Distribution of large lungworms (Nematoda: Dictyocaulidae) in free-roaming populations of red deer Cervus elaphus (L.) with the description of Dictyocaulus skrjabini n. sp.

Lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in domestic and wild ungulates. This study investigates the distribution, morphology and genetic diversity of D. cervi and a new lungworm species, Dictyocaulus skrjabini n. sp. infecting red deer Cervus elaphus, fallow deer Dama dama and moose Alces alces in Poland and Sweden. The study was conducted on 167 red deer from Poland and on the DNA of lungworms derived from 7 fallow deer, 4 red deer and 2 moose collected in Sweden. The prevalence of D. cervi and D. skrjabini n. sp. in dissected red deer in Poland was 31.1% and 7.2%, respectively. Moreover, D. skrjabini n. sp. was confirmed molecularly in 7 isolates of fallow deer lungworms and 1 isolate of red deer lungworms from Sweden. Dictyocaulus skrjabini n. sp. was established based on combination of their distinct molecular and morphological features; these included the length of cephalic vesicle, buccal capsule (BC), buccal capsule wall (BCW), distance from anterior extremity to the nerve ring, the width of head, oesophagus, cephalic vesicle, BC and BCW, as well as the dimensions of reproductive organs of male and female. Additionally, molecular analyses revealed 0.9% nucleotide sequence divergence for 1,605 bp SSU rDNA, and 16.5­17.3% nucleotide sequence divergence for 642 bp mitochondrial cytB between D. skrjabini n. sp. and D. cervi, respectively, and 18.7­19% between D. skrjabini n. sp. and D. eckerti, which translates into 18.2­18.7% amino acid sequence divergence between D. skrjabini n. sp. and both lungworms.

The Occurrence of Fascioloides Magna (Bassi, 1875) in the Wild Cervid Population in the Lower Silesian Wilderness - Epidemiological and Pathological Aspects.

Introduction: Fascioloides magna is a parasite of high veterinary importance due to its pathogenicity for wild and domestic ruminants. The aim of our study was to describe the presence of trematode infection in the red deer population in the Lower Silesian Wilderness, one of the established fascioloidosis foci in Central Europe, and to assess the overall prevalence of F. magna in the studied area. In order to achieve this, a coprological study of different cervid species was performed. Material and Methods: The livers of 99 red deer were collected over three years and examined for the presence of trematodes. Prevalence and infection intensity was estimated and a histopathological analysis was performed. In addition, 172 faecal samples from red deer, fallow deer and roe deer were examined. Results: By year, Fascioloides magna was isolated from the livers of 2/30 (6.7%), 9/34 (26.5%) and 10/35 (28.6%) red deer. Severe hepatic lesions, including massive tissue damage, extensive fibrosis, and cirrhotic changes in the liver parenchyma were observed. Faecal examination revealed the presence of F. magna eggs, with a prevalence of approximately 40%, 50% and 53% in roe deer, fallow deer and red deer, respectively. Conclusion: The eggs of F. magna may be commonly excreted in the faeces of roe deer, as well as those of red deer and fallow deer. The presence of F. magna throughout the cervid population in the Lower Silesian Wilderness favours the risk of the trematode's transmission to livestock or farmed deer.

A molecular survey of spotted fever group rickettsiae in introduced raccoons (Procyon lotor).

