Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar  but none of the 269 red deer . M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.Bovine tuberculosis (bTB) caused by  Mycobacterium tuberculosis complex (MTBC). The MTBC currently comprises M. bovis and M. caprae, the causal agents of bovine tuberculosis (bTB), M. microti, infecting mainly small wild mammals, M. pinnipedii, causing tuberculosis in marine mammals, M. mungi, recently described in mongooses, and the primarily human pathogens M. tuberculosis, M. africanum and M. canettiiTuberculosis is a chronic disease caused by bacteria of the M. bovis and, to a lesser extent, with M. caprae, constitutes a considerable obstacle to international cattle trade M. bovis infections are also of concern for the conservation of endangered species M. bovis and M. caprae have a zoonotic potential. In the European Union (EU), M. bovis accounted for 133 cases of human tuberculosis in 2009, with a case fatality rate of 5%, and sporadic cases of M. caprae infection in humans have been reported Bovine tuberculosis is a disease of global importance. Infection of livestock with Meles meles) and wild ungulates, respectively Eradication of bTB in cattle is hampered by the chronic nature of the disease and difficult in vivo testing A reservoir consists of a host population , within which a pathogen persists without the necessity of other species acting as external sources of infection  Trichosurus vulpecula) in New Zealand Syncerus caffer) in South Africa Odocoileus virginianus) in the USA Bison bison) and elk (Cervus elaphus manitobensis) in Canada Sus scrofa), red deer (Cervus elaphus elaphus) and fallow deer (Dama dama) have a reservoir status on the Iberian peninsula Among the best-known wildlife reservoirs for tuberculosis worldwide are the brushtail possum emerging disease in European wildlife, especially in wild ungulates e.g. . All couCapreolus capreolus capreolus), Alpine chamois (Rupicapra rupicapra rupicapra) and red deer M. bovis was absent in this region M. avium-intracellulare was isolated in all cases. However, the validity of this study was limited, as attempts to culture live organisms were restricted to these three samples that had macroscopic bTB-like lesions. In another study, attempts to culture M. bovis or M. tuberculosis from tissue pools of more than 320 farmed cervids from CH were unsuccessful In Switzerland (CH), the last documented cases of bTB in wildlife date back to the 1950′s, before the country officially gained TB-free status in 1960, and involved badgers, roe deer , no ethical approval or permit for animal experimentation was required.This study did not involve purposeful killing of animals. All samples originated from dead wildlife legally hunted during hunting season or legally shot because of severe debilitation. According to CH and FL legislation  and on the geographic proximity to neighboring countries where bTB had recently been reported in wildlife. CH is organized in political subunits (cantons) with different hunting regimes. The survey was carried out in the cantons of Geneva, Thurgovia, Saint Gall, Grisons and Tessin, and in FL . In the other areas, game wardens and hunters were asked to collect tissues and submit them to the FIWI immediately after collection. They were previously instructed for tissue identification and sample collection via lectures and demonstrations on carcasses. Tissue collection was carried out from the carcasses and organs after evisceration. Collected tissues per animal comprised the mandibular lymph nodes (ML), medial retropharyngeal lymph nodes (RL), palatine tonsils (PT), mediastinal lymph nodes (MedL), and the mesenteric lymph nodes (MesL). Sampling sets consisted of detailed sampling instructions, pre-labeled bags for each tissue, a pair of latex gloves, and a data sheet to gather information on species, sex, estimated age, body condition, date of death and location, and the presence of macroscopic lesions on the carcass. According to the age estimated by the submitter, animals were grouped into three categories: Juvenile (less than one year old), subadult (one to two years old) or adult (over two years old). Adequate packaging for mail delivery was provided, and shipping costs were covered by the project and the cantonal hunting offices.Calculation of sample size per species and sampling area was based on estimated population sizes derived from the regional hunting bags, and performed using WinEpiscope® 2.0 software In total, 434 free-ranging ungulates (165 wild boar and 269 red deer) were sampled and the targeted sample size was met or nearly met in all study areas . All reqWithin the existing national surveillance programs for wildlife health in CH and FL, game wardens and hunters are regularly encouraged to submit animals presenting disease signs or found dead to the FIWI for a post-mortem analysis free of charge. During the present project, the awareness of hunting authorities, game-wardens and hunters for bTB as a currently emerging disease was increased by articles published in hunting magazines and oral communications in the frame of courses and of the information campaign surrounding the cross-sectional study. Field partners were asked to systematically submit carcasses, organs or samples of all wild mammals presenting lesions suggestive of tuberculosis, independently of the species and geographic region.Capra ibex ibex) were included in the project: five animals were seen as potentially tuberculous by the submitter, and three were sent for bTB-unrelated routine diagnostics but presented tuberculosis-like lesions  at necropsy and were therefore sampled for further investigation. Tissue selection and collection for microbiological analyses were carried out according to the same protocol as for hunted, apparently healthy animals, except that organs with TBL were additionally collected. Organs presenting lesions were also systematically sampled for histology to determine the cause of disease. These samples were fixed in 10% buffered formalin and embedded in paraffin. Five-micron-thick tissue sections were obtained and stained with hematoxylin and eosin. Special stainings, including Ziehl-Neelsen, Gram, Grocott, and immunohistochemistry were applied to selected samples as needed. General bacteriological cultures were performed accordingly on selected samples following accredited protocols .Samples or carcasses of eight diseased animals  was evaluated macroscopically by qualified staff, either on-site if FIWI staff conducted sampling in the fields, or in the necropsy hall if samples were shipped by mail. Common lesions in tuberculous wild ungulates consist of caseo-granulomas of various sizes which can frequently become mineralized e.g., , or of pHalf of each lymph node and tonsil was pooled per animal for microbiological analysis. Between animals, instruments were thoroughly decontaminated with 5% Amocid®  and cutting board cover was changed. Subsequently, all samples were stored at −20°C and pools were sent frozen to the Swiss National Center for Mycobacteria at the Institute of Veterinary Bacteriology, University of Zurich, where microbiological analysis was performed.g and the pellet was frozen and preserved for PCR analysis. The remaining suspension was decontaminated for 15 minutes with 4 ml H2SO4 (4%) at room temperature, neutralized with 5.6 ml NaOH (1N) and buffered with 20 ml sterile phosphate buffered saline (PBS), according to document WHO/TB/98.258: Laboratory Services in Tuberculosis Control, Part III, Culture, pp. 37–42. After centrifugation for 20 minutes at 4000×g and 15°C, the supernatant was discharged and the pellet resuspended with 2 ml sterile PBS. Of this basal suspension, 0.5 ml were added into a vial of liquid medium BD BACTECTM MGITTM Tube  containing modified Middlebrook 7H9 broth base and an integrated fluorescent indicator. This mixture was enriched with 0.8 ml BD BACTECTM MGITTM 960-Supplement  containing PANTA  antibiotic mixture and growth supplement, and incubated in a BACTECTM MGITTM 320 incubator  at 37°C for eight weeks.All tissue handling was conducted in a laminar flow cabinet. A sterile set of surgical instruments and a new cutting surface were used for each pool. Of each tissue pool, about 2 g of material were taken for analysis, including gross lesions, if present. The tissue was mixed with 14 ml saline solution (0.9%), homogenized with an ULTRA-TURRAX® Tube Drive Workstation  and filtered through sterile gaze. Of this suspension, 1.5 ml were centrifuged for 20 minutes at 16000×Of the same basal suspension, 0.2 ml were inoculated on each Middlebrook 7H11 Medium slant agar  and Löwenstein-Jensen Medium slant agar , supplemented with Glycerin and PACT . Solid media were incubated at 37°C for at least eight weeks and checked regularly for growth. Cultures were considered positive if typical growth occurred and acid-fast bacilli were detected with Ziehl-Neelsen staining. In this case, DNA was extracted as described below. Media that showed no growth after twelve weeks were considered negative. All incubation procedures were conducted in a biosafety level 3 (BSL3) laboratory.Cystein-NaOH (NALC-NaOH) from the BD MycoPrepTM Specimen Digestion/Decontamination Kit  instead of H2SO4 and NaOH To verify the obtained results, analysis was repeated on all wild boar samples showing TBL (n = 17). Two modifications to the above mentioned protocol were made to enhance the chance for cultivation of slow-growing mycobacteria of the MTBC: Tissue material was decontaminated using N-Acetyl-L-DNA extraction from frozen tissue pellets was performed using