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.

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.

Molecular characteristics of representatives of the genus Brachylecithum Shtrom, 1940 (Digenea, Dicrocoeliidae) with comments on life cycle and host specificity.

The genus Brachylecithum was for the first time subject to molecular taxonomic phylogenetic analysis in order to ascertain relationships among its component taxa. We used two markers-the nuclear ribosomal 28S ribosomal DNA (rDNA) gene and the mitochondrial cox1 gene, for six species of the genus; 11 sequences of partial 28S rDNA and partial cox1 were obtained from adult B. capilliformis, B. glareoli, B. kakea, B. laniicola, B. lobatum, and B. strigis, and from larval stages obtained from snails of the genus Cepaea. We propose to synonymize B. strigis with B. lobatum, while the genetic differences in the 28S rDNA gene and mitochondrial cox1 gene confirm the species status of B. capilliformis and indicate a distinct group within Brachylecithum, including B. kakea and B. laniicola. Cercarial and metacercarial isolates from the snails showed 100 % similarity to B. lobatum; thus, it is the first record of Cepaea snails as intermediate hosts of this species and the first report on life cycle abbreviation within the Dicrocoeliidae.

Development of a multiplex PCR for identification of Dictyocaulus lungworms in domestic and wild ruminants.

Dictyocaulus lungworms are the causative agents of parasitic bronchitis (dictyocaulosis) characterised by coughing and severe lung pathology in domestic and wild ruminants. The objective of this study was to design a simple molecular test that could detect of lungworm DNA from both adult and larval lungworms and could distinguish between the most common Dictyocaulus species found in cattle and in some species of wild ruminants. A multiplex PCR test with four novel primers targeting species-specific regions of the second internal transcribed spacer (ITS2) was designed based on our own sequence data as well as on available sequence information in GenBank. After PCR amplification of lungworms from European bison (Bison bonasus), cattle (Bos taurus), moose (Alces alces), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), products were analysed with gel electrophoresis. This resulted in three specific bands of different size depending on the species analysed. Dictyocaulus viviparus collected from cattle or European bison resulted in a ca. 560 bp band, D. capreolus collected from roe deer produced a band ca. 400 bp and the longest DNA band (ca. 660 bp) was obtained with DNA from Dictyocaulus sp. collected from red deer and moose. Dictyocaulus eckerti bands with expected size of 714 bp were not observed in our study. The multiplex method produced consistent results with samples from both Sweden and Poland and overcame the limitations of traditional techniques based on differences in morphological features of parasites at different life stages.

Morphological and molecular characterisation of the nematode parasite Graphidioides affinis (Secernentea: Trichostrongylidae) in Patagonian maras, Dolichotis patagonum, kept in a zoo in Sofia, Bulgaria.

Introduction: Patagonian maras, rodents endemic to South America, are classified as a near-threatened species. Various factors affect their health including parasitic diseases. The aim of this study was to perform morphometric, molecular and phylogenetic characterisation of one such parasitic disease agent, the nematode Graphidioides affinis, specimens of which were found in captive Patagonian maras. Material and Methods: In March 2023, 18 Patagonian maras kept at the Sofia Zoo in Bulgaria were investigated with the use of coprological methods. Following the investigation, the animals were dewormed with the use of albendazole. Dead adult nematodes found in the faeces of dewormed maras were collected and preserved in 70% ethanol, and morphometrically, molecularly and phylogenetically analysed. Results: The morphometric analyses confirmed the nematodes to be Graphidioides affinis. The partial nucleotide sequences of the small subunit ribosomal rDNA (SSU), the internal transcribe spacer 2 (ITS2) and the large subunit ribosomal DNA (LSU) of G. affinis were obtained. These are the first available nucleotide sequences of this parasite. The phylogenetic analyses of the species showed its distinctiveness in comparison to other gastrointestinal nematodes, as it was grouped separately. Conclusion: The Patagonian maras kept in a European zoo retained their original parasitofauna which are related to South America.

Diversity of Anaplasma phagocytophilum Strains from Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus) in Poland.

