oct 1;57(5):584–98.

6. Adkins CA, p. S. Home ranges, movements and habitat associations of red foxes. Journal of Zoology. 1998 Mar 1;244(3):335–46.

7. Pence DB, Windbeg LA. Impact of a Sarcoptic Mange Epizootic on a Coyote Population. The Journal of Wildlife Managment. 1994 Oct;58(4):624–33.

8. Bak U, Hessellund K, Lykkegaard J. Sarcoptic mange in red fox. Denmark: Department of zoology University of Aarthus; 1997 p. 15–7.

9. Bornstein S, Mörner T, Samuel WM. Sarcoptes scabiei and Sarcoptic Mange [Internet]. Second Edition. Chapter 5; 2001. (Parasitic Diseases of Wild Mammals). Available from: https://doi.org/10.1002/9780470377000.ch5

10. Janulaitis Z, Juknelytė S, Griciuvienė L, Paulauskas A. Raccoon dog (Nyctereutes procyonoides) and native predators infection pathogens and parasites comparison. Biologija

39 | P a g e

[Internet]. 2014 Apr 29 [cited 2020 Nov 27];60(1). Available from: http://lmaleidykla.lt/ojs/index.php/biologija/article/view/2860

11. Hart BL, Hart LA, Thigpen AP, Tran A, Bain MJ. The paradox of canine conspecific coprophagy. Vet Med Sci. 2018 May;4(2):106–14.

12. Molnar B, Ciucci P, Mastrantonio G, Betschart B. Correlates of parasites and pseudoparasites in wolves (Canis lupus) across continents: A comparison among Yellowstone (USA), Abruzzo (IT) and Mercantour (FR) national parks. International Journal for Parasitology: Parasites and Wildlife. 2019 Dec;10:196–206.

13. Bermúdez S, Miranda R. Distribución de los ectoparásitos de Canis lupus familiaris L. (Carnivora: Canidae) de Panamá. Rev MVZ Córdoba [Internet]. 2011 Jan 1 [cited 2020 Dec 21]; Available from: https://revistas.unicordoba.edu.co/index.php/revistamvz/article/view/285

14. Sprent JFA. Observations on the development of Toxocara canis (Werner, 1782) in the dog. Parasitology. 1958;48(1–2):184–209.

15. Hellard E, Fouchet D, Vavre F, Pontier D. Parasite–Parasite Interactions in the Wild: How To Detect Them? Trends in Parasitology. 2015 Dec;31(12):640–52.

16. Martin AM, Fraser TA, Lesku JA, Simpson K, Roberts GL, Garvey J, et al. The cascading pathogenic consequences of Sarcoptes scabiei infection that manifest in host disease. R Soc open sci. 2018 Apr; 5(4):180018.

17. Burgess I. Sarcoptes scabiei and Scabies [Internet]. Medical Entomology Center, University of Cambridge; 1994. Available from: https://insectresearch.com/wp-content/uploads/simple-file- list/1994-Burgess-Sarcoptes-scabiei-and-scabies.-Advances-in-Parasitology-1994-33-235- 295.pdf

18. Traversa D, Di Cesare A, Lia RP, Castagna G, Meloni S, Heine J, et al. New Insights into Morphological and Biological Features of Capillaria aerophila (Trichocephalida, Trichuridae). Parasitology Research. 2011 Aug 1;109(1):97–104.

19. Portier J, Jouet D, Ferté H, Gibout O, Heckmann A, Boireau P, et al. New data in France on the trematode Alaria alata (Goeze, 1792) obtained during Trichinella inspections. Parasite. 2011 Aug;18(3):271–5.

40 | P a g e

20. Magi M, Macchioni F, Dell’Omodarme M, Prati MC, Calderini P, Gabrielli S, et al. Endoparasites of Red Fox (Vulpes vulpes) in Central Italy. : 5.

21. Smith LF. Internal Parasites of the Red Fox in Iowa. The Journal of Wildlife Management. 1943;7(2):174–8.

22. Swe PM, Reynolds SL, Fischer K. Parasitic scabies mites and associated bacteria joining forces against host complement defence. Parasite Immunology. 2014 Nov 1;36(11):585–93.

