Tick-borne pathogens in questing adults Dermacentor reticulatus from the Eastern European population (north-eastern Poland).

Dermacentor reticulatus is tick species with an expanding geographical range in Europe, which creates the possibility of spreading microorganisms of significant veterinary and medical importance. The study aimed to investigate the prevalence and genetic diversity of Rickettsia spp., Babesia spp., Borrelia spp. and Anaplasma phagocytophilum in adult D. reticulatus ticks from the Eastern European population in the urban and the natural biotopes of north-eastern Poland. Microorganisms were detected by PCR and identified by DNA sequencing. The overall infection rate of at least one of the pathogens was 29.6%. The predominantly was Rickettsia spp. (27.1%) (with R. raoultii-9.1%) followed by Babesia spp. (2.4%) with B. canis (1.5%) as the most frequent. Based on 18S rRNA gene sequence, three B. canis genotypes were revealed. The prevalence of R. raoultii and B. canis was significantly higher in ticks from natural biotopes. The infection rates of B. afzelii and A. phagocytophilum were determined at 0.9% and 0.3%, respectively. Co-infections were detected in 3.8% of infected ticks. In diagnosing tick-borne diseases in humans, tick-borne lymphadenopathy should not be excluded. The prevalence of different genotypes of B. canis suggests differences in the clinical picture of canine babesiosis in the area.

Cystidicola farionis, a Swim Bladder Parasite of European Smelt: Characterization of the Nematode Trehalose Strategy.

The molecular identification of Cystidicola farionis (a swim bladder nematode of European smelt from the Vistula Lagoon in Poland) was performed. Their prevalence level was determined, and changes in the trehalose synthesis pathway in larvae and adult nematodes were demonstrated. The trehalose level was almost four times higher in adult nematodes than in larvae. In contrast, the activity of both enzymes (trehalose 6-phosphate synthase, TPS and trehalose 6-phosphate phosphatase, TPP) involved in the synthesis of trehalose was higher in larvae than in adults under optimal conditions. The optimum pH for TPS isolated from larvae and adults was pH 7.0. The optimum pH for TPP from larvae and adults was pH 7.0 and pH 8.0, respectively. The optimal temperature was 20 °C, and Mg2+ ions were an activator for trehalose-synthetizing enzymes from both sources. Enzymes isolated from adult nematodes were less susceptible to divalent ion chelator and inorganic phosphate than larval enzymes. The dynamic transformation of trehalose in the nematode developing inside the swim bladder of the smelt appears to be an important metabolic pathway in the nematode survival strategy. These studies are aimed at a better understanding of the issue of the metabolic adaptation of parasites, which, in the future, may indirectly contribute to the elimination of the parasite from aquacultures, which will impact public health.

The Risk of Exposure to Ticks and Tick-Borne Pathogens in a Spa Town in Northern Poland.

The aim of this study was to determine the potential risk of human exposure to tick-borne infection in a recreation areas in a spa town located in northern Poland. Questing Ixodes ricinus and Dermacentor reticulatus ticks were collected in the spring of 2018. Tick-borne microorganisms were detected by PCR. Species were identified based on RFLP and the sequencing of DNA. In total, 38.3% of the ticks (34.6% of I. ricinus and 48.6% of D. reticulatus) were infected. The prevalence was 14.9% for Borrelia spp., 10.6% for Babesia spp. and 17.7% for Rickettsia spp. No Anaplasma phagocytophilum was detected. Spirochaetes B. afzelii, B. garinii and B. burgdorferi s.s. were detected only in I. ricinus ticks (20.2%). The differences in the infection rates of Babesia spp. between I. ricinus (7.7%) and D. reticulatus (18.9%) were not significant. DNA of B. canis and B. venatorum were identified in both tick species. B. microti were detected in D. reticulatus ticks. The prevalence of Rickettsia spp. was significantly higher in D. reticulatus (37.8%) than that in I. ricinus (10.6%). R. raoultii was identified only in D. reticulatus and R. helvetica in I. ricinus. Co-infections of at least two pathogens were recognized in 13% of positive ticks.

Questing Ixodes ricinus ticks (Acari, Ixodidae) as a vector of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in an urban area of north-eastern Poland.

