The New Data on the Serotonin and FMRFamide Localization in the Nervous System of Opisthorchis felineus Metacercaria.

BACKGROUND: Trematoda Opisthorchis felineus Rivolta, 1884 is the causative agent of dangerous parasite disease-opisthorchiasis, widespread in the Russian Federation. The details of the neuroanatomical localization of the serotoninergic and FMRFamidergic neurotransmitter elements as well as their functional roles remain not studied enough in both adult and larval forms of O. felineus. The studies in this area are important in term of the development of a new pharmacological strategy of the struggle with the causative agent of opisthorchiasis affecting the neuronal signal substances and the function of its nervous system. PURPOSE: The aim of this work was the immunocytochemical study of the neurotransmitters serotonin (5-HT, 5-Hydroxitryptamine) and neuropeptide FMRFamide localization in the nervous system of the opisthorchiasis causative agent-O. felineus metacercaria. To study the relationship between the detected neurotransmitters and the muscular elements of the parasite, the muscle staining was carried out simultaneously using fluorophore-conjugated phalloidin. METHODS: The localization of 5-HTergic and FMRFamidergic nerve structures was determined by immunocytochemical method. The staining samples were analyzed using a fluorescent and confocal laser scanning microscopies. RESULTS: The new data on the presence and distribution of the serotonin-immunopositive (IP)- and FMRFa-IP components in the central and peripheral departments of the nervous system of  O. felineus metacercaria has been obtained. Besides that a number of the new anatomical details of the nervous system organization and of the innervation of the organs and tissues in the investigated parasite have been revealed. CONCLUSION: The data obtained on the presence and localization of the 5-HTergic and peptidergic (FMRFamide) components in central and peripheral departments of the nervous system of O. felineus metacercaria elaborated and expanded the existing information about the nervous system as well as the innervations of the tissues and organs in the causative agent of opistchorchiasis.

MHC, parasites and antler development in red deer: no support for the Hamilton & Zuk hypothesis.

The Hamilton-Zuk hypothesis proposes that the genetic benefits of preferences for elaborated secondary sexual traits have their origins in the arms race between hosts and parasites, which maintains genetic variance in parasite resistance. Infection, in turn, can be reflected in the expression of costly sexual ornaments. However, the link between immune genes, infection and the expression of secondary sexual traits has rarely been investigated. Here, we explored whether the presence and identity of functional variants (supertypes) of the highly polymorphic major histocompatibility complex (MHC), which is responsible for the recognition of parasites, predict the load of lung and gut parasites and antler development in the red deer (Cervus elaphus). While we found MHC supertypes to be associated with infection by a number of parasite species, including debilitating lung nematodes, we did not find support for the Hamilton-Zuk hypothesis. On the contrary, we found that lung nematode load was positively associated with antler development. We also found that the supertypes that were associated with resistance to certain parasites at the same time cause susceptibility to others. Such trade-offs may undermine the potential genetic benefits of mate choice for resistant partners.

Prevalence and distribution of Dirofilaria repens Railliet et Henry, 1911 in dogs in Poland.

In 2011-2013 1588 samples of dogs' blood were examined for dirofilariosis using Knott method, as well as the Kingston and Morton method. The species of microfilariae was determined on the basis of morphometric characteristics. Samples were also examined using the Canine Heartworm Antigen Test. Positive samples were examined using a multiplex PCR assay for species confirmation. Microfilariae belonging to the species D. repens were found in the blood samples of dogs from all the provinces of Poland. The mean prevalence of this species observed in Poland was 11.7%. The range of intensity of infection was counted using the number of microfilariae found in 60 microl of blood amounted to between 1 and 158, and the mean intensity was 18 microfilariae. Microfilariae and antigens of D. immitis were not found in any examined blood samples.

Molecular identification of Fascioloides magna (Bassi, 1875) from red deer from South-Western Poland (Lower Silesian wilderness) on the basis of internal transcribed spacer 2 (ITS-2).

