Colorectal cancer and Blastocystis sp. infection.

BACKGROUND: Blastocystis sp. is a common intestinal protozoan found worldwide. Based on gene analysis, 17 subtypes (STs, ST1-ST17) have been identified, 9 of which have been isolated from humans. Differences in clinical consequences may depend on differences among the STs. Here, we evaluated the prevalence of Blastocystis sp. in patients with colorectal cancer (CRC) compared to a control group and assessed the relationships between Blastocystis sp. infection and sex; age; and CRC grade, stage, and location. METHODS: The study included 107 CRC patients (41 women and 66 men, median age 65 years); 124 subjects without colorectal cancer or a history of oncological disease comprised the control group (55 women and 69 men, median age 63). Stool samples were collected from patients before oncological treatment and examined using light microscopy (iodine-stained smear). Additionally, PCR-based identification of Blastocystis sp. was performed in 95 stool samples from CRC patients and 76 stool samples from the control group. RESULTS: Light microscopy showed that the prevalence of Blastocystis sp. was significantly higher in CRC patients than in the control group (12.15% and 2.42%, respectively; p = 0.0041). Multivariate analysis showed that the odds of Blastocystis sp. infection were fivefold higher in the CRC group than in the control group. PCR-based molecular examinations demonstrated that the proportion of patients infected with Blastocystis sp. was significantly higher in the CRC group than in the control group (12.63% and 2.63%, respectively; p = 0.023). The predominant ST in the CRC group was ST3, detected in nine patients (75%), followed by ST1 (2 patients, 16.7%) and ST2 (1 patient, 8.3%). No association was found between Blastocystis sp. infection and age, sex, or CRC stage, grade, or location. CONCLUSIONS: The results showed that CRC was associated with an increased risk of opportunistic Blastocystis sp. infection, even before oncological treatment. To the best of our knowledge, this is the first report estimating the prevalence of Blastocystis sp. infection in CRC patients before oncological treatment in Europe.

Acanthamoeba - pathogen and vector of highly pathogenic bacteria strains to healthy and immunocompromised individuals.

Acanthamoeba is a free-living protist pathogen, which is present in every place on Earth. 50 to 100 percent of the adult population has serum antibodies, specific for Acanthamoeba antigens. Acanthamoeba is an etiological agent of keratitis and encephalitis diagnosed in human. Acanthamoeba keratitis occurs in healthy persons and may lead to visual impairment and blindness, because corneal infection with this parasite fails to induce cell-mediated immune response due to the absence of resident antigen-presenting cells in the cornea. Systemic immunization with Acanthamoeba antigens induces Th1 cell-mediated immunity and serum IgG antibody, but do not prevent the development of keratitis. Immunization via mucosal surfaces stimulates IgA antibodies in tears and protects against the development of keratitis. Amoebae feed mainly on bacteria, fungi, and algae. By transferring intracellular bacteria, amoeba contributes to the spread of diseases dangerous to humans. Some microorganisms have evolved to become resistant to protist, since they are not internalized or able to survive, grow, and exit free-living protists after internalization. In many cases, the bacteria inside living amoebae survive longer, and multiply better, showing higher virulence. There is a hypothesis, which assumes that Acanthamoeba and symbiontic bacteria survive and multiply better in moist soil, rich in nitrogen compounds, particularly in the vicinity of the root systems of Alnus glutinosa, infected with nitrogen-fixing bacteria Frankia alni. Impact of soil environment created by nitrogen-fixing bacterium Frankia alni on specific relations between protists Acanthamoeba and highly pathogenic bacteria strains in Alnus glutinosa habitats in Poland continue to be established.

The role of companion animals in the environmental circulation of tick-borne bacterial pathogens.

