Of 467 participants enrolled, 361 (77.3%) completed questionnaires and had sufficient paired pre- and post-travel serum for testing; 58 (12.4%) were lost to follow-up; 21 had insufficient blood for testing; and 27 were excluded. There were 214 females (59.3%) and 147 males (40.7%). Pre- and post-travel specimens were collected at a median of 29 days prior to travel (range 0–265 days) and a median Olaparib molecular weight of 6 days following return to Australia (range 0–31 days). The

median travel duration was 21 days (range 7–326 days) with 74% <30 days. The major reasons for travel were tourism (73.1%), business (17.7%), and visiting friends and relatives (VFRs, 4.71%). Table 1 shows the demographic data and total traveler-days for the top 10 countries visited. Four of the 361 travelers (1.1%) demonstrated serological evidence of HCV infection. Two were past infections and two travelers had evidence of seroconversion, representing an incidence density of 1.8 new infections per 10,000 traveler-days (95% CI: 0.22–6.53). Both travelers with seroconversion were asymptomatic, and likely acquired selleck kinase inhibitor their infection in Vietnam (n = 1) or Thailand (n = 1) during short-term travel (14 days duration each). The traveler to Thailand was a 24-year-old female tourist who visited Koh Samui and Bangkok. The traveler to Vietnam (a 50-year-old male) traveled to the cities of Hanoi and Ho Chi Minh. None of the

four HCV seropositive travelers were viremic on testing of either pre- or post-travel sera. Six of the 361 travelers (1.77%) were anti-HBc antibody positive, consistent with evidence of HBV infection. Five of these infections were present before travel. One traveler showed evidence of seroconversion [pre-travel serum negative for anti-HBc immunoglobulin G (IgG) and IgM, anti-HBs, anti-HBe, HBsAg, and HBV DNA; post-travel anti-HBc IgG positive

but IgM negative, anti-HBs positive, HBsAg, HBeAg, anti-HBe, and HBV DNA negative]. The serological profile was consistent with self-limited primary infection. This traveler, a male aged 40, had evidence of seroconversion consistent with acquisition of HBV during his short business trip to China. He had his pre-travel blood collected 31 days prior to departure, traveled through China for 22 days, and Adenosine triphosphate had post-travel bloods taken 8 days post return to Australia. HBV PCR testing of sera from the entire cohort was negative; 56% of travelers (202/361) were HBV immune (anti-HBs ≥10 mIU/mL). The incidence density of HBV infection in nonimmune travelers was calculated as 2.19 per 10,000 traveler-days (95% CI: 0.07–12.19). This retrospective cohort study demonstrates that travelers are at risk of both HBV and HCV infection, and is the first to quantify the risk of HCV infection in travelers. While the number of seroconversions was small the identification of two HCV and one HBV seroconversion is notable and indicates potential exposure to other blood and bodily fluid-borne infections such as HIV.

92). The similarity was expected because both isolates belong to same forma specialis and geographical region. The most diverse (similarity coefficient value 0.12) isolates were Fol-6 and Foi-2. The dendrogram constructed based on similarity index resulted in two major clusters (Fig. 2). High bootstrap values were recorded with internodes, which indicate the robustness of the clustering. The first major cluster has been exclusively composed of Fom isolates, which is further divided into two subclusters having three Alectinib ic50 Fom isolates each. The second cluster having different subclusters comprises a mix of all the formae speciales taken into this study except Fom. The knowledge of abundance

and distribution of genetic variability within and among formae speciales of F. oxysporum PS-341 nmr is a prerequisite to study their genetic relationships (Bruns et al., 1991). In the present study, the relative density and relative abundance of SSRs in Fom was higher. So far, we do not have any strongly supported explanation for this. However, this discrepancy may be occurred because of transfer of lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account

for a quarter of the genome (Ma et al., 2010). It has been observed from genome-wide study that the distribution of microsatellites in the genome is not random. Coding regions are mostly dominated by tri and hexa-nucleotide Etofibrate repeats, whereas di, tetra, and penta nucleotide repeats are often found in abundance in noncoding region (Kim et al., 2008; Levdansky et al., 2008). Differential distribution in terms of abundance of SSRs has been reported in between intronic and intergenic regions, 5′ and 3′ UTRs, and in different chromosomes and lastly, different species have different frequencies of SSR types and repeat units (Li et al., 2004; Garnica et al., 2006; Lawson & Zhang, 2006). In our study, we observed similar pattern of distribution