The study was conducted in 2012-2013 on 75 fecal samples of red deer from the Lower Silesian Wilderness which were examined to determine the prevalence of Fascioloides magna in the game population. Finding liver fluke eggs in a single sample which were larger in size than Fasciola hepatica eggs indicated that further molecular analysis was necessarily. The partial sequence (116 bp long) of ITS-2 of the investigated eggs was identical to the sequences of F. magna from red deer (Cervus elaphus) (GenBank, EF534993; GenBank, EF534992) and from wapiti deer (Cervus elaphus canadensis) (GenBank, EF534994) from Slovakia, as well as from fallow deer (Dama dama) from the USA (GenBank, EF051080). This is the first molecular confirmation of the occurrence of F. magna in Poland.

Pathological lesions in European bison (Bison bonasus) with infestation by Ashworthius sidemi (Nematoda, Trichostrongylidae).

Asworthius sidemi Schulz, 1933 is a blood sucking gastrointestinal nematode, primarily typical for Asiatic deer. It was found for the first time in Poland in European bison in 1997. To estimate the level of invasion of A. sidemi and histopathological changes connected with the presence of the parasite in the years 2004-2007 parasitological and histopathological examinations of 54 European bison from Bialowieza Forest were carried out. Parasitological examination was carried out by the sedimentation method and A. sidemi were diagnosed under a binocular microscope. Samples for histological examination were collected from the abomasum and duodenum walls as well as from regional lymph nodes. Tissue samples were then fixed with 10% buffered formalin, embedded in paraffin, cut in to 5 microm thick sections and stained with hematoxylin and eosin (H&E). Parasitological examinations showed the presence of fourth stage larvae and juvenile forms of A. sidemi. The maximal intensity of invasion rose systematically from 4470 A. sidemi nematodes in 2004/2005 to 44310 in 2006/2007. Histopathological examinations showed infiltrations of inflammatory cells in the walls of abomasa and duodena at various levels of intensity (mainly lymphoid cells and eosinophils), hyperemiae, oedemae and lesions of mucosa and proliferation of lymphatic follicles. In individual cases of dysplasia of epithelial cells, atrophy or hyperplasia of glands and the presence of parasites in the lumen or walls of the abomasum/duodenum were observed. In one case, parasitic nodules were found. In regional lymph nodes proliferation of lymphatic follicles, presence of eosinophils and desolation of reproduction centers were observed. Intensification of histopathological changes was connected to a considerable degree with the developmental stage of A. sidemi as shown by parasitological examination.

Ashworthius sidemi (Nematoda, Trichostrongylidae) in wild ruminants in Bialowieza Forest.

Between 2003-2007, abomasa of 91 European bison (Bison bonasus), 4 red deer (Cervus elaphus) and 2 roe deer (Capreolus capreolus) shot in the Bialowieza Primeval Forest (Poland) were examined for worms presence. All the animals examined were infected with nematodes A. sidemi with an exception of one bison, that was shot in year 2003. There was much higher average intensity of invasion in bison (5529), than in red deer (85) and in roe deer (1837). The animals were shot in a period from December to March, and the nematodes found in them were fourth stage larvae and immature adult specimens. In the following years of examinations, a gradual increase in average intensity of infection was observed. In 2007, it reached in bison 10814 nematodes. Maximum intensity of invasion was found in this year as well and it reached 44310 nematodes in one bison. Pathological changes such as an oedema, hyperaemia and effusion in the abomasum and duodenum mucosa were most clearly seen in the calves that were highly infected. These changes probably lead to chronic diarrhoea, deterioration and deaths of young animals.

Relationships among ecology, demography and diseases of European bison (Bison bonasus).

The bison population in the Bialowieza Forest in Poland has now grown to approximately 300, while the herds in the Belarusian part of the forest total about 240 bison. The first signs of a health problem in these herds appeared in 1980, when two cases of balanoposthitis were detected in two bulls (2 and 5 years of age). Since 1980 research has been conducted to explain the cause of diseases, particularly balanoposthitis, and to monitor the health of bison in Bialowieza Forest. A total number of 614 bison (294 male and 320 female) of different ages was eliminated between 1980 and 2000. Not all the culled bison were examined (postmortem analysis, histopathological, bacteriological, virological and toxicological examinations, serological tests, molecular research). Based on the increase in numbers, reproduction in this population for the past 21 years is generally considered successful. Among 182 male bison eliminated during 1990-2000, only 85, or 47%, of the animals had balanoposthitis. Thus, the percentage of balanoposthitis cases went from 100% during the 1980s down to 47% in the past decade. It appears that the culling process has been a major factor leading to this decrease. It can be assumed that a set of factors is involved in the appearance of the disease (Corynebacterium spp., Bacillus sp., Pseudomonas aeruginosa, Escherichia coli, Ureoplasma spp, Fusobacterium necrophorum, Streptoccocus spp., Staphyloccocus spp.) while opportunistic infections including nematodes (Onchocerca spp.) are responsible for the occurrence of secondary lesions.

