The occurrence and molecular identification of Thelazia spp. in European bison (Bison bonasus) in the Bieszczady Mountains.

Infection with Thelazia nematodes results in eye disease in wild and domestic animals. The aim of the present study was to describe the occurrence of Thelazia nematodes in European bison, and to subject the isolated parasites to molecular identification and phylogenetical analysis. The eyeballs of 18 European bison from the Bieszczady Mountains, culled due to dysfunctional vision, were collected for study. The conjunctival sacs, tear ducts, corneal surface and nictitating membrane were rinsed with a saline solution. Any obtained nematodes were isolated under a stereoscopic microscope, and then identified as T. gulosa or T. skrjabini by molecular analysis of partial cox1 sequences. The prevalence of infection with Thelazia spp. was found to be 61%, with a 95% confidence interval (CI 95%) of 39-80%. Thelazia skrjabini was isolated from 56% (CI 95% 34-75%) of examined animals; T. gulosa was significantly less common (p = 0.038) with the prevalence of infection reaching 22% (CI 95% 9-45%). Three European bison were cross-infected with both T. gulosa and T. skrjabini. Phylogenetic analysis found the obtained sequences to be similar to those of Thelazia species from domestic ungulates in Europe. Infection intensity ranged from 1 to 16 nematodes per individual (median of three nematodes), and was significantly higher in females (6 nematodes) than in males (1 nematode; p = 0.019). A tendency for seasonal occurrence of nematodes in European bison was also observed. Our study provides further information regarding the patterns of Thelazia transmission in European bison in Poland.

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.

Molecular identification of Trypanosoma theileri complex in Eurasian moose Alces alces (L.).

Although the significance of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) as hosts and their role in the circulation of vector-borne pathogens in Europe is well described, the trypanosomes of moose (Alces alces) are poorly known. As heat sensitive ungulates, moose might be especially vulnerable to the effects of climate change and the associated rise in parasite pressure. Therefore, the aim of our study was to determine the prevalence of trypanosomes in moose in Poland, this being one of the largest populations in Central Europe since the 2001 hunting ban. Molecular analysis revealed the presence of Megatrypanum trypanosomes in almost half of the studied moose. As the population of moose in Central Europe has been recently growing, it is crucial to determine their role in the circulation of vector-borne pathogens in environment. This is the first study of the detection and molecular identification of Trypanosoma theileri complex in moose in central Europe.

Occurrence of chosen parasitic protozoa of roe deer (Capreolus capreolus) and red deer (Cervus elaphus) in Poland: a current review.

Both roe deer (Capreolus capreolus) and red deer (Cervus elaphus) are wild ruminants that are present in large numbers in Poland. Although both are very often infected with parasitic protozoa, the species composition of these parasites and their prevalence are relatively poorly known. The aim of the present work is to gather existing data on the occurrence and species diversity of parasitic protozoa of the genus Eimeria, Babesia, Theileria, Giardia, Cryptosporidium and family Sarcocystidae in red deer and roe deer in Poland and compare the findings with those from other European countries.

Rare case of presence of nematodes Dirofilaria repens in apleural cavity of a dog.

Dirofilariosis, caused by nematodes Dirofilaria repens, is a parasitosis of wild and domestic carnivores. Nematodes D. repens locate in the subcutaneous and intramuscular tissue. The aim of this article is to describe rare case of pleural infection of a dog with nematodes D. repens. 12-year-old golden retriever had been treated in the veterinary clinic due to emaciation, weakness and dehydration, observed for 2 weeks. Hematologic and biochemical tests revealed reduction in the number of leukocytes and erythrocytes, significant decrease of hemoglobin, hematocrit and albumins as well as increased ALAT, urea and creatinine levels. Parasitological examination of blood smear revealed the presence of multiple microfilariae. Increased echogenicity of renal parenchyma was visible during ultrasound examination. Necropsy of euthanized animal revealed adult nematodes D. repens, located in the pleural cavity. Additionally, enlarged kidneys with subcapsular petechiae and a single cyst in the renal cortex were observed. To our best knowledge, it is the first case of finding nematodes D. repens in a pleural cavity of a dog.

Gastrointestinal Helminths of a European Moose Population in Poland.

