Based on the abundance of E. multilocularis infection, the infected foxes were allocated into low (1-20 scolices), moderate (21-130) and high (more than 130) abundance groups. A new calculation based on the number of scolices and excess of gravid proglottid found in the samples increased the sensitivity of SCT by 19.61% (95% CI: 11.9-27.3) in low abundance, 53.39% (95% CI: 49.9-56.9) in moderate abundance and 6.73% (95% CI: 6.1-7.4) in high abundance group as compared to those of calculation based on the scolices of E. multilocularis only (p<0.05).

Keywords: Echinococcus multilocularis, SCT, red fox, epidemiology, prevalence, abundance, enumeration.

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6

SANTRAUKA

ECHINOCOCCUS MULTILOCULARIS PAS RUDĄSIAS LAPES (VULPES VULPES) LIETUVOJE: NAUJI TYRIMAI PAPLITIMUI ĮVERTINTI IR PATOBULINTO HELMINTŲ

SKAIČIAUS NUSTATYMO VERTINIMAS

Anni Takala

Magistro baigiamasis darbas

Darbo tikslas - nustatyti cestodo Echinococcus multilocularis paplitimą rudųjų lapių (Vulpes vulpes) populiacijoje Lietuvoje ir įvertinti patobulinto E. multilocularis skaičiaus nustatymo sedimentacijos metodu jautrumą. Nuo 2018 m. lapkričio iki 2020 m. gegužės mėn. buvo surinktos ir ištirtos (n=70) centrinė/rytinės Lietuvos 25 rajonuose sumedžiotos rudosios lapės, jos išskrostos ir sedimentacijos metodu įvertintas jų užsikrėtimas E. multilocularis.

E. multilocularis helmintai iš lapių žarnyno buvo išplauti ir surinkti sedimentacijos metodu (SCT). Nustatyta, kad 31,4% (22/70; 95% PI: 20,5–42,3) ištirtų lapių buvo užsikrėtę E. multilocularis.

Užsikrėtimo gausumas svyravo nuo 1 iki 2266 helmintų užkrėstai lapei.

Pagal užsikrėtimo E. multilocularis gausumą visos lapės buvo suskirstytos į mažo (1-20 helmintų), vidutionio (21-130 helmintų) ir didelio (>131 helmintų) užsikrėtimo grupes, visose grupėse vertinant patobulintą helmintų skaičiavimo metodiką. Nustatyta, kad patobulinta E. multilocularis skaičiavimo metodika, paremta mėginiuose rastų skoleksų ir atsidalijusių brandžių narelių pertekliumi, padidino (p<0,05) sedimentacijos metodo jautrumą 19,61% (95% PI: 11,9-27,3) mažo užsikrėtimo, 53,39% (95% PI: 49,9-56,9) vidutinio užsikrėtimo ir 6,73% (95% PI: 6,1-7,4) didelio užsikrėtimo grupėse palyginus su įprasta metodika, paremta skoleksų skaičiaus nustatymu.

Raktažodžiai: Echinococcus multilocularis, SCT, rudoji lapė, epidemiologija, paplitimas, užsikrėtimo gausumas, skaičiavimas.

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7

ABBREVIATIONS

LUHS - Lithuanian University of Health Sciences

OIE - Office International des Epizooties (World Organisation for Animal Health) WHO - World Health Organization

AE - alveolar echinococcosis

SCT - sedimentation and counting technique IST - intestinal scraping technique

PCR - Polymerase chain reaction PP - pooled prevalence

EU - European Union Fig. - Figure

Et al. - et alia I.e. - id est

CI - Confidence Interval

Used measurement units mm - millimeter

cm - centimeter

% - per cent

°C - degrees celsius

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8

INTRODUCTION

Echinococcus multilocularis along with other Echinococcus species is considered one of the most important cestode zoonoses worldwide and despite the research and control methods applied today, echinococcosis is still a significant risk to public health. E. multilocularis is considered endemic in many countries in the European Union, and this is largely due to the high distribution and occurrence of its main final host, the red fox (Vulpes vulpes), and the main intermediate hosts, small rodents from the subfamily of Arvicolinae (1–3).

The prevalence of E. multilocularis has been shown to vary among the adult and juvenile red foxes and according to many of the previous studies the juveniles have been more frequently and/or intensively infected than the adults (4), except in the case of previous study conducted in Lithuania where adult foxes were more abundantly infected than the juveniles (5).

The diagnosis of E. multilocularis in dead foxes is commonly performed by the intestinal scraping technique (IST) or a sedimentation and counting technique (SCT) from the small intestine, but more advanced methods to detect the excreted eggs are also available, such as coproantigen or copro- PCR detection (1,2,4,6–8). The method applied in this study is the sedimentation and counting technique that has been referred to as the “Gold standard” for the detection of E. multilocularis at necropsy (9,10).

Even though rarely mentioned, the common enumeration technique of E. multilocularis has been based on counting the scolices or intact cestodes in the samples (1,7,9,10). In this study we hypothesized that during the intestinal washing some scolices may remain attached to the intestine and therefore by calculating the scolices and any excess of detached gravid proglottids we may improve the evaluation of the abundance and even increase it.

This study evaluates the current prevalence of E. multilocularis in red foxes in Lithuania and investigates a novel, improved method for the enumeration to determine the abundance of infection in red foxes. The last studies made to investigate the prevalence of Echinococcus multilocularis in red foxes in Lithuania were published in 2007 and 2012 (5,11). The data presented in these studies was based on the samples collected in the period of 2001-2006 and covered a limited number of districts.

