Assessment of molecular methods as a tool for detecting pathogenic protozoa isolated from water bodies.

Several species belong to the Cryptosporidium and Giardia genus, the main parasitic protozoa occurring in water, but only some of them are infectious to humans. We investigated the occurrence of Cryptosporidium and Giardia and identified their species in the water samples collected from natural water bodies in north-western Poland. A total of 600 samples from water bodies used for bathing, sewage discharge, as drinking water sources and watering places for animals were screened. The samples were collected during a 3-year period in each of the four seasons and filtered using Filta-Max (IDEXX Laboratories, USA). Genomic DNA was extracted from all samples and used as a target sequence for polymerase chain reaction (PCR) and TaqMan real-time PCR, as well as for reverse line blotting (RLB) methods. PCR methods seem to be more sensitive to detect Giardia and Cryptosporidium DNA in water samples than RLB methods. All PCR products were sequenced and three were identified as C. parvum and four as G. intestinalis. The overall prevalence of C. parvum (0.5%) and G. intestinalis (0.6%) in the samples suggests that the risk of Cryptosporidium and Giardia infections in north-western Poland is minimal.

Recovery of DNA of Giardia intestinalis cysts from surface water concentrates measured with PCR and real time PCR.

The most important restriction for the detection in water samples is the low concentration of Giardia intestinalis cysts, additional difficulty is the presence of PCR inhibitors. We have carried out trials in order to assess the sensitivity of semi-nested PCR and TaqMan real time PCR on the basis of DNA extracted from G. intestinalis cysts coming from spiked environmental and distilled water samples, filtrated with the use of Filta-Max® equipment (1623 Method). Removal of inhibitors was carried out with addition of BSA in different concentrations. During the filtration and concentration of water samples, losses of cysts have been recorded. Moreover, addition of BSA to the PCR and real time PCR mix increases the sensitivity of reaction. The optimal concentration of BSA for semi­nested PCR was 15 and 20 ng/µl, whereas for real time PCR 5 ng/µl.

Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR.

The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR). Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts' wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 degrees C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN)--T kit--with an all night long incubation with proteinase K in 56 degrees C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of beta-giardin gene fragment and C(T) values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100% cases, was 100 cysts per 200 microl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.

Methods for parasitic protozoans detection in the environmental samples.

The environmental route of transmission of many parasitic protozoa and their potential for producing large numbers of transmissive stages constitute persistent threats to public and veterinary health. Conventional and new immunological and molecular methods enable to assess the occurrence, prevalence, levels and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne cysts and oocysts. These steps have been optimized to such an extent that low levels of naturally occurring (oo)cysts of protozoan can be efficiently recovered from water. Ten years have passed since the United States Environmental Protection Agency (USEPA) introduced the 1622 and 1623 methods and used them to concentrate and detect the oocysts of Cryptosporidium and cysts of Giardia in water samples. Nevertheless, the methods still need studies and improvements. Pre-PCR processing procedures have been developed and they are still improved to remove or reduce the effects of PCR inhibitors. The progress in molecular methods allows to more precise distinction of species or simultaneous detection of several parasites, however, they are still not routinely used and need standardization. Standardized methods are required to maximize public health surveillance.

An electron microscopic study of the phosphatases in the ciliate Balantidium coli.

The localisation and activity of D glucose-6-phosphatase (G-6-Pase) and alkaline phosphatase (AlP) in the trophozoites of Balantidium coli isolated from pig intestine content were investigated using ultrastructural and cytochemical methods. The activity of G-6-Pase was demonstrated on the membranes of the endoplasmic reticulum, particularly in the cortical part of the trophozoites. In addition, the product of the reaction to G-6-Pase was concentrated in the vesicular structures, which were distributed along the reticular membranes. These structures were described as vesicles similar to glycosomes, containing enzymes of glycogenolysis. It is very likely that hydrolases in B. coli are formed on the rough reticular membranes without the involvement of cisterns of the Golgi complex. The ultrastructural deposits of the reaction to G-6-Pase and AlP in the trophozoites of B. coli described here indicate that some membranes of the rough endoplasmic reticulum and small vacuoles with a strong reaction to these enzymes can play a similar role to the Golgi complex.

Molecular evidence of coinfection of Borrelia burgdorferi sensu lato, human granulocytic ehrlichiosis agent, and Babesia microti in ticks from northwestern Poland.

