Intestinal Helminth Communities of Grey Partridge Perdix perdix and Common Pheasant Phasianus colchicus in Poland.

The aim of this study was to describe the morphology and means of identification of helminths in native partridges (65) and introduced pheasants (32) in Poland and to determine the level of intestinal infection of these birds by helminths using parasitological and ecological indices. The birds were acquired during the hunting season in the years 2015-2017. Nematodes, Capillaria phasianina, cestodes, Railietina friedbergeri, and one trematode, Brachylaima sp. were recorded for the first time in partridges in Poland. Our findings indicate that parasites are more prevalent in pheasants (prevalence 70.4%) than in partridges (prevalence 50.0%). The component community and infracommunity of parasites of partridges are more diverse (Simpson's diversity index: 0.63 and mean Brillouin diversity index: 0.10 ± 0.17) and less dominated by a single parasite species (Capillaria sp., Berger-Parker dominance index: 0.53) than the pheasant parasite community (Simpson's diversity index: 0.07, mean Brillouin diversity index: 0.005 ± 0.02, dominant species Heterakis gallinarum, Berger-Parker dominance index: 0.96). There were statistically significant differences between partridges and pheasants in the Brillouin diversity index and in the prevalence of Heterakis gallinarum (55.6% in pheasants vs. 19.0 in partridges). There were significant differences between wild and farmed partridges in the prevalence of infection by Capillaria sp. (4.3% vs. 37.5%) and H. gallinarum (39.1 vs. 6.2%). In conclusion, the pheasant was shown to be a reservoir, carrier, and shedder of nematodes, which may increase the risk of infection in partridges.

Very low intraspecific sequence variation in selected nuclear and mitochondrial Parascaris univalens genes.

Equines were over decades considered to be infected by two morphologically virtually indistinguishable ascarid species, Parascaris univalens and Parascaris equorum. Reliable species discrimination is only possible using enzyme isoelectric focussing and karyotyping with P. univalens having one and P. equorum two chromosome pairs. However, presumably the complexity of both methods prevented their routine use in nearly all previous studies about prevalence and drug resistance of Parascaris spp. These have barely been performed on the species level although most studies stated presence of one or the other species. Recently, only P. univalens has been identified by karyotyping and the last published study identifying P. equorum dates back to 1989. In order to improve species-specific detection, molecular markers are required. Here, partial 12S rRNA, cytochrome oxidase I (COI) and complete internal transcribed spacer (ITS)-1 and - 2 sequences were obtained from 24 karyotyped Parascaris specimens from Poland and 6 German specimens (not karyotyped) and used in phylogenetic analyses with orthologous sequences from GenBank. All karyotyped specimens were identified as P. univalens. In the phylogenetic analysis, they formed very homogenous clusters for all target genes and in a multi-locus analysis. Within this cluster, almost all sequences from GenBank were also included, no matter if they had been assigned to P. univalens or P. equorum. However, a small number of P. univalens ITS and COI sequences originating from donkeys from a single farm in China formed a highly supported sister cluster suggesting that they might represent another Parascaris genotype or species. Our data also strongly suggest that nearly all ITS and COI sequences previously deposited in GenBank and assigned to P. equorum actually represent P. univalens. The fact that significantly different sequences can be found in Parascaris spp. suggests that PCR-based species diagnosis will be possible once molecular markers have been identified for P. equorum from karyotyped specimens.

The P-glycoprotein repertoire of the equine parasitic nematode Parascaris univalens.

P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the major site of expression but individual gene expression patterns were not conserved between the two nematodes. In P. univalens, PunPgp-9, PunPgp-11.1 and PunPgp-16.2 consistently exhibited the highest expression level in two independent transcriptome data sets. Using RNA-Seq, no significant upregulation of any Pgp was detected following in vitro incubation of adult P. univalens with ivermectin suggesting that drug-induced upregulation is not the mechanism of Pgp-mediated ML resistance. Expression and functional analyses of PunPgp-2 and PunPgp-9 in Saccharomyces cerevisiae provide evidence for an interaction with ketoconazole and ivermectin, but not thiabendazole. Overall, this study established reliable reference gene models with significantly improved annotation for the P. univalens Pgp repertoire and provides a foundation for a better understanding of Pgp-mediated anthelmintic resistance.