Green areas located within large cities, as natural ecotypes, are a convenient habitat for ticks and their use as recreational areas is associated with the potential risk of acquiring tick-borne diseases. This study estimated the I. ricinus tick density, prevalence of infection with Borrelia species and the diversity of these bacteria in a green urban area (Olsztyn) of north-eastern Poland, an endemic region of tick-borne diseases. The ticks were collected during spring and autumn of 2015, at sites differing in the degree of human pressure and habitat. Borrelia species detection, typing and a molecular phylogenetic analysis were carried out based on the sequenced flaB gene. The overall mean abundance of I. ricinus was 2.0 ± 1.55 ticks per 100 m2. The density of I. ricinus did not vary significantly between sites. According to semi-qualitative tick abundance categories, the collection sites were classified as 'very low' and 'low' tick abundance category. The overall infection rate of I. ricinus with Borrelia spirochaetes was 27.4%. The infection rate of adult ticks (42.0%) was three times higher than with nymphs (14.3%). Based on the restriction patterns and sequencing, B. afzelii (93.1%; 27/29), B. valaisiana 3.5% (1/29) and B. miyamotoi (3.5%; 1/29), related to the relapsing fever (RF) spirochaetes, were detected. No co-infections were found. Borrelia miyamotoi, detected for the first time in ticks in the north-eastern urban areas of Poland, was identical to isolates described as European-type. The Borrelia spirochaete infection rate of I. ricinus ticks in an urban area indicated a high risk of LB. Physicians should also be aware of B. miyamotoi infections among patients with a history of tick-bites in north-eastern Poland.

Dermacentor reticulatus ticks (Acari: Ixodidae) distribution in north-eastern Poland: an endemic area of tick-borne diseases.

Dermacentor reticulatus is the second most important tick species in Poland. Although the north-eastern region of Poland is considered typical for D. reticulatus and is treated as a contiguous area of Eastern populations of the meadow ticks, the occurrence of this tick species in this region to date has been recorded as separate foci. The present report supplements data on the geographical distribution of D. reticulatus in urban and natural biotopes of north-eastern Poland (Warmia and Mazury province). In 2015-2017 (during the springtime activity of ticks) adult questing D. reticulatus were found in 13 of 25 monitored localities. Six sites are located in urbanized areas, within the administrative borders of the city of Olsztyn and seven sites are in natural biotopes in the central part of Warmia and Mazury. A total of 398 adult D. reticulatus ticks, including 257 females and 141 males, were collected. A comparison of data grouped according to urban and natural type of area revealed no statistical differences between them. Taking into account the habitat type, the mean tick density was the highest in open landscapes. The identification of new foci D. reticulatus in the endemic areas of Lyme borreliosis, tick-borne encephalitis and canine babesiosis is crucial for determining the risk of diseases transmitted by ticks and taking proper preventive measures.

Helminths of European smelt Osmerus eperlanus (Linnaeus, 1758) in Lake Hancza and the Vistula Lagoon, with special regard to their zoonotic threats.

Reports published in recent years on the increased risk in the Baltic Sea of fish infection with larvae of nematodes potentially pathogenic to humans have prompted a study of European smelt Osmerus eperlanus with respect to the presence of these parasites in the Vistula Lagoon. Additionally, samples of this fish from Lake Hancza were comparatively considered. The body cavity, the surface of internal organs, the alimentary tract and the swimbladder of fish from two environmentally different water bodies were taken into account in the analysis. Only the tapeworm Proteocephalus longicollis was found in the alimentary tract of the fish from Lake Hancza. Depending on the period of study, the prevalence ranged from 60% to 100%, and the mean intensity from 11.5 to 42.0. The helminth fauna of smelt from the Vistula Lagoon was more diverse: Cystidicola farionis occurred with a prevalence from 25.9 to 75.0% and a mean intensity of infection from 2.7 to 66.0%, Proteocephalus longicollis and cystacanths of Corynosoma with a prevalence ranging in different years from 12.0 to 44.0% and from 14.8 to 50.0%, respectively. Zoonotic parasites, such as nematode larvae of the Anisakidae family (Contracaecum sp., Anisakis simplex), occurred in the intestine with a prevalence of 31%, 72% and 22% in consecutive years of the study.