the MagNA Pure LC DNA isolation kit II for mammalian tissue and the automated MagNA Pure LC instrument  according to the manufacturer's protocol, with an external Proteinase K digestion step. For mechanical disruption, 200 µl Tissue Lysis Buffer  was added to the pellet, and samples were homogenized twice using tubes containing ceramic beads  and a Precellys 24 homogenizer  for 45 sec. at 6.500 rpm. The samples were centrifuged for 2 min at 13.000 rpm and 80 µl of the supernatant were added to 20 µl Proteinase K  and incubated at 60°C for 30 minutes. After digestion, samples were centrifuged again for 1 minute at 8000 rpm and the supernatant was transferred to the sample cartridges. Setting of the MagNA Pure LC instrument was done according to the manufacturer's protocol.For DNA isolation from cultured bacteria, either one loop of colony material from slant agar suspended in 400 μl NaCl (0.9%), or 400 μl of liquid culture, were inactivated at 95°C for 30 minutes using a BioShake IQ . Bacteria were lysed by addition of 50 μl lysozyme (10 mg/ml) and an overnight incubation step at 37°C shaking at 900 rpm, followed by mechanical disruption as described above. DNA was extracted using the QIAGEN DNeasy blood and tissue kit  in accordance with the manufacturer's protocol.DNA extracts were analyzed at the Institute of Medical Microbiology, Zurich. PCR analysis for detection of MTBC was done with the COBAS® TaqMan® MTB Test kit  according to the manufacturer's instructions. Detection of MTBC DNA is based on primers amplifying a conserved region of the 16S ribosomal RNA gene in combination with a MTBC specific Taqman probe PCR amplification was carried out using the COBAS® TaqMan® 48 Analyzer  in 100 μl-reaction mixtures containing 50 μl of freshly made primer-master mix solution  and 50 μl of extracted DNA solution. If PCR inhibition was observed , the DNA samples were 5x diluted in lysis/elution buffer  of the Roche respiratory DNA extraction kit  and reanalyzed using the COBAS® TaqMan® MTB Test kit. Negative and positive control reactions were performed with material supplied in the test kit.If culture yielded acid-fast bacilli that were negative by PCR on grown colonies for MTBC DNA, these were classified as atypical mycobacteria and not further differentiated.2O and 5 μl of DNA positively tested in the PCR assay. Reverse hybridization of the amplified products was performed and the test strips were interpreted, both in accordance with the protocol provided by the manufacturer.If DNA specific for MTBC mycobacteria was successfully amplified from cultured material, genotyping was performed using the GenoType® MTBC kit  according to the manufacturer's protocol. Briefly, PCR amplification was carried out in reaction mixtures containing 35 μl of primer-nucleotide-mix , 5 μl 10x PCR buffer for HotStarTaq , 2 μl 25 mM MgCl2 solution , 0.2 μl HotStarTaq , 3 μl Hwww.Mbovis.orgSix DNA samples originating from tissue pellets were spoligotyped at the Animal Health and Veterinary Laboratories Agency, Weybridge, UK, according to the method of Kamerbeek et al. The two-tailed Fisher's Exact Test was used to compare the occurrence of TBL and atypical mycobacteria between wild boar and red deer, as well as among sexes, age categories and sampling areas within each species. Significance level for each test was set at <0.05. Statistical analysis, including the calculation of 95%-confidence intervals for bTB and MTBC prevalence, was performed using NCSS 2007 statistical software .Four online databases  were first searched for information on wildlife maintenance hosts for bTB worldwide, using the key words “bovine tuberculosis”, “wildlife” and “reservoir”. Scientific articles considered relevant according to the abstract were selected for detailed reading, and constituted the basis for further targeted search for articles documenting wildlife bTB reservoirs worldwide and risk factors favoring the maintenance of bTB in these host species. We defined a specific factor as a “risk factor” in this review, (1) if it had been shown to be associated with the prevalence or presence of bTB or TBL in the respective reservoir host , (2) if such a role was suggested by the author(s), or (3) if a factor was present that had been suggested or shown to play an important role in bTB maintenance in another country .Furthermore, we attempted to compile comparable data on bTB prevalence and population density of reservoir species and selected spillover hosts. Comparability required (1) availability of prevalence and density data from the same geographical area, and (2) use of the same methods for prevalence and density estimations, respectively, in the different regions. Since culture is considered the gold standard for mycobacterial diagnostics The situation regarding risk factors identified in the literature review was assessed for CH and FL. We reviewed the current legislation and conducted a telephone survey with officials of the hunting administrations of the study areas.We detected TBL in 17 wild boar (10.3%) and five red deer (1.9%). This difference between species was significant (p = 0.0002). We did not observe any case of generalized lesions in either species. In the majority of wild boar (n = 11), lesions consisted of focal to multifocal white-yellow calcified to caseo-calcified foci of 0.1–1.5 cm in diameter , frequenLesions in red deer consisted of purulent tonsillitis (n = 2) or miliary calcified lesions (n = 3), all confined to a single anatomical site. Differences among sexes, age categories and sampling areas were not significant.T; apparent MTBC prevalence: 3.6%, 95% CI 1.4–7.8%), all of which presented TBL (M. microti in two cases (TG435 and TG481), while an inconclusive banding pattern was obtained in the other four cases . Presence of MTBC mycobacteria was subsequently also detected by culture and PCRC for the two M. microti-positive animals and for TG422, but genotyping yielded inconclusive banding patterns. Culture material from TG422 was only weakly and transiently positive for MTBC mycobacteria, and thus no appropriate material could be obtained for further spoligotyping.Detailed laboratory results for wild boar and red deer are presented in nted TBL . SpoligoAtypical mycobacteria were cultured from 54 wild boar (32.7%), including all six MTBC-positive animals and six further animals with TBL. Culture growth was significantly more often observed in samples from wild boar with TBL than from individuals without visible lesions (p = 0.0009), while differences among sexes, age categories and sampling areas were not significant.T and PCRC . Atypical mycobacteria were cultured from 23 red deer (8.6%), including one animal with TBL. Overall, culture growth was more often observed in adults than in juveniles and subadults (p = 0.0479), but this difference was not significant when study areas were considered separately. Differences among sexes and sampling areas were not significant either. Atypical mycobacteria were significantly more often detected in wild boar than in red deer (p = 0.0000).In red deer, all samples were tested negative for mycobacteria of the MTBC both by PCRM. bovis and M. caprae were not found in the investigated samples, but detected MTBC mycobacteria could not be identified to species level in four wild boar.Overall, Combined macroscopic, histologic and bacteriologic examinations showed that the four red deer presented, respectively: a purulent lymphadenitis of a cervical lymph node; a severe bronchopneumonia; a lymphadenomegaly in association with myositis; and multifocal parasitic granulomas in the mesentery. The wild boar displayed multifocal mineralized foci within the liver parenchyma of presumptive parasitic origin. The chamois showed a granulomatous peritonitis and hepatitis that were due to presumptive parasitic infestation as well. In the ibex, the enlargement of mesenteric and pulmonary lymph nodes mentioned by the submitter was not confirmed at necropsy; this animal only presented a mild lymphoid hyperplasia of the pulmonary and mesenteric lymph nodes. The roe deer was diagnosed with a multisystemic lymphosarcoma.T and by mycobacterial culture.All eight cases were tested negative for mycobacteria both by PCRResults of the literature review are summarized in Part of the risk factors for aggregation and bTB maintenance directly or indirectly result from human influence: Intensive wildlife management (notably at hunting farms or clubs), including practices such as supplementary feeding, watering and fencing Data from our study areas on anthropogenic factors found to influence the risk of bTB maintenance in wildlife (as identified in the literature review) are summarized in M. bovis and M. caprae were not detected by a combined PCR and culture protocol in any of the sampled animals, and the development of a wildlife reservoir is currently unlikely. However, we report for the first time infections with a mycobacterium of the MTBC, M. microti, in Swiss wild boar.Our study is the first to assess the situation of bTB, a currently (re)-emerging disease in European wildlife, in the potential maintenance hosts red deer and wild boar in various areas of CH and FL. C for MTBC and thus classified as atypical mycobacteria. Contamination due to field sampling M. avium complex, known to occur in wild boar and common in domestic pigs Culture of mycobacteria succeeded in 77 animals, including 13 cases with macroscopic TBL, but the majority of these was negative by PCRT in six wild boar, all of which presented TBL, while only three of these animals were also positive by PCRC. However, the applied PCR