Therefore, an update to existing information on the prevalence and further distribution of E.

multilocularis becomes necessary.

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9 The aim of the present study is to examine the changes in prevalence of E. multilocularis in red foxes during the last sixteen years in Lithuania and to investigate a novel improved method for enumeration of E. multilocularis.

The research tasks:

1. Estimation of the prevalence of E. multilocularis based on the examination of small intestine samples by the sedimentation and counting technique (SCT).

2. Evaluation of the prevalence of E. multilocularis in relation to gender, age, district, and time of the death of the red foxes.

3. Evaluation of improved method for enumeration of E. multilocularis, collected using the sedimentation and counting technique (SCT), to calculate the abundance of infection in red foxes.

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10

1. LITERATURE REVIEW

1.1. Echinococcus multilocularis

1.1.1. Morphology and life cycle

Echinococcus multilocularis is a small cestode, only 1.2-4.5 mm in length and belongs to the genus Echinococcus. The adult tapeworm consists of a scolex and 4-5 segments of strobila, of which the last one is a gravid proglottid bearing thousands of eggs (fig. 1) (1,4).

The life cycle of Echinococcus multilocularis is a multi-host system commonly consisting of a carnivore such as red fox as a final host and a small rodent as an intermediate host. This is referred to as the sylvatic or natural cycle, but a domestic cycle exists as well and usually involves dogs or cats as the final host. Humans are considered mainly aberrant or accidental hosts (1).

During the life cycle the intermediate host ingests the eggs from the environment and the eggs develop into metacestodes in the host’s viscera. The final host, such as the red fox is infected when it eats the intermediate host or its viscera, containing the cysts. The protoscoleces within the cysts develop into mature worms in the final host’s small intestine and the life cycle continues as the eggs are passed with the feces into the environment (fig. 2) (1). However, there are records that even the assumed final hosts, such as dogs, can end up as an intermediate host and establish metacestodes in the liver. In some instances, E. multilocularis metacestodes have also been found in the livers of pigs (4,12).

The life expectancy of E. multilocularis in foxes' small intestine is recorded to be between 60 and 90 days and the highest egg excretions are between 33–39 days post infection (6,13).

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11 Fig. 1. Photomicrograph of E. multilocularis with arrows pointing to the genital pore (left picture) and uterus (right picture) (2).

1.1.2. Zoonotic importance

Echinococcus multilocularis is a zoonotic parasite, meaning it can be passed from animals to humans and cause a disease in humans. The disease caused by E. multilocularis in humans is called alveolar echinococcosis (AE) and can be fatal if not treated or diagnosed in time (14). The first human case of AE was diagnosed as early as 1852 in Germany (1).

The eggs of E. multilocularis become infectious when ingested by a suitable intermediate or aberrant host, such as humans. AE is characterised by the multiple small vesicles, about 3 cm in size, found in the viscera. These vesicles, or cysts, commonly reside in the liver but can also be found in other tissues such as lungs and brain (1). The cysts grow progressively, in a slow, cancer-like manner.

In humans it generally takes anywhere from 5-15 years until the clinical signs of the disease develop or the disease is diagnosed (15). The advanced infection leads to hepatomegaly and jaundice, and mortalities can be high despite aggressive treatments (16).

In Lithuania, 179 cases of AE in humans were diagnosed between 1997-2013, and the country has been ranked among the top 5 countries in Europe by the total number of cases (16,17).

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12 Fig. 2. The life cycle of Echinococcus multilocularis presenting both the sylvatic and domestic cycles (18).

1.1.3. Geographical distribution

Even though E. multilocularis has spread widely in the northern hemisphere and there are regions in all northern continents that are considered endemic, the geographic distribution of E.multilocularis depends on the existence of both intermediate and final hosts in those areas. The prevalence is also highly influenced by many factors such as the abundance of the hosts, the diets of final hosts and seasonal variations, especially in the case of the intermediate hosts, small rodents (1,4). A study investigating the effects of age, gender, season and habitat on the diet of the red fox in northeastern Poland found that the female foxes consumed more voles and that the males and juveniles had more diverse diets (3).

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13 In Europe, E. multilocularis was originally found to be endemic in central Europe; Germany, France, Austria and Switzerland, but has then been reported in many other countries too. At the moment, the European Union countries detected to have E. multilocularis extend from the mediterranean countries, such as Italy and Greece, until Scandinavia where Sweden and Denmark have reported the presence of E. multilocularis in red foxes. In the Arctic Archipelago of Svalbard in Norway, E. multilocularis was detected in the Arctic fox (Vulpes lagopus) in 1999. Studies conducted in the mainland of Norway, Finland, the UK and Ireland have so far reported an absence of E.

multilocularis in red foxes (1,15,16,18).

The study conducted by Oksanen et al. in 2016, grouped the affected countries into 3 groups according to their pooled prevalence (PP) of E. multilocularis in red foxes in European Union and adjacent countries (Fig. 3). Sweden, Denmark and Slovenia had a low PP (<1%), whereas countries of medium PP (>1 % to <10 %) included Austria, Belgium, Croatia, Hungary, Italy, Netherlands, Romania and Ukraine. Lithuania, along with Estonia, Latvia, Czech Republic, France, Germany, Poland, Slovakia, Liechtenstein and Switzerland were grouped as high PP (>10 %) countries (15).

