For the production, in particular, squid pen waste was first utilized successfully to produce a high yield of inexpensive EPSs from Paenibacillus sp. TKU023 and P. macerans TKU029. In addition, this technology for EPS production is prevailing because it is more environmentally friendly. The Paenibacillus spp. EPSs reported from various references constitute a structurally diverse class of biological macromolecules with different applications in the broad fields of pharmacy, cosmetics and bioremediation. The EPS produced by P. macerans TKU029 can increase in vivo skin hydration and may be a new source of natural moisturizers with potential value in cosmetics. However, the relationships between the structures and activities of these EPSs in many studies are not well established. The contents and data in this review will serve as useful references for further investigation, production, structure and application of Paenibacillus spp. EPSs in various fields.This review provides a comprehensive summary of the most recent developments of various aspects ( Agrobacterium species or Alcaligenes faecalis in initial studies [Natural polysaccharides are highly susceptible to natural biodegradation and are less harmful than synthetic polymers. Over the past few decades, the number of known polysaccharides produced by microbial fermentation has been gradually increasing. Most of the polysaccharides derived from microorganisms are of the exo-polysaccharide (EPS) type; these EPSs attach to the cell surfaces or are released into the extracellular medium ,2. The m studies . This bi studies . Microbi studies ,7 becaus studies ,9,10. Streptococcus equii and Streptococcus zooepidemicus [Bacillus subtilis and Bacillus polymyxa [Aureobasidium pullulans [Leuconostoc mesenteroides [Xanthomonas campestris [Sphingomonas paucimobilis [Pseudomonas species and Azotobacter chrococcum [Acetobacter xylinium [Agrobacterium and Alcaligenes species [Rhizobium [Paenibacillus spp. were only discovered in the past ten years.In recent years, significant progress has been made in the search for novel microbial EPSs that possess novel and highly functional properties . The difidemicus ; levan fpolymyxa ,13; pullullulans ; dextranteroides ; xanthanmpestris ; gellan imobilis ; alginatrococcum ; celluloxylinium ; curdlan species ; and sucPaenibacillus consists of over 89 species of facultative anaerobes, endospore-forming, neutrophilic, periflagellated heterotrophic and low G + C Gram-positive bacilli, which were originally included within the genus Bacillus and then reclassified as a separate genus in 1993 [Paenibacillus spp. produced a wide variety of different EPSs with diverse physiological and biotechnological functions. Paenibacillus spp. EPSs have also attracted great interest because of their biotechnological potential in different industrial processes and wastewater treatment [Paenibacillus spp., EPSs have shown that there is a growing interest in using Paenibacillus spp. EPSs as biomaterials; extensive research has been performed, leading to a large number of publications in recent years [Paenibacillus spp. EPSs. Furthermore, the applications of Paenibacillus spp. EPSs in various fields are extensively reviewed.The genus  in 1993 . Bacteri in 1993 . Paenibareatment ,30,31,32nt years ,30,31,32Paenibacillus spp. were discovered to produce various EPS. Some of the production conditions have been investigated to optimize the production for commercial usage. The production of EPS is not species-specific and each strain of the species produces different types of EPSs with different biotechnological properties. In our reviews, very recently, researchers observed that the culture of Paenibacillus spp. could produce two different, commercial, well-known EPSs. One of these polymers is a levan-type EPS from P. polymyxa EJS-3 [P. polymyxa ATCC 21830 [d-glucose-fructosyl transferase, EC 2.4.1.10) by microorganisms. Liu et al. [P. polymyxa EJS-3 grown in a culture medium  typically yielded 35.26 g/L of a levan-type EPS after cultivation for 60 h at 24 °C. Curdlan is a neutral, bacterial extracellular polysaccharide without branched chains, composed entirely of β-(1→3)-d-glycosidic linkages. Curdlan is a thickener with unique thermal gelling properties and is widely used in the food industry. Curdlan is biodegradable, nontoxic toward humans and the environment, edible and it has growing capacity in the pharmaceutical industry because of its potent biological activities [As mentioned earlier, xa EJS-3 , and theCC 21830 . Levan iCC 21830 . The micu et al. ,24,25,29Paenibacillus spp. EPSs fermentation processes, many investigators have studied the effects of various process factors on the maximal production of EPSs to optimize the fermentation conditions, such as medium composition, temperature, pH, and culture vessel [Paenibacillus spp. that have been reported in the literature. The EPS yields varied over a wide range from 3.44 to 41.25 g/L with the bacterial species and culture conditions. In particular, our group previously demonstrated that EPSs were induced from a squid pen powder (SPP)-containing medium by Paenibacillus sp. TKU023 [P. macerans TKU029 [Paenibacillus spp. Thus, there is a need to enhance the EPS productivity from Paenibacillus spp. through effective strategies of process intensification in the future.To improve the efficiency and productivity of e vessel ,30,31,32P. polymyxa strains, which may be responsible for the high yield of EPS with sucrose as a carbon source [N-acetylglucosamine. Therefore, to lower the production cost of EPSs and efficiently reutilize the chitin-containing fishery by-products, we screened and isolated EPS-producing bacteria using squid pen powder (SPP) as the sole C/N source. We first produced and characterized the EPSs from the SPP culture supernatant of two bacteria, Paenibacillus sp. TKU023 [P. macerans TKU029 [Paenibacillus sp. TKU023 and P. macerans TKU029 have a chitin hydrolysing mechanism because there is EPS production by TKU023 and TKU029 with SPP as the C/N source without sucrose  caused a decrease in their production [Paenibacillus spp. were in agreement with previous study that for EPS production by P. polymyxa KCTC 8648P, the optimum pH value of 7.0 was reported [P. polymyxa EJS-3, Liu et al. [P. polymyxa EJS-3 might be due to its living in the tissues of plants.Many results have also reported that the production of EPS is cell growth-associated ,31,32. I and 7.2  [6,7,22,reported . Howeverreported ,24,25,29u et al. ,24,25,29et al. [et al. reported that the optimal temperatures of P. polymyxa EJS-3 for cell growth and EPS production were 27 °C and 24 °C, respectively [P. polymyxa EJS-3 favoured lower temperature for EPS production compared with others.Incubation temperature is another critical factor for EPS biosynthesis . Rafigh et al.  found thet al. . Howeverectively . P. polyPaenibacillus spp. producing EPS was studied [et al. [P. polymyxa ATCC 21830 underwent significant enhancements as the agitation speed increased from 120 to 150 rpm. The highest yield of curdlan gum and biomass production was at an agitation speed of 150 rpm. The curdlan gum and biomass production were low at 120 rpm, which can be attributed to the limitation of oxygen transfer. However, at 180 rpm, lower levels of curdlan gum and biomass were observed, which can be ascribed to bacterial fragmentation mediated by several shearing mechanisms. However, for some other species of strains, higher agitation speed (600 rpm) has been used for curdlan production, e.g., from Agrobacterium sp. [Paenibacillus sp.The influence of aeration on the vital activity of  studied ,31,32. U studied . The fin [et al.  also indg for 20 min) to remove the remaining SPP and biomass. The supernatant was filtered through a 0.45-μm membrane filter, mixed with two volumes of methanol, stirred vigorously and kept overnight at 4 °C. The precipitate from the ethanol dispersion was collected by centrifugation at 12,000 g for 15 min, washed three times with sterilized distilled water, and then lyophilized to yield the crude EPS. This is the most common and convenient method for isolating EPS from culture supernatant [Paenibacillus spp.In our previous studies, the EPS sample after SPP fermentation was immediately autoclaved for 20 min to reduce the ropy condition of the culture and was centrifuged  seven times [The crude EPS was re-dissolved in distilled water and stirred vigorously at 80 °C for 30 min, mixed with four volumes of anhydrous ethanol, stirred vigorously and kept overnight at 4 °C. The precipitate from the ethanol dispersion was collected by centrifugation at 12,000× en times . The depet al. recently isolated EPS fractions from a fermentation medium by gradient ethanol precipitation [The deproteinized EPS was purified sequentially through column chromatography, including ion-exchange chromatography, gel filtration chromatography and affinity chromatography ,30,31,32pitation . Their rPaenibacillus spp. EPSs with different monosaccharide constituents and chemical structures have been isolated from their culture supernatant. Many research groups have elucidated the chemical structures of purified EPSs using infrared spectroscopy, liquid-state nuclear magnetic resonance (NMR) (one and two dimensions), solid-state NMR, gas chromatography (GC), GC-mass spectroscopy (GC-MS), high-performance liquid chromatography (HPLC), acid hydrolysis, methylation analysis, periodate-oxidation, and Smith degradation [Paenibacillus spp. showing various bioactivities have been investigated. The sources and chemical compositions of the EPSs are summarized in radation ,27,30,44Paenibacillus spp. varies in very broad ranges, from hundreds