Endoparasite loads and the efficacy of conventional anthelmintics against gastrointestinal nematodes in captive European bison.

Although little information exists on the efficacy of deworming in wild ruminants, gastrointestinal nematodes have been found to demonstrate increasing drug resistance. The spread of drug-resistant strains may be increased by transmission among livestock and susceptible wildlife species, thus posing a potential threat to endangered species, such as the European bison. The aim of the study was twofold: to identify the parasite loads in captive European bison with the use of coprological techniques, and to test the influence of other nearby ungulates on the richness of bison parasitofauna. Additionally, the efficacy of deworming procedures against gastrointestinal nematodes in bison was evaluated. The survey was based on a coprological investigation of 285 fecal samples from 156 European bison in 15 enclosures. The parasitofauna of the captive European bison was consistent with those of free-ranging populations. The highest prevalence was noted for Eimeria spp. oocysts (60.7%), strongyle eggs (50.9%), Fasciola hepatica eggs (13.1%), Dictyocaulus viviparus larvae (12.3%) and Trichuris sp. Eggs (9.47%). Moreover, the close proximity of other ungulate species resulted in a higher diversity of parasite species. In all cases, deworming with albendazole, fenbendazole and ivermectin proved to be ineffective against strongylids and Trichuris sp. The results of fecal egg count reduction test (FECRT) ranged from 37.2 to 99.6% (95% CI <90%) for albendazole; values >95% (95% CI = 41-100) were noted for fenbendazole, and FECRT ranged from 63.2 to 97.5 (95% CI = 0-99) for ivermectin. As the results of anthelmintic treatment are unsatisfactory, it seems justified to continue study in this area. Our study is the first large-scale attempt to evaluate the efficacy of anthelminthics in captive European bison. The potential sharing of parasite species between bison and other ungulates should also be further investigated from the perspective of minimizing the risk of the spread of drug-resistant parasite strains.

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.

Large lungworms (Nematoda: Dictyocaulidae) recovered from the European bison may represent a new nematode subspecies.

Although the Dictyocaulus lungworm, the agent of dictyocaulosis, is one of parasitological threats to European bison, its systematic position remains unclear. The aim of the present study was to evaluate the morphological features of the lungworm and the pathological lesions it induces, and to analyse mitochondrial (mt) genetic markers for systematic and molecular epidemiological studies. The morphological findings indicate that Dictyocaulus lungworms of European bison can be distinguished from those of cattle on the basis of differences in buccal capsule wall length, total body length, and spicules length in males, all of which were significantly longer in those of European bison. Nucleotide diversity calculated from pairwise sequence alignments of partial cytochrome c oxidase subunit 1 (cox1), cytochrome B (cytB) and NADH dehydrogenase subunit 5 (nad5) of specimens from cattle and European bison varied from 1.7% for nad5, 2.1% for cytB, to 3.7% for cox1 gene. Thus, among the lungworms of European bison and cattle, nad5 and cytB were the most conserved proteins, whereas cox1 was the most diverse. The mt cytB marker gene may be a suitable candidate for distinguishing between the two genotypes, as nad5 demonstrated the greatest within-genus sequence variation. The lung tissue of infected European bison manifests signs of verminous pneumonia characterized by interstitial pneumonia, bronchitis and bronchiolitis. Therefore, it appears that European bison and cattle are infected with slightly diverged, morphologically-different, genotypes of D. viviparus, indicating they belong to two separate worm populations. We propose, therefore, that the lungworm of European bison should be classified as D. viviparus subsp. bisontis.

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.

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.

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.

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.

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.

An assessment of the use of cox1 and cox3 mitochondrial genetic markers for the identification of Dictyocaulus spp. (Nematoda: Trichostrongyloidea) in wild ruminants.

Lungworms of the genus Dictyocaulus Railliet and Henry, 1907 (Nematoda: Trichostrongyloidea) are the causative agents of parasitic bronchitis (dictyocaulosis, husk) of various ungulate hosts, including domestic and wild ruminants. Correct diagnosis of lungworm species and a better understanding of the transmission patterns of Dictyocaulus spp. are crucial in minimising the risk of its cross transmission between wildlife and livestock, and for the control of dictyocaulosis. The study was conducted on large lungworms collected from European bison, roe deer and red deer. The study resulted in 14 sequences of the partial cox1 region of Dictyocaulus spp. and 10 novel DNA sequences of partial cox3 region, including the first available mt cox3 sequence, of the roe deer lungworm (D. capreolus). The European bison was infected with bison genotype of D. viviparus, whereas red deer and roe deer were infected with D. cervi and D. capreolus respectively. The current study revealed that the cox3 nucleotide sequences of D. capreolus and D. viviparus were 100% homologous to each other. Our findings indicate that the mt cox3 gene does not serve as an efficient mt marker for systematic, population genetic or molecular epidemiological studies of Dictyocaulus lungworms.