E. multilocularis is also found in Northern America, and the prevalence is influenced by multiple final hosts, such as the Arctic fox in the Alaskan regions, and red foxes and coyotes (Canis latrans) in the North Central Region. Russia and the adjacent countries as well as China and Japan are all considered endemic regions to E. multilocularis, and several carnivore species have been identified as potential final hosts (1,19).

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14 Fig. 3. The distribution and pooled prevalence of E. multilocularis in red and Arctic foxes in the EU and neighboring countries (15).

1.1.4. Prevalence in red foxes in neighboring countries

Many of the neighboring countries performed studies examining the prevalence of E.

multilocularis around the same time as did Bruzinskaite et al. in Lithuania. However, some countries such as Belarus and Poland have reported the occurrence of E. multilocularis long before this (17).

In Belarus, both adult and larval stages have been identified in animals and AE in humans already decades ago. A retrospective study was published in 2011 in which the red fox was identified as the definitive host. In 1995, Poland was already recognized as an endemic area and high levels of infected red foxes were recorded (up to 39.3%) in a study published in 2014 (17,20).

Several studies have been conducted in Latvia and the prevalences have varied among the studies quite significantly. In a study conducted between 2003-2008, only 45 red foxes were examined of which 35.6% were infected, with a worm burden varying between 1-1438 worms per infected fox. Two

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15 other studies were performed in 2007-2008 (42 foxes examined) and 2010-2015 (538 foxes examined) which revealed that the prevalence had almost dropped by half, to 17.1% (17,21,22).

Estonia reported the presence of E. multilocularis for the first time in 2003 with a prevalence of 29,4%; however, only 17 red foxes were examined. The worm burden ranged from 3 to 927. Another study published in 2015, revealed the prevalence to still be roughly the same (approximately 30%), even though a larger number of foxes were examined (n=137) (17,23,24).

In 2009 the Scandinavian mainland countries Sweden, Norway and Finland were still considered free from E. multilocularis (18). 3 years later, in 2011, the first E. multilocularis infection was detected in red fox in Sweden (25). After this, surveillance was increased in all Scandinavian countries but until today, the mainland of Norway and Finland have continued to be declared free of E. multilocularis (17–19,26).

1.1.5. Prevalence in red foxes in Lithuania

According to the studies conducted in Lithuania on the prevalence of E. multilocularis in red foxes, it was concluded that the tapeworm has become endemic and is highly prevalent, especially in certain regions of Lithuania. Before 2001, E. multilocularis had not been detected in foxes, however, this finding does not exclude the possibility that E. multilocularis has been present to Lithuania earlier (5,11).

The study published in 2007, examined 206 red foxes from various districts of Lithuania between October 2001 to April 2004. Of those examined foxes, 118 (57.3%) were positive for E. multilocularis.

In most of the regions, E. multilocularis was detected in the foxes but the highest prevalence was found in the Kaunas region (62.3%). The average number of these tapeworms was 56 per infected fox in Kaunas district (11).

The second study was published in 2012 and included the information gathered from the previous study published in 2007. The collection of foxes was extended until March 2006, and altogether the small intestines of 269 red foxes were examined. Of the 269 foxes, 158 (58.7%) were distinguished with a presence of E. multilocularis (5,11). The worm burdens varied greatly with a mean burden of 1309 (1-20 9249) worms per fox (17).

Between the 2001-2006, altogether 45 foxes were collected from the Kaunas region and of those 53% had an E. multilocularis infection (5).

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16 In both studies, the foxes` small intestines were examined by the "golden standard" sedimentation and counting technique (SCT) (27), and the parasites were identified as E. multilocularis according to the general size of the worm and the shape of the uterus of the last gravid segment (4,5).

It was found that foxes hunted in winter had lower numbers of E. multilocularis than those hunted in autumn. Also, lower abundances of E. multilocularis were recorded in juveniles compared to adults. The lower abundances of E. multilocularis found in foxes hunted in winter could be explained by the fact that the prey species (rodents) are not as plentifully available during the winter months and because of the life expectancy of the tapeworm in its final host is between 60 and 90 days, the infection would need to be a fairly recent one (5,13).

In a study published by Bruzinskaite et al. in 2009, two dogs were found to be infected with E.

multilocularis eggs. The feces of the village dogs were collected in the southwestern part of Lithuania.

Also, multiple necrotic and calcified lesions found in the livers of three family farmed pigs were identified by PCR as E. multilocularis (12).

1.1.6. Age and gender distribution among the infected red foxes

Bruzinskaite´s study from 2012 differs from other studies conducted in Europe by the conclusion that intestinal immunity would play a very small part in the development of level of infection. None of the juvenile foxes in Bruzinskaite´s study had heavy worm burdens, whereas 17% of the adult foxes had more than 1000 worms per animal. Previous studies have shown that juvenile foxes are more regularly and heavily infected than adult foxes. This has led to a suggestion that adult foxes can develop partial immunity in conditions where the infection pressure is high (4,5,13).

A 2019 study from Iran examined 23 red foxes of which 2 were infected with adult E.

multilocularis. It also reported that those 2 infected foxes were males and between 0-5 years old (youngsters), however, no adult male foxes were included in the study which may distort the results (2). In a study where 6 male and 9 female foxes were experimentally infected with E. multilocularis, both genders became equally infected (100%) and there were no specific differences in the amounts of eggs excreted by the different genders (13).