to thousands of kDa, depending on the culture strain, pH, cultivation techniques and C/N sources used. It has been reported that on a sucrose-containing medium, the bacterium produces high-molecular-weight EPS [P. polymyxa EJS-3 were estimated to be 1220 and 869 kDa, respectively [P. polymyxa ATCC 21830 was 170 kDa [Paenibacillus spp. varies significantly and is affected by variables such as temperature, pH and the initial carbon source concentration [Various techniques, such as viscometry, osmometry, sedimentation, and HPLC, have been used to determine the average polymer molecular weight (MW) and polydispersity index. Among them, high-performance gel permeation chromatography (HPGPC) is a common method for determining the MW of polysaccharides and has also been used by many researchers for the MW of EPSs. Size-exclusion chromatography with multi-angle laser light scatter detection is also an efficient method for the evaluation of the absolute MW of polysaccharides and provides greater resolution than traditional GPC ,30,45,46ight EPS . The molectively . The aventration . The monosaccharide composition analysis usually involves cleavage of glycosidic linkages by acid hydrolysis, derivatization, and detection and quantification by GC. In addition, high-performance anion-exchange chromatography with pulsed amperometric detection has been developed to supplement traditional methods because it does not require derivatization of the monosaccharide with high resolution . RecentlPaenibacillus spp. have been obtained; the monosaccharide composition is usually glucose, mannose, galactose and glucuronic acid in various mole ratios  linked fructan [P. polymyxa KCTC 8648P was composed of glucose, galactose, mannose, fucose and glucuronic acid [et al. reported that mannose, fructose and glucose were the constituents of the EPS from P. polymyxa EJS-3 [P. polymyxa SQR-21 was composed of mannose, glucose, fructose and glucuronic acid [et al. found that the EPS produced by P. elgii B69 was composed of glucose, glucuronic acid, xylose, and mannose [Paenibacillus strains. Xylose is rarely described as a main component of other bacterial polysaccharides. These dissimilarities reflect the species-specific production and biotechnological potential of EPS. Increasing numbers of microbial strains have been screened to produce bioactive EPS to replace synthetic chemicals, which have many side effects.Many different EPSs from e ratios . There i maltose . Madden pyruvate . Han and fructan . The EPSnic acid . Liu et  mannose . This fiPaenibacillus spp. are summarized in In recent years, microbial EPSs and their derivatives have found many applications in the food, pharmaceutical and other industries because they have different physiological activities from natural gums and synthetic polymers ,10. MoreCordyceps militaris SU5-08 [Fusarium solani SD5 [Pleurotus sajor-caju [Fomes fomentarius [Tremella fuciformis [Agrocybe cylindracea [Collybia maculate [Cordyceps jiangxiensis [Tremella mesenterica [Paenibacillus spp. EPSs. Free radicals are harmful to living organisms . To redus SU5-08 , Fusariulani SD5 , Pleurotjor-caju , Fomes fentarius , Tremellciformis , Agrocybindracea , Collybimaculate , Cordycegxiensis  and Trementerica . Howeverenterica ,60,61 arin vitro antioxidant capacities of EPS were evaluated using various assay methods and activity indices [Paenibacillus sp. TKU023 could produce EPS and antioxidant by using SPP as the sole C/N source. The culture supernatant incubated for four days using a baffled base flask showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing powers, ferrous ion chelating activity and high total phenolic content, but maximum EPS production was found at the fifth day when using a flat base flask. The production of two invaluable environmentally friendly biomaterials (EPS and antioxidant) is unprecedented. In addition, the use of SPP (waste) is green, which made the whole process more valuable and attractive [et al. reported that P. polymyxa SQR-21 produced one type of EPS using yeast extract and galactose as the best N and C sources, respectively. Their EPS showed good superoxide scavenging and moderate inhibition of lipid peroxidation and reducing activities [et al. studied the in vitro and in vivo antioxidant activity of the levan-type EPSs from endophytic bacterium P. polymyxa EJS-3. In antioxidant assays in vitro, both crude EPS and its purified fractions (EPS-1 and EPS-2) were found to have moderate DPPH radical scavenging activity, hydrogen peroxide scavenging activity, lipid peroxidation inhibition effects, and strong ferrous ion chelating activity. In antioxidant assays in vivo, mice were subcutaneously injected with d-galactose for six weeks and administered EPS-1 via gavage. As a result, administration of EPS-1 significantly increased the thymus and spleen indices of d-galactose in aging mice. Moreover, EPS-1 administration significantly enhanced the activities of antioxidant enzymes and the total antioxidant capacity and decreased the levels of malondialdehyde in both serums and livers of aging mice [et al. successfully acetylated, phosphorylated and benzylated the levan-type EPSs from P. polymyxa EJS-3 to obtain the derivatives of acetylated levan (AL), phosphorylated levan (PL) and benzylated levan (BL). For the antioxidant and antitumor activities in vitro of the natural polysaccharide and its derivatives, AL, BL and PL all exhibited higher reducing power, scavenging activity against superoxide radicals and scavenging activity of hydroxyl radicals compared to the natural polysaccharide, EPS-1. In addition, AL, BL and PL also exhibited higher antiproliferative activity against human gastric cancer BGC-823 cells in vitro than EPS-1. The enhanced activities of the derivatives were probably due to the introduction of acetyl, benzyl, or phosphoryl groups into the EPS-1 molecules, which increased the electron-donating ability and affinity with the receptors on immune cells. The results suggested that the derivatives could be explored as promising antioxidant and antitumor agents [Antioxidant activities have been attributed to various reactions and mechanisms. The  indices ,25,29,31tivities . Furthering mice . Very reP. macerans TKU029 can significantly increase skin hydration [i.e., 37.3% at 0 min and 36.2% at 180 min. The untreated control revealed an increase in skin hydration after 180 min of 8.1% for TKU029 EPSs and of approximately 0.8% for hyaluronic acid [P. macerans TKU029 can increase in vivo skin hydration and may be a new source of natural moisturizers with potential value in cosmetics.Polysaccharides of bacterial origin are very important in the cosmetic and pharmaceutical industries . One impydration . The samnic acid . The EPSPaenibacillus spp. Bioflocculation of high-ash Indian coals using P. polymyxa showed a 60% decrease in ash, suggesting that selective flocculation of coal is possible [P. polymyxa P13 was reported as an EPS producer that exhibited significant biosorption capacity of Cu2+ produced in several industries [P. polymyxa SQR-21 showed a high flocculating activity towards activated carbon [P. jamilae CP-38 was able to grow and produce EPS using olive mill wastewaters as the sole nutrient and energy source, with a concomitant reduction in the toxicity of the waste [P. polymyxa had cadmium sorption capacity in aqueous solution [et al. discovered a new EPS-based broad-spectrum bioflocculant produced by a newly isolated strain, P. elgii B69 [P. elgii B69 is a significant advantage [Chemical contamination of water from a wide range of toxic compounds, particularly dyeing pigments (from the textile industry), heavy metal ions, and other toxic suspended particles, remains a serious environmental problem causing serious risk to public health. Various physical and chemical processes have been developed for removing pollutants from wastewater. One of the most popular methods is flocculation due to its economic advantages and potency. Bioflocculants are more eco-friendly and biodegradable and are less harmful to the environment than inorganic flocculants and organic synthetic flocculants . Many repossible . P. polydustries . The EPSd carbon . P. jamihe waste . The EPSsolution . In partlgii B69 . This bidvantage .Paenibacillus spp. could produce different types of EPSs with different biotechnological properties. Many other important bioactivities of Paenibacillus spp. EPSs have also been investigated. P. polymyxa JB115 was isolated from Korean soil as a glucan producer for the development of animal feed additives showing activities as a biological response modifier, natural immuno-modulator and a potential anti-tumor agent for livestock [et al. demonstrated that P. polymyxa ATCC 21830 is capable of producing curdlan gum [As aforementioned, ivestock . Rafigh dlan gum . Curdlandlan gum ,67,68. Idlan gum . Paenibacillus spp. EPSs with applications in bioactivity and bioremediation. Economical, environmentally friendly and high quantity production of Paenibacillus spp. EPSs is necessary for various applications. Squid pen waste was utilized to produce high yield, inexpensive EPSs. The discovery of inexpensive EPSs not only solves environmental problems but also promotes the economic value of marine wastes. Furthermore, this EPS produced by P. macerans TKU029 using SPP can increase in vivo skin hydration and may be a new source of natural moisturizers with potential value in cosmetics. Novel uses of Paenibacillus spp. EPSs due to its inexpensive production and its bulk will be developed. In addition, the movement toward “greener” products and technologies that are more environmentally friendly is prevailing. Production methods of EPSs are eagerly anticipated in the future, and many have already reached the market. However, the relationship between the structural features, solution behavior, space conformation, and the bioactivity of Paenibacillus spp. EPSs is unclear due to the structural diversity and complexity of polysaccharide molecules. In addition, alteration of the chemical properties of the original EPSs will also greatly enhance their values and extend their range of applications. The elaboration of either biotechnological or technical procedures for the production of EPSs of diverse structures  and ultimate product functions  to meet the special demands of practical application are being launched and will soon provide a broad spectrum of new EPSs. These will be useful to understand the chemical structures, chain conformations and the biological activities for applications in various fields.This review contains the most recent information on the production of various