of SSR in the coding region where tri and hexanucleotide SSRs were predominant. These tri and hexanucleotide SSRs in the coding region are translated into amino-acid repeats, which possibly contribute to the biological function of the protein (Kim et al., 2008). Dinucleotide SSRs are often found in the exonic region of F. oxysporum; however, (GT)n and (AC)n repeats were common in all the three formae speciales. Stallings et al. (1991) reported that (GT)n repeat is able to enhance the gene activity from a distance independent of its orientation. However, more effective transcription enhancement resulted from the GT repeat being closer to promoter region. Similarly, (CA)n repeat can act as a bridge to bring the promoter into close proximity with a putative repressor protein bound downstream of the (CA)n SSR (Young et al., 2000).

Patients preferred shorter waiting times,[40] high satisfaction with pharmacy ratings, quality certificates and extended opening hours.[37] A study by Wellman and Vidican,[39] piloting the addition of medication management services in prescription benefit insurance models, demonstrated that respondents placed the greatest value on pharmacist provision of comprehensive medication management services. Several of the studies also indicated the existence of ‘status-quo’ bias (i.e. tendency to prefer their current service) among patients with respect to

pharmacy choices.[35-37] Four studies examined pharmacist preferences, of which only three elicited preferences with respect to pharmacy services,[41-43] while one was related to preferences for a specific new technology.[44] Grindrod et al.[42] investigated pharmacist preferences for specialised service provision and showed Epigenetics Compound Library that pharmacists preferred to provide medication and disease-management services but did not have significant preferences for screening services. This was contrary to Scott et al.[43] who investigated pharmacist preferences for extended roles in primary care. Significant predictors of pharmacists’ job choices included having an extended pharmacy team, integration with primary and secondary care as well as higher income whereas provision of chronic disease

management and health promotion services was not preferred. Using the latent class model, one study[42] showed the existence of preference heterogeneity in pharmacists’ preferences Alectinib research buy with pharmacists falling into three classes, indicating that groups of pharmacists may have different preferences with respect to specialised services provision. Pharmacist preferences were mainly investigated for process-related aspects such as duration of service, type or level of service provision, setting and integration with primary/secondary care and professional service/job-related aspects including job satisfaction, educational requirements and personal income. In the majority of the

studies eliciting Loperamide preferences for the delivery of specialised pharmacy services (medication or disease management), income was an important attribute, with pharmacists preferring higher incomes.[41-43] Only two studies investigated preferences of both patients and providers for haemophilia therapy.[45, 46] These were also the only studies that included health-outcome related attributes in addition to process-related outcomes. While patients and providers showed substantial consensus for some attributes (e.g. cost), preferences for other attributes were quite different. Patients were more focused on process-related attributes as compared to providers. The relatively few pharmacy DCE studies make it hard to make definitive conclusions about pharmacy services from both the provider and recipient viewpoints. However, a few findings may be highlighted.

Chromosomal clpP′-lacZ and clpX′-lacZ Venetoclax manufacturer transcriptional fusion strains were constructed by FLP-mediated site-specific recombination as described (Ellermeier et al., 2002). Cloning of rpoS including the 5′ untranslated region (UTR)(designated rpoS), the rpoS coding region alone without UTR (designated ΔUTRrpoS), and clpPX into the pHR718 vector was conducted as follows: the fragments of rpoS, ΔUTRrpoS, and clpPX were obtained by PCR amplification from chromosomal DNA of the MG1655 strain

using the pairs of primers listed in Table 2. The PCR fragments of rpoS and ΔUTRrpoS were digested with EcoRI and HindIII and then inserted into the EcoRI–HindIII region of the vector; the constructs were designated pHR718-rpoS and pHR718-ΔUTRrpoS, respectively. The PCR fragment of clpPX obtained was digested with MfeI and HindIII and then inserted into the EcoRI–HindIII region of the vector, yielding a plasmid designated pHR718-clpPX. Luria–Bertani

