For statistical analysis, the spss statistical package, version 1

For statistical analysis, the spss statistical package, version 16.0, was used. Deletion mutagenesis of blr1515 and blr1516 was performed by marker exchange as indicated in Fig. 1. The 5′ and 3′ flanking

regions of bdeA were PCR amplified using appropriate primers and subcloned in the pBluescript SK(+) vector (Stratagene, Afatinib supplier La Jolla, CA). Sequences of all primers used in this work are available from the authors upon request. A streptomycin-resistance cassette (Ω) excised from pBSL15Ω was inserted in between the two B. japonicum DNA fragments, yielding plasmid pRJ9587. pBSL15Ω had been constructed by cloning of the Ω cassette from pHP45Ω (Prentki & Krisch, 1984) on a 2.1-kb EcoRI fragment into the 2.6-kb backbone of EcoRI-digested plasmid pBSL15 (Alexeyev, 1995). The insert of pRJ9587 was cloned into the vector pSUP202pol6K (Zufferey et al., 1996), and the resulting plasmid pRJ9589 was then mobilized by conjugation from E. coli S17-1 (Simon et al., 1983) into B. japonicum strain 110spc4 (wild type) for marker replacement, yielding mutant strain 9589. A 6.1-kb B. japonicum genome fragment

containing the bdeAB genes including the flanking regions was PCR amplified with appropriate primers using the Phusion™ DNA Polymerase (Finnzymes, BioConcept, Allschwil, Switzerland). The PCR fragment was digested using intrinsic restriction sites for PstI and EcoRI (Fig. 1), subcloned for verification by sequencing, and eventually cloned into pSUP202pol6K. The resulting plasmid pRJ9638 was mobilized by conjugation into the B. japonicum mutant strain 9589, yielding strain 9589-38. Candidates this website with a single-recombination event were picked, and the correct integration of pRJ9638 upstream of the Ω cassette present in strain 9589 was verified by Southern blot analysis of genomic DNA. Potential growth-inhibitory compounds and antimicrobial peptides were purchased from Sigma Aldrich Co. (Buchs, Switzerland) and screened first for growth inhibition using the gradient agar plate technique (Szybalski

& Bryson, 1952). For agar plate diffusion assays, 15 ml of 0.9% PSY agar was warmed to 42 °C, inoculated with bacterial cell suspensions to 5 × 106 CFU ml−1 and quickly Mannose-binding protein-associated serine protease poured into round Petri dishes. Holes were punched into each plate using the end of a Pasteur glass pipette, and 15 μL of the test compound was pipetted into each hole. After 2 days of incubation at 30 °C, test plates were monitored for zones of growth inhibition on the bacterial lawn. The assays were repeated at least three times. Based on annotations compiled in the B. japonicum genome sequence database (http://bacteria.kazusa.or.jp/rhizobase/), genes blr1515 and blr1516 code for components of a multidrug efflux system, and their genomic organization (Fig. 1) suggests that they form an operon. Gene blr1515 is predicted to encode a protein of 397 amino acids (aa) with sequence similarity to Pseudomonas aeruginosa MexC (43%) and E. coli AcrA (40%) (Poole et al., 1993; Ma et al., 1995), for example.

For statistical analysis, the spss statistical package, version 1

For statistical analysis, the spss statistical package, version 16.0, was used. Deletion mutagenesis of blr1515 and blr1516 was performed by marker exchange as indicated in Fig. 1. The 5′ and 3′ flanking