[Infection of red deer with lung nematodes in Polish and Belorussian parts of Bialowieza Forest]. / Zarazenie jeleni nicieniami plucnymi w Polskiej i bialoruskiej czesci Puszczy bialowieskiej.

Fecal samples from red deer from polish and belorussian parts of Bialowieza Forest were examined by Baermann method. First stage larvae of three species of lung nematodes were found. In polish part in 100% of samples were larvae of E. cervi, in 50% V. sagittatus and in 21% D. noerneri, and in belorussian the level of infection was lower and larvae were in 92%, 36% and 12% of samples respectively. In significant part of samples were met mixtured invasion of two or three species. Finding of larvae of E. cervi is the first registration of this species in belorussian part of Bialowieza Forest, and V. sagittatus is a new species for the fauna of Byelorussia. Nematodes E. cervi are dangerous for domestic ruminants.

[Travassosius rufus (Trichostrongylidae)--new for Poland nematode of beavers and comments about helminthfauna of these animals]. / Travassosius rufus (Trichostrongylidae)--nowy dla Polski nicien bobrów i uwagi o helmintofaunie tych zwierzat.

Two females of beaver from north-east Poland were necropsied. Moreover, 15 samples of feaces were examinated. Both necropsied beavers were infected with 46 and 362 specimens of trematode Stichorchis subtriquetrus (RUDOLPHI 1814). In stomach of one beaver were found 2800 specimens of nematode Travassosius rufus Khalil, 1922. It was the first registration of this species in Poland. Coproscopical examination showed eggs of S. subtriquetrus in all 15 fecal samples and eggs T. rufus in 5 samples.

[Helminth fauna of free living European bison Bison bonasus (L.) in Bieszczady Mountains (Karpatian Mountains, Poland)]. / Helmintofauna zubrów Bison bonasus (L.) zyjacych na swobodzie w Bieszczadach (Karpaty, Polska).

Four free living European bison from Bialowieza/Caucasus line shot in February 1997 within Lutowiska Forestry District in the Bieszczady Mountains were used in investigations. All examined animals were infected with helminths. There were found 10 species of parasites. Five species of them Ostertagia leptospicularis, O. kolchida, Spiculopteragia boehmi, Cooperia pectinata and Ashworthius sidemi are thypical parasites of Cervides, and from them were infected bison in Bieszczady. In all necropsied animals was found invasion of nematode A. sidemi, with mean intensity 1542 specimens. It is the first registration of A. sidemi in Poland and European bison is a new host for this parasite. It seems that the source of infection A. sidemi in European bison in Bieszczady is the local population of deer, which during their migrations could bring this parasite from neighbouring Ukraine and Slovakia, where this nematode have been introduced together with deer Cervus nippon. There are also presented results of coproscopic examinations of 17 fecal samples from free living European bison in Bieszczady.

[Attempt of deworming of red and fallow deer in deer farm with rintal premix]. / Próba odrobaczenia cielat jeleni i danieli w hodowli fermowej preparatem rintal premix.

Rintal 2.4% premix (Bayer) in single dose 7.5 mg of active substance on kg body weight was given to the calves of red and fallow deer. Effectiveness of deworming against nematodes from family Trichostrongylidae was 85.2% in red and 97.8% in fallow deer. Against namatodes from genus Trichocephalus the effectiveness was higher extending in red deer 98.1%, and in fallow deer 99.5%. Anthelmintic was not effective against nematodes Varestrongylus sagittatus and Elaphostrongylus cervi.

[Occurrence of microfilariae Dipetalonema rugosicauda (Bohm and Supperer, 1953) in deer and selected hunting grounds]. / Wystepowanie mikrofilarii Dipetalonema rugosicauda (Böhm et Supperer, 1953) u sarn w wybrancych lowiskach.