protocol has been validated only for respiratory specimens and false-positive results have been reported from non-respiratory sample material T may be limited as only a small tissue portion is used for analysis and both the distribution and total amount of mycobacteria within an infected tissue may vary. Regarding culture, sensitivity may also be affected by various factors The PCR protocol we used for the detection of MTBC mycobacteria has shown high specificity Finally, only part of the required five lymphatic tissues were submitted for examination in 64% of the sampled animals; this may have decreased the sensitivity of our protocol because the presence of mycobacteria may be confined to certain regional lymph nodes only.Staphylococcus, Streptococcus, Actinobacillus or Actinomyces spp. Rhodococcus equi is increasingly detected in slaughter pigs, both with and without macroscopic TBL, and has been found in mandibular lymph nodes of 12.4% of sampled wild boar from Hungary Corynebacterium ulcerans have recently been reported in two wild boar from Southern Germany Nevertheless, because macroscopically identified TBL are non-specific for bTB, a number of differential diagnoses have to be considered for the observed lesions, such as granulomas of parasitic or fungal origin M. microti was confirmed by spoligotyping in two out of six PCRT positive animals. Spoligotyping was performed on DNA extracted directly from tissue, as we did not succeed in obtaining pure cultures of MTBC mycobacteria but growth of atypical mycobacteria likely outcompeted MTBC mycobacteria. Strains of M. microti can be particularly difficult to culture and slow-growing M. bovis, and thus overgrowth of M. microti with atypical mycobacteria is even more likely. However, our laboratory protocol was selected with regards to the detection of M. bovis and M. caprae, and other protocols may have been more suitable for the detection of M. microtiInfection with M. microti has been isolated from both wild boar and domestic pigs with TBL before M. bovis/M. caprae hosts  M. bovis or M. capraeDisease surveillance in wildlife in CH and FL consists of national scanning surveillance programs regularly complemented by targeted, risk-based investigations. Between 2006 and 2011 the FIWI staff has performed full necropsies, including histology in most cases, on 520 animals belonging to species known to be potential Overall, the combination of data from targeted and scanning surveillance obtained with various diagnostic protocols  does not suggest the occurrence of bTB in wildlife in CH and FL so far. However, as financial restrictions limited sample sizes per study area in the present study, the occurrence of bTB cannot be completely ruled out , highlighting the urgent need for harmonized procedures in wildlife health science It is not unlikely that the “hot spot concept” mentioned above is applicable to the Alpine situation: Despite (1) the presence of a current infection focus in Austria within a radius of about 50 km from the CH and FL borders The merit of early and adequate response to disease emergence was recently demonstrated by the reaction to an outbreak of bTB in Minnesota, USA, where the establishment of a disease reservoir in a potential maintenance host, the white-tailed deer, was successfully prevented


Results showed that different concentrations of Rosa damascena essential oil significantly reduced signs of morphine withdrawal compared to the control group in terms of number of jumps (p < 0.05 and p < 0.01), grooming, teeth chattering, rearing, climbing, wet dog shakes and writhing, but not for diarrhea (p < 0.05). In conclusion it seems that GABAergic activity induced by flavonoids from Rosa damascena essential oil can alleviate signs of morphine withdrawal, but further studies need to be done to better understand this mechanism. This research was done to test the effect of Rosa damascena essential oil on withdrawal signs of naloxone-precipitated morphine in male mice. Morphine dependence was induced by injection (IP) three times daily at doses of 50, 50 and 75 mg/kg, respectively, for 3 days. On day 4, after the last administration of morphine, Rosa damascena essential oil was administered at different concentrations  30 min before administration of naloxone . The following actions were taken as signs of withdrawal and records taken for jumping as a number and scores of 0 to 3 were given for incidences of grooming, teeth chattering, rearing, writing, diarrhea, wet dog shakes and climbing during a 30 min period R. damascena plant has also been used for medicinal applications and several in-vivo and in-vitro studies have been done on various products and isolated constituents from its flowers, petals and hips (seed-pot)  . This pleed-pot) -6. It iseed-pot) . Researceed-pot) , 7-11. μ receptor agonist, has been used to alleviate these withdrawal signs , weighing 20-30 g at the beginning of the experiment were used for these tests. Animals were housed eight per cage, maintained at 23± 1°C with a controlled 12 h light/dark cycle and with ad libitum food and water. Each mouse was used only once and each treatment group consistent of eight animals. They were acclimatized to the conditions of the experiment for at least 1-week prior to use. All procedures were performed during the light phase and done in accordance with guidelines authorized by the School of Medicine at Tehran University of Medical Science.DrugsThe following drugs were used for the tests: morphine sulfate , Naloxone HCL  and Diazepam HCL . All drugs were dissolved in saline and were injected intraperitoneally (IP) at volumes of 10 mL/kg.Preparation of the essential oil of Rosa damascena°C). To administer the essence it was reconstituted in normal sterile saline and filtered before injection . Rosa damascena flowers were collected from Kashan  in spring 2011. The plant species was identified by botanical specialists of the biological department of Barij Essence Pharmaceutical Co . Essential oil was obtained from fresh flowers taken from the plant by stem distillation (hydro-distilled) at the Barij Essence Pharmaceutical Co and stored in a refrigerator (- 4Induction of morphine dependenceMorphine was injected (IP) three times daily  at doses of 50, 50 and 75 mg/kg, respectively, for 3 days. On day 4, only a single morning dose of morphine (50 mg/kg) was injected before the naloxone injection  2h after the injection of morphine. Following the naloxone injection, animals were immediately placed individually on filter paper in a clear plastic cylinder (15 cm in diameter and 50 cm in height) for observation. The behavior of each animal was recorded for 30 minutes using a digital camera. Evaluations were taken for the following signs: jumping, climbing, wet dog shakes, grooming, teeth chattering, diarrhea, rearing and writhing. These signs were rated every 10 min using a scale from 0 to 3: A score of 0 indicated that a sign was absent during the 10 min observation period; a score of 1 indicated low intensity behavior; a score of 2 indicated average intensity and a score of 3 showed maximum intensity. Incidence of diarrhea was evaluated every 20 min and given a score accordingly .Grouping of animals and administration of extractsForty mice were randomly divided in to groups of eight. Morphine was administered to mice in all groups as discussed above. I: Control group (as a negative group): In this group a solvent of rose essential oil  was administered IP 30 min after the last dose of morphine and 30 min later, naloxone was injected.II: Diazepam group (as a positive group): In this group diazepam (5 mg/kg) was administered IP 30 min after the last dose of morphine and 30 min later, naloxone was injected. III: Rosa damascene essential oil group: In this group different concentrations of Rosa damascena essential oil  were administered IP 30 min after the last dose of morphine and 30 min later, naloxone was injected. Statistical analysis Data were expressed as mean ± SEM for eight mice in each group. One-way AONOVA followed by the Tukey test was used for comparisons of data. The Mann-Whitney U test was used for comparisons of checked signs data versus the control group. Difference of p < 0.05 was considered statically significant. Results showed that intraperitoneal administration of Rosa damascena essential oil at concentrations of 5, 20 and 40% significantly, according to the dose administered, reduced the number of jumping episodes in morphine dependent mice . DiazepThe results of this study indicate that Rosa damascena essential oil has component(s) that could attenuate the signs of morphine withdrawal in morphine dependent mice. Jumping is one of the most common signs used to assess the severity of morphine withdrawal. In agreement with our data, another study reported that Rosa damascena has laxative and prokinetic effects; thus applied doses of essential oil haven’t any significant effect in terms of decreasing incidence of diarrhea . Other rA and GABAB receptor agonists are involved in signs induced by morphine withdrawal. For example, several studies have reported that baclofen (GABAB agonist) and muscimol (GABAA agonist), both inhibited naloxone induced jumping behavior in morphine dependent mice in a dose-dependent manner (The family Rosaceae is a well known traditional folk medicine used for treating nervous system breakdown . Rosa dat manner , 27. In conclusion, it seems that flavonoids of Rosa damascena essential oil with GABAergic activity can attenuate morphine withdrawal signs. But further studies need to be carried out to better understand this mechanism.