regions of bdeA were PCR amplified using appropriate primers and subcloned in the pBluescript SK(+) vector (Stratagene, Roxadustat La Jolla, CA). Sequences of all primers used in this work are available from the authors upon request. A streptomycin-resistance cassette (Ω) excised from pBSL15Ω was inserted in between the two B. japonicum DNA fragments, yielding plasmid pRJ9587. pBSL15Ω had been constructed by cloning of the Ω cassette from pHP45Ω (Prentki & Krisch, 1984) on a 2.1-kb EcoRI fragment into the 2.6-kb backbone of EcoRI-digested plasmid pBSL15 (Alexeyev, 1995). The insert of pRJ9587 was cloned into the vector pSUP202pol6K (Zufferey et al., 1996), and the resulting plasmid pRJ9589 was then mobilized by conjugation from E. coli S17-1 (Simon et al., 1983) into B. japonicum strain 110spc4 (wild type) for marker replacement, yielding mutant strain 9589. A 6.1-kb B. japonicum genome fragment

containing the bdeAB genes including the flanking regions was PCR amplified with appropriate primers using the Phusion™ DNA Polymerase (Finnzymes, BioConcept, Allschwil, Switzerland). The PCR fragment was digested using intrinsic restriction sites for PstI and EcoRI (Fig. 1), subcloned for verification by sequencing, and eventually cloned into pSUP202pol6K. The resulting plasmid pRJ9638 was mobilized by conjugation into the B. japonicum mutant strain 9589, yielding strain 9589-38. Candidates BMS-907351 in vivo with a single-recombination event were picked, and the correct integration of pRJ9638 upstream of the Ω cassette present in strain 9589 was verified by Southern blot analysis of genomic DNA. Potential growth-inhibitory compounds and antimicrobial peptides were purchased from Sigma Aldrich Co. (Buchs, Switzerland) and screened first for growth inhibition using the gradient agar plate technique (Szybalski

& Bryson, 1952). For agar plate diffusion assays, 15 ml of 0.9% PSY agar was warmed to 42 °C, inoculated with bacterial cell suspensions to 5 × 106 CFU ml−1 and quickly VAV2 poured into round Petri dishes. Holes were punched into each plate using the end of a Pasteur glass pipette, and 15 μL of the test compound was pipetted into each hole. After 2 days of incubation at 30 °C, test plates were monitored for zones of growth inhibition on the bacterial lawn. The assays were repeated at least three times. Based on annotations compiled in the B. japonicum genome sequence database (http://bacteria.kazusa.or.jp/rhizobase/), genes blr1515 and blr1516 code for components of a multidrug efflux system, and their genomic organization (Fig. 1) suggests that they form an operon. Gene blr1515 is predicted to encode a protein of 397 amino acids (aa) with sequence similarity to Pseudomonas aeruginosa MexC (43%) and E. coli AcrA (40%) (Poole et al., 1993; Ma et al., 1995), for example.

Two reviewers (S-AP, IH) rated experimental and quasi-experimenta

Two reviewers (S-AP, IH) rated experimental and quasi-experimental studies for methodological quality to identify potential

sources of bias (study design, unit of randomisation, differences in baseline characteristics, objectivity of outcome measures, and completeness of follow-up; see Figure 1). We also noted whether statistical analyses were adjusted for clustering and whether the authors Venetoclax had mentioned possible contamination of the study groups. Due to heterogeneity in study methodology, comparison groups, setting, intervention targets and outcomes, we did not use traditional meta-analytic approaches to combine individual study results. Inconsistent reporting of means and standard deviations (SD) meant we could not calculate effect size measures such as Cohen’s d. We describe the impact of interventions on prescribing measures and clinical and patient outcomes as reported in the individual studies. Given the role of the pharmacist as an intermediary in medication management we also noted the frequency and nature of interactions between pharmacists and physicians and/or patients and the impact of CDSS on pharmacist workload and work patterns. Outcomes are summarised separately for each study and coded according to the following scheme: + (NS) means selleck that the intervention favoured CDSS (the outcome was

more consistent with the intentions of the CDSS) but was not statistically significant; – (NS) means that