Ticks are known as vectors of a wide range of pathogens of medical and veterinary importance; some of them of zoonotic concern constitute a hazard for the emergence of tick-borne diseases shared between humans and domestic animals and becoming a part of the 'One Health' concept. Canine and feline tick-borne diseases have emerged in recent years, performing an extensive geographic distribution and enlarged global prevalence. The present review focuses on the recent epidemiological studies on the emergence of tick-borne bacterial pathogens in dogs and cats, and the discussion whether pet ownership increases the risk of tick-borne diseases. A lot of data provide confirmation that dogs and cats themselves may substantially contribute to the circulation of the ticks and tick-borne bacterial pathogens in the environment. Molecular diagnostics of tick-borne pathogens infections generates a lot of problems like the choice of molecular methods and molecular markers for the detection of bacterial genomic DNA, but play an important role in the diagnosis of infections. The study provides some insight into molecular diagnostic techniques and new potentially recognized bacterial pathogens of this group. Protecting human and companion animal health from vector-borne infections requires controlling vector populations, containing development of novel, practicable strategies that will limit vectors and transmission of vector-borne disease pathogens.

Genetic diversity and pathogenicity of Blastocystis.

Blastocystis is a unicellular, anaerobic protist which lives in the intestinal tract of diverse animals, including humans. It was found that the host specificity and the pathogenic potential of different isolates are correlated with sequence variations in the SSU-rRNA gene. Identification of the organism to the species level is still an unresolved challenge. Genetic diversity revisions have led to the identification of 17 subtypes (STs) within the Blastocystis genus, and 9 (ST1 to ST9) have been reported in humans with varying prevalence. Since the members of the genus revealed a large genetic diversity, several molecular modalities of subtyping methods have been developed. Numerous studies on conveying the pathogenic potential to the molecular subtypes are available, but they could not be compared or analysed with the different molecular techniques employed. The use of different approaches may give false positives during diagnosis and the possibility of missed infections. A review of recent scientific literature indicates that the development of PCR assays is needed for molecular epidemiology and for mixed infections in health and disease cohorts, and also to help identify sources of Blastocystis transmission to humans, as well as to identify potential animal and environmental reservoirs. This review summarizes some of the recent progress and improvements in Blastocystis research on genetic diversity, taxonomy, molecular epidemiology, pathogenicity and subtyping methods.

Molecular detecting of piroplasms in feeding and questing Ixodes ricinus ticks

The purpose of this study was to detect piroplasms, which are pathogens of veterinary and zoonotic importance in ticks, that were collected from ponies and field vegetation and to determine the role of Shetland ponies as potential reservoir hosts for piroplasms. A total of 1737 feeding and 371 questing Ixodes ricinus collected from horses or vegetation were tested for the presence of Babesia and Theileria DNA. Piroplasm 18S rRNA gene amplification was conducted, and the obtained amplicons were sequenced. Babesia DNA was detected in only three ticks (one tick collected from a pony and two collected from vegetation), and all of the obtained sequences had 100% similarity to B. divergens. Theileria DNA was not present in the examined ticks. Thus, the above results indicate that ponies are probably not essential hosts for the detected species of piroplasms. Piroplasm species typical for horses (Babesia caballi and Theileria equi) were not detected because I. ricinus is not their vector. The low infection rate of I. ricinus with B. divergens shows that the disease risk for the local horse population and people associated with pony horses is low, but it demonstrates their possible role as a source of human infection in northern Poland.

Molecular evidence for Toxoplasma gondii in feeding and questing Ixodes ricinus ticks.

The aim of the present study was to detect Toxoplasma gondii in ticks collected from ponies and field vegetation and to determine the role of Shetland ponies as a potential reservoir host for T. gondii. A total of 1737 feeding Ixodes ricinus collected from 49 horses and 371 questing ticks were tested by PCR and sequencing for the presence and genotyping of T. gondii. All ticks were examined in a previous study to detect and identify pathogenic bacterial species. The aim of this study was also to detect co-infection of ticks with these bacteria and T. gondii. Genotyping of the sequenced B1 gene revealed that detected T. gondii strains represented genotype I, which is pathogenic for humans. T. gondii genotype I was detected in 4.5% of all I. ricinus, including in 2.99% of feeding ticks and in 10.24% of questing ticks; this difference was statistically significant. Thus, the above results indicate that ponies probably are not an essential host for the detected sporozoan. Infections with more than one pathogenic species were rare and involved mostly T. gondii and Borrelia burgdorferi sensu lato. Our results confirmed the presence of T. gondii in I. ricinus and showed a new geographical habitat of T. gondii occurring in I. ricinus ticks in Poland.