Parasitic infections have a negative impact on the fecundity and survival of wild ruminants, particularly moose; however, despite being more susceptible to parasitic diseases than other wild cervids, they remain poorly examined in this regard. Therefore, the aim of the present study was to identify gastrointestinal and liver helminth species of the moose population in central Europe, assess the factors contributing to infection intensities and examine their impact on moose health. Abomasum, small intestine, caecum and liver samples were collected from 46 moose in Poland and evaluated for helminth parasite fauna and histopathological changes. Additionally, 289 moose fecal samples were analyzed for the presence of eggs, oocysts and larvae of parasites. In total, 19 parasite taxa were identified. The most prevalent were Mazamastrongylus dagestanica and Ostertagia antipini, which are typical nematodes of moose, together with Spiculopteragia boehmi and O. leptospicularis, characteristic also of other cervids. Parasite species diversity and abomasal parasitic infection intensity were higher in adult moose than in yearlings and calves. The numbers of histopathological lesions depended on the intensity of parasitic infections, and were most severe in the livers of moose infected with Parafasciolopsis fasciolaemorpha. The analysis of fecal samples revealed several regional differences in the levels of parasite eggs, oocysts and larvae shedding. Our findings indicate an accumulation of parasite infections over time in moose, which may be related to high environmental parasite pressure, possibly connected with high moose density and the presence of wetlands; they also serve as the most comprehensive study of moose parasites in central Europe to date.

Biodiversity of lung helminths in terrestrial mammals from Eastern Europe.

This review provides the results of many years of original studies of the authors published in different journals and compilations taking into account an analysis of literature data. The studies on biological diversity of lung helminths of terrestrial mammals were performed by the authors in Armenia, Bulgaria and Poland. Literature data on European Russia were also taken into account. Sixteen animal species from the families Cervidae (six species), Bovidae (nine species) and Leporidae (one species) were investigated. In these animal taxons 27 helminth species from the families Dictyocaulidae (five species), Protostrongylidae (20 species), and Taeniidae (two species) have been found. Lung nematodes Muellerius capillaris and Protostrongylus hobmaieri, as well as zoonotic parasite E. granulosus were found to be common for all the countries studied. Also, relatively many species appeared common between several studied regions. Taking into account morphology, ecology and life cycles, we support a validity of genera Echinococcus and Alveococcus in the structure of the subfamily Echinococcinae of family Taeniidae.

First report of a newly-described lungworm, Dictyocaulus cervi (Nematoda: Trichostrongyloidea), in moose (Alces alces) in central Europe.

Lungworms from the genus Dictyocaulus are the causative agents of verminous pneumonia in domestic and wild ungulates. Recently, in 2017, a new species was isolated from red deer and described as Dictyocaulus cervi; however, little is known about its epidemiology and pathogenicity in other cervids. The aim of our study was to determine the extent of infection with Dictyocaulus nematodes in the moose population in Poland. Parasitological necropsies were performed in 18 moose and 249 faecal samples were analysed. A combination of multiplex PCR and analysis of the partial SSU, cox1 and cyt B regions revealed the presence of D. cervi infection in two of the necropsied moose. Histopathological examinations revealed changes, including multiple cross sections of larvae of nematodes in alveoli, massive pulmonary fibrosis, mononuclear cell infiltration and diffuse alveolar damage in the lungs of four animals. The lesions were more pronounced when adult Dictyocaulus nematodes were present in the bronchi and bronchioles. Some of the observed pathological changes could be attributed to co-infection by nematodes of the Protostrongylidae, whose larvae were found in all four animals with lung pathologies. In the faeces, Dictyocaulus sp. larvae only occurred together with Protostrongylidae larvae; in addition, higher numbers of Protostrongylidae larvae were excreted in the faeces of animals with dictyocaulosis. The present study is the first report of the presence of D. cervi in moose, and demonstrates the value of multiplex PCR in the identification of Dictyocaulus nematodes. Our findings indicate that co-infections with multiple species of lung nematodes in moose may be commonplace, and this should be considered as a factor aggravating the course of parasitosis.

The Nematodes Thelazia gulosa Railiet and Henry, 1910 and Thelazia skrjabini Erschov, 1928 as a Cause of Blindness in European Bison (Bison bonasus) in Poland.