Thelohanellus kitauei and comparative analysis with genomes of its two free-living cnidarian relatives revealed that T. kitauei has adapted to parasitism, as indicated by the streamlined metabolic repertoire and the tendency toward anabolism rather than catabolism. Thelohanellus kitauei mainly secretes proteases and protease inhibitors for nutrient digestion (parasite invasion), and depends on endocytosis (mainly low-density lipoprotein receptors-mediated type) and secondary carriers for nutrient absorption. Absence of both classic and complementary anaerobic pathways and gluconeogenesis, the lack of de novo synthesis and reduced activity in hydrolysis of fatty acids, amino acids, and nucleotides indicated that T. kitauei in this vertebrate host–parasite system has adapted to inhabit a physiological environment extremely rich in both oxygen and nutrients , which is consistent with its preferred parasitic site, that is, the host gut submucosa. Taking advantage of the genomic and transcriptomic information, 23 potential nutrition-related T. kitauei-specific chemotherapeutic targets were identified. This first genome sequence of a myxozoan will facilitate development of potential therapeutics for efficient control of myxozoan parasites and ultimately prevent myxozoan-induced fish-borne illnesses in humans.Members of Myxozoa, a parasitic metazoan taxon, have considerable detrimental effects on fish hosts and also have been associated with human food-borne illness. Little is known about their biology and metabolism. Analysis of the genome of  Along with the rapid growth in fish consumption, incidence of fish-borne parasitic zoonoses also increased  in T. kitauei-infected carp  in Wuqing, Tianjin, China in July 2007. After euthanasia, the intestines of infected fish were obtained by dissection. Cysts found on the surface of the intestines were carefully collected from the intestinal tissue and rinsed with sterile water. The cysts were cut open and spores were washed out with sterile phosphate-buffered saline (pH 7.0). The myxospore suspensions were further filtered with a 300 -µm mesh sieve to remove residual host fish tissue. The filtrates were centrifuged at 6,000 × g for 10 min, and the myxospore pellets were resuspended in 9.5 ml of sterile phosphate-buffered saline (pH 7.0). Host cells were lysed by addition of 0.5 ml of 20% sodium dodecyl sulfate and incubated at 25 °C for 10 min. After centrifugation at 8,000 × g for 10 min, the supernatants were discarded and spores were resuspended in 10 ml of sterile phosphate-buffered saline. The centrifugation and resuspension were repeated once. Finally, the myxospores were microscopically with care examined to ensure that host fish tissue and cells had been removed.Spore samples (2 g wet weight) were frozen in liquid nitrogen and then ground with a mortar and pestle. DNA was extracted as described previously . The purFor RNA extraction, purified spores (1 g wet weight) were frozen in liquid nitrogen and ground with a mortar and pestle. The lysate was transferred to a 1.5-ml RNase-free microcentrifuge tube and homogenized in 350 μl Buffer RLT Plus (Qiagen) with a vortex mixer . RNA was purified using the RNeasy Plus Mini kit (Qiagen). RNA concentration and purity were assessed with a Nanodrop spectrophotometer (Thermo Scientific).supplementary material and fig. S1, Supplementary Material online) and by phylogenetic analysis of 18S rDNA sequences focusing only on Thelohanellus spp. by RAxML version 7.2.6  were prepared using the GS FLX Titanium Library Preparation kit according to the manufacturer’s protocols . The purified genomic DNA is fragmented by hydrodynamic shearing to generate 8 kb and 20 kb span paired-end libraries. Quantitated DNA fragments from the paired-end library, flanked with proper amplification and sequencing adaptors, were immobilized onto microspheres (beads) and the entire bead-bound library was then emulsified with the amplification reagents (GS FLX Titanium LV emPCR kit). After the emPCR step, the DNA-carrying beads were cleaned and enriched before loaded into the wells of a PicoTiterPlate device (GS FLX Titanium PicoTiterPlate Kit [70 × 75]) at the density of no more than a single DNA bead per well. The loaded PTP device was then inserted into the Genome Sequencer FLX Instrument, and sequencing reagents were sequentially flowed over the plate. The sequencing was conducted using the GS FLX Titanium Sequencing Kit and the Genome Sequencer FLX Instrument strictly following the recommendations of the manufacturer . The raw output comprising a set of digital images (PIF files) was processed to obtain the sequence of the DNA library fragments/reads and then subjected to downstream analysis. The quality of the data obtained from the sequencing was verified using the Roche 454 Sequencing System Software version 2.6 , including normalization, correction, and quality filtering steps, to generate Standard Flowgram Format files containing the base called read sequences and per base quality scores as described in the Software manual.http://omictools.com/seqprep-s1982.html), and ConDe Tri_v2.1.pl (For short-insert (∼500 bp) DNA libraries, that is, standard DNA libraries, we followed the manufacture’s protocol (Illumina). Briefly, 5 μg of genomic DNA was fragmented by nebulization with compressed nitrogen gas. The DNA ends were polished and an “A” base was added to each end of the fragments. Next, the DNA adaptors (Illumina) with a single “T” overhang at the 3′-end were ligated to the products above. The ligation products were separated on a 2% agarose gel and an approximately 2-mm wide gel slice containing DNA of the desired size was excised (Qiagen Gel Extraction Kit). In this step, DNA fragments in a narrow size range were selected to facilitate the downstream assembly process. For long (∼3 Kb) mate-paired libraries, the manufacture’s mate pair library kit (Illumina) was used. Around 10–30 μg genomic DNA was fragmented by HydroShear and polished with biotin labeled dNTPs. The fragments in the size range of 2–5 Kb were selected by gel excision. The size-selected DNA fragments were circularized by intramolecular ligation of the blunt ends, and the remaining linear DNA fragments from the circularization reaction were digested by DNA exonuclease. Then, the circularized DNA was randomly fragmented to lengths of approximately 400 bp, and the fragments that contain the biotinylated ends of the original size-selected fragment were purified using streptavidin-coated magnetic beads. The fragments bound to the streptavidin beads were end-repaired and A-tailed, followed by attachment of the illumine paired-end oligo adapters by ligation. After the adapter ligation, PCR was carried out to simultaneously enrich and amplify those DNA fragments with adapter molecules on both ends. The product of the PCR amplication is a DNA smear of different fragment sizes. Fragments in the range of 350-650 bp were excised from the gel for cluster formation and sequencing. For quality control of Illumina sequence data, the adaptors were stripped with SeqPrep (_v2.1.pl  was used- and dATP. All the enzymes used above were included in the kit (mRNA-Seq Sample Pre Kit) from Illumina. Illumina adapters  were ligated onto the blunt-ended cDNA, and the library of DNA fragments (200–250 bp) was separated from unligated adapters by gel excision. Libraries were amplified by PCR with Phusion polymerase, denatured with 100 mM sodium hydroxide, and diluted in hybridization buffer before they were loaded onto a single lane of an Illumina GA flowcell. Cluster formation, primer hybridization, and paired-end sequencing (101 × 2 cycles) were performed using Illumina reagents. Expression level was determined using the mapped reads per kilobase of exon per million mapped reads (RPKM) in the spore cells.mRNA was isolated from total RNA using Oligo-dT beads (Invitrogen) and then fragmented by heating at 94 °C. First-strand complementary DNA (cDNA) was synthesized with random hexamer primers and second-strand cDNA synthesized with DNA polymerase. Double-stranded cDNA was end-repaired/blunt-ended with Klenow polymerase, T4 DNA polymerase, and T4 polynucleotide kinase. A single adenosine moiety was added to the cDNA using Klenow exohttp://soap.genomics.org.cn/soapdenovo.html) in iterative mode. The GC content of each assembled scaffold was calculated. The mean GC content was 28.8% with a standard deviation of 5.9%. Only sequences with GC content within three standard deviations of the mean were included in the genome assembly, and 70 scaffolds with GC content higher than 47% were discarded .The paired-end reads and mate-paired reads from Solexa sequencing were assembled using SOAPdenovo software, which uses the de Bruijn graph data structure