Anisakidae in beluga whales Delphinapterus leucas from Hudson Bay and Hudson Strait.

A total of 190 nematodes was isolated from the stomachs of 13 beluga whales Delphinapterus leucas from the Arctic part of Hudson Bay and Hudson Strait. Infection intensity ranged from 1 to 57 specimens and prevalence was 84.62%. Morphological examination of the nematodes revealed the presence of 3 species: Pseudoterranova decipiens sensu lato, Contracaecum osculatum s.l., and Anisakis simplex s.l. Molecular analysis by PCR-based restriction fragment length polymorphism (PCR-RFLP) resulted in the identification of 4 species: Pseudoterranova bulbosa, Contracaecum osculatum A and C, and Anisakis simplex sensu stricto. The nematodes were present in 3 developmental stages: L3 (159 specimens), L4 (16 larvae), and adults (15 worms: 11 males and 4 females).

Effect of temperature, NaCl concentration and aeration of solutions on the survivability of II stage larvae of Contracaecum rudolphii Hartwich, 1964.

Determinations were carried out for the effect of temperature, NaCl concentration and aeration of solutions on the survivability of stage II larvae of Contracaecum rudolphii nematode. Stage II larvae hatched from egg shells were placed in tap water as well as in 1% and 3% solutions of NaCl. Part of the culture was run on Petri dishes and aerated each day. The second part was transferred into high beakers and left non-aerated. All cultures were run in three replications at temperatures of 4, 10, 20 and 30 degrees C. Microscopic observations of the survivability and activity of the larvae were conducted every day in all samples. The larvae incubated for 30 days at a temperature of 4 degrees C were viable and non-motile. The longest life span was noted in the case of the larvae incubated in the aerated 1% solution of NaCl at a temperature of 10 degrees C, which also recorded the lowest mobility. The shortest life span was reported for the larvae incubated in the non-aerated 3% solution of NaCl at a temperature of 30 degrees C. The results obtained in this study indicate that the II stage larvae of C. rudolphii may live both in both fresh and salt waters and a significant factor determining their life span is water saturation with oxygen.

Effect of isolation method of Contracaecum rudolphii Hartwich, 1964 (Ascaridida: Anisakidae) eggs on embryonic development and the number of larvae hatched.

This study tested the isolation of C. rudolphii eggs using various methods and evaluated their course of embryogenesis and the number of larvae hatched. The pressing of eggs from the earlier-isolated uteruses was conducted in Petri dishes using bent preparatory needles to obtain eggs for a control culture. The experimental cultures contained incubated mature females in a culture medium which had been homogenized and digested by proteases. In the first experimental cultures, the females were incubated for three days at 40 degrees C in 10-ml plastic flasks. Eage's medium with the addition of 1% pepsin (pH 2) and 20% heat-inactivated foetal bovine serum was used as an incubation fluid. In the second method, the suspension of females homogenized with a blender was centrifuged for 3 minutes at 1000 rpm, the supernatant was removed and the sediment was then rinsed with a PS. In the third method, an attempt was undertaken to collect eggs by digestion of mature females with 0.1% and 1% aqueous solutions of pepsin adjusted to pH 2.0 with 1N HCl, as well as in 0.1% and 1% solutions of trypsin prepared in a Sörensen buffer (pH 7.6). The suspension obtained after complete digestion and still containing eggs was purified from proteases by washing several times with PS. In turn, no eggs were isolated by using the method of incubation of females in a culture medium nor by digestion with pepsin. The method of homogenization of whole nematodes resulted in egg damage. The best method of egg isolation was digestion with a 0.1% solution of trypsin. When the digestion was conducted with a 1% trypsin solution, the arrestment of embryogenesis was observed in a considerable percentage of the eggs, whereas eggs thecae were left by 31% of the larvae.

Experimental infection of Carassius auratus (L., 1758) with the second stage larvae of the nematode Contracaecum rudolphii Hartwich, 1964.