the intervention favoured the comparison group (the outcome of comparison groups was more consistent with the intentions of the CDSS) but was not significant; ++ means that the intervention favoured CDSS (the outcome was more consistent with the intentions of the CDSS) and was statistically significant; −− means that the intervention favoured the comparison group (the outcome of comparison groups was more consistent with the intentions of the CDSS) and was statistically significant; finally, 0 means that there was no difference between groups. We aggregated outcome data by reporting whether studies demonstrated at least one positive outcome (general trend in favour of CDSS for a prescribing, clinical or patient outcome) and statistically significant improvements in favour Decitabine in vivo of CDSS on the majority (≥ 50%) of outcomes (as used by Garg and colleagues[4]). We chose to report trends as well as significant results given the likelihood that some studies were underpowered to detect statistically significant differences in outcomes. We examined differences in the proportions of studies showing significant improvements on the majority of outcomes for our main research questions (i.e. differences between safety studies versus QUM studies, ambulatory versus institutional care, system- versus user-initiated studies, prescribing versus clinical outcomes) using Fisher’s exact test. All analyses were performed using StatsDirect (version 2.6.3).

As with studies on other acidophilic methanotrophs, culture purit

As with studies on other acidophilic methanotrophs, culture purity was MK-1775 price rigorously proven using a variety of microscopic and molecular analyses. Growth was greater on methane than on acetate (maximum OD410 nm of 0.8–1.0 and 0.25–0.30, respectively), as was the growth rate (μ=0.06 and 0.006 h−1, respectively). These data would suggest that methane is the preferred substrate of this strain. However, when both acetate and methane were used simultaneously, overall growth was enhanced, as first noted by Whittenbury et al. (1970) for other methanotrophs. Interestingly,

strain H2s was not found to grow significantly on any other organic acid or sugar (Table 1). With the finding of a facultative Methylocystis strain, Belova

et al. (2011) screened validly described Methylocystis species for facultative methanotrophic growth, and found that another acidophilic species with an optimal pH range of 5.8–6.2, Methylocystis heyeri H2, also grew significantly on acetate. Most mesophilic Methylocystis species (i.e. growth pH of 6.8) did not grow on acetate, with the exception of Methylocystis echinoides IMET10491 which grew in the presence of acetate selleck screening library from an initial OD410 nm of ∼0.03 to a final OD410 nm of 0.09 after 200 h of incubation. A second recent study supports the finding of facultative mesophilic Methylocystis species, with the characterization of Methylocystis strain SB2, a novel methanotroph that can only express pMMO (Im et al., 2011). This isolate, collected from a spring bog with an optimal growth pH of 6.8, was able to utilize methane, ethanol, or acetate as growth substrates. Growth was highest on methane followed by ethanol and acetate (maximum OD600 nm of 0.83, 0.45, and 0.26, respectively). Interestingly, growth on methane and ethanol followed standard exponential kinetics (μ=0.052 and 0.022 h−1, respectively), but growth on acetate

could be modeled as either exponential or linear growth. Such a finding supports the hypothesis that acetate is transported into Methylocystis strain SB2 as the undissociated acid, and at this growth pH, the proton-motive force is dissipated for acetate uptake (Axe & Bailey, 1995). Finally, as enough with other investigations of facultative methanotrophy, culture purity was verified using a variety of microscopic and molecular techniques. The recent findings of facultative methanotrophy raises some very interesting questions. Particularly, is the MMO expressed when these strains are grown on multicarbon compounds in the absence of methane? Interestingly, acetate has been shown to repress MMO expression in some facultative methanotrophs, while others constitutively express MMO regardless of the growth substrate. Specifically, when using acetate as an alternative substrate, M. silvestris was clearly shown to repress expression of the sMMO (the only form of MMO it expresses) in either the absence or presence of methane (Theisen et al., 2005).

68% NaCl containing 005% Tween 80 The fish in this experiment w

68% NaCl containing 0.05% Tween 80. The fish in this experiment were artificially grazed by one end of a wire netting in advance (this process was done by the same person).