The Prevalence of Endoparasites of Free Ranging Cats (Felis catus) from Urban Habitats in Southern Poland.

Growing urbanization leads to an increased risk of parasite spread in densely inhabited areas. Free-ranging cats can be locally numerous and come into frequent contact with both wildlife and humans. Cats are thus expected to contribute to parasitic disease transmission. In our study, we investigated the prevalence of endoparasites in free ranging cats in urban areas of Kraków city, based on necropsy of road-killed cats in relation to sex and diet of cat, season and habitat type. We found that 62% of 81 cats were infected with endoparasites with Toxocara cati being the most prevalent. In total, we identified seven parasite species. The number of parasite species was higher in suburban habitats and aside from Eucoleus aerophilus the prevalence of all parasites was higher in cats from suburban areas than in the individuals living in the city urban core. The prey of examined cats included mostly rodents, followed by soricomorphs and birds, which can all serve as paratenic hosts. Based on our results, we suggest that cats in urban areas should be considered as a serious potential zoonotic threat. Implementation of proper veterinary control and wider education on the topic is recommended.

Tarantobelus arachnicida (Nematoda: Rhabditida) invasion of exotic spiders in Poland

Nematodes of the Brevibuccidae family were stated among spiders of the Theraphosidae family (the South American species), which were bred in Poland. The first sign of infection was anorexia which led to gradually increasing lethargy progressed to a huddled posture. Additionally, a white discharge with nematodes between mouth and chelicerae was noted. All of the derived nematodes were morphologically identified and determined to the species Tarantobelus arachnicida. A molecular analysis covered amplification and sequencing of small subunit ribosomal RNA (18S rRNA). A post mortem examination demonstrated the presence of nematodes not only near the chelicerae, but also inside the intestine, hence the source of infection might be insects used as a food. The research showed that such kind of infection is an important disease, which poses a serious risk to the breeding spiders. To date there is no effective treatment, however, we demonstrated that usage of the Lugol's solution seems to be promising.

Endoparasites of pet reptiles based on coprosopic methods

Due to the growing popularity of reptiles as a household animals and the development of numerous reptile farms, they have become a common sight in veterinary clinics. As parasitic infections represent a serious problem among pet reptiles obtained by captive breeding and from pet shops, the purpose of the present study was to determine the species composition of parasites present in reptiles bred privately or in Cracow Zoological Garden, and those obtained from pet shops. Fecal samples collected from 91 reptiles (30 turtles, 40 lizards, and 21 snakes) were examined using the quantitative McMaster method. Parasite eggs or protozoan oocysts were identified in 59.3% of samples. These included the eggs of the Pharyngodonidae, Ascarididae and Rhabditoidea (Nematoda), and Trematoda, as well as oocysts of Isospora and Eimeria. In addition, pseudoparasites belonging to the Mesostigmata, Demodecidae and Myobiidae were found.

Equine strongyle communities are constrained by horse sex and species dipersal-fecundity trade-off.