1.1.7. SCT and enumeration of E. multilocularis

Even though the SCT has been considered as the "golden standard" for the detection and enumeration of E. multilocularis in carnivores, and its sensitivity has been described as close to 100%

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17 (7,8), it has also been investigated for its "limitations", especially for low abundance levels. A study conducted by Karamon et al. found that in an experimental test, the intestine samples inoculated with 30 worms were all detected positive at SCT (with a mean of 8.4 worms found). Samples inoculated with fewer worms had lower positive results. It should be noted, however, that the worms used for the study were entire tapeworms with gravid proglottid and were fixed with ethanol. Despite this, the study showed that SCT has the capacity to detect even low abundances of E. multilocularis in the intestines, but several factors still play a role such as the debris present in the sample, the possibility of the worms being washed out with the supernatant, as well as others, and consequently the mean abundance can be significantly lower than the real abundance (10).

In the majority of the studies the enumeration technique has not been discussed in detail, but the studies mentioning it, have mostly based it on counting the scolices and/or intact cestodes in samples (7,9,10,27).

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2. METHODOLOGY

2.1. Study design and research objectives

The study was formed to determine the prevalence of E. multilocularis in red foxes in Lithuania and to use a novel method of enumerating the parasites found per individual fox. The study was performed between November 2018 and May 2020 in the Department of Veterinary Pathobiology at LUHS in Kaunas, Lithuania. During the study, 70 foxes were dissected at the Pathology Center and the small intestines were collected and examined at a laboratory of the Department of Veterinary Pathobiology.

The enumeration of E. multilocularis was decided to be included in the study once it was observed that in many of the samples there were detached scolices and proglottids, and that counting only the scolices with strobilae might not give us accurate results and the worm burdens would probably be lower than what they were in reality.

The objective of this study was to redetermine the prevalence of E. multilocularis in red foxes and to compare the results with the previous studies. It would also be noted if the prevalence were influenced by factors, such as age, gender, district, and time of the death. The evaluation of two different enumeration methods was added to look for an improvement in the abundance of E.

multilocularis.

2.2. Materials and methods

2.2.1. Necropsy of red foxes and collection of samples

The red foxes used in the study were hunted by private hunters in various districts of Lithuania and supplied by the National Food and Veterinary Risk Assessment Institute of Lithuania to the Department of Veterinary Pathobiology of LUHS. Altogether 70 foxes were included in the present study.

The examined foxes were of random age and sex. During the necropsy, the age was determined from the teeth development (1) and the foxes were separated to adults (>1 year old) and juveniles (≤1 year old). The sex was determined visually. Other data recorded from the foxes and used in this study, included the identification number of each fox, date of death and the district where they were hunted.

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19 46 of the foxes were male and 23 female. The gender of one fox could not be determined. 45 of the foxes were adults and 24 juveniles. For one fox, the age was not determined.

The foxes were collected in 25 districts in the Eastern part of Lithuania. These included districts of Utena (n=10), Šalčininkai (n=6), Panevėžys (n=5), Kaunas (n=4), Vilnius (n=3), and 2 foxes from each district of Švenčionys, Širvintos, Kelmė, Ignalina, Raseiniai and Ukmergė, from 14 other districts there was only 1 fox per region recorded. For 16 individuals, the district was not recorded. Most of the foxes were hunted between September 2018 and December 2019 but for some, the time of culling was unclear and therefore the date was not recorded in our study.

The foxes were deep frozen at -80 °C for at least 3 days until the necropsy was carried out.

During the necropsy, the small intestines were ligated from both ends and stored by deep-freezing at - 80 °C for another 3 days at least, before being dissected and examined for parasites (27).

2.2.2. Parasitological examination of samples

For the detection of E. multilocularis, the sedimentation and counting technique (SCT) was used, and strict safety and precaution measures were followed (27). Either the whole sediment or the individual tapeworms were collected and stored in 96% ethanol solution for calculations performed later on. The samples with visibly low worm burdens were enumerated immediately.

Other parasites found during the SCT were collected and stored in tubes in 40% alcohol solution.

Intestinal samples from the first 12 foxes examined were left out of the study after the SCT, as no E.

multilocularis was identified. This was believed to be an error of not recognising the small parasite and including these could have possibly given us a falsely low prevalence of E. multilocularis.

2.2.3. Identification of E. multilocularis

The identification of E. multilocularis was based on the tapeworm´s characteristic morphology (1,4). The sediment samples were investigated macroscopically against a black background, which allowed E. multilocularis to be seen as small, white organisms. The samples were also investigated under a stereomicroscopy for the purpose of identification and to aid in the collection and enumeration of E. multilocularis (fig. 4).

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20 Fig. 4. One E. multilocularis scolex with a gravid proglottid (arrow), two scolices without gravid proglottid (arrowheads left) and one detached gravid proglottid (arrowhead right), pictured through stereomicroscopy.

2.2.4. Enumeration of E. multilocularis and grouping of abundances

For the enumeration, E. multilocularis in each sample were allocated into 3 groups that included 1) scolex with gravid proglottid, 2) scolex (+/- strobilae) without gravid proglottid, and 3) gravid detached proglottids (fig. 4 and fig. 5). The enumeration was done under the stereomicroscopy and with the help of Cellodiff which is used for counting blood cells.

