Molecular studies enabling the recognition of the role of game and ticks in the circulation of pathogens transmitted by ticks and detection of coinfections in order to estimate a risk which a contact with tissues of roe deer, red deer and wild boar from north-western Poland brings were the aim of this research. DNA isolated from the blood and spleen of game and from Ixodes ricinus were the study materials. The results shows that Capreolus capreolus and Cervus elaphus play an important role in the life cycle of Anaplasma phagocytophilum, two Bartonella species, Theileria and Babesia spp. Whereas in the isolates obtained from 50 representatives of Sus scrofa, the DNA of only one pathogen, A. phagocytophilum occurred. 63.5% of 74 PCR+ isolates from Capreolus capreolus showed a double coinfection and three isolates -triple. In the tissues of Cervus elaphus, the coinfections were triple in 38% of individuals, double in 40%, single in 84%.

Summary: Comparison of effiCienCy of various dna extraCtion methods from Giardia intestinalis Cysts measured by pCr and taqman real time pCrThe 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 ˚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 ˚C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of β-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 µl 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.

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.

Ticks of the genus Ixodes are vectors for many pathogens, including Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp., and may also serve as vectors for Bartonella spp. However, the role of ticks in Bartonella transmission requires additional studies. The aim of this study was to investigate whether coinfection with two or more vector-borne pathogens can occur in the following three groups of dogs: I - dogs with suspected borreliosis (N = 92), II - dogs considered healthy (N = 100), and III - dogs with diagnosed babesiosis (N = 50). Polymerase chain reactions were performed to detect DNA of Anaplasma phagocytophilum, Rickettsia spp. and Bartonella spp. in the blood of dogs. In dogs of Group I, the DNA of both A. phagocytophilum and Bartonella sp. was detected (14% and 1%, respectively). In eight dogs, coinfection was indicated: A. phagocytophilum or Bartonella sp. with B. burgdorferi s.l. (the presence of antibodies against and/or DNA B. burgdorferi s.l.). In the case of five dogs positive for A. phagocytophilum DNA, no coinfection with B. burgdorferi s.l. was shown. In Group II, the DNA of A. phagocytophilum was detected in four dogs. In Group III, no pathogenic agents possibly transmitted by ticks were confirmed. No DNA of R. helvetica was detected in any of the groups studied.

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