PURPOSE: The nematodes of the genus Thelazia are the cause of eye diseases of wild and domestic ruminants throughout the world. The aim of the study was to describe clinical cases of thelasiosis in European bison (Bison bonasus) in Poland, and provide morphometrical features of Thelazia gulosa Railiet and Henry, 1910 and Thelazia skrjabini Erschov, 1928 regarded as potentially useful for species differentiation METHODS: The conjunctival sacs, tear ducts, the surface of the cornea and nicitating membrane collected from bison were rinsed with saline solution. Any nematodes isolated from the sediment were subjected to morphometric analysis. RESULTS: Thirteen of the 16 examined European bison were infected with Thelazia nematodes, belonging to the species T. gulosa and T. skrjabini. The intensity of infection ranged from one to six (mean intensity 5), and four to 29 (mean intensity 14) nematodes T. skrjabini and T. gulosa respectively. Congestion of conjunctival sac, keratitis and corneal opacity, corneal ulceration and perforation as well as purulent eyeball inflammation were observed in infected animals. CONCLUSIONS: Thelazia gulosa and T. skrjabini can be identified by morphometrical features. As thelasiosis might be a serious threat for protected population of European bison, further studies are needed of the epidemiology and pathology of this emerging parasitosis in Poland.

Usefulness of PCR-RFLP of 18S rRNA gene for rapid post-mortem diagnostics of highly pathogenic Eimeria spp. (Apicomplexa: Eimeriidae) of European bison, Bison bonasus L. with histopathological correlation.

Eimeria spp. infection was investigated in 10 free-roaming European bison aged three months to 26 years by anatomopathological, histopathological, coproscopic and PCR-RFLP examination. The coproscopic study identified Eimeria oocysts in the faeces of five bison. The most prevalent morphotypes were E. bovis, present in all positive samples, and E. zuernii, in all but one. Additionally, mixed infections consisting of E. bovis, E. zuernii, E. alabamensis, E. auburnensis, E. canadensis, E. cylindrica, E. ellipsoidalis and E. subspherica were diagnosed in two bison calves. Besides being the most prevalent form, E. bovis also demonstrated the highest OPG (2,750). The presence of oocysts in the faeces was associated with those of macrogamonts, microgamonts and oocysts in the epithelium of the large intestine. Intestinal coccidiosis associated with lymphoplasmacytic enteritis was observed in many bison, not only those with positive OPG. Four animals with negative coproscopy results demonstrated early-stage gametogony in the large intestine; one case presented no endogenous stages of coccidians in the histopathological sections of the intestine, nor oocysts in the faecal samples. A 530 bp product of E. bovis 18S rDNA (GenBank: MK951685) was obtained from both the colon wall and oocysts; this was subjected to PCR-RFLP analysis based on AluI and Hin1II (NlaIII) restriction enzymes. Both samples yielded a consistent seven-band pattern, four of which (270 bp, 40 bp, 180 bp and 84 bp) were expected, and the other three represented undigested fragments. The obtained digestion pattern is indicative of Eimeria spp. infection, and can serve as a first-step diagnostic approach in detection of infection. The result of computer-based virtual digestion of the PCR product suggests that double digestion with Mval (BstNI) and KpnI restriction enzymes may be used as a second-step tool to distinguish between E. bovis, E. zuernii and E. alabamensis, all of which are highly-pathogenic species.

Patterns of parasite eggs, oocysts and larvae shedding by moose in the Biebrza marshland (NE Poland).

The study analyses patterns of endoparasite eggs, oocysts and larvae shedding by moose from the relict population in the Biebrza marshland, NE Poland, which has grown to be one of the largest in Central Europe since the ban on hunting imposed in 2001. The analysis identified 10 species or groups of parasites among 230 faecal moose samples collected over 16 consequent months. The most prevalent were the eggs of Trichostrongylidae, Trichuris spp., Nematodirella alcidis, Parafasciolopsis fasciolaemorpha and the larvae of Elaphostrongylus sp. Four parasite species were more prevalent in males, indicating male-biased parasitism, and the studied moose population exhibited a female-skewed sex ratio. Nematodirella alcidis eggs and Protostrongylid larvae were more prevalent during winter, which indicated their resistance to harsh weather conditions. The prevalence of Eimeria alces and Aonchotheca sp. increased during the growing season, as did the number of eggs per gram of faeces (EPG) of P. fasciolaemorpha, possibly due to the availability of water sources. Higher mean monthly temperature was also found to have a positive effect on the excretion of Trichostrongylidae and Moniezia spp. eggs. In addition, the time of infection and the specificity of the parasite life cycle, being sensitive to certain climatic conditions, also appeared to have a strong influence on eggs, oocysts and larvae shedding in this non-harvested moose population.