Three groups of these wounded yellow catfish were then immersed in FM07 suspension containing 106, 107 and 108 CFU mL−1, respectively, for 2 days and one group of fish used as control was immersed in oxygenated water. Three groups of yellow catfish were immersed in FM07 suspension containing 106, 107 and 108 CFU mL−1, respectively, and one group of fish used as control was immersed in oxygenated water. All the fish were healthy and no AZD2281 chemical structure wounds were found. These experimental infections lasted 8 weeks. Mortalities were recorded during the experimental infections. All fish were examined for gross pathological changes. Any dead or moribund fish were checked for the presence of the fungal pathogen. Live and moribund fish were killed with an overdose of tricaine methanesulfonate (MS-222). The tissues of the diseased fish from Niushan Lake and those with artificial infection were excised and fixed in Bouin’s fluid. Samples from healthy yellow catfish were also fixed in Bouin’s fluid as control. Part of the musculature was decalcified with formic acid–sodium citrate

solution. All tissues were dehydrated with ethanol, embedded in paraffin wax, and blocks sectioned at 6 μm with a rotary microtome. Slides were stained with Harris’ hematoxylin and eosin. The stained sections on the slide were covered with Canada balsam and photographed under a microscope (Zeiss Axioplan 2 imaging and Axiophot see more Dehydratase 2). The hyphae in the necrotic tissue were nonseptate, broad and branched. The color of the pure culture was white initially and soon became grayish brown. Microscopic examination revealed globose sporangia, measuring 30–78 μm in diameter (Fig. 1a). Columellae were subglobose to obovoid and collarettes were conspicuous

(Fig. 1b). Sporangiophores were either long and branched sympodially or shorter with slightly recurved lateral branches. Sporangiospores were hyaline, ellipsoidal to slightly asymmetrical or obovoidal and measured 5.0–7.0 μm in length and 3.2–5.5 μm in width. The sporangiospore walls were finely ornamented (Fig. 1c). Chlamydospores were produced in the basal mycelium, which were thick-walled, subglobose, oval or irregularly shaped, measuring 45 μm in length and 30 μm in width (Fig. 1d). Rhizoids and stolons were absent. The optimum growth temperature was 30 °C and there was no growth at 40 °C. There were no zygospores produced in test mating and this procedure was repeated with the same results. A 638-bp ITS rRNA gene fragment was amplified from the fungi and was deposited in GenBank under the accession number GQ415044. Compared with the sequences of ITS rRNA gene available in GenBank, the amplified nucleotide sequence showed 100% homology with the ITS rRNA gene sequence of M. circinelloides (accession number EF583641) (Fig. 2).

[51] From this starting point, the results from this research sug

[51] From this starting point, the results from this research suggest that the need to gain knowledge and understanding of each other’s roles through effective communication, is important. The current research suggests that while pharmacists may have thought about the role of different HCPs in asthma management, the GPs have not considered a role for the pharmacist that is beyond medications. The results

indicate that GPs would be open to a broader role for pharmacists, if they were confident that pharmacists had received the appropriate training. One way to gain confidence with one another is through interactions. The extent and means by which interactions occur between GPs and pharmacists may be different at different

stages of their relationship; however, having access to one another is obviously extremely ZD1839 in vitro important. In this research, a vast majority of participants were in close proximity to the nearest GP or pharmacist, but proximity was not specifically mentioned as an essential element to the relationship. However, pharmacists articulated face-to-face contact as an important enabler of the GP–pharmacist relationship. This is perhaps due to the heightened access/engagement that face-to-face contact provides[52] and the fact that it could be used as a means of ensuring engagement of both HCPs, rather than just ‘access’. At DAPT order the centre of the GP–pharmacist relationship was the act and nature of ‘communication’. It is clearly recognised by both HCP groups as an essential part of their relationship. Two clear aspects of communication were evident in this research:

the clinical content of the communication and the nature/style of the communication. GPs acknowledged the importance of the clinical content of the communication while pharmacists focused on the more personal aspect of the communication as was displayed in the nature and the style of communication between the HCPs. In both instances, the communication was clearly evaluated by the HCPs (Stage 2: a fragile point in the relationship where roles are being explored and tested) Ribonucleotide reductase and influenced future development of the relationship. It can be postulated that this particular point in the relationship is critical as the mismatch of expectations observed between GPs and pharmacists (in terms of the relationship, the purpose of communication, their respective roles in patient care, perceptions of the quality of disease management delivered and patient needs) could drive the relationship forwards or backwards. In fact, it might be at this point that the perceived barriers to collaboration, articulated both in this study and in the literature (including territorialism, attitudes, low morale, remuneration and patient engagement) may be most important[17,52–59] (Figure 1). Despite these challenges, there is a need to look beyond this critical point.

Design  Data were collected from 1057 children; validated questi

Design.  Data were collected from 1057 children; validated questionnaires were completed, Seliciclib in vivo and children were examined by trained dentist at ages 3 and 5. Logistic regression analyses were performed to explain dental attendance. Results.  At the age of 3, 62% and by 5 years, 21% had never visited the dentist. The first dental visit was considered a pleasant experience for the majority of children. Multivariable regression analyses revealed that children who were not first born, whose mothers had a higher educational level and whose parents had recently visited the

dentist, had significantly higher odds for having visited the dentist at young age. Conclusions.  Parents of young children need to be informed about and motivated for an early dental visit. Promotion campaigns should focus on firstborn children, children

from less educated parents, and parents who do not regularly see a dentist. “
“A wide range for the prevalence of Molar–Incisor–Hypomineralisation (MIH) has been found in regional studies. The aim of this IDH inhibitor study was to determine the prevalence of MIH in Germany and to compare the findings with other studies. In the compulsory dental school examination, the first permanent molars, permanent incisors, and second primary molars were examined according to EAPD criteria in 2395 children (8.1 ± 0.8 years) in four regions in Germany for the presence of MIH. Examinations were performed by five calibrated examiners (κ = 0.9) on clean teeth after toothbrushing. The prevalence of MIH at the four regions differed considerably (4.3–14.6%) with a mean prevalence of 10.1%. The

3-oxoacyl-(acyl-carrier-protein) reductase DMFT/dmft was generally low, but children with MIH exhibited statistically significant higher caries values. A total of 12.0% of the children with MIH also had at least one affected primary molar, which resulted in a statistically significant correlation between primary and permanent teeth. Most of the affected teeth had demarcated opacities, but more than half of the affected children showed at least one tooth with severe MIH. Molar–Incisor–Hypomineralisation is a prevalent finding in German school children. The prevalence varies highly in different regions, and the high rate of severe forms has clinically relevant implications. “
“International Journal of Paediatric Dentistry 2010; 20: 158–164 Background.  Caries is a disease that affects both primary and permanent dentitions, therefore new methods of caries diagnosis need to be tested on primary teeth as well as on permanent teeth. Aim.  This study reports the application of optical coherence tomography (OCT) to characterize sound dental structure and detect natural caries of human primary teeth. Design.  Six primary teeth were sectioned into thin slices (∼1.5 mm), and analysed perpendicular to the enamel surface by two home-made OCT systems operating around 1280 and 840 nm. The generated images were compared with histology as the gold standard. Results.

MRI at this point showed slight enlargement of the known lesions

MRI at this point showed slight enlargement of the known lesions and a worsening of cerebral edema (Figure 1). EEG confirmed right frontal epileptiform wave activity. Histopathological selleckchem reevaluation of the liver lesion confirmed AE. ABZ was reintroduced at 1200 mg/d and corticosteroid and carbamazepine doses were

optimized, resulting in clinical improvement and discharge from hospital. Several hospital admissions subsequently followed, with only slow improvement of neurological symptoms. Serum levels of ABZ and its prodrug ABZ-sulfoxide were determined by isocratic high-performance liquid chromatography using ultraviolet detection. Drug levels were examined 4 h after the morning dose and each time at the same time. They were repeatedly Rucaparib cost below the therapeutic range of 0.5–1.5 mg/L, despite increasing ABZ. To augment drug resorption from the gut daily fat intake was increased.1 Praziquantel and cimetidine were added to slow hepatic metabolization of ABZ2 (Figure 2). Levetiracetam was added for better seizure control. In February 2008, the patient presented again with worsening neurological symptoms due to progression of disease and cerebral edema with compression of the right lateral ventricle and midline shift, as shown on MRI (Figure 3). Clinical features of steroid-induced