BACKGROUND: Equine strongyles are a major health issue. Large strongyles can cause death of horses while cyathostomins (small strongyles) have shown increased resistance to anthelmintics worldwide. Description of strongyle communities have accumulated but little is known about the diversity of these communities and underpinning environmental factors. METHODS: Strongyles were recovered after ivermectin treatment from 48 horses located in six premises in Poland. Correlation between previously published species fecundity and the observed relative abundance and prevalence were estimated. Significance of horse sex was determined at the species level (prevalence, relative abundance) and at the community level (species richness and dissimilarity between communities). RESULTS: Strongyle species fell into two groups, contrasted by their prevalence and relative abundance. Six to nine horses were necessary to sample at least 90% of strongyle community diversity, providing a minimal cut-off to implement sampling trial in the field. Strongyle communities entertained a network of mostly positive interactions and species co-occurrence was found more often than expected by chance. In addition, species fecundity and prevalence were negatively correlated (Pearson's r = -0.71), suggesting functional trade-offs between species dispersal abilities and fecundity. This functional trade-off may underpin species coexistence. Horse sex was also a significant constraint shaping strongyle communities. Indeed, mares generally displayed more similar strongyle communities than stallions (P = 0.003) and Cylicostephanus calicatus was more abundant in stallions suggesting sex-specific interactions (P = 0.006). CONCLUSIONS: While niche partitioning is likely to explain some of the positive interactions between equine strongyle species, coexistence may also result from a functional trade-off between dispersal ability and fecundity. There is significant evidence that horse sex drives strongylid community structure, which may require differential control strategies between mares and stallions.

Cystic echinococcosis in Poland: genetic variability and the first record of Echinococcus granulosus sensu stricto (G1 genotype) in the country.

Cystic echinococcosis is one of the most important zoonotic diseases affecting humans and livestock worldwide, and is endemic in Poland. A set of six isolates on larval stages of Echinococcus granulosus sensu lato tapeworms collected from three humans, two pigs and one sheep from Polish foci of CE was examined by DNA sequencing of two mitochondrial genes (cox1, rrnS). The results demonstrated the presence of E. canadensis and E. granulosus sensu stricto in the investigated hydatid cysts. The former species was found in all five isolates from pigs and humans derived from central Poland. In a sheep hydatid cyst originating from Lesser Poland Voivodeship in southern Poland, E. granulosus s. s. (G1 genotype) was identified. This is the first report of an unambiguously autochthonous infection with E. granulosus s. s. in Poland. The global distribution and host affiliations of the commonly occurring G1 microvariant with nucleotide change 56C/T in cox1, detected here in Polish sheep, are discussed. The finding that sheep harboured E. granulosus s. s. may have important consequences for developing effective hydatid control programmes in Poland due to its longer maturation rate in dogs compared with E. canadensis G7. This may lead to greater expenditures for purchasing anthelmintics to provide an appropriate dosing regime in sheep-raising areas of the country.

Cephenemyia stimulator (Diptera) infection in roe deer (Capreolus capreolus) from Kraków area, southern Poland.

UNLABELLED: The aim of the study was to determine the prevalence of botfly (Diptera: Oestridae) larvae infection in roe deer populations (Capreolus capreolus) in the Kraków area on the basis of necropsy and questionnaire surveys. Hunters were surveyed about the age and sex of hunted animals, and the origin of their habitat. All parasite specimens were identified to species in the laboratory. The parasites were found in the nasal cavities, esophagus, and larynx of male roe deer aged 3-8 years, living in forest habitats. The level of infection was relatively low (13%), with the intensity ranging from 1 to 10 larvae per host. Although no fly larvae were found during the dissection of roe deer carcasses, the parasites received from the hunters were identified as Cephenemyia stimulator botflies. These findings are supported by the evidence drawn from the questionnaires completed by the hunters. KEY WORDS: parasites, Cephenemyia stimulator, Capreolus capreolus, necropsy, questionnaire study, Poland.

Endoparasites of exotic ungulates from the Giraffidae and Camelidae families kept ex situ.

Giraffes and camels are popular attractions at zoological gardens. In order to present the diversity of parasites infecting exotic ungulates from zoos, faecal samples from three giraffes and six camels from both the Silesian Zoological Garden in Chorzów, and Kraków Zoological Garden, were examined. The research was carried out over a ten-month period in 2013 and 2014. In total, 100 faecal samples from 18 animals were analysed with the use of the McMaster method. Moreover, coccidian oocysts were incubated to investigate their development and larvoscopic examination was conducted to detect the presence of nematode species. Giraffes were infected with coccidia from the genus Eimeria, and gastrointestinal nematodes from the Strongylida order, and Trichuris and Aonhotheca genera. One male giraffe was uninfected. The level of infection in giraffes was low when compared to camels kept in both of the zoos. Limited contact with other animal species contributed greatly to the lower level of infection in camels from Kraków Zoo than those from Chorzów, which were kept in the same enclosure as alpacas and Shetland ponies.