To assess the potential risk for tick-borne agents, Ixodes ricinus were collected from 2 sites in northwestern Poland. The ticks were tested by polymerase chain reaction for coinfection with Borrelia burgdorferi sensu lato (s. l.), human granulocytic ehrlichiosis (HGE) agent, and Babesia microti. Of the 533 processed ticks, 16.7% were positive for B. burgdorferi s. l., 13.3% for B. microti, and 4.5% for the HGE agent. Twenty ticks were coinfected with 2 or 3 of the pathogens.

Isolation and amplification by polymerase chain reaction DNA of Babesia microti and Babesia divergens in ticks in Poland.

Babesia microti and B. divergens, the etiological agents of human babesiosis, are transmitted by the bite of Ixodes ricinus. The purpose of this study was differentiation of those two species in ticks collected in urban woods in the city Szczecin (north-western Poland). The prevalence of the DNA of Babesia were investigated by PCR amplification with primers to the fragment from a gene encoding the nuclear small-subunit ribosomal RNA (SS-rDNA). We examined a total of 533 specimens of Ixodes ricinus. The mean infection rate was 16.3%. Our results indicate that a B. microti and B. divergens--specific PCR test may provide a sensitive tool also for the laboratory diagnosis of human babesiosis.

[Prevelance of the etiological agent of human ehrlichiosis (HGE) in ticks from west-north Poland]. / Wstepne badania czynnika etiologicznego ludzkiej ehrlichiozy (HGE) w kleszczach z zachodniopólnocnej Polski.

Ehrlichias occur in ticks in the cells of their haemolymph-hematocytes. They enter the vertebrate host organism with the saliva of the tick, during a blood meal. Humans can also be the hosts for this pathogen. Two pathogens cause a humane disease-monocytic ehrlichiasis (E. chaffensis) or granulocytic ehrlichiasis (HGE factor). The above disease units are difficult to diagnose because of their non-specific symptoms. A preliminary study has been conducted on the prevalence of the HGE factor in the ticks, Ixodes ricinus in the recreational areas of the West-Pomeranian Province. All forms of I. ricinus were collected from 3 sites. All the sites are known to be frequented by hikers and gatherers of forest mushrooms and berries. The site selection involved also careful consideration of the tree- and underbrush type. The ticks were collected twice a year in spring (May/June) and in autumn (August\September), which was associated with the biological activity of the collected acarines. A total of 1159 Ixodes ricinus ticks were collected, in this number 172 females, 167 males, 597 nymphs, and 223 larvae. Using the PCR technique, the 16SrRNA-gene fragment was amplified using primers specific for the HGE factor: EHR 790 and EHR 521. The studied population contained 3.7% infected females in spring and 2.7% in autumn, 0.68% infected males in spring, no infected in autumn. The nymphs were infected in spring (2.17%) and in autumn too (0.73%), but the larvae were not infected in both seasons. Analysing the above-mentioned results it can be concluded that the decisive majority of the individuals transmitting the HGE factor are the adult forms. The present study was only a preliminary one. In the future much more sites will be monitored, in the recreational areas of both the city of Szczecin and the entire province.

[Babesosis--difficulty of diagnosis]. / Babesjoza--trudnosci diagnostyczne.

Human babesiosis is caused predominantly by B. microti and B. divergens, a protozooan parasites of red blood cells. Both are transmitted by Ixodes ricinus ticks, also the primary vector of Lyme disease. Clinical manifestation varied widely from asymptomatic infection to a serve rapidly fatal disease. The diagnosis of babesiosis include examination of stained blood smers, serological evaluation indirect antibody tests and PCR. With the evolution PCR--based techniques, the diagnosis and monitoring of babesial infections became more sensitive and reliable.

The occurrence DNA of Babesia microti in ticks Ixodes ricinus in the forest areas of Szczecin.

Babesia microti, an intraerythrocytic protozoan and the etiological agent of human babesiosis, is transmitted by the bite of the tick, Ixodes ricinus. The aim of the present study was to confirm the presence of B. microti by detection of the DNA of these protozoans. The prevalence of B. microti was studied using the PCR method with primers complementary to the gene fragment encoding nuclear small-subunit ribosomal RNA (ss-rDNA). In the course of this study a total of 2095 ticks, Ixodes ricinus, were examined. The mean infection rate was 6.2%. Variable prevalance values were also obtained from six different locations and they were further modified by the seasons of the year. The results confirmed the competence of I. ricinus as a vector of B. microti and that a B. microti-specific PCR can provide a sensitive test for laboratory detection of babesiosis.

[Use of polymerase chain reaction (PCR) for detection of tick Borrelia burgdorferi sensulato in screening studies]. / Zastosowanie lancuchowej reakcji polimerazy (PCR) do wykrywania kretków Borrelia burgdorferi sensu lato w badaniach przesiewowych.