The morphological and molecular identification of the tapeworm, Taenia lynciscapreoli, in intermediate and definitive hosts in Poland.

The tapeworm Taenia lynciscapreoli is a new species of the genus Taenia described in 2016, and which remains poorly understood. The aim of the present study is to extend current knowledge regarding its, morphology and genome. Biological material was analysed from three species of wild animals: Eurasian lynx (Lynx lynx), roe deer (Capreolus capreolus) and moose (Alces alces). Twenty-four adult tapeworms and four larvae were obtained from Eurasian lynx and roe deer respectively; none were detected in the studied moose. On the basis of morphometric (hooks measurements) and molecular analysis (partial 780 bp cox 1 gene sequences), the analysed tapeworm was identified as Taenia lynciscapreoli species. The phylogenetic analysis of the obtained sequences identified two haplotypes. The obtained findings can be used to supplement the species description. To our knowledge this is the first morphological and molecular identification of T. lynciscapreoli in roe deer, intermediate host, in Poland.

Occurrence of tapeworms Moniezia benedeni (Moniez, 1879) in European bison Bison bonasus L. in Bialowieza Primeval Forest

Tapeworms Moniezia benedeni are cosmopolitan parasites of wild and domestic ruminants. The aim of this study was to examine the prevalence of those tapeworms in European bison in Bialowieza Primeval Forest. Parasitological necropsy of small intestine of 26 bison, aged from 3 months to 26 years was performed in years 2007­2011. Tapeworms M. benedeni were isolated from 11 examined animals and the prevalence of infection was estimated on 42.3%. Intensity of infection was ranging from 2 to 25 tapeworms and the mean intensity was estimated on 5.8 specimens. Prevalence in the group of calves reached 50% and in adult bison ­ 33.3%. Intensity of infection in calves was estimated on 2 to 5 tapeworms while in adult animals it was ranging from 1 to 2 parasites. High prevalence of tapeworms M. benedeni in European bison might result from poor condition or weak immunity of eliminated animals.

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.

A morphological and molecular comparison of Eimeria bovis-like oocysts (Apicomplexa: Eimeriidae) from European bison, Bison bonasus L., and cattle, Bos taurus L., and the development of two multiplex PCR assays for their identification.

The European bison, Bison bonasus is the largest terrestrial mammal in Europe; it is also on the red list, being recognized as vulnerable to extinction by the International Union for Conservation of Nature. The species suffers from low genetic variability, rendering it vulnerable to various environmental and biological threats. This study presents the first molecular confirmation of Eimeria bovis infection in European bison, and details a 1708 bp nucleotide sequence of the 18S rRNA gene in European bison-derived E. bovis (GenBank: MK691697). It also describes two multiplex PCR assays based on 18S rRNA gene for identifying Eimeria bovis oocysts and developmental stages in European bison and cattle. These yielded DNA banding patterns common for those of Eimeria spp. (250 bp for the first assay and 305 bp for the second assay) and species-specific E. bovis DNA in positive samples (344 bp and 586 bp, respectively). Both multiplex PCRs yielded bands characteristic of Eimeria spp. and E. bovis in samples containing DNA of oocysts from both bison and cattle. Moreover, convergent results were obtained for the DNA of the wall of colon in both assays, indicating the presence of developmental stages of Eimeria spp. other than E. bovis. Despite displaying the same sporulation time (four days), and similar general morphological features, the E. bovis oocysts derived from European bison were significantly narrower than those obtained from cattle (t = -6.19, p < 0.001), with a significantly higher shape index (length/width ratio) (t = 3.94, p <  0.001). The result provides further evidence for infection of European bison with a highly-pathogenic bovine protozoan, E. bovis.

First Description of Histopathological Lesions associated with a Fatal Infection of Moose (Alces Alces) with the Liver Fluke Parafasciolopsis Fasciolaemorpha Ejsmont, 1932.