BACKGROUND: The raccoon Procyon lotor (Linnaeus, 1758) (Carnivora; Procyonidae) is one of the most important and most intensively studied invasive mammal species in Europe. Within the last 30 years the raccoon has spread at an increasing rate, resulting in the establishment of local populations in various regions of Europe. In these newly colonised areas, gaps in knowledge of the raccoon's biology concern not only most aspects of its ecology in a broad sense, but also its pathogens and parasites. Most micropathogens recorded hitherto in the raccoons that have colonised Europe have documented epizootic and zoonotic potential. Thus, it is considered especially important to investigate the role played by the raccoon in the spread of pathogens through both animal-animal and animal-human pathways. METHODS: Tissue samples of raccoons from Poland and Germany were examined in this study. In total, 384 tissue samples from 220 raccoons (170 spleen samples, 82 liver biopsies, 132 ear biopsies) were examined using molecular methods. The presence of Rickettsia spp. DNA was screened through amplification of a fragment of the gltA gene. Samples that were PCR positive for gltA were tested for other rickettsial genes, ompB and a 17-kDa antigen. For taxonomic purposes, the obtained sequences were compared with corresponding sequences deposited in GenBank using the Basic Local Alignment Search Tool, and phylogenetic analyses were conducted using Bayesian inference implemented in MrBayes software. RESULTS: Rickettsia DNA was confirmed only in skin biopsies; no isolates from the spleen or liver were positive for Rickettsia DNA. With the exception of one sample from Germany, which was positive for Rickettsia helvetica DNA, all the samples positive for Rickettsia DNA derived from the Polish population of raccoons. DNA of Rickettsia spp. was detected in 25 samples, i.e. 11.4% of the tested raccoons, and R. helvetica was confirmed in 52% of the positive samples. Additionally, single cases of Rickettsia monacensis, Rickettsia raoultii, and Candidatus Rickettsia kotlanii-like were found, and in 32% of all the positive samples similarity was shown to different Rickettsia endosymbionts. Out of the samples that tested positive for gltA, amplicons of ompB and 17 kDa were successfully sequenced from 14 and three samples, respectively. CONCLUSIONS: To the best of our knowledge, this study provides, for the first time, evidence of the occurrence of Rickettsia pathogens and endosymbionts in the European population of raccoons. Further, broader research on different species of wild vertebrates, and ticks, as potential vectors and hosts for tick-borne pathogens, in natural as well as in peri-urban environments, is therefore required.

Copro-Molecular Identification of Tapeworms in Introduced Invasive Carnivores in Poland.

The raccoon (Procyon lotor) and the raccoon dog (Nyctereutes procyonoides) were introduced to Europe and, in the past decades, their populations have increased and adapted to synanthropic environments across Europe. In view of their possible further spread in Europe, the invasive species should be examined as potential reservoirs for helminths, including tapeworms. This study aims to investigate the prevalence and diversity of tapeworms in introduced wild carnivores in Poland by identifying cestode species based on copro-DNA analysis. A total of 214 individual fecal samples from non-native invasive carnivores, i.e., raccoons and raccoon dogs, and additionally 47 samples from native carnivores, i.e., European badgers (Meles meles), were analyzed for the presence of cestodes. PCR analysis of fecal samples targeting a fragment of mitochondrial (mt) 12S rRNA gene revealed the presence of cestode DNA in 19 of 103 (18.4%) raccoons, in 13 of 111 (11.7%) raccoon dogs and in 23 of 47 (48.9%) badgers. Sequence analysis demonstrated the presence of Mesocestoides litteratus in raccoons and raccoon dogs, while Mesocestoides lineatus was identified only in two samples derived from raccoon dogs. Moreover, in this study, Atriotaenia incisa was for the first time molecularly characterized by using fragments of mt 12S rRNA gene, and the DNA of this cestode species was detected in the fecal samples of all the examined host species.

Zoonotic Genotypes of Enterocytozoon bieneusi in Wild Living Invasive and Native Carnivores in Poland.

Wild carnivores, both introduced and native species, are able to adapt well to peri-urban environments, facilitating cross-species pathogen transmission with domestic animals, and potentially humans. The role of wild living reservoir hosts cannot be ignored because of their known carriage of E. bieneusi zoonotic genotypes. In the past decades, populations of wild living carnivores, i.e., native, such as red foxes, and invasive, such as raccoon dogs and raccoons, have increased and adapted to synanthropic environments across Europe, including Poland. The knowledge concerning E. bieneusi genotype identification and distribution in wild carnivores is limited worldwide. A total of 322 individual fecal samples from six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine and beech martens, were collected and then analysed for the presence of E. bieneusi using the nested PCR method. Overall prevalence of the pathogen was estimated to be as high as 27.3%. The infection rates for E. bieneusi varied between the carnivore species, from 13.7% in beech martens to 40.4% in raccoon dogs. Based on sequence analysis of the ITS region of the rRNA gene marker, we detected five known genotypes of E. bieneusi in examined animals. In the invasive species, E. bieneusi NCF2 and D genotypes have been identified, whereas in the native ones, E. bieneusi NCF2, D, C, EbCar2 and Type IV genotypes were identified. All E. bieneusi genotypes recorded in this survey clustered in Group 1, showing their zoonotic potential. Our results provide the first description of the occurrence and genotypes of the microsporidian E. bieneusi in wild living population of raccoon dogs in Europe. Our findings are important for the study of pathogen epidemiology and emphasize the fact that the invasive and the native wild living carnivores, both widely distributed, should be considered more seriously as significant sources of zoonotic pathogens hazardous to domestic and farmed animals and humans.