Daphnia magna, Thamnocephalus platyurus, Vibrio fischeri, and Raphidocelis subcapitata. Landfill leachate increased initial toxicity of wastewater. During biological treatment, significant decline of acute toxicity was observed, but still mixture of leachate and wastewater was harmful to all tested organisms.Combined treatment of landfill leachate and municipal wastewater was performed in order to investigate the changes of leachate toxicity during biological treatment. Three laboratory A2O lab-scale reactors were operating under the same parameters  except for the influent characteristic and load. The influent of reactor I consisted of municipal wastewater amended with leachate from postclosure landfill; influent of reactor II consisted of leachate collected from transient landfill and municipal wastewater; reactor III served as a control and its influent consisted of municipal wastewater only. Toxicity of raw and treated wastewater was determinted by four acute toxicity tests with  Waste volume is growing faster than the world's population, and management of wastes is a matter of considerable human concern , 2. Recy3 from each section daily. Samples were collected from the equalization basins.Leachate was collected from the Municipal Solid Waste Landfill in Zabrze (Poland). There are two sections in Zabrze Landfill: I: the “old,” reclaimed section; II: the “new” one receiving municipal waste since 2007. The landfill leachate from both sections is collected separately in equalization basins and recirculated to the waste dump. Excess of leachate is pumped to the sewage collection system. The leachate flow is 80 m3/d.Wastewater was collected from wastewater treatment plant in Zabrze-Mikulczyce (Poland). The place for wastewater collection was selected to ensure lack of earlier wastewater contamination by leachate. The daily flow of wastewater is average 5000 mA2O-activated sludge systems were composed of an anaerobic/anoxic/aerobic process with simultaneous nitrification-denitrification and biological phosphorus removal. Each system composed of three separate reactors with the following working volumes: anaerobic 2 L, anoxic 5 L, aerobic 7 L .The experiment was carried out in three activated sludge A2O systems: I, II, and III. Influent of system I consisted of mixture of wastewater and “old” landfill leachate. Influent of system II consisted of mixture of wastewater and new landfill leachate. System III served as a control and was fed with municipal wastewater. The scheme of influents composition in different periods of experiment was presented in All systems were operated under the same technical parameters  except fThe reactors were inoculated with an activated sludge sampled from municipal wastewater treatment plant. Activated sludge in reactors I and II was acclimated to the increasing concentration of landfill leachate in the influent (1 and 10%). Some earlier study showed that median share of landfill leachate in wastewater stream should be at 5% (v/v) level , 13. It Ammonium nitrogen as well as organic nitrogen was measured with Kjeltec 1026 analyzer. Chemical and biological oxygen demands (COD and BOD) were determined by standard methods , 15. CheWhole effluent toxicity tests were performed which means that the aggregate toxic effect of respectively influent or effluent was measured directly by a toxicity test.Following tests were proposed for toxicity evaluation.Vibrio fischeri were purchased from Azur Environmental . As a diluents, 2% NaCl was used. As the samples of wastewater were coloured, light absorbances were measured at 490 nm and colour correction procedure was applied.The test was carried out in the Microtox M500 toxicity analyzer according to the standard procedure , which iVibrio fischeri luminescence inhibition test was performed three times.Daphnia magna immobilization test was performed five times.Tests were carried out with neonates (<24 h). Five test dilutions were prepared in a 50% dilution series for each sample with three replicates of seven animals. The test volume was 20 mL. The animals were not fed during the experiment. Each test had a duration 48 h; the temperature was 24 ± 1°C. After an exposure, the number of immobile daphnids for each dilution was recorded. Tests were carried out according to the MicroBioTest Standard Operational Procedure. Readily hatched organisms were used for the test. Five test dilutions were prepared in a 50% dilution series.Each sample was with 3 replicates of 10 animals in disposable multiwell test plates. Test volume was 1 mL per well.Thamnocephalus platyurus acute toxicity test was performed five times.After 24 h in a 25°C incubator in the dark, the number of dead crustaceans was recorded. Raphidocelis subcapitata were exposed to the test sample in batch cultures over a period of 72 hours in 24 ± 1°C. The biomass in the control cultures increased exponentially by a factor of at least 16. Five test dilutions were prepared in a 50% dilution series with an initial biomass concentration 1 × 104 cells/mL. Each sample was with 3 replicates; growth inhibition test was performed five times.Exponentially growing Chemical parameters of leachate from both sampling sites are presented in Tables Biological treatment of leachate has been shown to be effective in removing organic and nitrogenous matter from immature effluent characterized by high BOD/COD ratio , 2. In pEffective ammonia and organic nitrogen removal was also observed in all three systems. Removal efficiency was within the range 85–99%. 10% amendment of leachate in wastewater stream decreased biodegradability of influent of system II . BOD/CODCombined treatment of landfill leachate and municipal wastewater was also investigated by Diamadopoulos et al.  in sequeSeveral authors revealed also the possibility of leachate treatment in combining aerobic-anaerobic conditions, which allowed to perform treatment with higher organic loading rates , 23. GomOrganic and ammonia nitrogen was effectively removed in systems I, II, and III—removal efficiency was as high as 86, 98, and 96 percent, respectively.50). The results of toxicity tests were presented in Toxicity of new landfill leachate was significantly higher than toxicity attributed to old landfill leachate. The overall toxicity of old leachate samples allowed to classify it as toxic, while new landfill leachate toxicity was more than ten times higher, and new leachate was classified as very toxic . 50 (or LC50): The results of toxicity tests were also examined for environmental relevance by calculating toxicity units (TUs) as reported in Tables If the mortality in a 100% effluent concentration was between 10% and 49%, the TUs were derived as follows:A toxic unit of zero was allocated to mortalities between 0% and 10% in 100% effluent exposure , 26.Daphnia magna (system II) and Raphidocelis subcapitata (system I). TU values of all influents towards tested organisms were below 10; therefore, influents were classified as toxic. After biological treatment, significant reduction of toxicity was observed. Slight residual toxicity of effluent was observed only for V. fischeri (system I and II). All effluents stimulated growth of algae due to still high content of nutrients (nitrate and phosphates). 1% amendment of landfill leachate in wastewater stream slightly increased whole influent toxicity . SignifiT. platyurus and V. fischeri. Toxicity of wastewater was successfully removed during biological treatment. Significant reduction of toxicity was also observed for effluent of system II, while effluent of system I was still characterized by important residual toxicity . Important increase of toxicity was observed in all tested bioassays while 10% of new landfill leachate was mixed with municipal wastewater (system II). 10% of old landfill leachate in wastewater stream resulted in important increase of influent toxicity towards Allium cepa root length inhibition was attributed to increase of leachate concentration within the range of 40–80%. The same authors also pointed that acute toxicity of treated leachate to Artemia salina was very low in a range of 10–80%, even though nondiluted leachate effected in 80% mortality of crustacean. Also Bialowiec et al. [Salix amygdalina leaf length and weight, despite exposition to landfill leachate concentration within the range of 0–12.5%. In the present study, battery of bioindicators was exposed to mixture of landfill leachate and raw wastewater. During the study, toxicity of wastewater and leachate was changing due to natural fluctuations in those samples' composition. It is commonly accepted that interaction between mixture components may result in antagonist or synergetic effects which cannot be solely predicted at the base of initial toxicity data of elements or chemical species. Average TU values of leachate from new landfill were about 10 times higher than TU values obtained for leachate from old landfill. However, while 10% of leachate was mixed with municipal wastewater, toxicity of systems I and II influents was at similar level . The reac et al.  did not Acute toxicity of landfill leachate is often attributed to high ammonium nitrogen concentration , 29, 30.In case of leachate from new landfill—at the base of present and some previous studies , 32—doseRaphidocelis subcapitata growth inhibition test, only acute toxicity tests were performed. Moreover, small group of 4 organisms served as biotest battery. Therefore, counting the hazardous concentration of landfill leachate, safety factor of 100 should be used. The highest leachate concentration, which after biological treatment would not be harmful for 95% of aquatic species, is 0.05%.  Investigation was performed for 1, 5, 10, and 15% of new landfill leachate cotreated with municipal wastewater. After biological treatment, toxic response of effluents significantly differed from the control for wastewater containing 5, 10, and 15%. of leachate. The maximum concentration of leachate, which, after biological cotreatment, would not be hazardous for more than 5% of species, could be roughly assessed with use of safety factors. For the toxicity measurement, except from Landfill leachate significantly disrupts biological treatment of wastewater. After biological treatment, wastewater enriched with 10% leachate did not meet the water quality standards and still was harmful to aquatic organisms. The calculated concentration of new landfill leachate, which after biological treatment would not be harmful for aquatic organisms, was 0. 05%.