INTRODUCTION: Parafasciolopsis fasciolaemorpha is a liver fluke typically parasitising moose in Central and Eastern Europe. The aim of our studies was to describe a case of fatal moose parafasciolopsosis, with special emphasis on the histopathological changes caused in the liver tissue by around 10,000 flukes. MATERIAL AND METHODS: A male moose, found dead in Polesie National Park, eastern Poland, was subjected to parasitological necropsy. Macroscopic and histopathological examination of the liver was performed. RESULTS: Over 10,000 flukes identified as P. fasciolaemorpha were isolated from the liver parenchyma. Histopathological examination of the liver revealed the presence of multiple cavities, which were filled with flukes and cellular detritus and encysted with a layered capsule of connective tissue. Extensive liver fibrosis with signs of incomplete septal cirrhosis was also observed. CONCLUSION: Parafasciolopsosis with accompanying diarrhoea was the most probable reason for the moose's death. However, it is possible that most moose are able to survive extremely intensive P. fasciolaemorpha infection by formation of extensive fibrosis, which isolates flukes from the liver parenchyma and therefore retards the failure of the organ. To the best of our knowledge, this is the first histopathological description of changes in the liver of a moose infected with P. fasciolaemorpha.

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.

Pathological lesions in the lungs of red deer Cervus elaphus (L.) induced by a newly-described Dictyocaulus cervi (Nematoda: Trichostrongyloidea).

The large lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in various ungulate hosts, including red deer. Recently, the red deer-derived lungworm D. cervi was described and separated from D. eckerti. Little is known of the transmission patterns, epidemiology, geographical distribution and pathogenicity of D. cervi. Histological examinations were performed on 22 formalin-fixed lung tissue samples of hunted red deer. Exclusively, D. cervi adults were derived from 15 red deer and confirmed molecularly (GenBank accession: MH183394). Dictyocaulus cervi infection was associated with various degrees of lung pathology, including interstitial pneumonia, bronchitis and bronchiolitis with an influx of eosinophils, lymphocytes, plasma cells and macrophages; massive hyperplasia of lymphoid follicles within bronchiolar tissue, and hyperplasia of the bronchial and bronchiolar epithelium. Furthermore, emphysema, atelectasis and lung tissue congestion were noted. Interestingly, interstitial and subpleural fibrosis was seen in adult Dictyocaulus-negative samples, suggesting either a prepatent phase of Dictyocaulus infection or infection/coinfection with protostrongylid nematodes.

Microfilariae Onchocerca alcis Bain et Rehbinder, 1986 ­ a new parasite of moose Alces alces (L.) in Poland

Onchocerca alcis Bain et Rehbinder, 1986 belongs to the subfamily Onchocercinae. Mature nematodes of O. alcis are located on the surface of hindlimb tendons. The aim of this article was to describe the occurrence of microfilariae of O. alcis in the skin of moose from Kampinos Forest. This is the first report of O. alcis in moose from Poland and the third finding of this rare species in the world.

Gastrointestinal parasites of captive European bison Bison bonasus (L.) with a sign of reduced efficacy of Haemonchus contortus to fenbendazole.

The history of European bison Bison bonasus Linnaeus, 1758 has been stormy since its extinction in the wild after the First World War. Due to the fact that the species was restored from just 12 founders, further expansion has suffered from low genetic variability, rendering the bison vulnerable to various pathogens due to inbreeding depression. Parasites are recognised as a key biological threat to bison population. Thus, parasitological examination including monitoring of the level of anthelmintic resistance in a herd should be a routine procedure involved in management and protection of European bison. This study was conducted in a group of 27 bison kept in a European bison breeding centre in Sweden. In April 2015, a faecal egg count reduction test (FECRT) was performed in animals with ≥ 100 gastrointestinal nematode (GIN) eggs per gram faeces, to determine effectiveness of fenbendazole (FBZ) treatment. Additionally, the third stage larvae were cultured for molecular examination by a conventional PCR as well as by real-time quantitative PCR (q-PCR) for detection of the blood-sucking nematode Haemonchus contortus. Faecal sampling was conducted 1 day before and 8 days after deworming each animal. Anthelmintic treatment turned to be entirely efficient toward intestinal nematodes of genera Nematodirus and Trichuris, whereas shedding of strongylid eggs from the subfamily Ostertagiinae was reduced from 81 to 30%. Polymerase chain reaction (PCR) on cultured third-stage larvae (L3) before treatment was positive for H. contortus, Ostertagia ostertagi and Cooperia oncophora, whereas post-treatment examination revealed exclusively the DNA of H. contortus. Thus, only H. contortus was involved in post-treatment faecal egg count (FEC). FECRT showed that the reduction in strongylid FEC to FBZ in the examined bison herd was 87% (95%-confidence intervals [95% CI] = 76-93), suggesting reduced efficacy of FBZ to strongylid GIN including mainly H. contortus.