The Complex Composition of Trans-resveratrol, Quercetin, Vitamin E and Selenium Inhibits the Growth of Colorectal Carcinoma.

BACKGROUND/AIM: Numerous studies have demonstrated an anti-cancer action of plant-derived polyphenols. Their action is mainly related to antioxidant, anti-inflammatory, immunomodulatory and inhibitory properties. It is expected that proper composition of nutrition factors with anti-cancer activity may prevent from cancer incidence or inhibit cancer progression. The aim of the study was to investigate the anti-cancer properties of a standardized composition of compounds: trans-resveratrol, quercetin, vitamin E and selenium (Neoplasmoxan, Vebiot) in a mouse model of CT26 colorectal carcinoma. MATERIALS AND METHODS: Colorectal carcinoma cells (CT26) were introduced subcutaneously (2×105/mouse) on the back of the mice. Neoplasmoxan suspension was administered intragastrically, daily, for 21 consecutive days. In collected tumors, the area occupied by tumor blood vessels and the number of immune cells; macrophages and CD8-positive cytotoxic T lymphocytes were evaluated. RESULTS: It was observed that administration of Neoplasmoxan inhibits the growth of colorectal carcinoma in mice. Tumor volume after Neoplasmoxan administration was 40% smaller than in control groups. No overall toxicity of Neoplasmoxan was observed. The area of blood vessels in tumors of mice that received Neoplasmoxan was reduced by approximately 20%. The area occupied by macrophages increased about 60% compared to the control group. However, no increased number of CD8-positive cytotoxic T lymphocytes was observed in the group that received Neoplasmoxan. CONCLUSION: A tendency of Neoplasmoxan to inhibit the growth of colorectal carcinoma was recorded. It also seems that additional combination of the tested preparation with standard chemotherapy or radiotherapy should bring a synergistic therapeutic effect.

Seroprevalence of Toxoplasma gondii infection in goats from the south-west region of Poland and the detection of T. gondii DNA in goat milk.

Toxoplasma gondii (Nicolle et Manceaux, 1908) is an obligatory intracellular protozoan parasite prevalent in animals and humans worldwide having medical and veterinary importance on account of causing abortion or congenital disease in intermediate hosts, including man. Since T. gondii has already been identified in the milk of goats, Capra aegagrus hircus (Linnaeus), the possibility of acquiring infection by ingesting unpasteurised goat milk should be taken into consideration. Thus, the aim of the present study was to determine the presence of T. gondii DNA in goat milk. First, 73 goats (females) from 36 farms located in Poland were examined serologically by direct agglutination test (DAT) to estimate the T. gondii serological status. Milk samples from 60 selected lactating females were examined for the presence of T. gondii DNA by Real time PCR and nested PCR (B1 gene). To estimate the clonal type of detected T. gondii, multiplex PCR was performed using 6 markers. In DAT, positive results were found in 70% of 73 goats. Among examined 60 milk samples, 65% were positive in Real time PCR and 43% in nested PCR. It is noteworthy that 11 samples positive in PCR were collected from seronegative goats. The multilocus PCR analysis mostly revealed the occurrence of genotype III, which is relatively rare in Europe. The recorded high prevalence of anti-Toxoplasma antibodies in tested goats (70%), associated with a high prevalence of T. gondii DNA in goat milk samples (65%), indicates a potential risk of the parasite transmission through goat milk ingestion.

Genetic diversity of Echinococcus multilocularis in red foxes in Poland: the first report of a haplotype of probable Asian origin.