Bacillus sp. SK13.002. Compared with the conventional enzymatic method for rutin transglycosylation (without microwave irradiation), microwave-assisted reaction (MAR) was much faster and thus more efficient. While the conventional reaction took dozens of hours to reach the highest conversion rate of rutin and yield of transglycosylated rutin, MAR of rutin transglycosylation completed within only 6 min providing almost the same conversion rate of rutin and yield of products consisting of mono-, di-, tri-, tetra-, penta-glucosylated rutins. The optimum transglycosylation conditions for microwave irradiation were 40 °C and 60 W with the reaction system consisting mainly of the mixture of 0.3 g rutin (0.49 mmol) pre-dissolved in 15 mL methanol, 1.8 g maltodextrin in 15 mL of 0.2 M sodium acetate buffer (pH 5.5) and CGTase (900 U). Results from this study indicated that MAR could be a potentially useful and economical technique for a faster and more efficient transglycosylation of rutin.Rutin was subjected to intermolecular transglycosylation assisted with microwave irradiation using cyclodextrin glucanotransferase (CGTase) produced from  Bacillus spp. and could be utilized to catalyze the enzymatic reaction which transfers glycosyl residues to compounds like rutin to form glycosyl-rutin . These transglycosylation reactions have been carried out using conventional techniques and were reported to be time-consuming [Rutin is one kind of bioflavonoid, which is used as a nutritive element or an antioxidant in the cosmetic, food and beverage industries. However, rutin is hardly soluble in water and not very stable, which limits its practical application in industries. To improve its physicochemical properties, many methods have been investigated, including the use of CGTase to carry out enzymatic transglycosylation to obtain transglycosylated rutin with improved properties such as good water solubility ,2. CGTasonsuming .Furthermore, because rutin has a low solubility in water but a high solubility in methanol, the latter solvent has been used for rutin transglycosylation in order to start with higher rutin concentrations. As a result, the enzymatic transglycosylation reaction system consists of nearly half part of methanol (v/v), which might induce an inhibitory effect on CGTase activity. This would probably explain the reason why it should take dozens of hours to complete the enzymatic reaction of rutin transglycosylation if the highest conversion rate of rutin is desired.Microwave irradiation has become an effective tool in synthetic organic chemistry, dramatically increasing reaction rates and yields –8. MicroBacillus sp. SK13.002 and maltodextrin as substrate to provide glycosyl residues.Since the time-course of rutin transglycosylation was extremely long (dozens of hours), a microwave-assisted reaction (MAR) technique was investigated in this study. To the best of our knowledge, enzymatic transglycosylation of rutin under microwave irradiation conditions has not been reported so far. The present study focused on comparing the transglycosylation rate of rutin under conventional conditions with that assisted with microwave irradiation using CGTase as a biocatalyst produced from Transglycosylation is an important process for structural modification of molecules to enhance their solubility and biological properties, which can also improve their properties and action in the body system –17. In tFirstly, it is well known that different enzymatic reactions would require also different reaction time periods if the highest yield of products is targeted. For the enzymatic transglycosylation of rutin, the long time-course probably depends on the nature of CGTase and the type of substrate.Secondly, methanol existing in the enzymatic reaction system might cause an inhibitory effect on the activity of CGTase during rutin transglycosylation. In order to test this assumption, the effect of methanol concentration on CGTase hydrolysis and cyclization activities was investigated. Results presented in As mentioned above, microwave irradiation might stimulate and accelerate chemical and enzymatic reactions. However, microwave irradiation did not always stimulate or accelerate chemical and enzymatic reactions ,19. It iTo answer this question, the effect of microwave irradiation on rutin transglycosylation was investigated. The results were positive and inspiring: Microwave irradiation could drastically accelerate the transglycosylation rate of rutin. Concretely, under specific microwave irradiation conditions, the reaction time to complete the transglycosylation of rutin decreased from dozens of hours to only several minutes.Different parameters including microwave power and reaction time were selected to standardize microwave-assisted reaction (MAR) for optimization. The effects of different microwave power levels and reaction time periods to complete the rutin transglycosylation are given in The release of glycosyl residues from the substrate  for transglycosylation is dependent on the availability of a particular energy level inside the microwave oven. In this study, the maximum yield of enzymatic reaction products (transglycosylated rutins) was obtained using 60 W as the irradiation power level and a 6 min reaction time.In order to optimize the transglycosylation of rutin under microwave irradiation conditions, the ratio of maltodextrin to rutin, as well as the reaction pH and temperature were also investigated. Results in For the conventional transglycosylation method, reaction parameters such as pH, temperature and time were investigated in order to optimize the yield of transglycosylated rutins (G-rutins). Results revealed that the optimal pH and temperature for the conventional enzymatic reaction were 5.5 and 35 °C, respectively (data not shown). In addition, G-rutins  increased gradually with time and reached the highest level after 24 h, but remained constant up to 32 h .Bacillus sp. SK13.002 CGTase and other catalysis characteristics were different from other CGTases. According to Suzuki et al. [et al. [Bacillus sp. SK13.002 has unique characteristics in terms of catalyzing capacity and optimal reaction conditions. Nevertheless, 24 h of time-course with Bacillus sp. SK13.002 CGTase in the conventional method for rutin transglycosylation is still too long to be economically applied. Therefore, MAR was selected as an alternative method to carry out the transglycosylation of rutin in a more competitive way.The optimal temperature for Compared with the conventional method for rutin transglycosylation, the microwave-assisted method dramatically accelerated the enzymatic reaction. As a result, the time required to complete the reaction decreased from dozens of hours to only several minutes . HoweverThe shortening of time-course for rutin transglycosylation using MAR technique indicates a positive effect on the enzymatic reaction. Similarly, microwave irradiation has been reported to exert positive effects on different kinds of enzymes ,12,20,21Although some researchers have tried to study the effects of microwave irradiation on the structure of enzymes ,27, furtBacillus sp. SK13.002 which was screened and kept by ourselves. Rutin was supplied by Zelang Medical Technology Co., Ltd . Methanol and acetonitrile were obtained from Sigma . Other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd .CGTase was obtained through cultivation of 2 and then precipitated with 70% saturated ammonium sulfate at 4 °C. The mixture was centrifuged at 10,000 rpm, 4 °C for 15 min and the pellets were dissolved in 20 mM Tris-HCl buffer, pH 8.0 and dialysed against the same buffer overnight at 4 °C. The solution obtained after dialysis was freeze-dried and the resulting CGTase powder was used to carry out all experiments.After cultivation for 96 h, cells were removed by centrifugation at 10,000 rpm and 4 °C for 15 min. The supernatant was used as crude enzyme solution, which was concentrated using Millipore ultra-filtration system  with a molecular cut-off point of 10 kDa. The concentrated enzyme solution was first adjusted to pH 7.0 using solid CaClThe starch hydrolyzing activity of CGTase was assayed according to the method of Shiosaka and Bunya , based o2CO3 was added to the reaction mixture. After 15 min, the decrease in colour intensity was measured at 550 nm. One unit of CGTase activity was defined as the amount of enzyme that forms 1 mmol β-cyclodextrin per min under the assay conditions.The cyclization activity of CGTase was tested using the phenolphthalein assay . A reactet al. [2 in the final solution. The conversion rate of rutin (%) was defined as follows: Conversion rate (%) = produced G-rutins/(remaining rutin + G-rutins) × 100.The transglycosylation of rutin by CGTase was carried out following the method of Suzuki et al.  with a fThe reaction system consisted of the same components mentioned above, with the enzymatic reaction occurring in a microwave oven (2450 MHz) at 40 °C and 60 W for several minutes. The reaction was stopped by heating in boiling water for 5 min.After the transglycosylation of rutin, samples were filtered through 0.45 μm membrane and analyzed by HPLC using Agilent ZORBAX Eclipse XDB-C18 column . The mobile phase contained a mixture of acetonitrile, water and formic acid  and the flow rate was 0.8 mL/min. UV detector was used at 254 nm, with the column temperature being 30 °C.All experiments were performed in triplicate and results are expressed as mean ± standard deviation.In the enzymatic transglycosylation of rutin, methanol used as solvent causes an inhibition of CGTase activities, which might result in dozens of hours of reaction time to achieve the highest conversion rate of rutin and yield of transglycosylated products, which are mono-, di-, tri-, tetra-, penta-glucosylated rutins when maltodextrin is used as the donor of glycosyl residues.Microwave irradiation accelerated dramatically the enzymatic reaction rate during rutin transglycosylation. Using 60 W at 40 °C in microwave-assisted reaction (MAR) system, the transglycosylation of rutin completed within only 6 min. Therefore, MAR could be a potentially useful and economical technique for a faster and more efficient transglycosylation of rutin.