Cushing’s syndrome were prominent. A brain biopsy, then performed because of lack of improvement of symptoms and imaging features, confirmed cerebral AE. Follow-ups in 2009 and 2010 showed progressive clinical improvement with minimal seizure activity and only residual weakness of the left foot, as well as improvement of MRI findings of the brain (Figure 4). In October 2011, under treatment with ABZ 1600 mg/d, praziquantel 6000 mg/d, dexamethasone 4 mg/d, and levetiracetam 3000 mg/d, physical examination showed mild left-sided weakness of the leg with concomitant hyperreflexia. MRI

findings have not improved further. Cerebral selleck chemicals llc AE is a rare and difficult-to-treat zoonosis caused by E multilocularis and is found only in the northern hemisphere. Natural definitive hosts, mainly foxes, and to a lesser extent wolves and domestic dogs, feed on infected rodents and carry the egg-producing adult worms. The larval metacestodes, that are able to wander to the liver or other organs, develop in small rodents, the intermediate hosts, and in humans, the aberrant intermediate hosts, who ingest the eggs either through contaminated food like fruit or water or through direct contact with definitive hosts. During the long incubation period of 5–15 years the patient is usually asymptomatic. The liver is the primary organ affected in 98% of cases. Metastasis formation in other organs has been described in 10–20%, and is usually associated with a long latency period in chronic disease. Spreading to the brain accounts for only 1% of AE cases described.3,4 Only 31 cases (0.04/100,000) of AE have been reported in Germany in 2010.

For women with a plasma VL of <50 HIV RNA copies/mL at 36 weeks,

For women with a plasma VL of <50 HIV RNA copies/mL at 36 weeks, and in the absence of obstetric contraindications, a planned vaginal delivery is recommended.     Angiogenesis inhibitor For women with a plasma VL of 50–399 HIV RNA copies/mL at 36 weeks,

pre-labour CS (PLCS) should be considered, taking into account the actual VL, the trajectory of the VL, length of time on treatment, adherence issues, obstetric factors and the woman’s views.     Where VL is ≥400 HIV RNA copies/mL at 36 weeks, PLCS is recommended.   7.2.2 In women in whom a vaginal delivery has been recommended and labour has commenced, obstetric management should follow the same guidelines as for the uninfected population. Grading: 1C 7.2.3 Vaginal birth after CS (VBAC) should be offered to women with a VL <50 copies/mL. Grading: 1D 7.2.4 Delivery by PLCS is recommended for women taking zidovudine monotherapy irrespective of plasma VL at the time of delivery (Grading: 1A) and for women with VL >400 HIV RNA copies/mL regardless of ART (see Recommendation 7.2.1) with the exception of elite controllers (see Section 5.5). Grading: 1D 7.2.5 Where this website the indication for PLCS is PMTCT, PLCS should be undertaken at between 38 and 39 weeks’ gestation. Grading: 1C 7.3.1 In all cases of term pre-labour spontaneous ROM, delivery should be expedited. Grading: 1C 7.3.2 If maternal HIV VL is <50