to construct contigs . SOAPdenhttp://www.repeatmasker.org/) with default parameter settings and by de novo repetitive sequence search using Repeat Modeler (http://www.repeatmasker.org/RepeatModeler.html).Repetitive sequences were identified by searching Repbase using RepeatMasker (http://cufflinks.cbcb.umd.edu/). For de novo assembly, transcripts were constructed using Trinity software (http://pasa.sourceforge.net/) and GMAP (http://evidencemodeler.sourceforge.net/).A strategy combining RNA-Seq and de novo approaches was used to predict protein-coding genes. First, the RNA-Seq reads were assembled to generate the training set for the de novo algorithms. Generally, a combination of mapping-based assembly (RNA-Seq reads were mapped to reference genome and assembled) and de novo methods were used. For mapping-based assembly, RNA-Seq reads were mapped to the assembled genome using SOAPsplice , allowinsoftware . The assand GMAP . The PASand GMAP , Augustuand GMAP , Glimmerand GMAP , and SNAand GMAP . The finhttp://blast.ncbi.nlm.nih.gov/Blast.cgi) against the eggNOG database (http://eggnog.embl.de), Kyoto Encyclopedia of Genes and Genomes (KEGG) reference database (http://www.genome.jp/kegg/), and the National Center for Biotechnology Information (NCBI) nonredundant protein sequence database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/). Gene Ontology (http://www.geneontology.org/) was used to classify and functionally annotate genes. Protein domains were predicted using the Pfam database (http://pfam.sanger.ac.uk/) for gene function assignment. The tRNA and rRNA genes were predicted with tRNASCAN-SE (http://lowelab.ucsc.edu/tRNAscan-SE/). Potential secreted proteins were predicted by SignalP v4.1  (http://www.predisi.de/) (http://merops.sanger.ac.uk/)  obtained against the Lipase Engineering database (http://www.led.uni-stuttgart.de/). Carbohydrate-active enzymes  were annotated using the dbCAN Web server (http://csbl.bmb.uga.edu/dbCAN/)  .Functions were assigned and classified by searching for sequence similarity with BLAST   and Predisi.de/) . Predict.ac.uk/)  with BLA/dbCAN/)  at an E porter/) . ReferenT. kitauei  and T. kitauei-Korea . Furthermore, the phylogenetic analysis showed that the parasite from common carp in this study is clustered with T. kitauei-China (HQ115585) and T. kitauei-Korea (HM624024) with 100% bootstrap confidence values . These findings, combining with the fact that this parasite showed the same host and tissue specificity as T. kitauei, confirmed our isolate as T. kitauei.Cysts of an unknown origin were collected from the intestines of common carp and examined for parasitic infection. Isolated myxosporean-like spores from the cysts showed morphological and morphometric features nearly indistinguishable from those of the previously described T. kitauei. We generated 1.48 Gb  from the Roche 454 sequencing and 5.5 Gb  and 2.5 Gb  from Illumina sequencing. A hybrid genome assembly of 454 and Illumina sequences was conducted by first assembling the Illumina mate-pair and paired-end data with SOAPdenovo, followed by fragmentation of the SOAPdenovo assembled contigs and assembly with the Roche 454 data using Newbler. This approach improves the performance of Newbler with Roche 454 data by correcting the homopolymer and insertion/deletion errors common to Roche 454 sequencing. Potential contamination of the parasite samples with carp DNA was excluded in four ways: 1) During sample collection, the carp intestinal tissue was carefully removed from the cysts, and the collected T. kitauei spores were repeatedly washed; 2) The purity of the T. kitauei DNA was checked by PCR with carp-specific primers, and no carp DNA was detected; 3) In the bioinformatics analysis, assembled scaffolds showing high similarity  to fish and bacterial sequences in GenBank were excluded; 4) A mean ± standard deviation (n = 3) approach was used to exclude potential carp scaffolds based on the fact that the mean GC content of T. kitauei  is lower than that of fish. After the filtering process, 150.7 Mb were assembled into 5,610 scaffolds (N50 = 170 kb), and over 90% of the genome assembly was included in 1,336 scaffolds.To optimize genome assembly, two next-generation sequencing platforms, Roche 454 and Illumina, were used to sequence the genome of T. kitauei was estimated based on 17-mer analysis . To further eliminate contaminating fish and bacterial reads, only the reads mapped to the draft assembly (∼1.3 Gb) were used. The peak of 17-mer frequency (M) correlates with sequencing depth (N), read length (L), and k-mer length (K), with the relationship expressed as M = N × (L − K + 1)/L. The genome size of T. kitauei was inferred to be 188.5 Mb, meaning that 80% of the genome sequences were present in our draft assembly of the T. kitauei genome. The loss of the remaining 20% of the genome sequences may be due to the presence of repeat sequences in the genome and/or the limitations of next-generation sequencing technologies.The genome size of T. kitauei genome was found to contain approximately 13.8% interspersed repeat sequences , and this number is likely underestimated owing to the limitations of genome sequencing strategies. Among the interspersed repeat sequences, approximately 7% were transposable DNA elements, including hAT-Charlie (2.3%) and TcMar-Tigger (0.2%); and some (0.07%) were long interspersed nuclear elements, while no short interspersed nuclear elements were observed. Moreover, a large number of DNA transposons and retrotransposons were found in the T. kitauei genome . These elements can lead to increased expression if inserted in the vicinity of virulence-related genes . One of these transposons encodes a retroviral aspartyl protease belonging to the expansive retroviral-like protease family, which may be involved in processing of polyproteins in T. kitauei.The assembled ed genes . ProteinT. kitauei genome. About 8,659 predicted genes, accounting for 51.5% of the total predicted genes , were expressed in the myxospores of T. kitauei. The numbers of genes with RPKM values in the range of 10–100, 100–1,000, and over 1,000 were 1,817, 715, and 121, respectively. Thereafter, we then compared the genome features of T. kitauei to those of the two closely related free-living cnidarian species H. magnipapillata and N. vectensis . The short introns may not allow alternative splicing , which are signaling pathways present in multicellular Metazoa but not in fungi or choanoflagellates. Together, these results are coincident with the fact that T. kitauei is a multicellular metazoan.Approximately 55% of the putative genes have homologs in the NCBI database and >3,500 gene products could be assigned to gene ontology terms. As shown in T. kitauei through the MEROPS database, constituting around 2.5% of the proteome . The number of proteases in T. kitauei is much greater than the number of putative lipid (22 including 1 secreted) and carbohydrate (44 including 4 secreted) digestive enzymes. The number of digestive enzymes in T. kitauei was considerably lower than that in the two free-living relatives, with the number in T. kitauei representing 42.8%/52.6%, 16.3%/37.3%, and 8.5%/19.4% of the numbers in N. vectensis/H. magnipapillata, for proteases, lipid and carbohydrate digestive enzymes, respectively. As noted, the scale of enzyme number reduction in T. kitauei as compared with the free-living relatives was lower for proteases than that of the lipid and carbohydrate digestive enzymes, suggesting that proteases are the main enzymes involved in nutrient digestion .We identified 422 protease (including 34 secreted) homologues in supplementary table S6, Supplementary Material online), the distribution of proteases in T. kitauei is clearly distinct: T. kitauei has a relatively large proportion of aspartic proteases (46% vs. 0−25%) but small proportion of cysteine proteases (13% vs. 12−38%) and metalloproteases . These proteins are highly related to hemoglobin catabolism, transposon multiplication, and processing of profilaggrin  were much lower than those in two free-living cnidirians . As astacin metalloproteases were reported to have the function for degradation of hydra extracellular matrix . Furthermore, around 40% of the protease families that were present in the two free-living cnidarians are absent in T. kitauei . Taken together, these observations indicated a different life styles of T. kitauei from its free-living relatives.Compared with 11 other free-living and parasitic fungi, protozoans, and metazoans , although the diversity (8 vs. 15–18 families) was lower in T. kitauei . Moreover, the serpin family (I04), which contains inhibitors of serine and cysteine endopeptidases, was greatly expanded in T. kitauei (182 vs. <5 in H. magnipapillata and N. vectensis).Relative to the number of proteases, there were more (206) putative protease inhibitors in supplementary table S8, Supplementary Material online). Within them, 5 of the 19 proteases in the C1A subfamily and 2 of the 5 kexin-type peptidases in the S8B subfamily are highly expressed  in the myxospore stage of T. kitauei. Nineteen protease inhibitors were putatively secretory in T. kitauei, with the majority (14) as serine and cysteine protease inhibitors belonging to the serpin, serine carboxypeptidase Y (I51), and Kunitz-type (I02) families, which may regulate the activities of host-derived serine and cysteine proteases. Notably, two of the secretory protease inhibitors from the I25A and I51 subfamily are highly expressed in the myxospore stage. Proteases and their inhibitors have been reported as important factors for invasion and immune evasion of parasites .Thirty-four proteases contain signal peptides . Although