Increased Parasitic Load in Captive-Released European Bison (Bison bonasus) has Important Implications for Reintroduction Programs.

Captive-bred animals, widely used in reintroduction programmes, are often immunologically naïve and more susceptible to pathogens. We analysed infection of invasive blood-sucking nematode Ashworthius sidemi in captive-bred European bison (Bison bonasus) released to the wild in the Bialowieza Forest (Poland). Mean A. sidemi infection intensity of released bison (29,137 nematodes) was over threefold higher than in wild bison (8756). It indicates a rapid acquisition and increase in the infection intensity in previously dewormed bison released from captivity. Thus, reintroduction programmes should consider the impact of pathogens and involve controlled exposure of captive animals to specific parasites prior to release.

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.

Lymphocyte responses of rats vaccinated with cDNA encoding a phosphoglycerate kinase of Fasciola hepatica (FhPGK) and F. hepatica infection.

Lymphocyte responses in the blood, peritoneal fluid and both mesenteric and hepatic lymph nodes of cDNA-FhPGK/pCMV vaccinated and/or Fasciola hepatica infected rats of both sexes were investigated to provide an insight into the immune responses that develop in different body compartments. The immune response that developed in cDNA-FhPGK/pCMV vaccinated females contributed to partial protection against F. hepatica infection (54% reduction in fluke recovery), while more liver flukes were found in the livers and bile ducts of cDNA-FhPGK/pCMV vaccinated male rats than in unvaccinated animals (increase of 13%). Rat sex not only affected the ultimate effectiveness of vaccination but also lymphocyte responses following vaccination and/or infection. Different CD4+ and CD8+ T cell profiles were noted in peritoneal fluid and lymph nodes, but not in blood, during acute and chronic fasciolosis. Moreover, independent lymphocyte responses developed in distinct body compartments. Immune responses of rats were polarized towards Th2/Treg with lymphocytes isolated from male rats showing higher IL-4 and IL-10 production than females. Lymphocyte proliferative capacities in response to mitogen (PHA) or vaccine antigen (FhPGK) were impaired in both sexes with a considerably higher reduction observed for males and restored lymphocyte proliferative capacities reported for females vaccinated with cDNA-FhPGK/pCMV during chronic fasciolosis.

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.

The first report of Aelurostrongylus falciformis (Schlegel, 1933) (Nematoda, Metastrongyloidea) in badger (Meles meles) in Poland

Aelurostrongylus falciformis belongs to the superfamily of Metastrongyloidea. This nematode occurs in European badgers and locates in lungs, in respiratory tract. Numerous species of land snails are intermediate hosts of the parasite. In 2015, parasitological necropsy of 9 badgers, shot in the Forest District Gleboki Bród in Augustowska Primeval Forest, was performed. Two examined animals were infected with nematodes A. falciformis. In the lungs of each badger two specimens of nematodes were detected (male and female). In the following article, description, morphometrical data and figures are presented. This is the first report of A. falciformis infection in badgers in Poland.

Interrelationships of Dictyocaulus spp. in Wild Ruminants with Morphological Description of Dictyocaulus cervi n. sp. (Nematoda: Trichostrongyloidea) from Red Deer, Cervus elaphus.