The role of ticks in transmission cycle of Toxoplasma gondii

Toxoplasmosis is globally distributed, water- and food borne zoonosis caused by the single protozoan Toxoplasma gondii and probably one-third of the world's human population is infected with this pathogen. Domestic and wild felids are definitive hosts of this pathogen and intermediate hosts for great variety of other homoeothermic animals. Human as other of the intermediate hosts may become infected in the main route of infection; it is the ingestion of parasite oocysts in contaminated water or soil and undercooked meat. However, the mechanism which this parasite uses to so large spread is not fully understood, because oral transmission does not explain the common event of this parasite in a variety of hosts, such as herbivorous animals or rodents and birds, as well as routes of spread to domestic hosts. Such a wide circle of hosts suggests a possibility of other paths of transmission and a role of ticks, the bloodseeking arthropods was considered in the transmission of T. gondii.

Identification of host blood-meal sources and Borrelia in field-collected Ixodes ricinus ticks in north-western Poland.

Forest animals play fundamental roles in the maintenance of Ixodes ricinus and Borrelia species in the forest biotope. To identify the forest vertebrate species that are host for I. ricinus and for the recognition of the reservoirs of Borrelia species, the blood-meal of 325 I. ricinus ticks collected at two forest sites in north-western Poland were analysed. Nested PCR was used to detect polymorphisms in a fragment of the mitochondrial 12S rRNA gene for the identification of the hosts species. The products were digested with the restriction enzymes, a combination that allows the identification of 60 vertebrate species, comprising 17 bird, 4 reptile and 39 mammalian species. Host DNA was detected in 244 (75%) I. ricinus individuals, with the species being detected and classified for 210 (86%) samples. The restriction patterns resulted in the identification of 14 vertebrate species, including 2 species of birds, lizard, badger, rabbit, deer; most of the samples contained DNA from wild boar (Sus scrofa), red fox (Vulpes vulpes), red deer (Cervus elaphus) and roe deer (Capreolus capreolus). Identification of Borrelia species was based on the flaB gene using nested PCR coupled to RFLP. This method allows the identification of all Borrelia species transmitted by I. ricinus in Europe, including B. miyamotoi and 3 genetic variants of B. garinii. In the studied isolates, 2 species belonging to B. burgdorferi sensu lato were identified--B. garinii and B. afzelii, and B. miyamotoi, which are related to relapsing fever borreliae.

Molecular evidence for bacterial pathogens in Ixodes ricinus ticks infesting Shetland ponies.

Ixodes ricinus has the potential to transmit zoonotic pathogens to humans and domestic animals. The feeding I. ricinus (n = 1737) collected from 49 Shetland ponies and questing ones from vegetation (n = 371) were tested for the presence and differentiation of the bacterial species. DNA of I. ricinus ticks was examined with PCR and sequencing analysis to identify species of Borrelia burgdorferi sensu lato (Bbsl), Anaplasma phagocytophilum and Rickettsia spp. Altogether, 24.3 % I. ricinus of the infested horses and 12.4 % ticks from vegetation carried at least one pathogen species. Horse-feeding ticks (19.2 %) were significantly more frequently infected with Borrelia spp. than questing ticks (4.8 %). Among Bbsl species, in I. ricinus infesting ponies, B. garinii, B. afzelii, B. burgdorferi sensu stricto, B. valaisiana and B. lusitanie and one species, B. miyamotoi related to relapsing fever group, were detected. The 73 flaB gene sequences of Borrelia obtained from feeding I. ricinus have been deposited in GenBank. Among Rickettsia species, two were identified: R. helvetica which was dominant and R. monacensis. Infections with more than one pathogenic species, involving mostly Bbsl and R. helvetica were detected in 6.3 % of infected ticks collected from horses. Shetland ponies may play an important role in the epidemiological cycle of Bbsl and probably could contribute to the natural cycle of A. phagocytophilum and R. helvetica as host for infected ticks. The awareness about these infectious agents in ticks from ponies might be an important criterion for the risk assessment of human diseases, especially as these animals are maintained for recreational purposes.