Lungworm (Nematoda: Protostrongylidae) infection in wild and domestic ruminants from Malopolska region of Poland.

The conducted study has focused on domestic, as well as wild ruminant species. The post mortem examination was carried out on 68 animals, including three wild species: roe deer (Capreolus capreolus) (25 indyviduals), red deer (Cervus elaphus) (6), fallow deer (Dama dama) (5) and two domestic: sheep (Ovis aries) (14) and cattle (Bos taurus) (18). Some of the species have also been investigated in the field by the coproscopical analyses. The faecal samples from roe deer (27), fallow deer (20), red deer (36) and moose (Alces alces) (10) were collected from the environment, while from sheep (10) and goat (Capra hircus) (10)--per rectum. Based on the obtained results the following values were calculated: prevalence, mean intensity and intensity range. The post mortem examination did not reveal pulmonary nematodes neither in domestic nor in wild ungulates, however, the larvae of aforementioned parasites were often stated in the stool samples taken from the environment. All wild species, except fallow deer were infected. Consequently, six species of lungworms have been identified. The first stage larvae of Varestrongylus capreoli occurred in 11 samples of roe deer and Varestrongylus alces in one moose. The larvae of Elaphostrongylus cervi were found in 19 red deer and Varestrongylus sagittatus in 3. Furthermore, Elaphostrongylus alces larvae were noted in 6 moose. Within domestic ruminants only one sheep and two goats were infected by Muellerius capillaris.

Parasite infections in red deer Cervus elaphus from Krakow area, southern Poland.

The study describes the parasitofauna of red deer Cervus elaphus from Kraków area (southern Poland). The study was done in years 2011-2012 and included altogether 6 animals. Five deer originated from the forest areas (hunted) and one came from the urban site (it died in a road accident). In addition to post-mortem examination, coproscopic analyses with sporulation were performed to define Eimeria species diversity. The deer were infected with four species of nematodes: Ashworthius sidemi, Spiculopteragia spiculoptera, Oesophagostomum venulosum and O. sikae, and two species of coccidia: E. elaphi and E. austriaca. The level of parasitie infections was low, but the results indicate the important role of deer as possible transmitter of A. sidemi to other ungulates in examined area.

Estimation of genetic parameters for resistance to gastro-intestinal nematodes in pure blood Arabian horses.

Equine internal parasites, mostly cyathostomins, affect both horse welfare and performance. The appearance of anthelmintic-resistant parasites creates a pressing need for optimising drenching schemes. This optimization may be achieved by identifying genetic markers associated with host susceptibility to infection and then to drench carriers of these markers. The aim of our study was to characterise the genetics of horse resistance to strongyle infection by estimating heritability of this trait in an Arabian pure blood population. A population of 789 Arabian pure blood horses from the Michalów stud farm, Poland were measured for strongyle egg excretion twice a year, over 8 years. Low repeatability values were found for faecal egg counts. Our analyses showed that less than 10% of the observed variation for strongyle faecal egg counts in this population had a genetic origin. However, additional analyses highlighted an age-dependent increase in heritability which was 0.04 (±0.02) in young horses (up to 3 years of age) but 0.21 (±0.04) in older ones. These results suggest that a significant part of the inter-individual variation has a genetic origin. This paves the way to a genomic dissection of horse-nematode interactions which might provide predictive markers of susceptibility, allowing individualised drenching schemes.

Zatorska goose - a subject of parasitological research.