Examinations were carried out in Bialowieska Forest, Borecka Forest, Lower Silesian Forest, woods in Olsztyn District, Slowinski National Park, and in experimental hunting areas of the Forest Research Institute in Ilawa and of the Warsaw Agricultural University in Rogów. Samples of the skin from the ear and nose were taken from 136 shot roe deers. Skin samples were cut into 1-2 mm pieces, incubated in a physiological solution in 25 degrees C for 24 hours and examined for the presence of microfilariae. Microfilariae of Dipetalonema rugosicauda were found in roe deers in all examined hunting grounds. Prevalence of invasion oscillated from 6.6% to 57.1%. The wide dispersion of D. rugosicauda in Poland is caused by common occurrence in our country of the intermediate host and vector of this nematode--tick Ixodes ricinus.

[Occurrence of larvae of Protostrongylidae family in terrestrial snails in the woods of Olsztyn and Silesian districts]. / Wystepowanie larw nicieni z rodziny Protostrongylidae w slimakach ladowych w lasach olsztynskich i slaskich.

During the years 1992-1993 terrestrial snails were examined to the infection with larvae of Protostrongylidae family in the South and North of Poland. Using compressor method 2267 terrestrial snails from two areas (Pszczyna and Ilawa) were examined. Collected gastropods belonged to the following species: Arion subfuscus (45 specimens), Bradybaena fruticum (9), Cepaea nemoralis (29), Succinea putris (2147), Zonitoides nitidus (37). Larvae of three species of Protostrongylidae family were found: Elaphostrongylus cervi, Varestrongylus sagittatus and V. capreoli. The highest extensiveness of invasion of Protostrongylidae was noticed in Succinea putris and reached 23.5% in one of plots in the Ilawa Forest District. The highest intensity of infection was found in S. putris and was as follows: E. cervi up to 150 larvae, V. sagittatus up to 41, and V. capreoli up to 132 larvae. Besides Z. nitidus (1 specimen) there were not noticed any larvae of Protostrongylidae in other species of terrestrial snails. Average extensiveness of invasion of Protostrongylidae in 1992 and 1993 amounted to 7.2 and 9.5% in Ilawa, and 13.9 and 9.4% in Pszczyna, respectively. The highest percentage of infested gastropods was noticed in June and July. Similar results were obtained in both biotopes examined.

[Long term survival of first stage larvae of Elaphostrongylus cervi (Nematoda, Metastrongyloidea) in a frozen state]. / Dlugotrala przyzywalnosc larw i stadium Elaphostrongylus cervi (Nematoda, Metastrongyloidea) w stanie zamrozenia.

Faeces of red deer from Bialowieza Forest containing first stage larvae of Elaphostrongylus cervi have been stored during five years in -20 degrees C. After this time 52% of the larvae survived. These larvae were fully alive and in experimentally infected snails Helix pomatia developed to the infectious stage.

[Attempt of experimental infection of sheep by invasive larvae of Elaphostrongylus cervi (Nematoda, Metastrongyloidea)]. / Próba doswiadczalnego zarazenia owiec larwami inwazyjnymi Elaphostrongylus cervi (Nematoda, Metastrongyloidea).

Three 1 year old sheep were infected per os with 1000, 2000, and 13,000 invasive larvae of Elaphostrongylus cervi. Animals did not show any clinical symptoms of invasion. No larvae of this nematode were found in the samples of faeces during the experiment. During post mortem examination of infected sheep no parasites or their larvae were found in tissues, and no pathological lesions caused by parasites were observed.

[New details on the morphology of Onchocerca lupi (Nematoda, Filarioidea)]. / Nowe szczególy morfologii Onchocerca lupi (Nematoda, Filarioidea).

Basing on the fragments of typical specimens new details of morphology of O. lupi females are given. The presence of cuticular thickenings in the form of half rings, with the bifurcated or turned ends is stated. On the surface of cuticula of microfilariae there is very delicate regular transversal striation. The morphometrical features of O. lupi with descriptions of close species were compared.

[Large-intestinal nematoda in bison]. / Nicienie jelita grubego zubrów.