For each infected fox, the total number of E. multilocularis was recorded using two methods. In method 1 we only calculated the scolices (+/- strobilae) (i.e. sum of enumeration group 1 and 2) as this has generally been the go-to method for calculating the abundance in previous studies. For method 2, we based the total abundance on the sum of scolices (+/- strobilae) (group 1 and 2) and added any excess of gravid proglottid left from group 3. These excess gravid proglottids were received when the scolices without the gravid proglottid (group 2) and the detached gravid proglottids (group 3) were summed up and if there was a larger number of gravid proglottids present, this was considered as excess of proglottids.

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21 Based on the calculation method 1, (i.e. the total of scolices found in each sample), we allocated the infected foxes into groups of low (range of 1-20 scolices), moderate (21-130 scolices) and high abundances (≥131 scolices). This allocation then allowed us to perform comparison of the two methods within each abundance group, and to see whether the abundance actually improved or increased when we used the calculation method 2 instead of method 1.

Fig. 5. Two E. multilocularis scolices and strobilae (arrows) and one scolex without a gravid proglottid (arrowhead) pictured through stereomicroscopy. These parasites were allocated into group 2 for the enumeration of E. multilocularis.

2.2.5. Statistical analysis

IBM SPSS Statistics 27 Software was used to perform the statistical analysis, yet the prevalence of E. multilocularis could be calculated manually. Microsoft Word and Excel programs were used to create the tables and the graphs. Two-tailed Fisher`s Exact Test, with a significance level of 95%, was used to determine the significance of association between the prevalence and the different variables such as the age, gender, district, and time of death. Values with p>0.05 were considered not significant.

Mann-Whitney Test and Independent Samples t Test were used to determine if there was a significant difference in the abundance by the gender or age of the foxes, with a significance level of 95%. 95% Confidence Intervals (95% CI) with upper and lower limits were calculated for the prevalence, worm burdens, as well as for the sensitivities of enumeration for method 1 and 2.

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22 A non-parametric Wilcoxon Signed Rank Test was used to compare the abundance of method 1 and method 2 within each abundance class (low, moderate and high), to see if there was any significant difference between the methods and whether the improved method 2 made any significant difference in the enumeration of worms. Values with p>0.05 were considered not significant.

Sensitivity was based on the mean abundance of the two methods and was calculated manually. It was also based on the assumption that method 2 yielded more accurate abundances of E. multilocularis as there was an excess of gravid proglottids and therefore the improved method 2 was considered as 100% sensitive in all abundance groups. The significance in the sensitivity between two enumeration methods was estimated from the likelihood profile, and was evaluated statistically significant if the sensitivity of the method was greater or less than the 95% CI of the other method within the same group and vice versa.

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3. RESULTS

3.1. Prevalence of E. multilocularis

The red foxes investigated for this study varied in gender, age and in origin as well as in the time of collection. A total of 22 of the 70 foxes examined were infected with E. multilocularis giving us a prevalence of 31.4% (95% CI: 20.5-42.3).

Most of the examined foxes were also infected with other helminth genera such as other cestodes, nematodes and trematodes. The infections were recorded and are presented in the table in the annexes (annex 1) but are not discussed in this study.

3.1.1. Prevalence of E. multilocularis among the districts

Among the 25 different districts included in our study, in 12 of them we found red foxes infected with E. multilocularis (fig. 6). On top of this, 16 foxes came from unknown districts (table 1).

Previously highly prevalent district, Kaunas, now only recorded a 25% prevalence (95% CI: 0.0- 67.4) (11), whereas Utena (95% CI: 9.6-70.4) and Panevėžys (95% CI: 0.0-82.9) had a prevalence of 40%.

Districts Širvintos (2/2), Pakruojis (1/1), Prienai (1/1) and Kazlų Rūda (1/1), all recorded a prevalence of 100% (95% CI: 100.0-100.0), although only 1 or 2 foxes were examined from these regions. 50% prevalence was found in Kelmė, Švenčionys and Ukmergė (95% CI: 0.0-100.0).

Šalčininkai had a prevalence of 16.7% (95% CI: 0.0-46.5). The prevalence for unknown regions was 37.5% (95% CI: 13.8-61.2), which meant that the true prevalences within the districts could be very different if we knew where these foxes came from. Additionally, the prevalence did not show any correlation with the districts (p=0.823).

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24 Fig. 6. Prevalence (%) of E. multilocularis within the districts where red foxes were hunted in and the total prevalence within Lithuania.

Table 1. The number of examined and infected red foxes per district and their prevalence with 95% CI.

District No. infected/

No. examined

Prevalence, %

(95% CI) District No. infected/

No. examined

Prevalence, % (95% CI)

Kaunas 1/4 25

(0.0-67.4) Kelmė 1/2 50

(0.0-100.0)

Elektrėnai 0/1 0

(0.0-0.0) Jurbarkas 0/1 0

(0.0-0.0)

Mažeikiai 0/1 0

(0.0-0.0) Švenčionys 1/2 50

(0.0-100.0)

Pakruojis 1/1 100

(100.0-100.0) Zarasai 0/1 0

(0.0-0.0)

Trakai 0/1 0

(0.0-0.0) Unknown 6/16 37.5

(13.8-61.2)

Panevėžys 2/5 40

(0.0-82.9) Klaipėda 0/1 0

(0.0-0.0)

Širvintos 2/2 100

100.0-100.0 Plungė 0/1 0

(0.0-0.0)