Agaricomycotina using several sets of new primers. Large introns (∼1500 bp) at variable locations were detected in ∼5% of the sequences we obtained. We suspect that widespread presence of large introns is responsible for our low PCR success (∼30%) with this locus. We also sequenced the nuclear internal transcribed spacer rDNA regions (ITS) to compare with COI. Among the small proportion of taxa for which COI could be sequenced, COI and ITS perform similarly as a barcode. However, in a densely sampled set of closely related taxa, COI was less divergent than ITS and failed to distinguish all terminal clades. Given our results and the wealth of ITS data already available in public databases, we recommend that COI be abandoned in favor of ITS as the primary DNA barcode locus in mushrooms.DNA barcoding is an approach to rapidly identify species using short, standard genetic markers. The mitochondrial cytochrome oxidase I gene (COI) has been proposed as the universal barcode locus, but its utility for barcoding in mushrooms  has not been established. We succeeded in generating 167 partial COI sequences (∼450 bp) representing ∼100 morphospecies from ∼650 collections of  DNA barcoding is an approach to rapidly identify species using short, standard genetic markers http://www.barcoding.si.edu) has identified several alternative loci that may be used together for a multilocus barcode for land plants To qualify as a DNA barcode region, a locus should be easy to amplify from most or all species in the target group using universal primers and show low intra-specific and high inter-specific divergence (creating a “barcode gap”). For animals, the mitochondrial cytochrome oxidase I (COI) locus appears to satisfy these criteria for most groups Ascomycota, COI was shown to be more effective than ITS, but less than beta tubulin A, for distinguishing among species of Penicillium subgenus PenicilliumNeohumicola COI and ITS provided similar resolution Aspergillus and Fusarium, respectively, and more critically, detected COI paralogs. In the Basidiomycota, Vialle et al. Urediniomycetes) they examined due to the presence of introns in the priming and sequencing regions and, when they did obtain sequences, variation was inferior to ITS and LSU. At least four introns occur in the COI coding region of the mushroom Agrocybe aegeritaTrametes cingulataAgaricus bisporusAgrocybe aegerita), two (Crinipellis perniciosa), or eight (A. bisporus) introns have been reported from the 600 bp “barcoding region” at the 5′-end of the gene Although the idea of DNA barcoding is both essential to and already an informal part of much current research with fungi, only a few studies exist that examined the effectiveness of COI as a DNA barcode Basidiomycota:Agaricomycotina), representing about one-third of all known species of fungi , by evaluating COI as a DNA barcode for the group. The criteria we used included the ability to design universal primers, amplification success, and capacity to diagnose species using a phylogenetic approach. We also compared COI with ITS, the latter being an informal “standard” marker for species resolution in Agaricomycotina.Here, we set out to facilitate the formalization of a DNA barcoding effort in mushrooms and their relatives  and the permit for collecting specimens in Algonquin Provincial Park was provided by Ontario Parks to Jean-Marc Moncalvo (s.n.).Agaricomycotina  were newly collected from Ontario, Québec, and Maryland for this study. Species identifications were made comparing macro-and micro-morphological features with species descriptions in the relevant taxonomic literature (too numerous to list). Vouchers of all newly collected specimens are deposited in the fungal herbarium of the Royal Ontario Museum (TRTC). Additional specimens used in this study were borrowed from other herbaria .Approximately six hundred and fifty mushrooms in three classes of Cryptococcus neoformans AY560609, Moniliophthora perniciosa NC005927, Schizophyllum commune AF402141, Agrocybe aegerita AF010257). PCR amplification was achieved using a cycling program with an initial denaturation of 95C for 2 min, followed by 30 cycles of denaturation at 94 C for 45 sec, annealing at 60 C for 45 sec, and extension at 72 C for 1 min 10 sec, followed by a final extension at 72 C for 7 min and an indefinite refrigeration at 4 C. New primers “COXBOL1-F” and “COXBOL1-R” were also designed for improved amplification of COI from genomic DNA for Boletales , 55 C (30 sec), and 72 C (1 min), followed by 25 cycles of 94 C (30 sec), 50 C (30 sec), and 72 C (1 min), and ending with a final extension step at 72 C for 7 min. The ITS region was amplified and sequenced with primers ITS1F and ITS4 using standard protocols www.barcodinglife.org; 33] and through GenBank . Additional sequences obtained from GenBank that were used in this study include the following accession numbers: EU231946, EU231948, EU231949, EU231954, EU231958, EU231966, EU231969, EU231971, EU231980, EU231982, EU231983, EU231984, EU231985, EU231990, EU231991, EU231992.DNA was extracted following various standard protocols oletales  based onIntron sequences from COI were removed before aligning the exon regions using ClustalW2 gat)CATTA— and —GACCT(caaa...)-3′ to facilitate alignment. These motifs correspond to the conserved 3′ and 5′ termini of the flanking SSU and LSU genes, respectively. Because ITS sequences are length-variable regions with rapid substitution rates, a global alignment cannot be unambiguously constructed. Therefore, in order to ensure proper assessment of character homology between sequences, we identified “alignment groups” of sequences with 80% or greater similarity, a threshold that enabled unambiguous alignment. Any sequences that were greater than 20% dissimilar to each other were assumed to represent different species . More than 90% of specimens (144 spp. in 49 genera) are members of the principal gilled-mushroom subclass Marasmius androsaceus, and Lepiota clypeolaria have an intron inserted at position 4), but these locations were not conserved within genera . In addition, presence/absence of intron was not always consistent between multiple specimens of the same species .Only 204 specimens (31.4%) yielded COI sequences, representing 57.7% and 61.3% of all species and genera sampled, respectively. The most successful primer combinations for COI amplification were 11F/2eR (615 bp amplicon), 12F/4R (529 bp amplicon), and 8F/2eR (666 bp amplicon). Most COI sequences used for this study were generated using combinations 11F/2eR or 12F/4R and trimmed to approximately 450 bp. Introns were found in 14 sequences (∼7% of those that amplified) in 18 species from 12 genera. Introns occurred in nine different locations . IdenticAgaricomycetidae, including 97 taxa belonging to the Boletales and Agaricales and 5 taxa belonging to the Polyporales , Russulales (Albatrellus), and Thelephorales (Sarcodon), were selected to compare COI with ITS. Thirty-six of the 102 morphospecies were represented by more than one sequence (2–9 sequences/species). Phylogenetic analysis of the COI dataset resulted in a moderately supported tree (aLRT≥0.95) and generally reflects the known topology of the Agaricomycetidae, except for the placement of Polyporus badius near Suillus and a non-monophyletic Boletales.A total of 167 specimens from 102 morphospecies of Intra- and inter-specific divergence was calculated for 30 species represented by more than one sequence in both the COI and ITS datasets based on global alignments of each gene . For COIBoletus spp., all ratios of inter- to intra-specific divergences for COI and ITS were greater than one, the minimum threshold for a barcode locus  and 0.64% for COI . The lowest minimum inter-specific divergence for ITS was 0.96% (Boletus regineus) and 0.20% for COI (Boletus spp.).A side-by-side comparison reveals that COI and ITS distances are qualitatively similar . Except de locus . In geneBoletus where intraspecific sampling was most extensive. In one case, ITS recovers B. nobilissimus as distinct from B. quercophilus, but COI does not. Similarly, ITS recovers a monophyletic B. edulis sensu stricto B. rex-veris with one specimen of B. edulis.COI and ITS largely agree on terminal taxa, which corresponds well with morphospecies . DiscrepFusariumLike Vialle et al. Boletus edulis and B. nobilissimus) that are recovered as reciprocally monophyletic by ITS, in agreement with a recent multigene phylogeny Overall, the performances of COI and ITS as barcode markers for mushrooms are similar. Both generally exhibit low intra-specific and high inter-specific divergences in our dataset and both were able to distinguish most of the morphospecies we sampled ; Fig. 2.Entoloma aff. sericeum ALG-07-108) from one of the contig folders. Our dummy sequences ranged from 1–20% divergent from the original. Except for two situations, where gaps are randomly distributed across over 15% or more of the sequence and when gaps are evenly distributed across 12% or more of the sequences (an extra base inserted every 8th position), Sequencher always correctly placed the two closest sequences together. Although the two cases where Sequencher did not correctly assign the two closest sequences together is of some concern, these synthetic scenarios are not very likely to be encountered with real biological material and we did not observe any misleading bias using the real data.The inability to produce a reliable alignment of ITS sequences across a divergent set of taxa is a major disadvantage of using ITS as a barcode. We avoided relying on a global alignment of ITS sequences by first identifying “alignment groups” where assessment of homologous characters for a set of sequences is trivial using widely used alignment software. Our approach relied on the contig assembly algorithm employed by Sequencher, which is normally used to assemble complementary sequence trace files output from ABI sequencers. By eliminating the ∼120 bp of the largely invariable 5.8S RNA subunit, increasing the minimum overlap to the maximum (100 bp), and using a threshold of 80% similarity, we avoided the problem of underestimating sequence divergence among a set of sequences, thereby minimizing ambiguous homology assessment. However, there are two related problems with this method: 1) contig folders must contain sets of sequences with 80% or better similarity, but some sequences may be 80% similar to two or more sets of sequences and could legitimately reside in more than one alignment group, and 2) the contig assembly algorithm is likely to be order-dependent , so when a sequence could reside in two folders it may always preferentially be placed in the first contig folder encountered even if its closest relative is in another folder. To explore whether or not the algorithm employed by Sequencher was biasing our contig assembly, we added to the ITS dataset dummy sequences where we modified the number of substitutions (randomly distributed) and gaps (randomly and evenly distributed or at a single site of extension) of the sequence with the greatest inter-specific divergence (Ideally, a global alignment would be possible for phylogenetic-based methods of identification and this is still a characteristic that may be sought in alternative barcoding loci for mushrooms. But the competency of ITS as a barcode demonstrated here and elsewhere, as well as the wealth of ITS data that can be mined from publicly accessible databases such as GenBank and UNITE The barcode locus that was been chosen for animals, COI, will not work for mushrooms, rusts