The aim of the study was to determine the level of gastrointestinal parasites in a native breed of geese - Zatorska goose - based on coproscopic testing. Faecal samples were collected from 90 young geese in three age groups (5, 7 and 9 weeks old) in 2014. The geese were kept indoors on deep litter and pastured from spring to autumn. The area of the pastures around the buildings where the geese grazed was about 1 hectare, divided into quarters for different age groups. Before grazing, the birds were dewormed with fenbendazole (Fenbenat powder 4%, Naturan). As additional treatment for coccidiosis, coccidiostats were added to the feed. The study was conducted using the McMaster quantitative method with centrifugation (flotation liquid: NaCl and glucose). The birds were shown to be infected with coccidia and nematodes. The prevalence of Eimeria sp. infection (mean 40%) and the number of oocysts per gram of faeces (reaching 5,300 OPG) were highest in the youngest age group of geese. The level of Amidostomum anseris infection was similar in the three age groups, with prevalence from 40% to 50% (nematode egg output ranged from 50 to 350 eggs per gram of faeces, EPG). Capillaria anatis was observed only in 5- and 7-week-old geese.

The Alice - "Follow the White Rabbit" - parasites of farm rabbits based on coproscopy.

The aim of the study, conducted in the years 2011-2013, was to determine the level of gastrointestinal parasites infection in New Zealand White rabbits, kept at the Experimental Station of the University of Agriculture in Krakow. The study showed rabbits protozoan infection with the genus Eimeria, belonging - based on the sporulation method - to the following species: E. magna, E. media, E. perforans, E. stiedae and E. irresidua. The highest prevalence of infection, as well as the intensity of oocysts output (OPG - oocysts per gram of faeces), was noted for E. magna and E. media - respectively 31.4 % (19477.3 OPG), and 40.0 % (14256.07 OPG). The infection of rabbits with Eimeria spp. differed significantly between years. With regard to oocysts output, the level of infection was strongly connected with the age of rabbits, being higher in young animals. However, the range of infection was highest among adults. Among nematodes, Passalurus ambiguus pinworm was regularly found (prevalence reached 21.9%), other species - Trichuris leporis, and Graphidium strigosum were rarely noted. The overall infection with nematodes did not differ between years. Similarly, as in the case of Eimeria older individuals were more often infected by nematodes. We observed some trends in parasite oocysts/eggs output; the protozoan oocysts were recorded more often in faecal samples collected in the evenings, whereas the nematodes eggs occurred frequently in the mornings. This situation may be related to the phenomenon of coprophagy occurring in the mammals of Lagomorpha order. The results of the study indicate that especially coccidiosis constitute permanently throughout the years an important problem in the rabbitry examined.

Presence of blood-sucking mesostigmatic mites in rodents and birds kept in pet stores in the Cracow area, Poland.

The aim of the study was to investigate the occurrence of zoonotic arthropod parasites in small animals sold in selected pet stores in the Cracow area. The research was conducted in seven pet stores, keeping a total of six species of rodents and three species of birds. In two shops, two species of mites of the order Mesostigmata were detected on the animals and in their surrounding: Dermanyssus gallinae, the poultry red mite, and Ornithonyssus bacoti, the rat mite. Both observed species of mites may be harmful to animals, as well as to people working in the shops or potential pet owners. This study discusses the possible origin of the parasites and their importance to human health.

Herbal formulations as feed additives in the course of rabbit subclinical coccidiosis.

Two simultaneous experiments were carried out in a breeding farm of New Zealand White rabbits (Oryctolagus cuniculus f. domesticus) to determine the feasibility of replacing coccidiostats with garlic and oregano preparation. The research took place during June and July, the period of the greatest threat of coccidiosis caused by Eimeria spp. (Apicomplexa: Eimeriidae). In one investigation, 40 rabbits aged 1-3 months were divided into four groups of ten animals: Group A being a control which received no coccidiostats in feed, Group B receiving the coccidiostat Baycox in water once at weaning, Group C receiving the coccidiostat robenidine in feed, and group D receiving herbal extracts in feed. In the second trial, six mated females were allocated equally to three groups analogous to A, C, and D above during pregnancy and lactation. Bulk stool samples were collected from each group of rabbits at weekly intervals for coproscopic analysis, and the production results of the animals were recorded. In the young rabbits, both the faecal coccidia oocyst counts and body weight gains were more favourable in group D than the remaining groups. Also, the female rabbits of group D were the least infected. The results demonstrate that garlic and oregano feed additives exert a positive influence on the level and course of coccidia infection, with regard to maintaining a good level of animal productivity, and these herbal extracts appear to have potential value in coccidiosis prophylaxy.