Within the last few years, the incidence of Lyme disease has rapidly increased in Europe, with the causative agent of the disease is Borrelia burgdorferi--a spirochete. In Poland, Lyme borreliosis is being identified, mainly, based on the clinical symptoms, epidemiological anamnesis, and serological tests. On the other hand, it is evident from the foreign publications, that in many cases representing different phases of Lyme disease, a reliable and totally accurate identification tool of Borrelia burgdorferi is amplification of bacterial DNA using PCR method. The main goal of the present studies has been implementation of the DNA amplification method into diagnostic procedures of Lyme. Although we dealt with DNA of B. burgdorferi isolated from tics, it would not make a difference because the method of DNA isolation is the same for human samples. The results acquired from the preliminary studies, suggest that amplification of a fragment of fla gene, may be useful in Lyme disease diagnostics. Spirochetes of B. burgdorferi sensu lato were detected in tics Ixodes ricinus using PCR method in both individual animals and tick pools. The latter version of the method seems to be very useful in so called screening studies, because of minimizing the cost and duration of the procedure.

[Genetic diversity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in north-west Poland]. / Genetyczna zmiennosc Borrelia burgdorferi s. l. u kleszczy Ixodes ricinus zebranych w pólnocnozachodniej Polsce.

Borrelia burgdorferi sensu lato (s. l.), the etiological agent of Lyme diesease, is transmitted by the bite of Ixodes ricinus. During May and September 1999, field surveys on Lyme disease spirochetes were conducted in three locations of a region of north-west Poland, known as recreational districts visited by many people. The ticks Ixodes ricinus were collected in natural habitats by dragging a flanel cloth over the vegetation. Sex and developmental stage of each tick were determined. Based on a polymerase chain reaction test with primers that recognize a chromosomal gene of all strains, out of the total 1414 specimens collected, 126 (8.9%) were found to be infected. The species B. burgdorferi s. l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s. s.), Borrelia garinii, and Borerelia afzelii, witch could be distinguished in nested-PCR tests with species-specific primers. B. burgdorferi s. s. was most prevalent (96% of infected ticks), followed by B. garinii (1.3%), and B. afzelii. was not found. Of the infected ticks, over the 99% were infected with a single species, one specimens was infected with two species. For 4 ticks, the infecting species could not be identied. The difference in rates of prevalence was observed among the tree locations (17%--5.3%--3.2%).

[The occurrence of Ixodes ricinus in the selected recreative areas in the province of Szczecin. Part II]. / Wystepowanie Ixodes ricinus na wybranych terenach rekreacyjnych b. województwa szczecinskiego. Czesc II.

The aim of the study was to estimate the occurrence (the quantative and rate percent) of Ixodes ricinus in the popular recreation areas in Szczecin (Arkonka, Osów, Glebokie, Landscape Park of Szczecin, Dabie Forest Park, Zdroje Forest Park) and in province of Szczecin (Forest of Goleniów, Insko, Pobierowo, Chojna). Investigations were performed in 1998 year, twice on each places; in May/June and repeted in September/October. The temperature and humidity of air were measured. Obtained specimens were regard of sex and growing stage during each collection. A total of 2.055 specimens collected 49% were nymphs, 13.9% female, 11.3% male and 25.8% larvaes. The nymphs the most frequently were in spring when humidity of air was 55% and temperature 24 degrees C. The larvaes, in autumn were most frequently (31.4%) then in spring (20.5%) when the temperature of the air was 18-22 degrees C, and the humidity from 60 to 85% during the collections.

[Isolation of Borrelia burgdorferi sensu lato in ticks Ixodes ricinus by polymerase chain reaction (PCR)]. / Wykrywanie Borrelia burgdorferi sensu lato w kleszczach Ixodes ricinus metoda lancuchowej reakcji polimerazy (PCR).

Attempts were made to identify the causative orgamsm of Lyme disease in Szczecin from tick Ixodes ricinus as a vector. Ticks were collected in 1997 year in forest areas of Szczecin, from localites associated with numerous attendance of people. The method used in this study was the polymerase chain reaction (PCR) on the flagellin structural gene fla of Borrelia burgdorferi sensu stricto. The flagellin PCR primer set reaction was conservative for B. burgdorferi sensu stricto, B. afzelii and B. garinii. The overall prevalence of B. burgdorferi sensu lato, in tick population studied was 8.8%. The female, nymphs and larves of Ixodes ricinus were infected almost just the some--about 10%, when the male 2.5% only.