MTHFR C677T and A1298C, are associated with several cancers. This study aimed to evaluate the effects of MTHFR polymorphisms on colon cancer risk and possible interactions with environmental factors in a population from northeastern Thailand.Polymorphisms in methylenetetrahydrofolate reductase (MTHFR), such as MTHFR C677T and A1298C polymorphisms by polymerase chain reaction with restriction fragment length polymorphism techniques. Associations between variables of interest and colon cancer were assessed using conditional logistic regression.This hospital-based case–control study was conducted during 2002–2006; 130 colon cancer cases and 130 age- and sex-matched controls were enrolled. Information was collected and blood samples were obtained for assay of adj = 3.5; 95% CI: 1.19–10.25 and ORadj = 1.71; 95% CI: 0.74–3.96, respectively). The risk was also higher in subjects with frequent constipation  and occasional constipation . An interaction was observed between the MTHFR C677T polymorphism and freshwater fish consumption on colon cancer risk . Interactions were observed between the MTHFR A1298C polymorphism and bowel habits, family history of cancer, alcohol consumption, and beef consumption on colon cancer risk .Increased risk of colon cancer was associated with alcohol consumption and bowel habits. Alcohol drinkers who consumed ≤0.50 or >0.50 units of alcohol per day had elevated risks (ORMTHFR polymorphisms and environmental factors were also observed.In a Thai population, colon cancer risk was associated with alcohol and beef consumption, bowel habits, and family history of cancer. Interactions between  The population-based cancer registry of Khon Kaen, a province in northeastern Thailand, showed that the incidence of colorectal cancer during 1998–2000 was 8.6 and 7.0 per 100 000 population in males and females, respectively.1–4 Of these incident cases, approximately 60% were colon cancers and 40% were rectal cancers. An increasing trend was observed for both.In Thailand, colorectal cancer is one of the 10 most common cancers, and incidence has been increasing in both sexes in all areas during 1988–2000.5–16 Of these, obesity, physical inactivity, and alcohol consumption have been most consistently associated with increased risk of colon cancer. On the other hand, use of nonsteroidal anti-inflammatory drugs and high consumption of vegetables and fruits are generally associated with a decreased risk of colorectal cancer. The findings on smoking are not consistent. In epidemiological studies, diets low in folate have been found to increase the risk of colon cancer.17–19 Other dietary factors, including methionine and vitamins B6 and B12, have been associated with colon cancer in some but not all epidemiological studies.7,11,19–21 Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in folate metabolism; it catalyzes the conversion of 5,10-methylenetetrahydrofolate  to 5-methyltetrahydrofolate (5-methyl-THF).22 Two common polymorphisms in the MTHFR gene have been characterized.23,24 C677T causes an alanine to valine substitution in the N-terminal catalytic domain, which reduces enzyme activity and leads to lower levels of circulating folate (5-methyl-THF), accumulation of 5,10-methylene-THF, and increased plasma homocysteine.25,26A number of behavioral and environmental factors have been linked with colorectal cancer risk.MTHFR C677T and A1298C are associated with decreased risks of colon cancer27–29 and acute lymphocytic leukemia.30 However, the same variants have also been linked with an increased risk of endometrial cancer,31 cervical intraepithelial neoplasia,32 esophageal squamous cell carcinoma,33 gastric cancer,34 and bladder cancer.35 To our knowledge, no studies of this topic have been conducted in Thailand.This functional polymorphism has attracted a great deal of attention with regard to cancer risk, but the results have been conflicting. Several studies have shown that the low-activity variants of MTHFR C677T and A1298C.As part of the multicenter “International collaborative epidemiological study of host and environmental factors for stomach and colorectal cancers in Southeast Asian Countries”, we examined putative risk factors for colon cancer in a population from northeastern Thailand, with a focus on both environmental parameters and polymorphisms in MTHFR gene.A total of 130 new cases of colon cancer were recruited from Srinagarind Hospital and Khon Kaen Regional Hospital, Khon Kaen Province, between October 2002 and October 2006. All patients were from Khon Kaen Province or neighboring provinces and were histologically confirmed to have colon cancer. The patients were interviewed within 3 months of diagnosis. During the same period, 1 control matched for sex, age (±3 years), and province of residence was recruited for each case. Subjects with gastrointestinal disease or other cancers were excluded. All gave informed consent for their participation in the study. Subjects who refused to complete the interview or were unable to do so because of advanced age or other reasons were excluded. The controls had a variety of illnesses, the most common of which were inflammation, and diseases and disorders of the eye and genitourinary system. A 5-ml blood sample was obtained from all cases and their matched controls, and transferred to the laboratory for investigation of polymorphisms in the 36 For beverage consumption, there were 2 groups: alcoholic beverages and tea/coffee. The questions for each item requested information on whether the participant did or did not drink the beverage, the frequency of drinking, and the amount consumed per occasion.Subjects were interviewed by 2 trained interviewers using a structured questionnaire comprising 2 sections. The first section included demographic and socioeconomic status, smoking history, family history of cancer, past history of illness, and bowel habits. The second section was a food frequency questionnaire structured by meal. There were 9 categories of food items. The questions for each item consisted of frequency of consumption  and amount consumed per unit of frequency. Support for the validity of this dietary method is provided by our previous study.The interview requested information on habits 1 year before the subjects became sick with their present illness. For bowel habits, the interviewers asked the subjects to recollect these habits beginning from adolescence to working age, until 1 year before the present illness, and asked the subjects to determine if their bowel habits had changed.Genomic DNA was extracted from buffy coat fractions of the cases and their matched controls using a standard technique at Nagoya City University Medical School, Nagoya, Japan. Gene amplification and polymorphism analyses were performed in the Microbiology Laboratory at the Faculty of Medicine, Khon Kaen University, Thailand.23 Briefly, amplification of MTHFR C677T used 2 primers, [F]:5′-TGA AGG AGA AGG TGT CTG CGG GA-3′ and [R]:5′-AGG ACG GTG CGG TGA GAG TG-3′. The PCR product (198 bp) was digested with 10 units of HinfI restriction enzyme (Fermentas Life Sciences) in a 20-µl reaction mixture containing 5 µl of PCR fragments and 2 µl of 10× buffer R at 37°C overnight. Digestion products were visualized after electrophoresis on 8% polyacrylamide with ethidium bromide. Results of the HinfI RFLP analysis of the MTHFR C677T polymorphism are illustrated in Figure The polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) technique was modified as previously described. product 98 bp wasMTHFR A1298C polymorphism was performed as previously described.24 The primers for PCR amplification were [F]:5′-AGG ACG GTG CGG TGA GAG TG-3′ and [R]:5′-CAC TTT GTG ACC ATT CCG GTT TG-3′. The PCR product of A1298C (163 bp) was digested with 10 units of MboII restriction enzyme (Fermentas Life Sciences) in a 20-µl reaction mixture containing 5 µl of PCR fragments and 2 µl of 10× buffer R at 37°C overnight. Digestion products were visualized after electrophoresis on 12% polyacrylamide with ethidium bromide. The MboII RFLP analysis for MTHFR A1298C polymorphism is illustrated in Figure Analysis of the MTHFR C677T and MTHFR A1298C were also analyzed.Associations between colon cancer and potential risk factors were evaluated using odds ratios (ORs) and 95% confidence intervals (95% CIs) derived from conditional logistic regression. Crude and adjusted odds ratios were estimated for each independent variable. Factors included in the multivariate analysis by conditional logistic regression were those found to be strongly associated with colon cancer in univariate analysis and those that were not found to be strongly associated with colon cancer in univariate analysis but were shown in previous reports to play an important role in colon cancer risk. Possible modifications of the effects of potential risk factors by polymorphisms in MTHFR C677T and A1298C polymorphisms were analyzed in the colon cancer cases and their matched controls by using the McNemar test; 2 × 2 tables were employed to compare genotype distributions between any 2 groups. Both tests were utilized to compare the cases and their matched control subjects with regard to genotype frequencies and potential risk factors for colon cancer such as demographic characteristics, diet, smoking, and alcohol drinking.The distributions of alleles/genotypes for the MTHFR C677T and MTHFR A1298C polymorphisms. All statistical analyses were performed using STATA version 10, and a P value <0.05 was considered statistically significant.To investigate the gene–environment and lifestyle interactions, ORs and their 95% CI were calculated using the McNemar test. The ORs were adjusted for age and sex according to the yamuan (homemade cigarette-sized cigars). Duration of smoking and average number of cigarettes per year were computed based on all smoking periods reported, and patients were dichotomized using the median for the controls. Average number of cigarettes was calculated as annual cigarette consumption (filtered and unfiltered) plus 1.5 times annual yamuan consumption. The 1.5 correction factor was used because yamuan are larger than regular cigarettes. The amount was categorized based on the 50th percentile of the controls and dichotomized into low and high levels.For the analysis of cigarette smoking, the subjects were categorized as smokers or nonsmokers. Smokers included those who smoked filtered cigarettes, unfiltered cigarettes, or sato], white whiskey, Thai and other whiskies) at least once a month. Those who did not drink, or consumed alcoholic beverages less than once a month, were categorized as nondrinkers. The alcohol consumption of each subject was calculated as alcohol units. A unit of alcohol was defined as 10 milliliters (or approximately 8 grams) of ethyl alcohol. The number of units of alcohol in a drink was determined by multiplying the volume of the drink (in milliliters) by its alcohol