BACKGROUND: The Gram-negative bacterium Anaplasma phagocytophilum is an intracellular pathogen and an etiological agent of human and animal anaplasmosis. Its natural reservoir comprises free-ranging ungulates, including roe deer (Capreolus capreolus) and red deer (Cervus elaphus). These two species of deer also constitute the largest group of game animals in Poland. The aim of the study was to genotype and perform a phylogenetic analysis of A. phagocytophilum strains from roe deer and red deer. METHODS: Samples were subjected to PCR amplification, sequencing, and phylogenetic analysis of strain-specific genetic markers (groEL, ankA). RESULTS: Five haplotypes of the groEL gene from A. phagocytophilum and seven haplotypes of ankA were obtained. The phylogenetic analysis classified the groEL into ecotypes I and II. Sequences of the ankA gene were classified into clusters I, II, and III. CONCLUSIONS: Strains of A. phagocytophilum from red deer were in the same ecotype and cluster as strains isolated from humans. Strains of A. phagocytophilum from roe deer represented ecotypes (I, II) and clusters (II, III) that were different from those isolated from red deer, and these strains did not show similarity to bacteria from humans. However, roe deer can harbor nonspecific strains of A. phagocytophilum more characteristic to red deer. It appears that the genetic variants from red deer can be pathogenic to humans, but the significance of the variants from roe deer requires more study.

A comprehensive analysis of chemical and biological pollutants (natural and anthropogenic origin) of soil and dandelion (Taraxacum officinale) samples.

A range of analytical methods (GC-MS, LC-MS, voltammetry, microbiological and microscopic techniques, PCR) was used to assay a range of potential chemical and biological contaminants in soil and dandelion samples. The results provide the first comprehensive safety analysis of dandelion as a herbal product. Samples were collected from three different sites in Poland where the local population collects dandelion plants for their own consumption: Rudenka (a mountain meadow in the European Ecological Network of Natura 2000 protection area, free of agrotechnical treatments for over 30 years), Warszawa 1 (dense single-family housing with heavy traffic), and Warszawa 2 (recreation area with heavy traffic near a coal-fired heat and power plant). The assays of heavy metals and other chemical pollutants (PAHs, PCBs, dioxins, pesticides, mycotoxins) confirm that all collected soil and dandelion samples were chemically pure; however, 95 species of pathogenic bacteria were detected, including "carnivorous" Vibrio vulnificus, zoonotic Pasteurella pneumotropica, Pasteurella canis, Staphylococcus pseudintermedius, Staphylococcus lentus and Francisella tularensis as well as 14 species of pathogenic fungi and one protozoan parasite (Giardia intestinalis). The discovery of septicemia agents V. vulnificus, Fusobacterium mortiferum and Rahnella aquatilis in the soil surrounding dandelion roots and in the flowers, G. intestinalis in dandelion leaves and roots samples, all collected in Warsaw, is highly disturbing. This finding underlines the need for increased caution when collecting dandelion in densely populated areas with a large population of pets. Thorough washing of the harvested plants is necessary before using them for consumption, especially in the case of making salads from fresh dandelion leaves, which is becoming increasingly popular among people leading healthy and an environmentally friendly lifestyle.

Prevalence and Genotyping of Anaplasma phagocytophilum Strains from Wild Animals, European Bison (Bison bonasus) and Eurasian Moose (Alces alces) in Poland.

Wild large ungulates, like European bison (Bison bonasus) and Eurasian moose (Alces alces), form an important part of the circulation of Anaplasma phagocytophilum, a Gram-negative, intracellular, tick-transmitted bacterium, in the natural environment. Bison and moose tissue samples were subjected to 16S rDNA, groEL and ankA partial gene marker amplification with specific primers using various variants of PCR. Out of 42 examined individuals, Anaplasma sp. were detected in 4/13 Eurasian moose (31%) and 7/29 European bison (24%). In addition, 12 groEL and 5 ankA partial gene positive samples were obtained from the examined animals. The phylogenetic analysis of the groEL partial gene classified samples from European bison to ecotype I, and samples from Eurasian moose to ecotype I and II; the analysis of the ankA partial gene assigned the samples to clusters I and IV. This study extends knowledge about A. phagocytophilum in wild large ungulates in Poland. This is the first report about the occurrence of Anaplasma sp. in one of the largest populations of free living European bison in the world. Our findings confirm that strains of A. phagocytophilum from Bison bonasus and Alces alces may constitute a natural reservoir of pathogenic HGA Anaplasma strains.