Lungworms from the genus Dictyocaulus cause parasitic bronchitis (dictyocaulosis) characterized by coughing and severe lung pathology in both domestic and wild ruminants. In this study we investigated the interrelationships of Dictyocaulus spp. from European bison (Bison bonasus L.), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) by nucleotide sequence analysis spanning the 18S RNA gene (small subunit [SSU]) and internal transcribed spacer 2 (ITS2) regions of the ribosomal gene array as well as the mitochondrial cytochrome c oxidase subunit 1 (cox1). Molecular analyses of sequence data obtained partly with novel primers from between 10 and 50 specimens from each host were carried out. Bayesian inference analysis revealed that each host species was infected with different genotypes. Analysis of cox1 sequence data showed a diverse genetic background and high evolutionary potential of Dictyocaulus taxa. Data from lungworms of European bison revealed a distinct genotype of Dictyocaulus viviparus, whereas Dictyocaulus capreolus was only found in roe deer. In contrast, red deer were infected with a taxon with unique SSU, ITS2, and cox1 sequences. These results indicate the occurrence of a novel genotype from red deer, which differs significantly from the National Center for Biotechnology Information reference sequence of Dictyocaulus eckerti. The molecular evidence was consistent with a morphological study with description and imaging of Dictyocaulus cervi n. sp. recovered from red deer. Dictyocaulus cervi n. sp. can be distinguished from D. eckerti on the basis of the absence of cervical papillae, the occurrence of a single ring of 4 symmetrical submedian cephalic papillae, length of the tail in females, morphometry of the female reproductive system, and measurements of gubernacula in males. In conclusion, our findings further strengthen the idea that the genetic complexity and diversity among Dictyocaulus lungworms infecting wildlife ruminants is larger than previously believed and warrants further investigation.

A massive invasion of Parafasciolopsis fasciolaemorpha in elk (Alces alces) in Lublin Province, Poland.

UNLABELLED: Liver fluke Parafasciolopsis fasciolaemorpha is a parasite typical of elk (Alces alces). The present study describes the identification of a massive invasion of the liver fluke P. fasciolaemorpha in an elk in the Sobibór Landscape Park, Eastern Poland. In February 2016, samples of liver and faeces were collected from the elk during postmorte examination. A section of liver tissue and three grams of faeces were examined for the presence of flukes or fluke eggs by decantation. In total, 11,150 juvenile and mature flukes of P. fasciolaemorpha were found in the examined liver. Multiple cavities with distinct walls, filled with dark liquid, trematodes, fluke eggs and cellular detritus were seen in the cross section of the parenchyma. Additionally, some of the bile ducts were plugged with calcerous deposits. The three grams of examined faeces were found to contain 322 grey and golden-coloured eggs of P. fasciolaemorpha. The presence of such a massive infection of P. fasciolaemorpha in the examined elk has a significant impact on the health of the individual. We can assume P. fasciolaemorpha has a significant influence on the health and abundance of the entire elk population in Poland. KEY WORDS: Parafasciolopsis fasciolaemorpha, elk, Sobibór Landscape Park, Poland.

Coprology of a single Northern white-breasted hedgehog (Erinaceus roumanicus): first report of Isospora rastegaievae in Poland.

Coprological analysis of a sample from one free-living hedgehog was done with the use of a direct flotation method with additional incubation of fecal material. The study revealed three types of eggs and oocysts in the feces. The most commonly diagnosed were oocysts of Isospora rastegaievae (543/3g), while oocysts of Monocystis sp. (267/3g) and eggs of Aonchotheca/Eucoleus spp. (52/3g) were seen less often. This is the first report of coccidia I. rastegaievae (Apicomplexa: Eimeriida) and acephaline gregarine Monocystis sp. (Apicomplexa: Eugregarinida) infection in a hedgehog in Poland.

Immune responses in rats and sheep induced by a DNA vaccine containing the phosphoglycerate kinase gene of Fasciola hepatica and liver fluke infection.

Immune responses of rats and sheep following vaccination with cDNA encoding phosphoglycerate kinase of Fasciola hepatica (cDNA-FhPGK/pCMV) and F. hepatica infection were investigated in the present study. cDNA-FhPGK/pCMV vaccinated female Sprague-Dawley rats were better protected by vaccination than their male counterparts - 48% reduction in fluke burden for females and no protection for males when compared with appropriate infection control groups. Moreover, male rats developed marked leukocytosis during the study with higher neutrophil, eosinophil and monocyte responses than females. Additionally, dynamics of eosinophil and monocyte responses varied between sexes. Increased titres of anti-FhPGK IgG1 and IgG2a correlated with the protective effect of vaccination that was observed among female rats. In the case of male sheep, no differences in worm burdens and in the course of the immune response were observed following vaccination. Titres of specific antibodies detected were low, and cellular responses were not significant. Apparently, sheep immune responses induced by cDNA-FhPGK/pCMV vaccination are not effective at controlling F. hepatica infection. Poor immunogenicity of DNA vaccines in large animals is still a major obstacle of this technology that has to be overcome.