Why is There Still no Human Vaccine Against Lyme Borreliosis?

Lyme disease, transmitted by ticks, is a complex illness that can be difficult to diagnose but easy to treat in most early cases, yet difficult in its latest stage. Every year, infections with Borrelia burgdorferi sensu lato spirochetes cause thousands of new cases of illness around the world, including people with a normal immunological reaction. Prevention in the form of vaccines is difficult due to e.g. very high variability of Borrelia antigen proteins, which precludes the construction of an effective vaccine. After the withdrawal of the OspA vaccine (LYMErix) in the USA, despite promising results, no vaccine protecting humans against all pathogenic species from the B. burgdorferi s.l. group is available. Recent data indicate that an effective vaccine may require a combination of several antigens or multiple epitopes based on vector-borne proteins and several outer membrane proteins of Borrelia. With the discontinuance of Lyme vaccines, personal protective behavior and the avoidance of exposure in high-risk areas remain necessary resources of prevention.

Differentiation Borrelia Species in Environmental Samples with High-Resolution DNA Melting Analysis.

BACKGROUND: Borrelia burgdorferi sensu lato includes at least 20 species in the world, and half of these are found in Europe. The usefulness of high resolution melting (HRM) analysis of DNA denaturation curves has been assessed for differentiation of Borrelia species. METHODS: HRM protocol for Borrelia species was used to examine the 77 DNA extracts selected from earlier studies with the use of three different molecular markers: flaB, rplL, and groEL. RESULTS: The studies revealed that the best marker is the groEL gene, which enables identification of 8 Borrelia species, including B. miyamotoi from the relapsing fever borreliae group and 7 of B. burgdorferi s.l. complex (B. garinii, B, afzelii, B. burgdorferi s.s., B. valaisiana, B. lusitaniae, B. bissetii, B. spielmanii). CONCLUSIONS: The HRM method, when compared with other PCR variants with regard to the reduced time of analysis, is an alternative for the procedures used in the molecular diagnostics of borreliosis including testing of blood samples or saved Ixodes ticks for the presence and genotyping of Borrelia burgdorferi after biting a patient.

Thermophilic potentially pathogenic amoebae isolated from natural water bodies in Poland and their molecular characterization.

The free-living amoebae (FLA) may live in the environment and also within other organisms as parasites and then they are called amphizoic. They are potentially pathogenic for humans and animals and are found in water that is a source of infection. The aim of this study was molecular detection and identification of these FLA in natural water bodies in North-Western Poland to evaluate the risk of the pathogenic amoebae infections. We examined surface water samples collected from 50 sites and first, the tolerance thermic test was performed in order to select thermophilic, potentially pathogenic strains. For molecular identification of FLA, regions of 18S rDNA, 16S rDNA and intergenic spacers were amplified. Acanthamoeba T4 and T16 genotypes of 18S rDNA gene and 18S rDNA of H. vermiformis were detected. We identified two variants of Acanthamoeba T4 genotype, two variants of Acanthamoeba T16 genotype and one variant of H. vermiformis. Identification of the T16 genotype and H. vermiformis in water was for the first time in Poland. Additionally, we made attempts to adapt the RLB method for detection and differentiation of FLA species and strains. PCR seems to be more sensitive than RLB hybridization, though.

Why are there several species of Borrelia burgdorferi sensu lato detected in dogs and humans?