Quantifying the functional disparity in pigment spot-background egg colour ICP-OES-based eggshell ionome at two extremes of avian embryonic development.

It is known that a developing avian embryo resorbs micronutrients (calcium and other chemical elements) from the inner layer of the eggshell, inducing thinning and overall changes in the shell's chemical composition. However, an aspect yet to be explored relates to the local changes in the multi-elemental composition (ionome) of the pigment spot and adjacent background colour regions of eggshells resulting from avian embryogenesis (with respect to two extremes of embryonic growth: the maternal level at the moment of egg laying, and after the completion of embryonic growth). To address this problem, we used inductively-coupled plasma optical emission spectrometry (ICP-OES) to establish the elemental profiles of microsamples from the cryptic eggs of Capercaillie Tetrao urogallus and Black Grouse Tetrao tetrix, representing the background colour and pigment spot regions of the shell. We then related these to the developmental stage of the eggs (non-embryonated eggs vs. post-hatched eggshells) and their origin (wild vs. captive hens). Our results show an apparent local disparity between the pigment spot and background colour regions in the distribution of chemical elements: most elements tended to be at higher levels in the speckled regions of the shell, these differences becoming less pronounced in post-hatched eggshells. The trends of changes following embryonic eggshell etching between the pigment spot and background colour shell regions were conflicting and varied between the two species. We hypothesized that one potential working explanation for these interspecific differences could be based on the variable composition of elements (mostly of Ca and Mg), which are the result of the varying thickness of the individual shell layers, especially as the relative difference in shell thickness in the pigment spots and background colour regions was less in Black Grouse eggs. Overall, this investigation strongly suggests that egg maculation plays a functional role in the physiological deactivation of trace elements by incorporating them into the less calcified external shell layer but without participating in micronutrient resorption. Our major critical conclusion is that all research involving the chemical analysis of eggshells requires standardized eggshell sampling procedures in order to unify their colouration and embryonic status.

Ashworthius sidemi in cattle and wild ruminants in Poland - the current state of play.

Ashworthius sidemi, a blood-sucking abomasal nematode, has been identified in various wild ruminants, including deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama) and moose (Alces alces). Although it has been observed throughout Poland, most sightings have been in the eastern part of the country. However, more recently, A. sidemi has been confirmed in the Ruszów Forest District (Lower Silesian Wilderness). It is now possible to test the faeces of cattle for the DNA of the third-stage infectious larvae (L3) of A. sidemi. The present paper describes such a molecular study of 120 faecal samples collected from cattle grazed in the Ruszów Forest District and Biebrza Marshland, where A. sidemi had previously been detected in wildlife. In this study, no A. sidemi DNA was identified in any of the examined samples.

Supporting dataset and methods for egg sizes, eggshell thicknesses and metal concentrations measured in the shells and contents of eggs of Capercaillies Tetrao urogallus.

The dataset presented in this data paper supports "Eggshell resorption, and embryonic mobilization and accumulation of calcium and metals in eggs of wild and captive Capercaillies Tetrao urogallus" (Orlowski et al., 2019) [1]. Here we present the supplementary data on the following: (1) egg sizes, regional eggshell thicknesses (at the equator, sharp pole and blunt pole) as well as the concentrations of two major micronutrients (Ca and Mg) and eight trace elements (Cr, Cu, Mn, Fe, Co, Cd, Pb and Zn), measured in the shells and contents of eggs of captive-bred and wild Capercaillies. (2) How the proportions of elements sequestered into eggshells become depleted during embryogenesis expressed as the %change of concentrations of various elements measured in the shells and contents of eggs at different stages of embryonic advancement (early dead embryos, late dead embryos and hatched eggs). (3) The relationships between the age of dead embryos and three regional eggshell thicknesses and concentrations of different elements measured in the shells and contents of these eggs.

Eggshell resorption, and embryonic mobilization and accumulation of calcium and metals in eggs of wild and captive Capercaillies Tetrao urogallus.