Genetic variants and increased expression of Parascaris equorum P-glycoprotein-11 in populations with decreased ivermectin susceptibility.

Macrocyclic lactones (MLs) represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum. Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps) are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and identified as orthologs of Caenorhabditis elegans Ppg-11 and an unnamed Caenorhabditis briggsae Pgp designated as Pgp-16 using phylogenetic analysis. Quantitative real-time PCR was used to compare expression between tissues. Significantly higher PeqPgp-11 expression was found in the gut for both genders, whereas for PeqPgp-16 the body wall was identified as predominant expression site. Furthermore, Pgps were analyzed regarding their participation in resistance development. Using SeqDoC analyses, Pgp-sequences of P. equorum populations with different ML susceptibility were compared. This approach revealed three single nucleotide polymorphisms (SNPs) causing missense mutations in the PeqPgp-11 sequence which correlated with decreased ML susceptibility. However, no resistance associated differences in mRNA expression levels were detected between embryonated eggs of these populations. In contrast, comparison of two pre-adult groups with different ivermectin (IVM) susceptibility revealed the presence of the three SNPs and in addition statistically significant PeqPgp-11 overexpression in the group of worms with reduced susceptibility. These results indicate that Pgp-11 might be involved in IVM resistance in P. equorum as it shows increased expression in an IVM exposed life-cycle stage of an IVM resistant population as well as occurrence of putatively resistance associated SNPs in populations with reduced IVM susceptibility. These SNPs are promising diagnostic candidates for detection of ML resistance with potential also for other parasitic nematode species.

Setaria tundra in roe deer (Capreolus capreolus)--new findings in Poland.

Setaria tundra is a filarioid nematode transmitted by mosquitoes, which is sporadically observed in Poland. Some ecological factors, such as the synurbisation of some wild ungulates and the climate changes observed in recent years, could affect the possibility of infection, especially with parasites transmitted by arthropod vectors. The study was conducted to evaluate the presence of Setaria sp. in roe deer populations in different environments. Parasitological dissections of 53 roe deer were performed. The animals were taken from the area of the Cracow agglomeration, and from arable and forest lands near the city. During the autopsy, nematode specimens (one to twelve per host) were found only in the abdominal cavity of roe deer from the urban area. All parasites were classified to Setaria tundra species on the basis of morphology and dimensions. The infection observed was probably associated with the specific environmental conditions of the urban area.

Occurrence of internal parasites in stone martens (Martes foina) from Cracow and suburbs.

The pine marten Martes martes and the stone marten Martes foina are the only representatives of the genus Martes in Poland. During the last few decades, an intensive synurbisation of stone martens has been recorded. The aim of the study was to assess the level of infection by internal parasites of Martes foina in the Cracow urban area. Eleven necropsies were performed of stone martens killed during animal-vehicle collisions. Additionally, coprological examination of 129 stone marten scats was included. For further analysis, two standard methods, i.e. flotation and decantation, were used. The intensity of infection by parasites was low. Taenia martis was found to be present in one marten individual by necropsy, while Taeniidae helminths and two genera of nematodes, Capillaria and Toxocara, were identified during coprodiagnosis. The diet analysis of stone marten scats revealed a high proportion of two food categories. Percentage frequency of occurrence for plant material and anthropogenic was calculated as 42.3% and 15.4%, respectively. This type of food composition might suggest lower infection by internal parasites in particular species, especially with an indirect life cycle.