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.

Helminth Assemblages of the Antarctic Black Rockcod, Notothenia coriiceps (Actinopterygii: Nototheniidae) in Coastal Waters near Galindez Island (Argentine Islands, West Antarctic): Temporal Changes in the Endoparasite Community.

PURPOSE: Analysis and comparison of the helminth assemblages in Antarctic rockcod Notothenia coriiceps collected near the UAS "Akademik Vernadsky" (Argentine Islands, West Antarctica) in 2002 and 2014-2015 were performed to characterise the parasite community and investigate the temporal changes in helminth assemblages and infection parameters. METHODS: All specimens of N. coriiceps (n = 194) were caught at depths of 10-30 m. Parasites (22,856 helminth specimens and 15,057 cysts) were collected manually and identified based on their morphology. Statistical analysis of the quantitative data was performed using the Quantitative Parasitology 3.0 (QP 3.0), Paleontological Statistics (PAST v. 3.1), and PRIMER 6 software. RESULTS: Twenty-seven species of four taxonomic groups were recorded: trematodes (8 species), cestodes (4), nematodes (5), and acanthocephalans (10). Helminth samples collected in 2002 and 2014-2015 showed a rather high similarity in species composition. The species richness was higher in the sample collected in 2014-2015, while the evenness and diversity in the two samples were similar. The dissimilarity between helminth infracommunities in the two samples appeared to be statistically significant. Larval cestodes Diphyllobotrium sp., the acanthocephalan Metacanthocephalus rennicki, and the trematode Neoleoburia antarctica were found to make the most significant impact on the dissimilarity. CONCLUSIONS: The analysis of the composition and structure of helminth community in N. coriiceps revealed the changes that have happened during the last decade. At least some of the changes are attributed to the changes in marine ecosystems in Western Antarctica.

Molecular Detection of Bartonella spp. in Rodents in Chernobyl Exclusion Zone, Ukraine.

PURPOSE: Bacteria of the genus Bartonella are obligate parasites of vertebrates. Their distribution range covers almost the entire world, from the Americas to Europe and Asia. Many Bartonella species use rodents as reservoirs, and while much is known about Bartonella infection of rodents in central Europe, its extent is poorly understood in Eastern Europe. METHODS: The present study examines five rodent species (Apodemus flavicollis, Myodes glareolus, Microtus arvalis, Apodemus agrarius, Apodemus sylvaticus) in the Chernobyl Exclusion Zone in Ukraine. Total of 36 small mammals were captured in September 2017. RESULTS: The overall prevalence of Bartonella spp. was 38.9% (14/36) in rodents. Obtained four sequences from Apodemus flavicollis, were identical to Bartonella grahamii and B. taylorii. CONCLUSION: This is the first report to confirm the presence of Bartonella spp. in rodents in the Chernobyl Exclusion Zone, Ukraine by molecular methods. The sequences show similarity to Bartonella strains occurring in Europe.

Detection of Anaplasma phagocytophilum in Wild and Farmed Cervids in Poland.