(LB) medium containing 1% Tryptone (Difco), 0.5% yeast extract (Difco), and 1% NaCl (Miller, 1972) PF-562271 solubility dmso was used with the following supplements when required (per liter): 50 mg ampicillin, 25 mg kanamycin, and 50 mg spectinomycin. Cells were grown at 37 °C and growth was monitored using a Spectomonitor BACT-2000 (Nissho Electronics, Tokyo). Cells were pelleted and immediately resuspended in sodium dodecyl sulfate (SDS) loading buffer in a volume with equal OD540 nm. After boiling for 5 min, equal volumes (25- or 5-μg protein) were loaded on SDS-12% polyacrylamide gels. After electrophoresis, proteins were transferred to polyvinyliden difluoride membranes and probed with a 1 : 5000 dilution of anti-σS antibody (Tanaka et al., 1993). As the secondary antibody, peroxidase-conjugated affinity pure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch) was used at a dilution

of 1 : 2000. The bands were detected using the ECL antibody detection kit (GE Healthcare Bioscience) and an X-ray film (Fujimedical X-ray Film RX). The half-life of σS was determined using the method described previously (Tu et al., 2006). To cultures in the logarithmic growth phase (OD540 nm=0.35), chloramphenicol (200 μg mL−1) was added, and 750-μL culture samples were withdrawn at 1 and 2 min (pgsA+) and Baf-A1 order 5 and 10 min (pgsA3). The cells were precipitated with 5% ice-cold trichloroacetic acid. Precipitated pellets were washed with 80% cold acetone and then suspended in SDS sample buffer. SDS-polyacrylamide gel electrophoresis and Western blotting analysis were carried out as described above. The activity of β-galactosidase was assayed using o-nitrophenyl-β-d-galactopyranoside as the substrate. The specific activity was recorded as μmol of o-nitrophenol min−1 (mg cellular protein)−1 (Miller, 1972). Cells were grown as described above, and aliquots were removed from exponential-phase cultures (OD540 nm=0.35) to prepare total RNA.

The questionnaire explored the behaviour, ‘giving information to medical counter assistants’. Respondents were categorised

as ‘information givers’ or ‘non-givers’ according to their response to the question ‘Last time you bought a pharmacy medicine, did you tell a member of the pharmacy staff: what your health problem was; what product you wanted; both (health problem and product); something else’. Respondents who answered ‘health problem’ or ‘both’ were categorised as ‘information givers’. Those who answered ‘product’ were categorised as ‘non-givers’. Responses of ‘something click here else’ (n = 44) or missing responses (n = 122) were excluded from the analysis as they could not be classified accurately into an information giver or non-giver. Behavioural intention for giving information (BI) was measured using three items: The next time I buy a pharmacy medicine: I intend to give the MCA information; I want to give the MCA information; I expect to give the MCA information (rated on a 7-point scale (7 = strongly disagree, 1 = strongly agree) then reverse scored). BI to give the information sought by WWHAM (BI-WWHAM) was rated on a 7-point scale (7 = strongly disagree,

1 = strongly agree) then reverse scored for each of the five WWHAM items (Table 2). For each measure, item scores were summed and higher scores reflected stronger intention to give information and to give WWHAM information. Attitude was measured by summing scales on four statements http://www.selleck.co.jp/products/erastin.html (‘The next time I buy a pharmacy medicine, for me to give information to the

selleck chemicals MCA will be … good/bad, worthless/worthwhile’, etc.) using bipolar scales (1 to 7 with 1 = good and 7 = bad). Subjective norm was measured by two statements (‘People who are important to me will think I should give information to the MCA’, ‘I feel under pressure from other people to give information to the MCA’) using a 7-point scale, strongly agree to strongly disagree (1 to 7), which was then reverse scored. PBC was measured by summing scales on two statements (‘The next time I buy… . , for me to give information to the MCA will be difficult/easy, impossible/possible’) using bipolar scales (1 to 7 with 1 = difficult/7 = easy), which were then reverse scored. The beliefs investigated were behavioural (n = 4), control (n = 11) and normative (n = 4). They were assessed using 7-point scales from ‘strongly agree’ to ‘strongly disagree’ (Tables 2 and 5). The full questionnaire was piloted with 30 individuals randomly selected from the electoral roll sample. A response of 28.6% (n = 8) was achieved. A second pilot using a shorter version gave a higher response rate of 47.3% (n = 14/30). In the main study, the two versions were sent to half the sample each (on the basis of random selection), i.e. direct measures, and direct measures plus salient beliefs, to allow further investigation of the trade-off between response rate and length of questionnaire.