percentage, and dividing by 1000.37For the analysis of alcohol drinking, there were 2 categories: drinkers and nondrinkers. Ever drinkers were defined as those who consumed at least 1 type of alcoholic beverage  defined as 5.0% for beer, 7.0% for For the analyses of dietary intake within the previous year , 2 levels were used—low and high. Frequencies of each dietary intake, and the amount of intake per year, were computed based on each type of dietary intake reported. Patients were then dichotomized using the median for the controls.Table MTHFR C677T polymorphism, the prevalence of the T allele was 20% and 27.7%, respectively. For the MTHFR A1298C polymorphism, the frequency of the C allele was 66.9% in cases and 58.5% in controls (Table Among the cases 130) and controls (130) genotyped for the 30 and coMTHFR 677 C/C genotype, those with the MTHFR 677 C/T genotype had a tendency toward reduced risk . Those with the MTHFR 1298 A/C genotype had a tendency toward higher risk of colon cancer than did those with the 1298 A/A genotype , whereas those with the MTHFR 1298 C/C genotype had a tendency for decreased risk, as compared with patients with the 1298 A/A genotype .Table There were no associations between colon cancer risk and occupation, smoking status, or tea/coffee drinking. There was an association between alcohol consumption and colon cancer, as shown in Table On univariate analyses of dietary intake based on the food frequency questionnaire, with the low level as the referent group, there was no significant association between overall food consumption and colon cancer risk. Only beef consumption showed a possible link with colon cancer risk .P value <0.10), and factors that have been reported to have a strong relationship with colon cancer, were included in the multivariate analysis by conditional logistic regression. These factors were bowel habits; family history of cancer; alcohol consumed per day; and consumption of beef, freshwater fish, offal, and fruit.Factors found to have a strong association with colon cancer on univariate analysis . For the MTHFR A1298C polymorphism, there were interactions with bowel habits, family history of cancer, and beef consumption .Risk factors that were identified as significant were studied for interaction with polymorphisms in 4 although absolute values are much lower than those in Japan and European countries.38 In the present study, the authors recruited cases from 2 hospitals: Srinagarind Hospital, which is a teaching hospital, and Khon Kaen Regional Hospital, which specializes in cancer treatment. A great number of cancer patients come to these 2 hospitals for treatment. Therefore, we were able to recruit a sufficient number of cases for this study.In northeastern Thailand, colon cancer is one of the 10 most common cancers and the incidence rate is increasing,10 The strongest association in the present study was between constipation and colon cancer. Sonnenberg and Muller39 conducted a systematic review of 9 case–control studies published before 1992, and evaluated the risk for colorectal cancer associated with self-reported constipation and bowel movement frequency: constipation was associated with a significant increase in the risk of colorectal cancer . Although we only investigated colon cancer, our findings are consonant with those of earlier studies. It has been hypothesized that constipation increases colon cancer risk because it results in longer transit times in the colon, which increases the duration of contact between the colonic mucosa and concentrated carcinogens in the lumen.40,41 Possible carcinogenic agents within stool include bile acids,42,43 fecapentaene,44 and ammonium acetate.45 Constipation is also a major symptom of colorectal cancer; however, in this study the authors informed participants that they should focus on bowel habits that began in adolescence through working age until 1 year before illness, and reminded them to think of any changes in bowel habits, to avoid information bias.Our present results augment earlier findings on lifestyle-related risk factors for colorectal cancer in northeastern Thailand.6 Important sources of known heterocyclic amine carcinogens with established links to colorectal cancer46 include cooked meat (which contains heterocyclic amines and nitrosamines). In our study, there was a trend toward increasing risk of colon cancer with high beef consumption in both univariate and multivariate analyses, but the associations were not significant. However, in the study of possible gene–environment interactions between the MTHFR A1298C polymorphism and beef consumption, an interaction was observed . This finding highlights an important public health concern, especially for those who eat beef.A number of studies have found an association between beef consumption and the risk of colorectal cancer.1–4,47 which might be partly due to the lack of effective cancer screening in Thailand. Northeastern Thailand has, for many decades, been the most impoverished part of the country. Until the introduction of various industries in the last 20 years, the population was largely rural, relying on cultivation of rice as the staple food. The genetic background of this region has not changed, so the increase in the incidence of colon cancer must be due to lifestyle or environment changes.An analysis of cancer registries in Thailand shows that most patients present at a late stage,5,12,13 Ethanol may act as a solvent or have cytotoxic effects on tissues. It may also cause deficiencies in nutrients, particularly folate.7,48 In this study, there was a trend toward an association between alcohol consumption and the risk of colon cancer, with a possible link to the MTHFR A1298C polymorphism.A number of studies have reported that high alcohol consumption increases the risk of colon cancer and that the association is likely dose-dependent, irrespective of the type of alcohol consumed.6,49 Although our results showed only a tendency for protection against colon cancer, vegetables and fruits possess antioxidants that can reduce DNA damage.46 In addition, a key constituent of vegetables and fruits is folate. There is evidence that consumption of folate is related to the risk of adenomas and other types of neoplasms. Folate and MTHFR may be important in influencing the availability of S-adenosylmethionine (SAM), the universal methyl donor, thereby affecting both DNA methylation and the nucleotide pool.24 A deficiency of folate in tissues with rapidly replicating cells results in ineffective DNA synthesis, which reduces cell proliferation, impairs cellular physiology, and alters cell morphology. The MTHFR enzyme plays a central role in the metabolism of folate, a nutrient that has been inversely associated with colorectal cancer risk.Several epidemiological studies have provided evidence that high consumption of vegetables and fruits lowers the risk of colon cancer.MTHFR C677T polymorphism is associated with a reduced risk of colorectal cancer, especially in people who consume high levels of folate27,29,50–55 or take multivitamin supplements that include B2, B6, and B12,29,56 but an increased risk has been noted in those who consume a large amount of alcohol. The importance of folate was confirmed by an in vitro study of HCT116 cells, in which the MTHFR C677T mutation was associated with significantly increased genomic DNA methylation when the folate supply was adequate or high. However, in cases of folate insufficiency, the mutation was associated with significantly decreased genomic DNA methylation.57Several reports have shown that the MTHFR 677 was 27.7% in controls, which corresponds to other studies.27,50–54,56,58,59 This polymorphism has been shown to modify the risk for several cancers in a site-specific manner.60,61 Epidemiologic evidence indicates that this polymorphism has a protective effect in individuals with adequate or high levels of folate and other nutrients involved in 1-carbon metabolism.62–64 In those with inadequate levels of folate and related nutrients, the protective effect conferred by this polymorphism appears to be lower; in some cases, an increased risk of colorectal cancer has been observed.64In this present study, the genotype frequency of MTHFR 1298 polymorphism, the genotype distribution in the controls deviated from the Hardy–Weinberg equilibrium. The rate for the A1298C polymorphism in controls was higher than rates noted in reports from other countries. Our figures may be representative for the Thai population; however, there have been no previous reports. The possibility of genotyping error should be acknowledged. Genotyping error in MTHFR 1298 polymorphism studies has been reported when genotyping was performed by digesting PCR products with the enzyme Mboll, which recognizes the sequence GAAGAN8 and digests this sequence on wild allele 1298A (A/A). If both 1298A and 1317C are present on a chromosome, the fragment size of digestion does not differ and it is thus difficult to differentiate heterozygous individuals (1298 A/C genotype) from wild homozygous individuals (1298 A/A genotype), even when a positive control is included in all the electrophoresis runs to compare the sample results.65,66 Therefore, fragment size analysis by sequencing and comparison of the sequence in GenBank is necessary to confirm the result. In a comparison of RFLP using the enzyme MboII and direct sequencing techniques for identification of polymorphism in A1298C, an 8.6% difference in genotyping was found.67In our analysis of the As is the case with any case–control study, the possibility that cases and controls differentially reported inaccurate information should be considered when interpreting the results. A differential reluctance on the part of cases or controls to accurately report average levels of drinking may have occurred in this study. One might expect the underestimation of true alcohol drinking to have occurred at some level above 1 drink per day , which would result in inconsistency in the association of colon cancer with the level of alcohol consumption.Associations of various types of cancer with food groups and nutrient compositions have been studied elsewhere. Although we made great efforts to use available investigative tools in this study, there is no standardized food frequency questionnaire for northeastern Thai food. Thai eating habits and foods are quite different from those in the West, so it is difficult at present to quantify foods and calculate the precise intakes of vitamins and nutrients.One other limitation in this study is that its relatively small size probably had some impact on the analysis of interactions, as evidenced by the absence of data for some variables in the tables. A future study with a larger sample size is warranted.MTHFR C677T and MTHFR A1298C polymorphisms were found but require additional investigation. Therefore, further research on the mechanisms underlying carcinogenesis in the Thai population is required.We conclude that bowel habits and family history of cancer are strongly associated with the risk of colon cancer in this population. The findings also suggest that individuals who consume alcohol and beef are at increased risk. Interactions of lifestyle factors with