Ukmergė 1/2 50

(0.0-100.0) Vilnius 0/3 0

(0.0-0.0)

Raseiniai 0/2 0

(0.0-0.0) Alytus 0/1 0

(0.0-0.0) 25

0 0 100

0 40

100

50

0 40

16.7 100

0 50

0 37.5

0 0 0 0 0 100

0 0 31.4

0 10 20 30 40 50 60 70 80 90 100

Prevalence (%)

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25 District No. infected/

No. examined

Prevalence, %

(95% CI) District No. infected/

No. examined

Prevalence, % (95% CI)

Utena 4/10 40

(9.6-70.4) Šakiai 0/1 0

(0.0-0.0)

Šalčininkai 1/6 16.7

(0.0-46.5) Kazlų Rūda 1/1 100

(100.0-100.0)

Prienai 1/1 100

(100.0-100.0) Anykščiai 0/1 0

(0.0-0.0)

Ignalina 0/2 0

(0.0-0.0) Jonava 0/1 0

(0.0-0.0)

Lithuania 22/70 31.4

(20.5-42.3) Continuation of table 1.

3.1.2. The infection incidence in relation to gender

9 female foxes were infected with E. multilocularis, giving us a prevalence of 39.1% (95% CI:

19.2-59.1), and of males 26.1% (95% CI: 13.4-38.8) were infected. Female adults also had higher prevalence (42.9%; 95% CI: 16.9-68.8) than the male adults (29.0%; 95% CI: 13.1-45.0). In addition, in youngsters, the prevalence was also higher in females (33.3%; 95% CI: 2.5-64.1) than in males (21.4%; 95% CI: 0.0-42.9) (fig. 5).

However, the prevalence didn´t show any significant correlation in relation to the gender (males and females) (p=0.157).

Fig. 5. Prevalence (%) of E. multilocularis in red foxes of different age and gender.

33.33

42.86

39.13

21.43

29.03 26.09 31.43

0 10 20 30 40 50 60

Juvenile female

foxes

Adult female

foxes

Female foxes (total)

Juvenile male foxes

Adult male foxes

Male foxes (total)

All foxes (total)

Prevalence (%)

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26 3.1.3. Prevalence of E. multilocularis in adult and juvenile foxes

Of all the examined foxes, 33.3% (95% CI: 19.6-47.1) of the adults were infected with E.

multilocularis and 29.2% (95% CI: 10.1-47.4) of the juveniles were infected. Statistically the prevalence among the different ages (adults and juveniles) was not significant (p=0.857).

3.1.4. Seasonal variation in the prevalence of E. multilocularis

The red foxes were hunted between 17.09.2018-21.12.2019, but for 11 foxes the time of death was not recorded and of those foxes, 4 were found to be infected with E. multilocularis. All together 36 different dates were recorded as a time of death. During the statistical comparison, the prevalence in relation to time of death did not differ significantly with p=0.893.

3.2. Abundance of E. multilocularis in red foxes

The worm burden varied from 1-2266 worms per infected fox. The mean abundance (the level of infection) was 307 (95% CI: 56.87-556.86) worms per infected fox. The abundance in youngsters, in general, was 401 (95% CI: -366.31-1168.88) worms per fox with the infection varying from 3-2266 worms per fox. In adults, the mean intensity was 263 (1-1255, 95% CI: 31.06-494.54) (table 2). The abundance of E. multilocularis and the age of the foxes did not show any significant correlation (p=0.832).

Among the female foxes, the youngsters had only 14 worms on average (95% CI: -2.29-30.29), whereas the adults had 365 (95% CI: -205.30-935.30) worms. This took the mean abundance in females, in general, to 248 (95% CI: -108.73-604.73) parasites per infected fox. However, there was no significant difference in the abundance between the female adults and youngsters with p=0.714.

In males, the youngsters were clearly more heavily infected than the adults, with an average of 921 (95% CI: -1971.95-3814.62) worms per infected fox. In male adults the abundance was 195 (95%

CI: -58.92-448.25). Altogether, the mean abundance in male foxes was 376 (95% CI: -42.15-794.81).

There was no significant difference in the abundance between the male adults and youngster (p=0.100).

Overall, the abundance did not show any correlation with the gender (females and males) of the foxes with p=0.337. There was also no significant difference in the abundance between the female and male adults (p=1.000), or female and male youngsters (p=0.310).

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27 Table 2. The prevalence of E. multilocularis in examined red foxes with the upper and lower 95%

confidence interval (CI), and the mean abundance of E. multilocularis infection in foxes of different gender and age with the upper and lower 95% CI.

No. of foxes infected with E.

multilocularis/

No. of foxes examined

Prevalence of E.

multilocularis,

% (95% CI)

The total number of E.

multilocularis found

Mean abundance

(min-max)

95% CI

FEMALE RED FOXES

Adult 6/14 42.9

(16.9-68.8) 2190 365

(1-1255) -205.30-935.30

Youngster 3/9 33.3

(2.5-64.1) 42 14 (7-20) -2.29-30.29

Total 9/23 39.1

(19.2-59.1) 2232 248

(1-1255) -108.73-604.73 MALE RED FOXES

Adult 9/31 29.0

(13.1-45.0) 1752 195

(2-930) -58.92-448.25

Youngster 3/14 21.4

(0.0-42.9) 2769 921

(228-2266)