Seroepidemiologic studies of influenza among well-returned travelers indicate seroconversion in 2.8%,6 and among febrile returning travelers, the incidence of influenza is estimated to be between 5 and 15%.13 Thus, our findings likely represent a significant underestimate of cases of influenza among ill-returned travelers. High hospitalization rates potentially indicate that only more severe infections Antidiabetic Compound Library ic50 were evaluated at a GeoSentinel site, thereby further underestimating the burden of influenza in travelers. Third, during influenza season in temperate countries, confirmatory diagnostic tests are not often sent once influenza is circulating within a community,

and this study included only confirmed or probable diagnoses. Fourth, absence of immunization history limits our ability to quantify true potential influenza preventability in this cohort. Fifth, given the short incubation period of influenza, we cannot exclude the possibility that some travelers, especially those returning home during their influenza see more season, or residing in the tropics or ESEACN, became infected en

route home or after travel. Influenza acquired abroad versus from the country of residence would be impossible to distinguish clinically. Finally, our data do not permit estimation of incidence rates or destination-specific numerical risks for influenza.7,14 This is the single largest analysis of latitudinal patterns of influenza in travelers, to date, and is derived from a multicenter, heterogeneous population, reflecting the spectrum of travel demographics and destinations over a 10-year period. Alternate hemisphere and out-of-season influenza vaccine availability may benefit a small proportion of travelers. As noted previously, while knowledge of influenza prevention among travelers appears to be good, translation of this knowledge into uptake of prevention measures such as vaccination, antiviral prophylaxis, and hand hygiene among travelers remains low.15,16 Proportionate morbidity estimates by region of travel

can inform pre-travel consultation and emphasize the selleck chemicals ease of acquisition of infections such as influenza during travel. These data can inform broad-level decision-making in travel medicine, public health, and health care policy. GeoSentinel: the Global Surveillance Network of the International Society of Travel Medicine is supported by Cooperative Agreement U50/CCU412347 from the Centers for Disease Control and Prevention. The funding source (the Centers for Disease Control and Prevention) had no role in study design, data analysis and interpretation, or in writing the manuscript. A. K. B., F. v. S., P. L. L., E. S., and M. E. W. state that they have no conflicts of interest to declare. F. C. has received an honorarium to attend the Tamiflu Advisory Board once in 2006. P. G. was sponsored by Sanofi-Pasteur to attend conferences. J. T.

g., heart

muscle and eyes) or organs of the excretory system (e.g., kidneys and bladder) before arthritis onset (day 2).[20] Epigenetics Compound Library cell line However, additional 18F-FDG signaling can be detected in the joints of fore and hind paws in acute experimental arthritis at day 13, suggesting a specific 18F-FDG uptake in inflamed joints. PET/CT imaging showed hot spots of inflammatory metabolic activity in wrist and ankle joints. After treatment of human sTNFR (etanercept) or saline with G6PI-induced mice, PET/CT found a marked 4.9-fold decrease of total 18F-FDG uptake in sTNFR (etanercept)-treated arthritic mice. Comparable results were obtained using histopathological assessment of therapeutic intervention.[20] Thus, PET/CT is a convenient technique

for monitoring disease activity or efficacy of treatment in experimental arthritis. It is noteworthy that FDG PET/CT may help predict therapeutic response to novel treatments. In a group of active RA patients, before infliximab treatment, all patients indicated enhanced 18F-FDG uptake in at least one metacarpophalangeal region or wrist.[49] After 14 and 22 weeks, selleck chemical DAS decreased to 4.3 ± 1.5 and 3.9 ± 1.3, respectively. The change in mean SUV after 2 weeks of infliximab treatment correlated markedly with DAS at 14 and 22 weeks, respectively.[49] The study found a strong correlation between early changes in 18F-FDG for uptake in hand joints and clinical disease activity after 14 and 22 weeks of treatment. At a group level, the findings suggest that 18F-FDG

