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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.

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 , specimens of which were found in captive Patagonian maras. 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. The morphometric analyses confirmed the nematodes to be . The partial nucleotide sequences of the small subunit ribosomal rDNA ( ), the internal transcribe spacer 2 ( ) and the large subunit ribosomal DNA ( ) of 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. The Patagonian maras kept in a European zoo retained their original parasitofauna which are related to South America.

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.

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 16.5–17.3% nucleotide sequence divergence for 642 bp mitochondrial cytB between D. skrjabini n. sp. and D. cervi, respectively, and 18.7–19% between D. skrjabini n. sp. and D. eckerti, which translates into 18.2–18.7% amino acid sequence divergence between D. skrjabini n. sp. and both lungworms.

Background European bison, Bison bonasus , is a strictly protected species of large mammal, with 25% of the world’s population living in Poland. The most numerous populations of European bison live in the Białowieża Primeval Forest, northeastern Poland, and in the Bieszczady Mountains, southeastern Poland. The purpose of this study was to investigate the occurrence of Anaplasma spp. in B. bonasus from Poland. Methods Tissue samples were collected from 45 European bisons between 2021 and 2024 in the Białowieża and Bieszczady areas. Two genetic markers, 16S ribosomal DNA (rDNA) and msp4 , were used for the detection, genotyping, and phylogenetic analysis of bacteria from the genus Anaplasma . Results The prevalence of Anaplasma spp. was 40% (18/45) in the examined samples. Anaplasma phagocytophilum was detected in 10 samples, and eight samples were found to be positive for the presence of Anaplasma marginale DNA. Conclusions This study is the first report of A. marginale occurrence in Poland and the first report of A. marginale occurrence in B. bonasus in Europe. Infection by the pathogenic A. marginale in strictly protected species such as the European bison may have an impact on the health, ecology, and conservation of this endangered species. Graphical abstract

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.

Background The bacteria of the genus Bartonella are obligate parasites of vertebrates. Their distribution range covers almost the entire world from America, Europe, Asia to Africa and Australia. Some species of Bartonella are pathogenic for humans. Their main vectors are blood-sucking arthropods such as fleas, ticks and blood-feeding flies. One such dipteran able to transfer vector-borne pathogens is the deer ked ( Lipoptena cervi ) of the family Hippoboscidae. This species acts as a transmitter of Bartonella spp. in cervid hosts in Europe. Methods In the present study, 217 specimens of deer ked ( Lipoptena cervi ) were collected from 26 red deer ( Cervus elaphus ) hunted in January 2014. A short fragment (333 bp) of the rpoB gene was used as a marker to identify Bartonella spp. in deer ked tissue by PCR test. A longer fragment (850 bp) of the rpoB gene was amplified from 21 of the positive samples, sequenced and used for phylogenetic analysis. Results The overall prevalence of Lipoptena cervi infection with Bartonella spp. was 75.12% (163/217); 86.67% (104/120) of females and 60.82% (59/97) of males collected from red deer hunted in the Strzałowo Forest District in Poland (53°45′57.03″N, 21°25′17.79″E) were infected. The nucleotide sequences from 14 isolates ( Bartonella sp. 1) showed close similarity to Bartonella schoenbuchensis isolated from moose blood from Sweden (GenBank: KB915628) and human blood from France (GenBank: HG977196); Bartonella sp. 2 (5 isolates) and Bartonella sp. 3 (one isolate) were similar to Bartonella sp. from Japanese sika deer (GenBank: AB703149), and Bartonella sp. 4 (one isolate) was almost identical to Bartonella sp. isolated from Japanese sika deer from Japan (GenBank: AB703146). Conclusions To the best of our knowledge, this is the first report to confirm the presence of Bartonella spp. in deer keds ( Lipoptena cervi ) in Poland by molecular methods. Bartonella sp. 1 isolates were most closely related to B. schoenbuchensis isolated from moose from Sweden and human blood from France. The rest of our isolates ( Bartonella spp. 2–4) were similar to Bartonella spp. isolated from Japanese sika deer from Japan.

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.

Background Echinostomes are cosmopolitan digenean parasites which infect many different warm-blooded hosts. Their classification is extremely confused; the host spectrum is wide, and morphological similarities often result in misidentification. During our long-term studies on the helminth fauna of rodents and carnivores we have collected 27 collar-spined echinostomes which differ in morphology to an extent that suggests the presence of more than one species. Here, we describe this material, and the extent of host-related variation in this parasite. Methods Specimens of Isthmiophora isolated from four host species (badger, American mink, hedgehog, striped field mouse) were subject to morphological and molecular examination; the data were statistically analysed. Results Our results show that genetically all the Isthmiophora specimens obtained from all the examined hosts are conspecific and represent I. melis . On the other hand, the individuals isolated from Apodemus agrarius are morphologically distinct and, based on this criterion alone, should be described as a new species. Conclusions The morphological traits of Isthmiophora melis are much variable and host-dependent; without molecular analysis they would suggest a necessity to describe a new species or even genus. Such a high level of intraspecific variability may be affected by the host’s longevity.

Anaplasma phagocytophilum is a pathogen of veterinary and medical importance. It is the causative agent of tick-borne fever (TBF) in ruminants (also known as bovine or ovine granulocytic anaplasmosis), and of human granulocytic anaplasmosis (HGA) in humans. In Europe, A. phagocytophilum is transmitted by Ixodes ricinus (Linnaeus 1758) ticks. The aim of this study was to confirm the presence of A. phagocytophilum DNA in bloodsucking flies belonging to the Tabanidae family using molecular methods. It represents the first detection of this pathogen in Haematopota pluvialis (Linnaeus 1758), Tabanus bromius (Linnaeus 1758), and Tabanus distinguendus (Verrall 1909) in Europe.