HIV RNA copies/mL immediate induction of labour is recommended, Benzatropine with a low threshold for treatment of intrapartum pyrexia. Grading: 1C   For women with a last measured plasma VL of 50–999 HIV RNA copies/mL, immediate CS should be considered, taking into account the actual VL, the trajectory of VL, length of time on treatment, adherence issues, obstetric factors and the woman’s views. Grading: 1C 7.3.4 If maternal HIV VL is ≥1000 RNA copies/mL plasma immediate CS is recommended. Grading: 1C 7.3.5 The management of prolonged premature ROMs (PPROM) at ≥34 weeks is the same as term ROM except women who are 34–37 weeks’ gestation will require group B streptococcus prophylaxis in line with national guidelines. Grading: 1C

7.3.6 When PPROM occurs at <34 weeks. Grading: 1C   Intramuscular steroids should be administered in accordance with national guidelines     Virological control should be optimized     There should be multidisciplinary discussion about the timing and mode of delivery 7.4.1 Intrapartum intravenous zidovudine infusion is recommended in the following circumstances:     For women with a VL >10 000 HIV RNA copies/mL plasma who present in labour, or with ROMs or who are admitted for planned CS Grading: 1C   For untreated women presenting in labour or with ROMs in whom the current VL is not known. Grading: 1C   In women on zidovudine monotherapy undergoing a PLCS intravenous zidovudine can be considered. Continued oral dosing is a reasonable alternative. Grading: 1B 8.1.

3) To investigate whether the uptake of SpHtp1 can be caused by

3). To investigate whether the uptake of SpHtp1 can be caused by physical disruption of the membranes by Saprolegnia, a His-tagged SpHtp1 fusion protein without the putative signal peptide, SpHtp124-198-His, was synthesized in E. coli, purified and characterized (Fig. S6). Treatment with the final bleed SpHtp1 antibody in combination with the secondary antibody Fluor 488 showed no fluorescence in or on RTG-2 cells.

When the RTG-2 cells were treated with SpHtp124-198-His, no fluorescence was detected when the preimmune antiserum was used in combination KU-60019 with the secondary antibody Fluor 488, also showing that the treatment of SpHtp1-His did not affect the fish cells (Fig. 4). However, SpHtp124-198-His and final bleed SpHtp1 antibody-treated RTG-2

cells showed SpHtp124-198-His localization on the surface of the fish cells and also inside the fish cells, surrounding the nucleus (Fig. Selleckchem DAPT 4). Furthermore, when the fish cells were incubated with SpHtp124-198-His and only labelled with the primary or the secondary antibody, no fluorescence was observed inside or outside the cells. Identical results were observed when an anti-His antibody was used for the immunolocalization studies (Fig. S7). Setting up a model infection system without having to sacrifice animals has many obvious advantages as it helps to fulfil the ultimate goal of the three Rs, whereby the aim is to reduce, refine and replace animals in experimental research. Here, we have shown that the trout RTG-2 cell line represents an excellent in vitro system for studying the very early interactions between

fish cells and S. parasitica, and that it can also be used to study the molecular mechanism of infection. Analysis of ESTs from zoospores and germinated cysts resulted in the identification of a putative RxLR effector protein, demonstrating that these types of proteins are possibly not only present in plant pathogenic oomycetes but also in animal pathogenic Florfenicol oomycetes. SpHtp1 is expressed in the preinfection and the very early infection stages of S. parasitica, as are many RxLR effector genes from P. sojae and P. infestans (Whisson et al., 2007; Dong et al., 2009). Analysis of the protein sequence revealed that SpHtp1 lacks the ‘so-called’ EER motif, which is found closely behind the RxLR motif in about 500 putative P. infestans RxLR effector proteins (Whisson et al., 2007) (Fig. 1a and b). The EER motif in the PiAvr3a and PsAvr1b proteins seems to be required for effector translocation of P. infestans and P. sojae, respectively (Whisson et al., 2007; Dou et al., 2008a). However, another intracellularly recognized RxLR effector protein, Atr13 of Hyaloperonospora arabidopsidis, lacks the EER motif (Allen et al., 2004), suggesting that the presence of an EER motif is not always essential for the translocation of every RxLR effector into host cells, or for inducing a hypersensitive response.