The aim of the present study was to estimate the genetic diversity of the cestode Echinococcus multilocularis Leuckart, 1863 in Poland based on sequence analysis of the mitochondrial genes of worms isolated from red foxes, Vulpes vulpes (Linnaeus). Overall, 83 adults of E. multilocularis from the same number of foxes in different parts of Poland were used for analysis. Sequences of the three mitochondrial genes, cytochrome b (cob), NADH dehydrogenase subunit 2 (nad2) and cytochrome c oxidase subunit 1 (cox1), were analysed. Seventy-four individual biological samples were successfully sequenced. Combined sequence analysis of these three genes exhibited fifteen Polish haplotypes (EmPL1-EmPL15). Most isolates (n = 29; 39%) were classified to the EmPL1 haplotype, which occurred mainly in the east, north and centre of Poland. Haplotype EmPL4 (n = 14; 19%) and other haplotypes appeared predominantly in the south and west area. Fourteen haplotypes were grouped in the European clade. One Polish haplotype (EmPL9) (n = 7, 10%) was assigned to the Asian clade with haplotypes from Japan and Kazakhstan. This haplotype was found only in northeast Poland and this is the westernmost report of haplotype of E. multilocularis belonging to the Asian clade in Europe. The investigation demonstrated that populations of E. multilocularis in Poland (and probably also in eastern Europe) included not only different European haplotypes but also those of the Asian origin.

Epidemiological survey in Leczynsko-Wlodawskie Lake District of eastern Poland reveals new evidence of zoonotic potential of Giardia intestinalis.

Faecal samples from 297 farm animals were collected from 18 households in distinct sites of the Leczynsko-Wlodawskie Lake District of eastern Poland. They included samples from 86 cattle (Bos taurus), 84 pigs (Sus scrofa f. domestica), 81 sheep (Ovis aries), 10 horses (Equus caballus), and 36 dogs (Canis lupus familiaris). The samples were examined for the presence of Giardia intestinalis by the Direct Fluorescence Assay (DFA) and semi-nested PCR. All amplicons were sequenced on both strands. By DFA, cysts of Giardia spp. were detected in 66 of 297 faecal samples (22.2%). Positive specimens for Giardia spp. were derived from 29.8% of examined pigs, 21.0% of sheep, 18.6% of cattle, 10% of horses, and 19.4% of dogs. Based on the detection of the ß-giardin gene by PCR, 39 (13.1%) of the 297 examined samples were recognized as positive. Detection of the presence of Giardia cysts by DFA test was overall significantly higher compared to PCR (p=0.0045). By PCR, Giardia was found in 28.1% of sheep, 11.6% of cattle, 10% of horses, 9.5% of pigs and 5.6% of dogs. Partial ß-giardin gene sequences were obtained for 73.7% of the PCR positive samples. From sequenced samples derived from the studied animals, Giardia were identified as assemblage A (8 samples), B (1 sample) and E (18 samples). As assemblages A and B may be zoonotic, the farm animals living in eastern Poland could be regarded as a potential source of Giardia infection for humans.

Prevalence and molecular typing of Giardia duodenalis in wildlife from eastern Poland.

Faecal samples from 162 wild animals were collected from 32 distinct sites of Leczynsko-Wlodawskie Lakeland (eastern Poland). The presence of Giardia duodenalis (Stiles, 1902) was assessed by a Direct Fluorescence Assay (DFA) and by Polymerase Chain Reaction (PCR) and sequencing of a fragment of the beta-giardin gene. DFA showed the presence of cysts of G. duodenalis in 12 of 162 faecal samples (7%), namely in four wild boars (15%), four foxes (19%), two roe deer (4%), and two wolves (29%). PCR identified 34 of the 162 (21%) samples as positive, including 11 wild boars (41%), five red deer (18%), 11 roe deer (23%), four moose (17%), two wolves (29%) and a single sample from the European badger. Thus, PCR detected a significantly higher number of infection than DFA (P = 0.0005). However, 14 of 34 PCR products could not be sequenced because of their insufficient amount; the low number of cysts, poor conservation of the faeces or presence of PCR inhibitors may have contributed to weak DNA amplification. Sequence analysis of the remaining 20 products showed the presence of assemblage B in wild boars, red deer and roe deer, whereas samples from wolves were identified as assemblage D. This is the first detection of assemblage B in wild boars and deer. As assemblage B has zoonotic potential, wild animals from eastern Poland may act as reservoirs of cysts of G. duodenalis infectious for humans.