Borrelia burgdorferi sensu lato is a group of spirochete bacteria species some of which cause borreliosis in humans and dogs. Humans and dogs are susceptible to illness from many of the same tick-borne pathogens, including B. burgdorferi s.l. (Bbsl). Little is known about the pathogenic role of the species of Bbsl in canines. The molecular methods which detect and amplify the DNA of borreliae and allow differentiating borreliae species or strains have not been used in canine diagnostics yet. Until now, it has been believed that in European dogs, like in humans, at least three pathogenic species occur but the most frequently described symptoms may be associated with the infection caused by B. burgdorferi sensu stricto species. A dog as well as a human is a host for many species of Bbsl, because borreliacidal ability of serum of dogs and humans is evident only in certain genospecies of Bbsl. Therefore both a dog and a human harbor more species than in case of some wild animal species which create older phylogenetic Bbsl species-host systems and these animals may act even as a non-competent reservoir host. Apart from many genospecies of Bbsl, a dog harbors other tick-borne agents and dual or triple infections may occur.

Host and pathogen DNA identification in blood meals of nymphal Ixodes ricinus ticks from forest parks and rural forests of Poland.

DNA analysis of blood meals from unfed nymphal Ixodes ricinus allows for the identification of tick host and tick-borne pathogens in the host species. The recognition of host species for tick larvae and the reservoirs of Borrelia, Rickettsia and Anaplasma species were simultaneously carried out by analysis of the blood meals of 880 questing nymphal I. ricinus ticks collected in forest parks of Szczecin city and rural forests in northwestern Poland that are endemic areas for Lyme borreliosis. The results obtained from the study indicate that I. ricinus larvae feed not only on small or medium animals but also on large animals and they (i.e. roe deer, red deer and wild boars) were the most prevalent in all study areas as the essential hosts for larvae of I. ricinus. The composition of medium and small vertebrates (carnivores, rodents, birds and lizards) provided a more diverse picture depending on study site. The reservoir species that contain the most pathogens are the European roe deer Capreolus capreolus, in which two species of Rickettsia and two species of Borrelia were identified, and Sus scrofa, in which one Rickettsia and three Borrelia species were identified. Rickettsia helvetica was the most common pathogen detected, and other included species were the B. burgdorferi s.l. group and B. miyamotoi related to relapsing fever group. Our results confirmed a general association of B. garinii with birds but also suggested that such associations may be less common in the transmission cycle in natural habitats than what was thought previously.

Rapid identification of Borrelia by high resolution melting analysis of the groEL gene.

This study examined the possibility of applying a new diagnostic method, high resolution analysis of DNA denaturation curve (high resolution melting - HRM), for identification of Borrelia species. DNA samples were obtained from Ixodes ricinus ticks collected from vegetation and removed from hunted roe deer. For differentiation of Borrelia species, the HRM protocol based on the analysis of the groEL gene was applied. A product characteristic for Borrelia was obtained in 19/123 samples (15.4%). The studied isolates were classified as four species: B. garinii, B. valaisiana, B. afzelii and B. miyamotoi. Two separate groups of isolates within the B. afzelii species were also found. The results show that the groEL gene is useful for rapid differentiation of B. burgdorferi sensu lato with the HRM method from different extracts of DNA and it also allows precise differentiation of Borrelia species and strains. The HRM method shortened and simplified detection and differentiation of Borrelia species from different biological sources.

Association of the ADRB2 Gly16Arg and Glu27Gln polymorphisms with athlete status.

The ß-adrenergic receptors (ß-ARs) have known functional roles in cardiovascular and pulmonary responses as well as the appropriate substrate metabolism required for athletic ability. Thus, the ß-AR genes are plausible candidates for the variations observed in strength/power and endurance performance levels. The aims of the present study were to compare the frequency distribution of the ADRB2 Gly16Arg and ADRB2 Glu27Gln polymorphisms among athletes of sports with different metabolic and cardiopulmonary demands (endurance vs. strength/power) and to test the association between the Gly16Arg and Glu27Gln genotypes and athlete status. The study was performed in a group of 223 Polish athletes of the highest nationally competitive standard (123 endurance-oriented athletes and 100 strength/power athletes). Control samples were prepared from 354 unrelated, sedentary volunteers. The χ² test of independence revealed that the frequencies of the Gly16 and Glu27 alleles were significantly higher in the strength/power athletes than in the controls (69.0% vs. 59.7%; df = 1, P = 0.017 and 51% vs. 41.5%; df = 1 P = 0.017, respectively). The study showed that ADRB2 Gly16Arg and Glu27Gln markers are associated with athlete status in Polish athletes. An excess of Gly16 and Glu27 alleles and the Gly16:Glu27 haplotype observed in the strength/power athlete subgroup suggests that the Gly16 and Glu27 alleles might increase the probability of becoming a strength/power athlete rather than an endurance-oriented athlete.