23. Sakalauskas P, Lipatova I, Radzijevskaja J, Paulauskas A. Pathogen screening in the red fox (Vulpes Vulpes) from Lithuania. biologija [Internet]. 2020 Jan 4 [cited 2020 Nov 29];65(4). Available from: https://www.lmaleidykla.lt/ojs/index.php/biologija/article/view/4122

24. Romashov BV. Three Capillariid species (Nematoda, Capillariidae) of carnivores (Carnivora) and discussion of system and evolution of the nematode family Capillariidae. 1. Redescription of Eucoleus aerophilus and E-boehmi. Zoologicheskii Zhurnal. 2000 Dec 1;79:1390–1.

25. Willingham AL, Ockens NW, Kapel CMO, Monrad J. A helminthological survey of wild red foxes (Vulpes vulpes) from the metropolitan area of Copenhagen. Journal of Helminthology. 2009/06/05 ed. 1996;70(3):259–63.

26. ITIS USF and WS. ITIS Standard Report Page: Vulpes vulpes [Internet]. ITIS U.S. Fish and Wildlife Service; 2020. Available from: https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&anchorLocation=Subor dinateTaxa&credibilitySort=TWG%20standards%20met&rankName=Subspecies&search_val ue=180604&print_version=SCR&source=from_print#SubordinateTaxa

27. Zienius D. RETROSPEKTYVINIAI PASIUTLIGĖS PREVENCIJOS IR KONTROLĖS ASPEKTAI LIETUVOS LAUKINIŲ GYVŪNŲ POPULIACIJOJE 1995 – 2002 METAIS. 2004; 7.

28. Lietuvos medžiotojų ir žvejų draugija. Sumedžiotų gyvūnų apskaita (hunted animals) 1965- 2013 [Internet]. Lietuvos medžiotojų ir žvejų draugija; 2014 p. 3. (Sumedžiotų gyvūnų apskaita (hunted animals) 1965-2013). Available from: http://www.lmzd.lt/files/uploaded/faunos-statistika/sumedziojimas-iki-2014.pdf

29. ITIS USF and WS. Nyctereutes procyonoides (Gray, 1834) Taxonomic Serial No.: 183821 [Internet]. ITIS U.S. Fish and Wildlife Service; 2020. Available from:

41 | P a g e

https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=183821# null

30. Kauhala K. Reproductive strategies of the raccoon dog and the red fox in Finland. 1996. :8.

31. Warrell M. Rabies and other lyssavirus diseases. The Lancet. 2014 Mar 20;363(9413):959–69.

32. Zienius D, Sereika V, Lele R. Rabies occurrence in red fox and raccoon dog population in Lithuania. :7.

33. Bochkov AV. A review of mammal-associated Psoroptidia (Acariformes: Astigmata ) [Internet]. 1Zoological Institute of the Russian Academy of Sciences,; 2010. Available from: https://www.researchgate.net/publication/258371685_A_review_of_mammal- associated_Psoroptidia_Acariformes_Astigmata

34. Baker EW, Camin JH, Cunliffe F, Wooley TA, Yunker CE. Guide to the families of mites. Department of Zoology, University of Maryland; 1958.

35. Li L, Gibson DI, Zhang L-P. An annotated catalogue of the ascaridoid nematode parasites of Chinese vertebrates. Systematic Parasitology. 2016 Jan 1; 93(1):1–35.

36. Dow C, Jarrett WFH, Jennings FW, Mcintyre WIM, Mulligan W. The production of active immunity against the canine hookworm Uncinaria stenocephala. Journal of the American Veterinary Medical Association. 1959;135(8):407–11.

37. Hill RLJ, Roberson EL. Temperature-induced separation of larvae of Uncinaria stenocephala from a mixed fecal culture containing Ancylostoma caninum. J Parasitol. 1985 Jun;71(3):390–1.

38. Schurer J, Davenport L, Wagner B, Jenkins E. Effects of sub-zero storage temperatures on endoparasites in canine and equine feces. Veterinary Parasitology. 2014 Aug 29;204(3):310–5.

39. Holmes PR, Kelly JD. Capillaria aerophila in the domestic cat in Australia. Australian Veterinary Journal. 1973;49(10):472–3.

40. Curtsinger DK, Carpenter JL, Turner JL. Gastritis caused by Aonchotheca putorii in a domestic cat. J Am Vet Med Assoc. 1993 Oct 15;203(8):1153–4.