failed to map to KEGG endocytosis pathway, the extensive collection of genes in low-density lipoprotein receptor family (122 genes) detected in T. kitauei suggests that low-density lipoprotein receptor-mediated endocytosis (a type of clathrin-dependent pathway) may play an important role in T. kitauei uptake of fish lipids . Although caveolin, the central component of caveolae-dependent endocytosis (a type of clathrin-independent pathway), was not detected in both T. kitauei and the two relatives, folate receptors, which are alternatively related to the clathrin-independent pathway, were identified. In addition, actin β/γ 1 is expressed at relatively high level  in the transcriptome of myxospore-stage T. kitauei, and might be essential for phagocytosis , its CD63 antigen , Niemann-Pick C1 protein 1 (RPKMs of 2 of 7 genes are 355.94 and 589.40), cysteine protease cathepsin L , and lipase LYPLA3 (RPKM of 1 of 3 genes is 610.83) are highly expressed in the myxospore stage. These results indicate that T. kitauei mainly carries out endocytosis, especially low-density lipoprotein receptor-mediated endocytosis, to acquire main nutrients such as lipids .Endocytosis  has been confirmed to be central in the pathogenesis of many parasites, including a gondii . Like thT. kitauei has a limited repertoire of membrane transporters . Thelohanellus kitauei seems to rely primarily on secondary carriers (56%) and to a lesser degree on primary active transporters (30%)  and the amino acid/polyamine/organocation superfamilies (10%) , which together are involved in the utilization of carbohydrates, phosphates, and amino acids (T. kitauei are mainly composed of ATP-binding-cassette transporters (14%), which involve in exporting drug  of the putative anion transporters are involved in phosphate transport . Therefore, the low blood phosphorus and calcium levels observed in T. kitauei infected carp might be attributed to the powerful capability of T. kitauei in transporting host calcium and phosphate  . The secno acids . On the ing drug , and P-ting drug . Nearly hosphate .Fig. 4.T. kitauei genome revealed extensive reductions in overall metabolic capacity, with fewer genes involved in the eight primary metabolic processes, that is, carbohydrate, energy, nucleotide, amino acid, lipid, cofactor/vitamin, glycan, and other amino acid metabolism .To date, the metabolic pathways of myxozoans have not been well characterized. In this study, comprehensive, genome-wide metabolic pathway reconstruction for a myxozoan was firstly conducted. Compared with the two free-living relatives, E. histolytica, Plasmodium falciparum, and tapeworms  for conversion of malate to pyruvate , as well as the propionyl-CoA cycle which converts succinate to propionate, suggesting a less importance of the anaerobic malate dismutation pathway producing succinate in T. kitauei  and oxidative phosphorylation  were highly expressed in the myxospore stage of T. kitauei  further suggesting that T. kitauei may have greater metabolic dependency on respiration than on fermentation.Anaerobic fermentation plays an important role in the energy metabolism of many parasites, such as apeworms . Both T.T. kitauei has a reduced ability to de novo synthesize and hydrolyze amino acids , which is similar to Schistosoma mansoni and tapeworms . The polyamines and their derivatives mediate a variety of important functions and are therefore essential to cell growth and proliferation  that converts ornithine to the polyamine putrescine was highly expressed  in the myxospore stage of T. kitauei. Other amino acids that can be de novo synthesized  or partially synthesized  and catabolized by the two free-living cnidarians are unlikely synthesized or catabolized in T. kitauei .Compared with the two free-living cnidarians, apeworms . Thelohaferation . ThelohaT. kitauei and the two free-living relatives lack the pathways for fatty acid biosynthesis, they are capable of fatty acid elongation . Unlike the two free-living relatives, T. kitauei has an incomplete β-oxidation pathway in that it lacks dehydrogenases specific for either short- or medium-chain acyl-CoA , suggesting that the lipid metabolism in T. kitauei inclined to anabolism rather than catabolism.Although both T. kitauei. The two free-living cnidarians have the complete pentose phosphate pathway, whereas T. kitauei appears to have a transaldolase-deficient pentose phosphate pathway, like in P. falciparum . Nevertheless, T. kitauei seems to utilize an alternative pathway that contains three enzymes, namely phosphofructokinase, transketolase, and aldolase for hexose-pentose interconversion, which maintains cytosolic NADP+/NADPH and H+ balance. Notably, ribose-phosphate pyrophosphokinase, which converts d-ribose-5′-phosphate to phosphoribosyl pyrophosphate, is highly expressed  in the myxospore stage of T. kitauei. Adenosine monophospates (AMP) can be converted to high energy molecule adenosine triphosphate (ATP) and its cyclic structure cyclic AMP plays an important role in intracellular signaling. The product phosphoribosyl pyrophosphate seems to mainly enter the adenine salvage pathway to form AMP, due to the absence of enzymes  for either de novo nucleotide synthesis or histidine metabolism, and the presence of adenine phosphoribosyltransferase in T. kitauei . The genomes of the two free-living cnidarians indicate that they can synthesize all glycolytic metabolites by gluconeogenesis. Thelohanellus kitauei, however, lacks fructose-bisphosphatase , implying the absence of gluconeogenesis and the inability to convert fatty acids and amino acids into glucose. Actually, the fructose-bisphosphatase was also absent in other intestinal parasites such as G. lamblia, E. histolytica, Cryptosporidium paryum, and Cryptosporidium hominis (http://www.genome.jp/kegg-bin/get_htext?htext=br08601_map00010.keg&hier=5). Taken together, the absence of gluconeogenesis, high proportion of sugar transporters , and the high proportion of glycosyltransferases  may underlie the evolution of T. kitauei’s ability to more efficiently harvest carbohydrates (mainly glucose) from the host.The reduced ability to hydrolyze fatty acids and amino acids, and the completeness of key carbon metabolism pathways (glycolysis and the tricarboxylic acid cycle) suggest that carbohydrates are the primary energy source for T. kitauei using the dbCAN Web server . However, T. kitauei lacks both chitinases and chitosanases . Therefore, T. kitauei either degrade chitin through endoglucanases, endo-β-N-acetylglucosaminidases, or other enzymes containing chitin/chitopentaose-binding modules that were identified in T. kitauei genome . Alternatively, it is not impossible that T. kitauei does not have the capability for chitin degradation. Further investigations are necessary to uncover the detailed model of chitin metabolism in T. kitauei.The genomes of the two nonparasitic relatives suggest that they are capable of synthesizing and hydrolyzing chitin. Although failed to map to KEGG metabolic pathways, ten genes encoding cellulose/chitin synthases were annotated in Thelohanellus kitauei seems to have lost the ATP-expensive pathways for de novo biosynthesis of inosine 5′-phosphate and uridine 5′-phosphate. Therefore, it must rely on salvage pathways, which is similar to other parasites such as E. histolytica, G. lamblia, and Trichomonas vaginalis . In contrast to the two free-living cnidarians, T. kitauei lacks the enzymes for degradation of free purine and pyrimidine bases.T. kitauei is a convergent trait among endoparasites  requirement and the predominant aerobic respiration are consistent with the parasitic site of T. kitauei, that is, the loose connective tissue of the intestinal submucosa, where a high abundance of capillaries may supply sufficient oxygen and nutrients, especially glucose.The reduced level of metabolic complexity observed in arasites . The lacSome antiprotozoan drugs have shown efficacy against myxosporean infections, but these compounds either are toxic to fish or have shown limited efficacy or poor palatability . Also, mT. kitauei in its carp host. To identify potential drug targets, we first selected the 2,653 most highly expressed genes (RPKMs ≥ 10) out of 8,659 expressed ones in the T. kitauei spore transcriptome. The putative products of these genes were searched against known drug target proteins in the ChEMBL database (https://www.ebi.ac.uk/chembl/), and 513 were found to be homologs of existing drug targets .The myxospore stage is tied with disease processes of T. gondii (http://zfgenomics.zfscreens.com/sub/carp). Fourty-seven T. kitauei-specific target genes that lack orthologs in the host were selected , with 23 of them related to nutritional and metabolic pathways  and metabolism of carbohydrates (4), cofactors (3), glycan (1), nucleotides (1), and amino acids (1)  . Homolog targets , suggest targets . Moreoveydrolase , implyinydrolase . Actuallydrolase .Table 2T. kitauei genome to provide a useful reference for comparative genomics of eukaryotic fish parasites, particularly to identify the relative importance of shared genes among various evolutionarily distant fish parasites such as Ichthyophthirius multifiliis. Insights gained from this study will promote experimental approaches for myxozoan research and facilitate the development of control strategies for severe fish parasite infections and may ultimately prevent myxozoan infection in humans.The characterization of the first genome of a myxozoan will provide a platform for exploring basic questions on the biology and evolution of myxozoans. We also expect the Supplementary figures S1–S5, and tables S1–S16 are available at Genome Biology and Evolution online .