PCR and real time PCR for the detection of Cryptosporidium parvum oocyst DNA.

Three DNA extraction kits were used, all without preliminary procedures, then DNA extraction was preceded with freeze/thaw cycles in three versions. A lack of desired effect resulted in the application of liquid nitrogen/water bath cycles before the use of the extractions in further experiments. The effectiveness of DNA extraction was measured by PCR signal and C(T) values of real time PCR. A comparison of the efficiency of various Cryptosporidium parvum undiluted oocyst treatments prior to DNA extraction with the use of three kits has shown that the best results were obtained after extraction of DNA with the QIAamp DNA Tissue Mini Kit (T kit), preceded by triple liquid nitrogen/water bath in 100 degrees C for 2 minutes and with overnight proteinase K digestion. After extraction with the T kit, the detection limit was 50 oocysts per 200 microl when effectiveness was evaluated with PCR and 10 oocysts in the case of real time PCR.

Progress in the molecular methods for the detection and genetic characterization of Cryptosporidium in water samples.

Cryptosporidium, the protozoan parasite, has several transmission routes, including anthroponotic and zoonotic transmission, as well as the foodborne way, but mainly by water. The oocysts, the resistant stage produced by Cryptosporidium, are remarkably stable, and can survive for weeks or even months in the environment. Furthermore, the infective dose is low, probably even a single oocyst can cause infection. The Cryptosporidium genus includes at least 16 species; nevertheless, only a few can cause cryptosporidiosis, an intestinal disease in human and domestic mammals. Thus, the genetic characteristics of different Cryptosporidium species became fundamental in the diagnosis, monitoring, prevention and control of infections caused by this pathogen. Unfortunately, the traditional phenotypic techniques meet with difficulties in the specific diagnosis of cryptosporidiosis, therefore the new molecular tools must be applied. The RT -qPCR method can be used to differentiate viable and dead Cryptosporidium oocysts, and the LAMP assays have advantages for detection of organisms at relatively low concentration in environmental samples; however, the NAS BA assay specifically detects as few as one oocyst of a viable human pathogenic Cryptosporidium species. Reverse line blot hybridization (RL B) has been successfully used for specific identification and for differentiation of Cryptosporidium species. Described techniques are the most promising methods for the sensitive and accurate detection, but require a considerable selection of appropriate tools, genetic markers and analytical techniques for interpretations of database. However, the applicability of most of these methods to detect Cryptosporidium species or genotypes from environmental samples needs to be evaluated and standardized.

A comparative analysis of molecular markers for the detection and identification of Borrelia spirochaetes in Ixodes ricinus.

Borrelia burgdorferi sensu lato, carried by Ixodes ticks, is one of the most significant human pathogens, causing Lyme disease. As there is no standardized PCR method for detection and identification of spirochaete DNA, we carried out a comparative analysis using a set of complementary primers for three regions in the genomic DNA of these bacteria (genes fla and rrs and the non-coding rrs-rrlA region). DNA extracted from 579 Ixodes ricinus ticks was subjected to nested PCR. DNA of the examined spirochaetes was detected in 43 (7.4 %) lysates when the fla gene was used as a molecular marker, in 7 (1.2 %) lysates when using primers complementary to the rrs gene, and in 12 (2.1 %) lysates using primers complementary to the non-coding rrs-rrlA sequence. RFLP analysis based on the fla gene helped identify species from the B. burgdorferi sensu lato complex (B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana), detect co-infections, and also identify Borrelia miyamotoi. Therefore, the fla gene is the most sensitive and specific molecular marker for the detection and identification of Borrelia spirochaetes in I. ricinus.