In the Bialowieza Forest the parasitological autopsy of large intestine of 6 bisons was made at the beginning of winter of 1987-1988, and 9 dissections were made in the latter part of both seasons. Four species of nematodes were found to occur together: Chabertia ovina, Oesophagostomum radiatum, Oe. venulosum and Trichocephalus ovis. At the end of wintering in bisons the invasion of Ch. ovina appeared in high percent of animals, but the intensity of it was low. However, the extensity and intensity of invasion of three remaining species of nematodes distinctly arose. In comparison with bisons in closed reserves, free living bisons assimilated Oe. venulosum and Ch. ovina. It is likely that the winter quarters are the source of invasion. It can be explained by the permanent accumulation of feaces with invasive forms of nematodes, and by feeding bisons on the great amount of hay, in which the invasive forms of nematodes may also occur.

[Harboring of helminth parasites by the European bison (Bison bonasus L.) and deer (Cervidae) in the Bialowieza forest]. / Ksztaltowanie sie helmintofauny zubrów (Bison bonasus L.) i jeleniowatych (Cervidae) w Puszczy Bialowieskiej.

Parasitological autopsy of 31 european bison , 9 red-deer and 3 roe-deer was made in Bialowieza Forest in the period 1983-1988. Together 20 species of nematodes were found, from them as much as 18 in bison , 6 in red-deer and 8 in roe-deer . The highest intensity and 100% of extensity was shown by nematodes from subfamily Ostertagiinae. Four species of this subfamily (O. leptospicularis, O. kolchida, S. boehmi i S. mathevossiani) have been permanently adapted by bison from cervids, however two species of nematodes typical for bison and cattle (O. ostertagii and O. lyrata) have not been adopted by deer . Besides, bison adopted from deer the following species of nematodes: T. capricola, C. pectinata, N. roscidus, N. europaeus and from moose N. alcidis. Bison gave to red-deer only C. bilobata, and roe-deer have not adopted any nematodes from bison . B. trigonocephalum and S. dagestanica found in roe-deer are in Poland typical parasites of moose .

[Occurrence of tissue Filarioidea in ruminants in Poland]. / Nicienie tkankowe z nadrodziny Filarioidea wystepujace u przezuwaczy w Polsce.

In the years 1985-1988 in the Bialowieza Forest and in the woods in the vicinity of Olsztyn, 130 deer, 136 roe-deer, 52 bisons and 40 cattle were subject to parasitological section. Seven species of tissue nematoda of the superfamily Filarioidea were detected. Onchocerca jakutensis, O. garmsi and Cutifilaria wenki in deer, Dipetalonema rugosicauda in roe-deer, and Onchocerca lienalis and O. gutturosa in bison and cattle are first records of these species in Poland. The bison is a new host for O. lienalis and O. gutturosa, the roe-deer--for O. flexuosa.

[Experimental infection of 2 species of laboratory rodents with invasive larvae of Elaphostrongylus cervi (Nematoda, Metastrongyloidea)]. / Doswiadczalne zarazenie larwami inwazyjnymi Elaphostrongylus cervi (Nematoda, Metastrongyloidea) dwóch gatunków gryzoni laboratoryjnych.

Single doses (from 300 to 1000 larvi per an animal) of invasive larvae E. cervi Cameron, 1931, obtained from experimentally infected snails Helix pomatia L. were given to 17 guinea pigs and 17 golden hamsters. Clinical nervous symptoms in the form of paresis and paralysis of limbs occurred only in the guinea pigs which were given a dose of 1000 larvi. These animals died in the period from the 75th to 117th day of infection. From their central nervous system single adult males and females of E. cervi were isolated. In the lungs and mesenteries of 2 dead pigs live larvae of E. cervi were found. This fact proves that the guinea pig can fulfil the role of a final and a paratenic host of E. cervi. No clinical symptoms were noticed in any hamster. In hamsters dissected on the 7th day of infection live larvae of E. cervi were found in the mesentery and in the fleshy part of the diaphgram. After 14 days the larvae found both in the mesentery and in the diaphragm were dead and surrounded by cellular infiltration. A strong tissue reaction of the hamster after the administration of E. cervi larvae is responsible for the larvae destruction and resorption.