Susceptibility of the goldfish to infection with the newly hatched second stage larvae of Contracaecum rudolphii was investigated under laboratory condition. A week after the larvae had been placed in the fish tank, the autopsied goldfish showed the nematodes in their intestine lumen. The majority of the larvae were dead. In week 2 and 3 of exposure, the larvae were being found exclusively in the intestinal wall; in week 4 and 5, the larvae, in addition to being present in the intestinal wall, were also found in the body cavity. No larvae were found in the fish autopsied in subsequent weeks.

Leeches (Hirudinida: Piscicolidae)--parasites of Antarctic fish from Channichthyidae family.

There has hitherto been very few research projects focusing on ectoparasites of Antarctic fishes. The presently reported study provides data on the prevalence and the intensity of leeches (Hirudinida: Piscicolidae) infecting fishes. The materials were collected in December-February 1986/87 off the Elephant Island, South Georgia, Joinville Island, and South Shetlands. The following leech taxa were recorded in the Antarctic fishes of the family Channichthyidae: Trulliobdella capitis (Brinkmann, 1947); Cryobdella antarctica Epstein, 1970; Nototheniobdella sawyeri Utevsky, 1997; and Cryobdella sp. The above findings constitute new geographic records from off Elephant and Joinville Island and South Georgia.

Seasonal activity of the common European tick, Ixodes ricinus (Linnaeus, 1758), in the forested areas of the city of Olsztyn and its surroundings.

UNLABELLED: Common European ticks, Ixodes ricinus, have been found in forest areas situated within the boundaries of big cities, and in areas changed by anthropogenic pressure. Monitoring I. ricinus, in the areas used by the public for recreation and leisure, makes it possible to assess the risk of infection with the diseases they transmit. The objective of this study was the comparison of the seasonal activity of ticks, I. ricinus, in the afforested areas of the city of Olsztyn and its surroundings. MATERIAL AND METHODS: This survey was conducted at sites located in the afforested areas of Olsztyn and within the Warminski Forest situated about 15 km from Olsztyn. Ticks, I. ricinus, were caught every 10-14 days from April to October 2001 by the commonly applied method of flagging. During each catch, the temperature (T) and relative humidity (%RH) was measured with a thermohygrometer, 1 m above the duff level. RESULTS: The tick population density at the Warminski Forest site was higher than that at the sites within the city limits (116.8 and 20.5 ticks per 100 m2, respectively). Two peaks of tick activity were recorded in this area--the spring peak in June and the autumn peak (much lower) in September. The activity of ticks in the afforested area within the city limits was much lower--only one (spring) peak occurred, which at most sites was observed in June. At all sites, included in the study, the peak activity of ticks was observed in April. CONCLUSION: The study suggests that the afforested areas outside the city are more dangerous to people than the area within the city because of the large number of ticks there.

Activity of selected hydrolases in excretion-secretion products and extracts of adult Contracaecum rudolphii.

BACKGROUND: Enzymes contained in excretion-secretion (ES) products of parasites released to the environment play multiple roles: they facilitate hatching and moulting of larvae, enable a parasite to migrate within tissues, inhibit blood coagulation, defend the parasite from host's immunological response, and enhance feeding and nutrition. The aim of the study was to determine hydrolase activity in ES products and extracts from adult Contracaecum rudolphii. MATERIALS AND METHODS: Adult nematodes were isolated from intestines of black cormorants nesting at Katy Rybackie (the Vistula Lagoon). Nematode batches of 30 individuals each were placed in 5 ml portions of antibiotic-enriched physiological salt solution and incubated for 24 hours at 37 degrees C. After incubation, the solutions containing ES products were collected and dialysed for 24 h at 4 degrees C against distilled water. Extracts were obtained by homogenising the nematodes with the physiological salt solution (0.9% NaCl). The homogenate was centrifuged for 10 minutes at 3000xg. Enzyme activities were assayed in the supernatant. The enzymatic activity in ES products and homogenates was determined with the API ZYM kit (Bio Merieux S.A., Lyon, France). Hydrolase activities were expressed in volumetric units (nmol) of the hydrolysed substrate. RESULTS: The nematode ES products showed 10 hydrolases to be active. The highest activity was that of esterases, except for lipase the activity of which was not detected. Among glucosidases, the highest activity was shown by alpha-glucosidase, much lower activities being typical of beta-galactosidase and N-acetyl-beta-glucosaminidase. The remaining glucosidases proved inactive. Among proteases, leucine arylamidase and valine arylamidase were found to be active only. The nematode extracts revealed activities of 15 hydrolases. The highest activity was typical of esterases. Among glucosidases, the highest activity was typical of alpha-glucosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase. Activities of the remaining glucosidases were much lower. No activity of alpha-galactosidase was detected. Among proteolytic enzymes, leucine arylamidase proved the most active, while activities of valine arylamidase and chymotrypsin were much lower. The remaining proteases revealed no detectable activity. CONCLUSIONS: The ES products of adult C. rudolphii were found to contain active hydrolases which may damage the epithelium lining the host's alimentary tract. Activities of almost all glucosidases in the parasite's extracts suggests that, like in most nematodes, the parasite's main energy source is derived from carbohydrate metabolism.