42 | P a g e

41. Nugaraitė D, Mažeika V, Paulauskas A. Helminths of mustelids (Mustelidae) in Lithuania. biologija [Internet]. 2014 Oct 29 [cited 2020 Dec 6]; 60(3). Available from: http://lmaleidykla.lt/ojs/index.php/biologija/article/view/2970

42. Nemzek JA, Lester PA, Wolfe AM, Dysko RC, Myers DD. Chapter 12 - Biology and Diseases of Dogs. In: Fox JG, Anderson LC, Otto GM, Pritchett-Corning KR, Whary MT, editors. Laboratory Animal Medicine (Third Edition) [Internet]. Boston: Academic Press; 2015. p. 511–54. Available from: http://www.sciencedirect.com/science/article/pii/B9780124095274000122

43. Areekul P, Putaporntip C, Pattanawong U, Sitthicharoenchai P, Jongwutiwes S. Trichuris vulpis and T. trichiura infections among schoolchildren of a rural community in northwestern Thailand: the possible role of dogs in disease transmission. Asian Biomedicine. 2010 Feb 1; 4(1):49–60.

44. Tylkowska A, Pilarczyk B, Pilarczyk R, Zyśko M, Tomza-Marciniak A. Presence of Tapeworms (Cestoda) in Red Fox (Vulpes Vulpes) in North-western Poland, with Particular Emphasis on Echinococcus Multilocularis. Journal of Veterinary Research. 2019 Mar 22; 63:71–8.

45. Kharchenko V, Kornyushin V, Varodi E, Malega O. Occurrence of Echinococcus multilocularis (Cestoda, Taeniidae) in red foxes (Vulpes vulpes) from Western Ukraine. Acta Parasitologica [Internet]. 2008 Jan 1 [cited 2020 Dec 6];53(1). Available from: https://www.degruyter.com/doi/10.2478/s11686-008-0008-9

46. Borgsteede FHM. Helminth parasites of wild foxes (Vulpes vulpes L.) in The Netherlands. Zeitschrift für Parasitenkunde. 1984 May 1; 70(3):281–5.

47. Juokslahti T, Korhonen T, Oksanen A. Coccidiosis in farmed silver foxes (Vulpes vulpes) and blue foxes (Alopex lagopus) in Finland: a case report: Acta Vet Scand. 2010 Oct;52(S1):S18, 1751-0147-52-S1–18.

48. M. B. Hildreth, D. S. Blunt, J. A. Oaks. LETHAL EFFECTS OF FREEZING ECHINOCOCCUS MULTILOCULARIS EGGS AT ULTRALOW TEMPERATURES. Journal of Parasitology. 2004 Aug 1; 90(4):841–4.

43 | P a g e

49. Šarkūnas M, Vienažindienė Ž, Rojas CAA, Radziulis K, Deplazes P. Praziquantel treatment of dogs for four consecutive years decreased the transmission of Echinococcus intermedius G7 to pigs in villages in Lithuania. Food and Waterborne Parasitology. 2019 Jun; 15:e00043.

50. Zienius D, Pridotkas G, Lelesius R, Sereika V. Raccoon dog rabies surveillance and post- vaccination monitoring in Lithuania 2006 to 2010. Acta Vet Scand. 2011 Dec; 53(1):58.

51. Volume contents. Molecular Ecology. 2006 Aug 14; 15(14): i–xviii.

52. Goretzki J. Der Fuchs breitet sich zu stark aus : Risiken für Mensch und Tier. :2.

53. Vienažindienė Ž, Deplazes P, Šarkūnas M. The Effect of removal of feces on subsequent excretion of intestinal parasite eggs in Lithuanian village dogs. Tarptautinė mokslinė konferencija “Gyvūnų fiziologijos ir patologijos aktualijos” : programa ir tezės : Kaunas, 2017 rugsėjo 28-29 d. = International scientific conference “Actualities in animal physiology and pathology” : programme and abstracts : Kaunas, 28-29 September, 2017 / Lietuvos sveikatos mokslų universitetas. Veterinarijos akademija. [Virškinimo fiziologijos ir patologijos mokslinis centras] / Lithuanian University of Health Science. Veterinary Academy. Veterinarijos akademija. [The Research Center of Digestive Physiology and Pathology]. Kaunas : Terra Publica, 2017. Veterinarinės patobiologijos katedra; 2017.

44 | P a g e