The formation of primary and secondary lysosomes in Balantidium coli, Ciliata.

Trophozoites, vegetative forms of Balantidum coli isolated from pigs affected by acute and asymptomatic balantidiasis were studied. Lysosomes and food vacuoles were revealed by cytochemical detection of lysosomal marker, acid phosphatase. The cytoplasm of all the B. coli trophozoites examined was found to contain numerous structures which differed widely in shape, size and location in the cells. One of them was located among the rough endoplasmic reticulum membranes and another one in the vicinity of endosomes. Those structures were regarded as the primary lysosomes. The two types of vesicular structures most probably represent two stages of the primary lysosome formation. Trophozoites were also found to contain secondary lysosomes which are formed by fusion of several primary lysosomes with phagosomes. The ultrathin sections of B. coli trohozoites showed the presence of two types of phagosomes. They were divided, based on their contents, into auto- and heterophagosomes.

[The occurrence of Ixodes ricinus in the select recreative areas in the Province of Szczecin. Part I]. / Wystepowanie Ixodes ricinus na wybranych terenach rekreacyjnych województwa szczecinskiego. Czesc I.

Among Polish ticks species the most common Ixodes ricinus has the biggest medical importance. Within the last few years, the incidence of disease transmitted by ticks has rapidly increased. We have made a thorough analysis of the quantative and rate per cent of occurrence of various stages Ixodes ricinus in the forest areas of some places in Szczecin province and in the parks of Szczecin, that are known as highly recreative and frequently visted by many people. A total of 426 (68% numphs) specimens collected there show that ticks frequently occupy habitats closely associated with man.

Cytochemical identification of mucocysts in Balantidium coli trophozoites.

The mucocyst ultrastructure in B. coli has not been described so far. As demonstrated in this work, cytoenzymatic assays on B. coli with the use of a reaction-detecting membrane-coupled hydrolase, i.e., ATP-ase, permitted identification of the mucocysts in the ciliate studied. The shape, size, and location of mucocysts in B. coli trophozoites were found to correspond to descriptions of these structures in other ciliates. The mucocysts were more numerous in B. coli trophozoites isolated from the symptomatic balantidiosis-affected pigs (Group I), and the product of reaction to ATP-ase was more copious than in Group II trophozoites. However, not all the bubble-like structures with similar morphological features reacted positively to the enzyme. The discrepancy was explained by the cytoenzymatic reaction to Beta-GR. The reaction product was visible in the vesicular structures, situated above the plasmolemma, although some of them contained no reaction product. Thus the presence of two types of secretory structure can be inferred: the mucocysts, with ATP-ase in their membranes, and other extrusomes containing active Beta-GR.

[Growth of Balantidium coli populations under in vivo and in vitro conditions]. / Wzrost populacji Balantidium coli w warunkach in vivo i in vitro.

Trophozoites of Balantidium coli were isolated from the pig's caecum and cultivated in vitro. Pigs were divided into two groups: one with the acute balantidiosis and the second--with the asymptomatic balantidiosis. In the first case the biotic potential of protozoans turned out to be higher (the most intensive divisions occurred 24 hours after the second passage). Protozoans isolated from pigs with the asymptomatic balantidiosis had lower biotic potential (the most intensive divisions occurred 24 hours after the third passage). This fact indicates the differentiation of the investigated populations of B. coli.

Ultrastructural and cytochemical identification of peroxisomes in Balantidium coli, Ciliophora.

Peroxisomes of the trophozoites of Balantidium coli isolated from pig intestine content were investigated, using ultrastructural and cytochemical techniques. The peroxisomes of B. coli trophozoites from pigs with subclinical balantidiasis are less then 0.8 mm in diameter whereas those from pigs with acute balantidiasis are greater than 0.8 micron in diameter. In all the trophozoites peroxisomes are round, oval or dumb-bell shaped. Catalase as an indicative enzyme was detected by cytochemical techniques in B. coli peroxisomes.

A comparison of nucleic acid content in Balantidium coli trophozoites from different isolates.

Cytophotometric assays were performed on Balantidium coli trophozoites isolated from 30 pigs affected by acute balantidiasis (Group I) and from 30 pigs with symptom-free balantidiasis (Group II). Trophozoites from cultures obtained from Group I and II pig isolates were assayed for comparison. Comparative cytophotometric studies on nucleic acids of B. coli trophozoites isolated from acute and symptomless balantidiasis-affected pigs as well as from in vitro cultured trophozoites showed differences which could have resulted from differences between populations in the trophozoans under investigation.