PET may therefore be a valuable technique for predicting the efficacy of infliximab therapy as early as 2 weeks after initiation of treatment. Rituximab, a chimeric monoclonal antibody against the CD20 antigen, has been approved for the treatment of RA patients.[50] Tran et al.[51] radiolabeled rituximab with 124Iodine (124I) for PET imaging. Results showed that patients who did not receive pre-treatment with unlabeled rituximab indicated localization of nearly all radioconjugate in the spleen and to a lesser extent bone marrow, examined by PET/CT imaging 10 min after administration of 124I-rituximab.[51] Findings after 24 h indicated that the uptake in the spleen was largely diminished while the radioactivity accumulated in the thyroid.[51] In contrast, 124I-rituximab has favorable pharmacokinetics for targeting pathological B cells after pre-treatment with unlabeled rituximab, where patients predosed with unlabeled rituximab indicated persistent tracer availability in the central circulation for multiple days, with almost no splenic uptake.[51] Furthermore, PET imaging of patients received 124I-rituximab at 24 h and later exhibited accumulation of the tracer in joints (e.g.

4). Apart from PLFA nor16:0, the highest proportions of the plant litter-derived 13C were detected in 18:2ω6,9 and 18:1ω7 (Table S2). Readily available C of both plant litter types thus promoted mainly fungi and Gram-negative bacteria, which is in accordance with recent studies. The rapid labelling of the fungal biomass after only 1 month of incubation was recently explained by fungal hyphae that grow into the litter from the mineral soil layer (Moore-Kucera & Dick, 2008). Twelve weeks after litter application, a large difference between L. corniculatus and C. epigejos was observed as a result of the

lack of Gram-positive bacteria in L. corniculatus (nor14:0, iso15:0, ant15:0), but also because of a decreased proportion of Selleck INK128 13C in fungi (18:2ω6,9) in C. epigejos. This result underlines the competition between fungi and Gram-positive bacteria as discussed above; in C. epigejos treatments, a decrease in fungi results in

a decreased litter-degrading activity, which in turn promotes Gram-positive bacteria in the decomposition process. In both treatments, an increased proportion of litter-derived 13C was detected in Gram-negative bacteria, indicated by 16:1ω5, 18:1ω7, 18:1ω9 and cy19:0 (Table S2; Fig. 4). These results generally confirm the recent findings of Kramer & Gleixner (2006), who postulated a preferential uptake of litter C by Gram-negative bacteria, while Gram-positive bacteria utilized soil-derived C. The low C content of the soil might explain the outcompetition of Gram-positive bacteria mainly in the L. corniculatus treatments by fungi as long as available N from the litter material is present. Forty weeks after the application of litter selleck compound material, samples from L. corniculatus and C. epigejos treatments again showed a similar 13C distribution among the PLFA biomarkers. In contrast to the samples at 12 weeks, an increase of 13C in a number of Gram-positive cAMP (iso15:0, iso16:0, iso17:0, 10ME17:0) and Gram-negative biomarkers (17:1ω8, 16:1ω5)

was observed in both treatments. This result is in accordance with experiments performed with soils from climax ecosystems, where, in the later phase of litter decomposition, the 13C distribution among a high diversity of microbial communities indicates a system in a steady state, where incorporated litter C has been recycled throughout the microbial community structure (Rubino et al., 2010). Both Gram-negative as well as Gram-positive bacteria have been found in context with complex and recalcitrant litter material decomposition (Peacock et al., 2000; Elfstrand et al., 2008). Overall, the results of the present study show (1) a stronger influence of litter quality on biological interactions between bacteria and fungi during the decomposition process compared with litter degradation in climax ecosystems, which in turn alters the process of litter decomposition and results in different rates of litter degradation of the two colonizer plants L. corniculatus and C. epigejos.

Overall there was a modest benefit in terms

of delaying the decline in CD4 cell count, or time from seroconversion, to requiring initiation of lifelong ART following a 48- [16] or 60- [15] week course of ART. A post hoc analysis from the SPARTAC RG7204 clinical trial trial [16] showed a non-significant trend towards benefit in time to CD4 cell count <350 cells/μL when ART was initiated closer to the time of infection (HR 0.48; P = 0.09). This randomized study supported cohort studies in which a more rapid rate of CD4 cell loss was seen in individuals presenting within 12 weeks of a negative HIV antibody test [17, 18]. For this reason, we suggest that the following are discussed with those presenting with a very short