BACKGROUND: The role of cervids in the circulation of A. phagocytophilum has not yet been clearly determined; however, several species of wild and farm cervids may be a natural reservoir of this bacteria. METHODS: Spleen and liver tissue samples were taken from 207 wild (red deer, roe deer, fallow deer and moose) and farmed cervids (red deer and fallow deer) from five geographical areas. These were tested for the A. phagocytophilum16S rDNA partial gene by nested PCR. RESULTS: Anaplasma spp. were detected in 91 of 207 examined cervids (prevalence 43.9%). Three different variants of 16S rDNA partial gene were reported, one for the first time. Anaplasma phagocytophilum was more often detected in young specimens than in adults and more often in the spleen than in the liver. CONCLUSIONS: Cervids from the four sites across Poland were found to be major natural reservoirs of various strains of A. phagocytophilum. This is the first study to use spleen and liver as biological material to detect A. phagocytophilum in moose in Poland.

Occurrence of Echinococcus spp. in red foxes and wolves in the protected area of the Tatra National Park in southern Poland - a threat to human health.

INTRODUCTION: Echinococcus multilocularis has been endemic in red foxes in eastern and central parts of Europe, and E. granulosus s. l. identified in wolves in some countries. In recent years, wolves hale emerged as potentially important definitive hosts of E. multilocularis. OBJECTIVE: This aim of the survey was to record indirectly using nested-PCR test with faecal samples the presence of Echinococcus multilocularis and E. granulosus s. l. in the two species of wild canids in the protected area of the Tatra National Park (TNP) in Western Carpathian, southern mountainous part of Poland. MATERIAL AND METHODS: From February to June 2019, experienced staff of TNP randomly collected fox and wolf faeces on and off hiking trails at altitudes from 850 m to 2,000 m above sea level. In total, 91 faecal samples from red foxes and 19 from wolves were collected. Genomic DNA was obtained by direct extraction from faecal samples using a commercial kit, and from taeniid eggs retrieved from the same samples after flotation. RESULTS: A nested PCR screening of 91 red fox faeces indicated the prevalence of E. multilocularis of 4.4%. Positive samples were confirmed by sequencing parts of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Neither genomic DNA of E. multilocularis nor of E. granulosus s.l. was obtained from 19 wolves faeces, nor from taeniid eggs retrieved from these samples by initial flotation. CONCLUSIONS: The current results show that humans might be exposed to a risk of fox tapeworm infection in nature, even at high altitude inan alpine zone, in an environment contaminated by roaming red foxes encouraged by food leftovers on mountain trails.

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.

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.

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.

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.

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.

Molecular detection of Anaplasma phagocytophilum in wild carnivores in north-eastern Poland.

BACKGROUND: Anaplasma phagocytophilum is an obligate parasitic intracellular bacterium. It is the causative agent of granulocytic anaplasmosis, with effects on human and animal health. In Europe, the pathogen is mainly transmitted among a wide range of vertebrate hosts by blood-sucking arthropods. The aim of this study was to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dogs (Nyctereutes procyonoides), badgers (Meles meles), foxes (Vulpes vulpes), martens (Martes sp.) and European polecats (Mustela putorius), using molecular methods. METHODS: In the present study, 174 spleen samples were collected from adult, wild carnivores hunted in the years 2013-2016. A short fragment (383 bp) of the 16S ribosomal RNA gene partial sequence was used as a marker to identify A. phagocytophilum in spleen samples collected from carnivores using nested PCR. RESULTS: The prevalence of A. phagocytophilum in wild carnivores was 31.61% (55/174). Seven sequences of A. phagocytophilum were generated from two raccoon dogs, two badgers, one marten, one red fox and one European polecat. Six identical nucleotide sequences were obtained from one raccoon dog, two badgers, one marten, one red fox and one European polecat (A. phagocytophilum sequences 1: MH328205-MH328209, MH328211), and these were identical to many A. phagocytophilum sequences in the GenBank database (100% similarity). The second sequence (A. phagocytophilum sequence 2: MH328210) obtained from the raccoon dog shared 99.74% identity with A. phagocytophilum sequence 1. CONCLUSIONS: To our knowledge, this is the first study to use molecular methods to determine the presence of A. phagocytophilum in wild carnivores, viz raccoon dog, badger, marten and European polecat, in Poland. The detected A. phagocytophilum sequences (1 and 2) were closely related with those of A. phagocytophilum occurring in a wide range of wild and domestic animals and vectors.