-1971.95- 3814.62

Not defined 0/1 0.0-0.0 0 0 0.0-0.0

Total 12/46 26.1

(13.4-38.8) 4521 376

(2-2266) -42.15-794.81 TOTAL RED FOXES

Adult 15/45 33.3

(19.6-47.1) 3942 263

(1-1255) 31.06-494.54

Youngster 7/24 29.2

(10.1-47.4) 2814 401

(3-2266) -366.31-1168.88

Not defined 0/1 0.0-0.0 0 0 0.0-0.0

Total 22/70 31.4

(20.5-42.3) 6756 307

(1-2266) 56.87-556.86

3.3. Enumeration of E. multilocularis and the abundance groups

11 foxes infected with E. multilocularis were allocated to a low abundance group based on the number of scolices found. This included foxes with a range of 1-20 scolices in the sample. 6 foxes were allocated to a moderate abundance group with a range of 21-130 scolices and, lastly, 5 foxes to the high

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28 abundance group (more than 130 scolices found). The total and mean in each enumeration group was calculated along with 95% CI, standard deviation and margin of error (table 3).

In the present study, 9 out of the 22 foxes (40.9%; 95% CI: 20.4-61.5) had an excess of gravid proglottids in the sample. When we allocated the foxes into the three different abundance groups, we had an excess of gravid proglottids in 2 (18.2%; 95% CI: 0.0-40.9) samples in the low abundance group, in 6 (100%; 95% CI: 100.0-100.0) samples in the moderate abundance group and in the high abundance group, one sample (20%; 95% CI: 0.0-55.1) had an excess of gravid proglottids.

As an example, if we would have only counted the group 1 (i.e. entire worms) in individual fox samples, we would have missed the infection altogether in 3 foxes.

Method 1, which was based on counting only the scolices gave us a mean abundance of 7.45 (1- 20, 95% CI: 3.59-11.3) worms in low abundance group, as compared to method 2 giving a mean abundance of 9.27 (1-20, 95% CI: 5.27-13.3) worms. In the moderate abundance group, the method 1 had a mean worm count of 61.83 (28-127, 95% CI: 34.4-89.2), while with the method 2 the mean abundance was 132.67 (30-270, 95% CI: 60.7-205). Based on method 1, foxes in the high abundance group had a mean of 1091.80 (569-2266, 95% CI: 566-1620) worms, while the mean abundance in method 2 was 1170.60 (569-2266, 95% CI: 654-1690) worms.

A significant difference was found between method 1 (total scolices) and method 2 (total abundance) in the moderate abundance group with p=0.028. Also, the sensitivity of method 1 was only 46.61% (95% CI: 43.15-50.07), whereas the sensitivity of method 2 was 100% (95% CI: 93.05-106.95) in the moderate abundance group. Method 1 had lower sensitivity also in the low (80.39%; 95% CI:

72.69-88.10; p=0.180) and high (93.27%; 95% CI: 92.63-93.91; p=3.17) abundance groups as compared to those of method 2 (100%; 95% CI: 80.59-119.41 and 97.44-102.56) in both respective groups.

Sensitivity of SCT, when using the method 2, improved in the low abundance group by 19.61%

(95% CI: 11.9-27.3), by 53.39% (95% CI: 49.9-56.9) in the moderate abundance and by 6.73% (95%

CI: 6.1-7.4) in the high abundance group. Increased sensitivities of method 2 were considered statistically significant (p<0.05) in all abundance groups.

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29 Table 3. The total and fractionated abundances of E. multilocularis within the low, moderate, and high abundance groups in red foxes, and the sensitivities and significant differences of the calculation for method 1 and 2. In method 2, the total abundance was based on the calculation of total number of scolices summed up with the excess of gravid proglottids (the difference between the number of scolices without gravid proglottids (group 2) and number of detached gravid proglottids).

* Significantly higher total abundance as compared to those of total no. of scolices in the method 1 (p<0.05) within the same abundance group.

** Statistical significance estimated based on the 95% CI limits as the sensitivity of both of the methods are outside of the 95% CI limits of the other method within the same abundance group.

SD, standard deviation; 95% CI, 95% confidence interval with upper and lower limit.

Method 1 Method 2

Group 1:

Scolex with gravid proglottid

Group 2:

Scolices with strobilae/

without gravid proglottid

Total no.

of scolices (min-max)

Sensitivity,

% (95% CI)

Group 3:

No. of gravid detached proglottids

Excess of gravid proglottids

Total abundance (min-max)

Sensitivity,

% (95% CI)