test interval (≤12 weeks), in particular, those with severe symptoms of seroconversion such as rash, fever, weight loss, persistent lymphadenopathy, diarrhoea >4 days, malaise, headaches or laboratory evidence of acute HIV infection (e.g. as defined in SPARTAC [16]). A 48-week course of ART showed a benefit in surrogate markers of HIV-disease progression: delaying CD4 decline and lowering viral set point up to 60 weeks after stopping therapy. There was no such benefit from 12 weeks of ART. In those individuals presenting within 12 weeks of infection, this effect was more marked; however, there is no clear evidence of long-term clinical benefit of ART in this setting. No study has learn more examined whether ART started during, or soon after, PHI should be continued long term, but most clinicians

would recommend that irrespective of indication to start ART, once initiated, it should be continued indefinitely. Discontinuation of ART in the context of treatment of PHI was not commonly associated with morbidity, however [15, 16]. Initiation of a PI-based regimen is recommended if therapy is started before the availability of a genotype result, based on the prevalence of transmitted rates of drug resistance in the UK [19]. There is no specific evidence to support the role of ART in PHI to prevent onward transmission of virus but there is little reason to consider that ART is any less effective in reducing infectivity at this time, so long as viral suppression has been achieved [20]. Patients with recently diagnosed PHI may be in a Sclareol particularly vulnerable psychological state, and thus ill-prepared to commit to starting long-term treatment. We recommend the evidence that treatment with ART lowers the risk of transmission is discussed with all patients, and an assessment of the current risk of transmission to others is made at the time of this discussion (GPP). We recommend following discussion, if a patient with a CD4 cell count >350 cells/μL wishes to start ART to reduce the risk of transmission to partners, this decision is respected and ART is started (GPP).

Thus, 660 000 cases

of sepsis occur in the United States each year, and combined with the high mortality, it ranks as a leading cause of death. Staphylococci, including methicillin-resistant Staphylococcus aureus, have become the most frequently isolated bacteria in nosocomial infections, giving rise, according to some reports, to more than 50% of the cases (Bearman & Wenzel, 2005). Severe sepsis occurs when sepsis (the combined events of infection and the systemic inflammatory response syndrome, i.e. SIRS) is complicated by organ dysfunction, hypotension or hypoperfusion; septic shock is characterized by persistent arterial hypotension despite adequate fluid resuscitation (Bone et al., 1992; Levy et al., 2003). The Sequential Organ Failure Assessment (SOFA) score, which evaluates the respiratory, blood clotting, hepatic, cardiovascular, central nervous and renal systems, has been advocated as a simple (bedside) method to monitor organ BMN 673 molecular weight dysfunction and guiding supportive therapy of critically ill patients, including those with sepsis (Vincent et al., 1996, 1998; Afessa et al., 2007). Several papers have demonstrated that the SOFA scoring system predicts mortality, which increases with an increasing

score and the number of failing organs. Dysfunction or failure of the cardiovascular system, followed by the renal and central nervous systems, had the single most serious impact on severity in patients in intensive care (Moreno et al., 1999), as also evidenced in septic shock patients, who suffer the highest mortality (Vincent et al., 2006). The mean CHIR 99021 time to reach the maximum SOFA scores was the highest for the liver; dysfunction of the liver, however, did not contribute to an increased risk of death (Moreno et al., 1999). The liver thus seems to be an organ on which the sum of ailments converges, making failure a late and secondary event. In a previous study (Nielsen et al., 2009b), we showed that an intravenous inoculation of S. aureus

in pigs induces acute pyaemia, with the formation of microabscesses in various organs 4– 6 h after the challenge. However, no mafosfamide systemic inflammatory response, and thus sepsis, was induced, probably due to the short duration of the experiment. The aim of the present study was to further characterize the pig as a model for human S. aureus-induced sepsis and severe sepsis. Therefore, the inoculated pigs in the present study were kept for up to 48 h, inoculation was repeated in some pigs and the study included the evaluation of the possible late event of liver dysfunction or failure. Details on the experimental animals, the bacterial inocula and the design of the study have been published previously (Jensen et al., 2010). Briefly, 12 clinically healthy female specific pathogen-free (SPF) Yorkshire–Landrace crossbreed 8-week-old pigs with a body weight (BW) of 20–25 kg were used. The pigs, which remained clinically healthy during the acclimatization period of 7 days, were randomly assigned to three groups.