Occurrence and seasonal activity of European ticks Ixodes ricinus (Linnaeus, 1758) in the forest areas of Olsztyn.

A total of 667 ticks (82% nymphs, 10.5% females and 7.5% males) were caught at 7 selected sites within the Las Miejski in Olsztyn. The average population of ticks was 18.6 individuals/100 m2. Two peaks of tick activity were recorded: the spring peak in May and the autumn peak in August.

Activity of hydrolases in the reproductive system and in consecutive stages of Ascaris suum development.

The activity of hydrolases in the eggs isolated from the uterus of A. suum is in most cases similar to that in the reproductive system. The activity of hydrolases was low in the stages of cleavage and gastrulation, and it grew until the larva stage.

The effect of low temperatures on the development of eggs of Ascaris suum Goeze, 1782.

Eggs of A. suum were kept at -10 and -20 degrees C for 12 weeks and subsequently incubated in a thermostat at 28 degrees C. During the incubation, their development was checked every 5 days. It was found that storage at low temperatures slows down their development and reduces the number of eggs successfully completing their embryogenesis.

Effects of ivermectin and albendazole against Anisakis simplex in vitro and in guinea pigs.

The activity of ivermectin and albendazole against larval Anisakis simplex was tested in vitro and in experimentally infected guinea pigs. Before drug exposure the medium for half of the larvae was adjusted to pH 2.0 with 1 N HCl, whereas the other half was held at pH 7.0. To these solutions, ivermectin was added to full concentrations of 1, 2, 5, 10, 50, 100, or 200 microg/ml, and for albendazole, 300, 400, and 500 microg/ml. Animals from group I were given 0.1 ml of 1% (3.3 mg/kg) ivermectin, whereas guinea pigs from group II were each given 5-7 mg (16.6-23.3 mg/kg) of albendazole orally. The efficacy of both drugs against L, A. simplex was high in vitro and in vivo against the larvae in different organs of guinea pigs.

Influence of Anisakis simplex stage III larvae upon the activity of proteases under in vitro conditions.

The larvae of Anisakis simplex had the largest influence upon decreasing the activity of porcine pepsin. The activity of that enzyme in tests, where the larvae were present during the entire period of incubation, was lower than in the controls. No similar trends were observed in case of the solutions with bovine and porcine trypsin. The activity of those enzymes in the solutions containing the larvae was higher than in the controls. Only the activity of porcine trypsin after 10 h of incubation was slightly lower in the experimental sample than in the control, however, during the later hours the dynamics of the activity decrease of that enzyme in the controls was higher than in the experimental samples. The recorded activity of papain in the samples containing the larvae was higher than that in the controls during the entire time of the experiment.

Effect of different contents of proteins and vitamin B2 in the feed on the prevalence and infection intensity of Ascaridia galli in chickens.

A group of 10-day-old chickens was experimentally infected with a dose of 500 infective eggs of Ascaridia galli (Schrank, 1788) (Nematoda). Forty-nine days post infection the chickens were necropsied and the infection intensity and prevalence were determined. The group maintained on the feed containing 11% of protein showed higher infection intensity and prevalence compared to the chickens fed a diet containing 19% of protein. The addition of vitamin B2 to the feed containing 11% protein resulted in lower intensity and prevalence of the infection. In both series of experiments no effect of Zn-bacitracin or vitamin B2 on infection intensity or prevalence was observed for chickens fed feed containing 19% of protein.