Although it is fairly well known that eggshells are the major source of Ca and other micronutrients for developing avian embryos, potential anatomical and/or ecological correlates explaining the variation in embryo-induced depletion of micronutrients from eggshells remain to be addressed. Using unhatched eggs at various developmental stages and post-hatched eggshells of wild and captive-bred birds of a large precocial species, the Capercaillie Tetrao urogallus, we explored two major questions. (i) Do egg size, regional eggshell thickness as well as levels of two major micronutrients (Ca and Mg) and eight trace elements (Cr, Cu, Mn, Fe, Co, Cd, Pb and Zn) measured in egg shells and contents vary with embryonic age within and between wild and captive-bred bird eggs? (ii) How the proportions of eggshell elements become depleted during the entire process of embryogenesis? The eggshells of wild Capercaillies were thinner at the equator and the sharp pole, and contained more Mg and Ca, while in their contents there was more Cu, Cd and Pb compared with captive birds. All ten metal concentrations measured in the egg contents increased (from 15% to 285% more) with the age of embryos. The concentrations of six elements measured in eggshells (Cu, Mn, Fe, Co, Cd and Zn) decreased consistently (from 15 to 70% less) during embryogenesis. This suggests the presence of these elements in the innermost decalcified shell layer and their further mobilization into the embryos/egg contents. Our results strongly imply that most trace elements present in the egg contents/developing embryos are mobilized from the eggshells. We highlight the fact that knowledge of the magnitude of embryogenesis-related functional changes in the physical (such as shell thickness) and chemical traits of the eggs and eggshells of oviparous vertebrates is fundamental for drawing correct inferences regarding the response of an individual to variable environmental conditions.

A further increase in the prevalence and intensity of infection with Ashworthius sidemi nematodes in red deer in the Lower Silesian Wilderness

The nematode Ashworthius sidemi is a typical parasite of Asiatic deer, particularly sika deer (Cervus nippon). It was probably first carried into Poland by migrating red deer. The aim of the present study was to determine the prevalence and intensity of A. sidemi infection in roe deer and red deer in the Lower Silesian Wilderness in the winter season 2015/2016. Parasitological necropsy of 34 abomasa of red deer from Forest District Ruszów was performed. The prevalence of A. sidemi infection was found to be 91.1%, and the intensity to range from 10 to 2680 nematodes, with a mean intensity of 394 parasites. This prevalence of ashworthiosis and maximum intensity of infection were respectively 17.2% and 38% higher than observed in the previous season (2013/2014). The mean intensity was also slightly higher than reported previously.

The occurrence of Anaplasmataceae in European populations of invasive carnivores.

The raccoon (Procyon lotor) and the raccoon dog (Nyctereutes procyonoides) belong to a group of the invasive species. The introduced species as potential reservoirs for vector-borne pathogens have been the subject of recent research, though there are still no data with reference to the European population of the raccoon, and few studies concern only the raccoon dog. This study shows the occurrence of Anaplasmataceae representatives in raccoons and a sympatric population of the raccoon dogs obtained from the area of Poland and Germany. During the study, the occurrence of Anaplasma phagocytophilum ecotype I in the introduced raccoon in northwestern Poland was revealed. Additionally, Candidatus Neoehrlichia sp. (FU98) was identified for the first time in the raccoon dog in Central Europe and thereby the raccoon dog is a new host for this pathogen.

Expansion of Ashworthius sidemi in red deer and roe deer from the Lower Silesian Wilderness and its impact on infection with other gastrointestinal nematodes.

Nematodes Ashworthius sidemi are typical parasites of Asiatic cervids, mostly sika deer (Cervus nippon). Migrating red deer have probably introduced this parasite to the area of Poland from neighbouring countries. The aim of the study was to determine prevalence and intensity of infection of red deer and roe deer from the Lower Silesian Wilderness with A. sidemi after one year from previous studies and to examine the influence of these parasites on other species of gastrointestinal nematodes. 31 abomasa of red deer and 3 abomasa of roe deer from Forest Dictrict of Ruszów have been examined on the presence of parasites. In comparison with previous studies, red deer showed significant, over triple increase of prevalence of A. sidemi and over fivefold increase of mean intensity of infection. Maximum intensity of infection has increased over 24 times. These results indicate on dynamic spread of this new aswortiosis foci. Apart from previously registered parasite species, four new nematodes have been found in examined red deer and roe deer during this year studies. Red deer have been infected with Spiculopteragia assymmetrica, typical for fallow deer and Haemonchus placei - typical for bovine, whereas roe deer have been infected with Trichostrongylus axei and Trichostrongylus capricola, parasites of sheep, goats and bovine. Prevalence of red deer infection with nematodes S. boehmi and Ostertagia leptospicularis was lower than two years ago while prevalence of infection with S. mathevossiani and O. kolchida was higher. Intensity of infection with these nematodes has increased.