RED FOXES WITH LOW ABUNDANCES (1-20; Range of scolices), N=11 Total

abundance 50 32 82 43 20 102

Mean

abundance 4.55 2.91 7.45

(1-20) 80.39%

(72.69-88.10) 3.91 1.82 9.27

(1-20) 100% **

(80.59-119.41) 95% CI 1.68-7.42 -0.55-6.37 3.59-11.3 0.61-7.21 -0.92-4.56 5.27-13.3

SD 4.85 5.85 6.54 5.58 4.63 6.77

Margin of

error 2.87 3.46 3.86 3.3 2.74 4

RED FOXES WITH MODERATE ABUNDANCES (21-130), N=6 Total

abundance 254 117 371 542 425 796 *

Mean

abundance 42.33 19.5 61.83

(28-127) 46.61%

(43.15-50.07) 90.33 70.83 132.67

(30-270) 100% **

(93.05-106.95) 95% CI 22.8-61.8 8-31 34.4-89.2 17.3-163 0.43-141 60.7-205

SD 24.36 14.34 34.23 91.25 87.97 90.02

Margin of

error 19.5 11.5 27.4 73 70.4 72

RED FOXES WITH HIGH ABUNDANCES (more than 131), N=5 Total

abundance 3229 2230 5459 1651 394 5853

Mean

abundance 645.80 446.00 1091.80

(569-2266) 93.27%

(92.63-93.91) 330.20 78.80 1170.60

(569-2266) 100% **

(97.44-102.56) 95% CI 355-937 176-716 566-1620 157-503 -59.2-217 654-1690

SD 332.29 308.50 599.75 197.49 157.6 590.05

Margin of

error 291 270 526 173 138 517

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30

4. DISCUSSION

According to our study, the prevalence of E. multilocularis in red foxes in Lithuania has decreased from the 2012 prevalence of 58.7% (5) to 31.4% (95% CI: 20.5-42.3) in the present study, which means that it has decreased by almost half since the last study. In Latvia, a similar trend was observed and the prevalence from 2008 (35.6%) had halved by 2014 (17.1%) (17,21,22). The prevalence in the present study also correlates with prevalences reported in some of the neighboring countries. In a study performed in Poland in 2014 (20), 39.3% of red foxes were infected with E.

multilocularis, and in Estonia, the prevalence in the same animal species was approximately 30% in 2015 (17,24). The reasons for the decreased prevalence in Lithuania cannot be unambiguously determined, but in Latvia it was thought to be related to a decline in red fox populations in recent years (17).

Even though the number of examined red foxes was less (n=70) in our study than in the previous study by Bruzinskaite (n=269), the method for isolation of E. multilocularis (the sedimentation and counting technique) was the same. In Bruzinskaite´s study (5) it was not specified what was the distribution among the gender and age of the examined foxes. Previously the district of Kaunas had the highest prevalence of infected red foxes, but in our study no definite conclusions could be drawn on the prevalences among the districts or whether some regions were truly more heavily infected than the others. This was due to a lower sample size than that of the earlier study and the high number of foxes coming from unknown districts (37.5%; 95% CI: 13.8-61.2).

During present study, there were no significant differences seen between the prevalence and the age of the foxes (adults and youngsters) (p=0.857), which corresponds to the finding recorded by Hofer et al. (27) and Bagrade et al. (22). Both Kapel et al. (13) and Hofer et al. (27) also found no significant difference between the prevalence and the gender, which was also the case in our study with p=0.157.

However, as seen from our results, the females (39.1%; 95% CI: 19.2-59.1) seemed to be more frequently infected than the males (26.1%; 95% CI: 13.4-38.8). This could be explained by a finding in Kidawa´s study (3) where female foxes' diet was found to consist mostly of voles, while male foxes had a more diverse diet and were found to prey on other mammals and birds more frequently.

Due to the fact that 12 foxes had to be left out from the study in the beginning because it was believed that there was a problem with recognising the small parasite, the identification of E.

multilocularis could be improved by more advanced methods, such as the PCR. This kind of method

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31 would remove the possible human error related to visual identification as other Echinococcus spp. have similar structures. A good example of this is E. granulosus that also used to be endemic in some areas in Lithuania (12). Other variables that could have been included in our survey include other parasitic infections (other helminths and mange) and their relationship with E. multilocularis. However, adding these in the study would have required a lot of extra time for the identification and enumeration of the parasites and therefore they simply could not be included in the time frame given for this study.

The enumeration method used in our study showed that without dividing E. multilocularis into groups and not considering all these groups during the enumeration, we could have missed the infection altogether in 3 foxes. In the majority of the studies where the worm burdens were recorded it did not become clear how the enumeration of the parasite was done in detail, but in the study by Hofer et al.

(27) it was presumably done manually and in case the worm burdens were high (more than 100 worms per fox), the total number was counted from the count of one subsample. The juvenile stages (only the scolices) were also included in the calculations. The sensitivity of this method was not discussed.

Despite the fact that the abundance of E. multilocularis showed no significant correlation among the age or the gender of the foxes (p>0.05), the worm burdens in most of the studies correlated with the results of the present study. Hofer et al. (27) found that on average the mean abundance was more than twice as high in juveniles than in adults and this trend was also seen in our study with adults harboring 263 (1-1255, 95% CI: 31.06-494.54) worms on average and the juveniles 401 (3-2266, 95% CI: - 366.31-1168.88). This was especially true in the case of male juveniles which had the highest worm burdens of 921 worms per fox (228-2266, 95% CI: -1971.95-3814.62), whereas the male adults had a mean intensity of 195 (95% CI: -58.92-448.25). However, in the case of females, adults had higher worm burdens (365, 95% CI: -205.30-935.30) than the juveniles, who only had 14 (95% CI: -2.29- 30.29) worms on average. The 3 infected juvenile females in our study were hunted in July, September, and October, whereas 4 out of the 6 infected female adults were hunted between March and April. As the highest worm burdens are usually expected between 33–39 days post infection (6,13), the ingestion of infected intermediate host could be expected to have happened during the breeding season which was also found to be the time when the consumption of small mammals increased in red foxes, according to a study published in northeastern Poland in 2011 (3). The same study also found that the period between September and November was commonly the time when red foxes preyed mostly on either large mammals (ungulates) or consumed plant material. This finding could to some extent explain the differences in abundance of female adults and juveniles in our study.

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