Assessment of viability of the nematode eggs (Ascaris, Toxocara, Trichuris) in sewage sludge with the use of LIVE/DEAD Bacterial Viability Kit.

Sewage sludges from wastewater treatment plants may contain live parasite eggs, which can be a source of humans and animals infection. According to the current rules, parasitological examination includes detection of the Ascaris spp., Trichuris spp. and Toxocara spp. eggs and estimation of their viability. The viability assessment based only on the incubation and observation of isolated egg is long and imprecise. The aim of this study was to develop sensitive and less labour-intensive methods for assessing viability of Ascaris spp., Toxocara spp. and Trichuris spp. eggs in sewage sludge. For this purpose, LIVE/DEAD Kit was used. Firstly, the possibility of distinguishing between live and dead eggs in water was assessed. Secondly, an appropriate amount of dyeing mixture needed to distinguish the live and dead eggs in the sewage sludge was determined using experimentally enriched samples and naturally contaminated samples of sludge. Eggs were isolated from the samples by own method which was a combination of flotation and sedimentation, preceded by a long mixing. After the last stage of the procedure, sediment containing the eggs of parasites was stained by LIVE/DEAD kit according to the manufacturer instructions, but with the use of different variants of dyes mixture concentration. The investigation showed that live and dead eggs of these three parasites could be differed by this method with the use of proper concentration of dyes. Live eggs were stained in green (Ascaris and Trichuris) and green-blue (Toxocara). However, all types of dead eggs were red coloured. The study demonstrated that after some modifications (resulted from the nature of the samples) the LIVE/DEAD kit is useful for assessing the viability of Toxocara, Ascaris and Trichuris eggs occurring in the sludge.

Occurrence of Cryptosporidium oocysts and Giardia cysts in effluent from sewage treatment plant from eastern Poland.

Cryptosporidium spp. and Giardia lamblia (synonyms: Giardia duodenalis, Giardia intestinalis) are emerging protozoa causing disease in humans and animals worldwide. These parasites can pose a serious threat to immunocompromised people, for whom the symptoms are more severe and may include abdominal pain, watery diarrhoea, nausea, headaches, malaise, and fever. One of the sources of these parasites can be treated wastewater from wastewater treatment plants (WTPs). Samples of treated wastewater (effluent), each of 10 L volume, were collected from 13 municipal WTPs located in eastern Poland. Cryptosporidium oocysts and Giardia cysts were separated by the immunomagnetic method. The presence and/or concentration of protozoan (oo)cysts in effluent samples were determined by direct immunofluorescent microscopy, nested PCR and Real Time PCR. Viability of (oo)cysts was determined by double-staining with the use of Live/Dead BacLight kit (Invitrogen). Cryptosporidium spp. oocysts were detected in 8 WTPs (61.5%) and Giardia spp. cysts in 11 WTPs (84.6%) by microscopic analysis. Both pathogens were detected in samples from 7 WTPs. Median concentrations of Cryptosporidium and Giardia (oo)cysts in 13 examined samples were 2.2/L and 6.6/L, respectively, while mean concentrations were 28.5/L and 113.6/L, respectively. In positive samples, Cryptosporidium oocysts concentrations ranged from 0.4 - 154.1 oocysts per litre, and Giardia cysts concentrations ranged from 0.7 - 660 cysts per litre. By nested PCR, Giardia DNA was detected in 4 samples of the 13 examined, (30.8%) while Cryptosporidium DNA was never detected. In Real Time PCR, positive results for Giardia were obtained in 5 samples (38.5%) and in none of the samples for Cryptosporidium, with the exception of one equivocal result. Viable (oo)cysts of Cryptosporidium and Giardia were detected in 3 out of 4 samples examined, in the ranges of 12.5 - 60% and 50 - 100% of total (oo)cysts, respectively. In view of our preliminary study, the presence of oocysts and cysts (largely viable) in effluents from WTPs imply a risk of transmission of waterborne protozoan parasites to humans. Therefore, additional wastewater purification procedures are necessary.