Conservation genetics of the capercaillie in Poland - Delineation of conservation units.

The capercaillie (Tetrao urogallus) is one of Poland's most endangered bird species, with an estimated population of 380-500 individuals in four isolated areas. To study these natural populations in Poland further, more than 900 non-invasive genetic samples were collected, along with samples from 59 birds representing large, continuous populations in Sweden and Russia; and from two centres in Poland breeding capercaillie. Microsatellite polymorphism at nine loci was then analysed to estimate within-population genetic diversity and genetic differentiation among populations. The results confirmed that isolation of populations and recent decreases in their sizes have reduced genetic diversity among capercaillie in Poland, with all the country's natural populations found to be experiencing the genetic after-effects of demographic bottlenecks. The results of analyses of genetic differentiation and structure further suggest the presence of a 'lowland' cluster (encompassing birds of the Augustowska and Solska Primaeval Forests in Poland, and of Sweden and Russia), and a Carpathian cluster. Capercaillie from Sweden and Russia are also found to differ markedly. The Polish lowland populations seem more closely related to birds from Scandinavia. Our genetic analysis also indicates that the stocks at breeding centres are of a high genetic diversity effectively reflecting the origins of founder individuals, though identification of ancestry requires further study in the case of some birds. Overall, the results sustain the conclusion that the Polish populations of capercaillie from the Carpathians and the lowlands should be treated as independent Management Units (MUs). This is to say that the breeding lines associated with these two sources should be maintained separately at breeding centres. The high level of genetic differentiation of birds from the Solska Primaeval Forest suggests that this population should also be assigned the status of independent MU.

Levels of metals in kidney, liver and muscle tissue and their relation to the occurrence of parasites in the red fox in the Lower Silesian Forest in Europe.

Together with the occurrence of parasites, increased concentrations of xenobiotics, to which scavengers are greatly exposed, may significantly influence the physiology of red foxes. It is also suspected that these two factors interact. The accumulation of various metals (Ca, Cd, Cu, Fe, Hg, K, Mg, Ni, Pb, Zn) in kidney, liver and muscle tissue was investigated, as well as the occurrence of parasites, and the potential link to the presence of metals. Generally speaking, neither sex nor age influenced these concentrations. K, Mg and Fe were found in the highest concentrations and Hg was found in the lowest. Various relationships between the concentrations of metals were observed in the tissues. 34% of the specimens studied were hosts to parasites. No clear, significant connection between the concentrations and the occurrence of parasites was noted, but the discernible trend confirmed by the logistic regression, needs further study.

Factors affecting the level of damage by wild boar in farmland in north-eastern Poland.

BACKGROUND: There was an unchecked increase in wild boar population and in the damaged crops in Poland in 2010, and the amount of compensation reached €12.4 million. The objective of the present study was to establish the factors affecting boar-related damage and to verify the effectiveness of protective strips used again wild boar pressure upon fields. RESULTS: Damage was positively correlated with the wild boar population density, the farmland-forest ecotone index, the proportion of deciduous stands, the Simpson index for dominant tree species and the age class of forest stands. The relationship between level of damage and feeding strips was not significant. Stepwise regression explains 55.6% of the variability of a given feature affected by the wild boar density, the forest-field ecotone index and the Simpson index for the age structure of the forest. During 5 years, the compensation for damage caused by boar amounted to €34.2 million, whereas the revenue from carcasses sold amounted to €9.5 million. CONCLUSIONS: Protective strips do not reduce the damage to crops, and so the offer of high-protein food on these strips, which increases reproduction rates among wild boar, should be discontinued. Reduction in the level of damage can only be achieved by a radical reduction in numbers of wild boar.