Toxoplasma gondii (T. gondii). The parasite has cosmopolitan distribution, infecting almost all species of warm-blooded animals. Latent T. gondii infection in HIV/AIDS patients is a risk for development of cerebral toxoplasmosis (CT). The aim of this study is to determine seroprevalence of latent T. gondii infection and assess its associated factors among individuals infected with HIV in Arba Minch Hospital, south Ethiopia. A facility-based cross-sectional study involving 170 HIV-infected individuals attending Arba Minch Hospital antiretroviral therapy (ART) clinic was conducted from April to June 2013. Data on demographic profile of the study participants and factors associated with T. gondii infection were gathered using a questionnaire. Serum was tested for IgG anti-T. gondii antibody by enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS version 20 software. Seroprevalence of latent T. gondii infection among the study participants was 88.2%. Consumption of raw meat  and involvement in farming/gardening activities  were independent predictors of T. gondii seropositivity. This study revealed high prevalence of latent T. gondii infection, similar to other studies. Monitoring of the patients to prevent reactivation of the latent T. gondii infection is recommended.Toxoplasmosis is a parasitic disease caused by  Toxoplasma gondii (T. gondii), an obligate intracellular parasite infecting virtually all species of warm-blooded animals. The parasite has a worldwide distribution with an estimated one-third of the world's population being infected. It has a complex life cycle, undergoing sexual phase in the feline definitive host and asexual phase in its intermediate hosts. The parasite is transmitted to humans mainly by accidental ingestion of its oocysts from cat faeces, consumption of infected raw meat, and rarely vertical transmission during pregnancy [Toxoplasmosis is a disease caused byregnancy . Clinicaregnancy . Howeverregnancy .T. gondii is one of the opportunistic parasites causing morbidity and mortality in HIV-infected individuals. Primary infection with the parasite initially results in IgM immune response, followed by specific IgG anti-T. gondii response. Following immune response of the host against T. gondii, cyst stage of the parasite which contains slow replicating bradyzoites forms in skeletal muscles and neuronal tissues. However, in immunocompromised individuals, reactivation of the latent T. gondii as a result of conversion of the bradyzoites to the proliferative tachyzoite stage may result in toxoplasmic encephalitis (TE) [tis (TE) . The ristis (TE) . T. gondii infection in HIV-infected individuals. Seroprevalence of T. gondii in HIV-infected individuals is often high in most of the reports, with substantial incidence of TE in AIDS patients not receiving prophylaxis [ T. gondii infection in HIV-infected individuals in Ethiopia reported prevalence of more than 75% [ T. gondii seroprevalence of 81.4% among women of child bearing age in central Ethiopia [ T. gondii is similarly high in most of the sub-Saharan African countries [Several studies documented varying magnitude of latentphylaxis . Most ofthan 75% . MoreoveEthiopia  and 83.6Ethiopia  were alsountries –15. T. gondii infection in HIV-infected individuals is not a routine practice in health care facilities in Ethiopia. Moreover, data on seroprevalence of T. gondii is limited in Ethiopia, and published reports on magnitude of T. gondii infection in HIV-infected individuals in the study area have not been obtained. Therefore, this study determined seroprevalence of latent T. gondii infection and assessed associated risk factors among HIV-infected individuals attending Arba Minch Hospital antiretroviral therapy (ART) clinic.Laboratory diagnosis ofA facility-based cross-sectional study was conducted in Arba Minch Hospital found in Arba Minch town. The town is located 505 km south of the capital Addis Ababa. Arba Minch is located at altitude of 1200–1300 meters above sea level, with average annual temperature of 29°C. The hospital serves more than 2 million people in the region. The study was conducted from April to June 2013.A total of 170 HIV seropositive individuals who were on follow-up at Arba Minch Hospital ART clinic during the months April to June 2013 were included in the study. The study participants were prior diagnosed with HIV and under follow-up at the ART clinic of the hospital. The sample size was estimated using the general formula for single population proportion, with the following assumptions: prevalence (p) of 87.4% [ T. gondii infection. The questions administered for assessing predisposing factors of T. gondii infection include habit of eating raw meat and vegetables/fruits, farming/gardening activities, sources of drinking water, history of blood transfusion, and presence of domestic cats at household. The questionnaire data were collected by a trained nurse. Following the interview, approximately 2 mL of venous blood was collected from each consenting study participant by experienced phlebotomist. The blood was processed following standard procedures. Briefly, serum was separated from red blood cells and stored at −20°C prior to assay. It was then tested for anti-T. gondii IgG antibody using ELISA test kit  at Arba Minch Regional Laboratory Department, following the manufacturer's instruction. The CD4+ T cell count of the study participants was obtained from log book of the ART clinic.A pretested questionnaire was used to gather sociodemographic information and data on factors predisposing to T. gondii infection. Variables with P value <0.25 by the bivariate analysis were entered into multivariate model. A P value <0.05 was set as statistically significant during the analysis.Data were first collected in hardcopy. After checking for completeness and consistency, the data were entered into computer, cleaned, and analyzed using SPSS version 20.0 software package. Descriptive statistics were performed to describe demographic profile of the study participants. Bivariate and multivariate logistic regression analyses were utilized to identify factors predisposing toEthical clearance was obtained from Jimma University Research Ethical Review Committee. Official permission was sought from Arba Minch Zonal Health Bureau and Arba Minch Hospital Administration. Moreover, written informed consent was obtained from all study participants prior to enrollment in the study. Confidentiality of the collected information and laboratory test results was maintained. Laboratory test results were communicated to the attending physician for further management of the cases.A total of 170 HIV seropositive individuals attending Arba Minch Hospital ART clinic had participated in this study. The majority (64.1%) of the study participants were female. Mean age of the participants was 35.54 years. The vast majority of the study participants (86.5%) were urban residents. Sociodemographic profile of the study participants is demonstrated in T. gondii antibody seropositive.Overall, out of the total 170 HIV seropositive study participants included in this study, 150 (88.2%) were IgG anti- T. gondii infection were also assessed. More than one-third (37.6%) of the study participants were within the age group 35–44 with T. gondii seroprevalence of 90.6%. IgG anti-T. gondii seropositivity increased as age group of the study participants increases. The difference was significant  with gender of the study participants. Similarly, no significant difference in latent T. gondii infection with place of residence, educational status, consumption of raw vegetables or fruits, source of drinking water, and history of blood transfusion was observed in this study  (Data on CD4+ T lymphocyte count were available for 136 of the study participants. The CD4+ T cell count ranged from 55 to 1574 cells/ < 0.05) . T. gondii infection  in the household did not significantly affect T. gondii seropositivity. Presence of domestic cats at household as a risk factor was documented in school children elsewhere [Despite the significant association of farming/gardening activities withlsewhere . Differe T. gondii. In the current study, 6.5% of the study participants responded to having had previous blood transfusion, 90.9% of whom were T. gondii seropositive. There was no significant difference in T. gondii seropositivity with history of blood transfusion. The risk of T. gondii infection through blood transfusion is low, probably due to the low prevalence of acute T. gondii infection in blood donors.Blood transfusion is also a possible means of transmission of T. gondii seropositivity between study participants with CD4 T-cell count below 200 and ≥200 cells/μL. This finding is consistent with a report from Malaysia [μL. The vast majority of these were IgG anti-T. gondii seropositive. HIV-infected individuals with low CD4+ T cell count coinfected with T. gondii are at higher risk of reactivating the latent infection [ T. gondii infection, and incidence of the disease is associated with T. gondii IgG seropositivity and low CD4+ T cell count [In this study, CD4 + T lymphocyte count of 80% of the study participants was available during the data collection period. There was no significant difference inMalaysia . In the nfection . It has ll count . T. gondii seropositivity. As age group of the study participants increases, a corresponding increase in T. gondii seropositivity was noted. This is probably related to prolonged exposure time as age increases.Among the sociodemographic and other factors assessed in this study, only age was significantly associated with T. gondii infection is high among the study participants, similar to most of the studies. The rate of T. gondii seropositivity significantly increased with age. Self-reported consumption of raw meat and history of involvement in farming/gardening activities were the main predictors of T. gondii seropositivity among the study participants. Creating awareness about T. gondii infection and follow-up of their status is recommended. Moreover, screening of T. gondii infection in HIV-infected individuals should be considered. Further studies are required to determine incidence of TE in HIV-infected individuals in the area.In conclusion, seroprevalence of latent


Pieris rapae is one of the most destructive pests of Brassicaceae. Yet little is understood about its genes involved in development. To facilitate research on P. rapae, we sequenced the transcriptome of P. rapae during six developmental stages, including the egg, three larval stages, the pupa, and the adult. In total, 240 million high-quality reads were obtained. De novo assembly generated 96,069 unigenes with an average length of 1353 nt. Of these, 31,629 unigenes had homologs as determined by a blastx search against the NR database with a cut-off e-value of 10−5. Clusters of Orthologous Groups of proteins (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to functionally annotate those genes. Then, 849 genes involved in seven canonical development signaling pathway were identified, including dozens of key genes such as Hippo, Notch, and JAK2. A total of 21,883 differentially expressed (cut-off of 2-fold) unigenes were detected across the developmental stages, most of which were found between the egg and first larval stages. Interestingly, only 34 differentially expressed unigenes, most of which are cuticle protein related genes, were detected with a cut-off of 210-fold. Furthermore, we identified 32 heat shock protein (Hsp) genes that were expressed with complete open reading frames. Based on phylogenetic trees of the Hsp genes, we found that Hsp genes with close evolutionary relationships had similar expression pattern. Additionally, partial pattern recognition receptors genes were found to be developmental regulated. This study provides comprehensive sequence resources for P. rapae and numerous differential expressed genes, and these findings will lay the foundation for future functional genomics studies on this species.The small white butterfly  Pieris rapae (Lepidoptera: Pieridae) is one of the most destructive agricultural pests in the Brassicaceae family, causing considerable economic loss in China [P. rapae and its natural enemy Pteromalus puparum provide an important physiological interaction model, for the study of innate immunity. Dozens of immunity-related genes have been cloned, including the serotonin receptor [The cabbage butterfly, in China . P. rapareceptor , the ryareceptor , and proreceptor . Althougreceptor , the nucAthetis lepigone egg and larva [In Lepidoptera, the genome sequences of more than five species and transcriptomes of dozens of species  have beend larva . The mosnd larva . In addind larva .P. rapae. We hoped to obtain high quality assembled transcripts and to discover development related genes during various periods of insect growth by performing de novo transcriptome assembly. The result produced a high-quality reference transcript set for future research on this species.We attempted to obtain expression data throughout all developmental stages, by performing comprehensive RNA sequencing on P. rapae, which is not a protected or endangered insect species. There are no ethical issues involved in this research.There are no specific permits required for collection of P. rapae larvae were collected from Shanghai, China in May 2014 and reared at 25 ± 1°C, a relative humidity of 80 ± 5%, and a photoperiod of 10D:14L on cabbage leaves until they developed into pupae. Pupae were maintained in the same conditions as described above until emergence. Samples were collected and washed twice in ddH2O in filter paper. Clean samples were reserved in RNAlater  and stored at -20°C until RNA extraction. In total, 40 eggs, six larvae six pupae and three pairs of adults were used as sequencing samples.P. rapae using an RNeasy MinElute Cleanup Kit  according to the manufacturer's instructions. The RNA concentration was assessed using a Qubit fluorometer (Invitrogen Corp. USA) and the quality was confirmed using a 2100 Bioanalyzer . Stage-specific samples from RNA extractions were pooled at an equal concentration. cDNA libraries were constructed using a TruSeqTM RNA Sample Preparation Kit v2 according to the manual . Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads with two rounds of purification. During the second elution of the poly-A RNA, the RNA was also fragmented and primed for the first-strand cDNA synthesis. The RNA templates were removed and the second-strand cDNA was synthesized to generate double-stranded (ds) cDNA. Ds cDNA ends were repaired and a single ‘A’ nucleotide was added to the 3’ ends of the fragment. Then, the adapters were ligated to the end of the ds cDNA, and prepared for hybridization onto a flow cell. Finally, PCR was use to selectively enrich DNA fragments with adapter molecules on both ends and to amplify the amount of DNA in the library. The libraries were sequenced on an Illumina GAIIx  platform with sequence runs of 2 × 125 bp paired-ends at the laboratory of WuXi AppTec .Total RNA was isolated from the samples at six developmental stages of de novo transcriptome assembly using Trinity software [We performed a rigid filtering process with a cut-off Q-value of 30 using the NGS QC Toolkit . High-qusoftware , 16, withttp://weizhongli-lab.org/metagenomic-analysis/server/). Unigenes were translated into amino acid sequences by TransDecoder software, which is part of the Trinity software [Unigenes were first blasted against the NCBI non-redundant (NR) protein database using Blastx with an e-value cutoff of 1E-5. The best blast hits were used to describe the features of unigenes, such as the gene name, species homology, and identity distribution. Next, blast results were functionally annotated with Gene Ontology (GO) terms using Blast2GO software  and the software , 16.2(fold change) was used to compare two stages. Three criteria were used to describe differential gene expression with a false discovery rate (FDR) less than 0.001. First, log2(fold change) > 2 between any adjacent stages was used to identify significantly differential expression. Second, log2(fold change) > 2 between all of the adjacent stages was used to identify genes with frequently changed expression levels. Finally, genes expressed in unique stages were identified as stage-specific unigenes.To obtain gene expression information, we calculated abundance estimation for each sample against assembly sequences using RSEM software . Then, tMega 5.0 software  was usedP. rapae  reads pooled from all stages. The assembly generated 238,738 transcripts with an N50 value of 2471 nt and 96,069 unigenes with an average length of 1, 353 nt. Reads libraries from each stage were also assembled separately, resulting in lower numbers of unigenes in larva and higher number in adults  and Microplitis demolitor (30.4%). The distribution of unigenes and transcripts was analyzed in a 500 nt window in Using the Trinity assembly program , 16, then adults . The GC P. rapae unigenes was performed using BlastX against non-redundant databases. Longer unigenes tend to have more homologs compared to short ones , B. mori (23.3%), Plutella xylostella (13.1%), P. xuthus (2.2%), and Papilio polytes (0.6%), had the top five counts of unigenes with NR annotation , developed by the Gene Ontology Consortium, was used for standard gene function annotation, providing a dynamic, controlled vocabulary that can be applied to multiple species [P. rapae was successfully mapped to three main functional processes, which were composed of 51 GO terms (The GO (gene ontology) database ( species . Using BGO terms . Based oClusters of orthologous groups of proteins (COGs) represent a phylogenetic classification of the protein database involving eukaryotes (KOG) and prokaryotes (COG) , 24. BefP. rapae, and approximately 849 unigenes were associated with seven signaling pathways. The largest signaling pathway was MAPK (231 genes), followed by Wnt, Fox, Hippo, Hedgehog, and Jak-STAT  pathways involving more than 300 unigenes. The top three pathways were as follows: metabolic pathways (3620 genes), biosynthesis of secondary metabolites (1214 genes), and biosynthesis of antibiotics (789 genes). Considerable numbers of unigenes were involved in the development related pathway of Jak-STAT . Some maJak-STAT .D. melanogaster [RSEM  was usednogaster . The topTo identify transcripts that were uniquely expressed in the egg, L1, L3, L5, pupae, or the adult stages, we used custom scripts to filter transcripts expressed in any two stages. A total of 9724 transcripts were found to be stage-specific. Among them, most stage-specific transcripts were found in the egg stage . PartialD. plexippus, B. mori, P. polytes, Biston betularia, and P. xuthus. One of 33 was predicted to be arylalkylamine N-acetyltransferase, which is involved in the day/night rhythmic production of melatonin. The rest of the annotated genes are uncharacterized.To further investigate the frequently changed gene expression level, we found 39 transcripts that were significantly differentially expressed at all pairwise adjacent developmental stages. Then, 33 of them were annotated . InteresHsp genes with complete open reading frames in P. rapae were identified as members of six Hsp families, including sHsp, Hsp10, Hsp40, Hsp60, Hsp70, and Hsp90. All of the best blast hits were found in Lepidoptera. The numbers of identified genes in the sHsp, Hsp10, Hsp40, Hsp60, Hsp70, and Hsp90 families of P. rapae were 17, 1, 3, 2, 5 and 4, respectively. Detailed information about Hsp genes in P. rapae including their unigene IDs and the deduced amino acid sequences is listed in After searching the automated database and a careful manual review, a total of 32 P. rapae  genes are found to be significantly expressed during the development, including Peptidoglycan recognition proteins (PPRPs), hemolin, thioester-containing proteins (TEP), and β-1,3-glucan recognition proteins (βGRP)  . P. rapaP. rapae is a major pest of Cruciferae and is involved in the important host-parasitoid model of P. rapae and P. puparum, its genetics have not been well studied. In the absence of complete genome sequences, deep transcriptome analysis can provide a basis for performing future gene expression and functional analysis on P. rapae and can improve our understanding of its biological processes and molecular mechanisms. In the current study, mRNA-seq technology was applied to reveal the developmental transcriptome of P. rapae. The de novo assembly of short reads without a reference genome remains a challenge with the emergence of bioinformatics tools [P. rapae. The principal metric of an assembly is the length of the transcripts. Depends on approximately 35 Gb of raw data from Illumina sequencing, the 2431 bp of N50 is about three times and four times as length of that of A. lepigone [P. xylostella [Although cs tools . Trinitycs tools . Using tlepigone  and P. xlostella , respectP. rapae, P. xylostella [A. lepigone [de novo assembly. The 31,629 unigenes annotated by the NR database showed that the Monarch butterfly (D. plexippus) shared the highest similarity with the cabbage butterfly (P. rapae) rather than the silkworm (B. mori). This similarity may be because P. rapae is phylogenetically closer to D. plexippus than to B. mori, which indicates a common selection of genes between closely related insects. This pattern is different from the transcriptome studies of Lepidoptera P. xylostella [A.lepigone [Nilaparvata lugens [Sogatella furcifera [Tribolium castaneum. These patterns are likely due to the insufficient quantity of Lepidoptera transcriptome data published during the study period.The numbers of unigenes annotated in the NR database appear to be similar in three Lepidoptera insects lostella  and A. llepigone  after delostella  and A.lelepigone , as wellurcifera . They alP. rapae. The ratio of the number of unigenes annotated by the three main GO categories were roughly equal to another Lepidoptera insect A.lepigone [D. melanogaster, no more than 30% have been evaluated by direct assay, expression pattern, genetic interaction, or mutant phenotype [D. melanogaster were mostly inferred from the computational or indirect evidence, these annotations are generally thought to be of lower quality than those derived from experiments. For other non-model insects, GO annotations were almost inferred from the computational evidence, which may have led to an abundance of false positive. Moreover, based on the KEGG database, both species have over 10,000 genes annotated in approximate 300 canonical pathways. For the seven conserved development-related signaling pathways, such as Notch [P. rapae orthologs of these key genes were previously unknown. The discovery of these genes may help us elucidate the metabolic mechanism of P. rapae during development. However, the KEGG annotation is clearly based on orthology-function conjectures, which are known to be false in at least some cases [P. rapae.The GO and KEGG annotations allowed us to categorize genes and examine the major metabolic pathways for lepigone , and washenotype . Becauseas Notch , almost me cases . TherefoP. rapae. Dramatically changed expression levels between eggs and larvae were also previously found in Lepidoptera Cnaphalocrocis medinalis [A. lepigone [Spodoptera litura [PGRPs, hemolin, and βGRP. Hemolins have only been found in Lepidoptera, and has not been identified in other insect orders. In P. rapae, hemolin is a 46.97 kDa protein, which composed of four immunoglobulin domains. The extremely up-regulation at the adult stage, indicating developmentally regulated, which is similar in M. sexta [hemolin may activate by the steroid hormone 20-hydroxyecdysone [P. rapae in these two developmental stages.βGRP2 transcripts become highly abundant prior to pupation is a similar with M. sexta [M. sexta [Anopheles gambiae [D. melanogaster [Numerous differentially expressed genes were detected in the pair-wise comparisons of the six developmental stages. Most of the differentially expressed genes were down-regulated in the L1 stage compared to the egg stage, resulting in the largest gene expression differences during the development of edinalis . It may edinalis . Cuticullepigone  and Spoda litura . The cuta litura . These pa litura . FurtherM. sexta . The devecdysone . βGRPs wM. sexta . PGRPs, M. sexta , Anophel gambiae , and D. nogaster . A systeP. rapae, and their expression was confirmed to be changed in response to parasitization by the parasite P. puparum. In the present transcriptome, six Hsp families including 32 members were obtained, whereas Hsp100 and Hsp110 were not found. Of the 32 Hsp genes, 29 were newly identified and 3 of them were blast hits for previously identified Hsp genes [D. melanogaster [Liriomyza sativa [P. rapae, indicates that genes within a functional group tend to be expressed at similar times [Heat shock proteins are highly conserved chaperones that respond to environmental changes by facilitating protein folding and preventing protein denaturation. Hsp proteins could be classified into eight families based on homology and molecular weight as follows: sHsp, Hsp10, Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and Hsp110 . In our sp genes . Considesp genes , 48, thenogaster  and Liria sativa . Similarar times . Hsp genar times . A genomP. rapae genes and their function. Development-related genes such as cuticular protein, play an indispensable role in the insect cuticle integration, resistance, and innate immunity. If the expression of a key cuticular protein gene inhibited by RNAi technology lead to the death of P. rapae, this may form the basis of a novel pest control strategy.A total of 240 million reads were obtained by transcriptome sequencing and the de novo assembly yield 96,069 unigenes with an average length of 1353 nt. Based on similarity searches against databases, 31,629 unigenes were matched to known proteins. The data presented in this study will provide important reference points for further studies of S1 FigThe X- axis represents the COG term. The Y-axis shows the number of unigenes.(TIF)Click here for additional data file.S2 Fig−ΔΔCT method. The significant difference (P<0.5) of each gene expression between other stages and the L1 stage are indicated with asterisks.Transcript levels for all samples were assessed by real-time PCR. The experiment was perfomred in triplicae (mean ± standard deviation of the mean). All mRNA expression data were normalized to the control gene 18S RNA. None of the selected genes can be detected in the egg stage. Samples from the L1 stage were used as control. The relative expression level to the control using the 2(TIF)Click here for additional data file.S1 File(TXT)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 FileNasonia vitripennis), Am (Apis mellifera), Bt (Bombus terrestris), Cv , Md (Microplitis demolitor), Oc (Oxya chinensis), Dm (Drosophila melanogaster), Tc (Tribolium castaneum), Pr (Pieris rapae), Px (Papilio xuthus), Dp (Danaus plexippus), Bm (Bombyx mori), Cs , Pv (Polypedilum vanderplanki), Fa (Fopius arisanus), and Hs .The trees were generated using the NJ method by Mega5 software. Expression levels were compared with their RPKM values. The used amino acid sequences of other Hsps are from Nv ((PDF)Click here for additional data file.S1 TableP. pieris, respectively.E, L1, L3, L5, L, P, and A refer to the egg, the first stage of larva, the third stage of larva, the fifth stage of larva, pupa, and the adult of (DOC)Click here for additional data file.S2 TablePartial nodes genes were listed in (XLS)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(DOC)Click here for additional data file.


Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants  and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations. Chlamydia abortus is an obligate intracellular bacterium that can colonize the placenta of several animal species causing abortion or stillbirth [C. abortus is endemic among small ruminants and is the most common cause of infectious abortion in sheep and goats in many countries worldwide [illbirth –3. This orldwide .C. abortus is classified as a member of the family Chlamydiaceae that currently comprises the single genus Chlamydia, which contains 11 species  and a novel candidate species named C. ibidis [C. abortus is low. Methods based on the cross reactivity of monoclonal antibodies, restriction patterns of the ompA gene and the phylogenetic analysis of rRNA genes, resulted in little or no evidence of genetic diversity with regard to the host, associated disease or geographical origin of strains [ompA sequences were highly conserved [C. abortus strains, named LLG and POS, isolated in Greece from an aborted goat and sheep, respectively [C. abortus strains circulating in the same area. These strains were characterized as variants on the basis of unique inclusion morphology, differences in polypeptide profiles and antibody cross-reactivity, diversity of rRNA, ompA and pmp sequences, and different behavior and ability to colonize the placenta and fetus compared to other wild-type strains [C. abortus reference strain S26/3 revealed notable differences in the pseudogene content [C. abortus strains, to such an extent that "subspecies" status has been suggested for them [. ibidis –6. Studi strains –9. Howevonserved . Furtherectively , were fo strains . Compari content ,19. rRNAfor them .C. abortus strains into distinct genotypes [C. abortus strains into six genotypes [C. abortus strains examined [C. abortus strains, this study aimed to determine the suitability of MLST for genotyping C. abortus in comparison to MLVA. To achieve this, a well-referenced collection of C. abortus strains of known MLVA genotypes, along with two other collections of field isolates and samples were genotyped. In addition, we aimed to explore and delineate the C. abortus clonal lineages to obtain new insights into how clones or lineages of particular epidemiological relevance emerge and diversify.Interestingly, a recent study using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), as well as a different approach using a multilocus sequence typing (MLST) system, has allowed the differentiation of enotypes ,22. The enotypes . In contenotypes , recogniC. abortus genomic DNAs were analyzed. These comprised: (i) a collection of 33 strains (panel A) that were representative of all C. abortus genotypes, as determined by MLVA [C. abortus field isolates belonging to the predominant MLVA genotype MT2 [In this study a total of 94  by MLVA ; (ii) a type MT2 ; and  from the first or second culture passage of the original strains and isolates. Additional field samples originated from different regional veterinary diagnostic laboratories or veterinary services in France, Greece and Italy, from abortion cases between 2005 and 2011. The origin and source of the C. abortus strains, isolates and samples investigated are presented in Tables The C. abortus with a species-specific real-time PCR assay targeting the ompA gene [All DNA samples were confirmed to be mpA gene  prior tompA gene ,26 were MLVA genotyping was performed by targeting five tandem repeat loci, as previously described . RepeatsgatA, oppA, hflX, gidA, enoA, hemN and fumC, as previously described [Chlamydiales MLST website . Sequencing of both DNA strands was performed by Eurofins (Germany). Numbers for alleles and sequence types (STs) were assigned in accordance with the Chlamydiales MLST Database.MLST genotyping targeted seven housekeeping genes, namely escribed . Target MLST and MLVA results were entered into BioNumerics software v7.1 (Applied Maths) for minimum-spanning-tree analysis. Priority rules within the BioNumerics software were set to assign the primary founder  as the ST that initially would diversify to produce variants that differ at only one of the seven loci, as was previously described for the eBURST algorithm for inferring patterns of evolutionary descent from MLST data . The cloC. abortus ST, the sequences of all seven loci were concatenated to produce an in-frame sequence of 3,098 bp, and a maximum likelihood tree was constructed. The HKY85 model of nucleotide substitution, assuming a discrete gamma distribution with eight categories, was used for tree reconstruction using MEGA5 software [dN/dS ratios were computed by the method of Nei and Gojobori [Maximum likelihood trees for each chlamydial MLST locus were reconstructed to determine the extent by which the phylogenetic signal varied between gene loci, testing for possible recombination ,29. For software . dN/dS rGojobori  as impleGojobori . An unweInitially, the MLST scheme was applied to the panel A strains to assess the genetic relatedness of strains with particular MLVA profiles. Specifically, 32 representative strains belonging to the six MLVA genotypes (MT1 to MT6)  were anaChlamydiales MLST website. This new allelic profile (ST86) was detected in five strains belonging to MT2 , their allelic profiles were analyzed for all field isolates and samples . Altogether, six STs were identified, namely ST19, ST25, ST29, ST30, ST86 and ST87. As expected, the ST30 profile was detected and confirmed in one field sample   . Minimumof ST19) . The simof ST19)  and was hemN_21 allele  and differed at only a single nucleotide site from the allele in the putative ancestral ST , charact1 allele .C. abortus STs and the avian ST36 previously designated as C. abortus [C. abortus clonal complex and other chlamydial clonal lineages, especially the ruminant ST30 and avian ST36 singletons, the degree of phylogenetic consistency between MLST locus trees was examined  in France, and ST87 as well as the singleton ST30 in Greece.The st three . Some C.C. abortus STs seem to be diverse across geographic distances, as exemplified by the different ST distribution patterns among C. abortus strains, isolates and samples originating from France and Greece  of this lineage  over time and/or geographical distribution, the population of ST19 and, to a lesser extent ST29, diversified into multiple MT subpopulations Figs .C. abortus populations in defined areas  evolve slowly and are highly conserved, the resolution provided by MLST is low for the investigation of recent evolution and, above all, for short-term epidemiological studies. Unlike MLST, MLVA targets several types of markers with variation in the numbers of repeats at particular loci, to be used by some bacteria as a means of genomic and phenotypic adaptation to the environment . In our ed areas . MeanwhiC. abortus strains, isolates and field samples collected from 1950 to 2011 from diverse geographic locations reveals new insights into the population structure, genetic diversity and potentially the evolutionary history of C. abortus. Taken together, MLST and MLVA analyses demonstrated that C. abortus, similarly to other genetically monomorphic bacteria, has a predominantly clonal population structure consisting of subpopulations, many of which seem to be associated with particular geographic regions. The analyses further revealed that ruminant C. abortus strains are more genetically diverse than generally recognized, since novel genotypes have been detected. Both techniques effectively distinguished the "LLG/POS variant" lineage from the "clonal complex" one, supporting the notion that possibly two separate host niche adaptations of C. abortus have occurred through time. In combination, MLST and MLVA may provide additional information into the origins and evolutionary relationships of circulating C. abortus populations.In this study the molecular characterization of 94 ruminant S1 FigC. abortus strains AB7 (A), S26/3 (B), POS (C) and CY71 (D). A 100-bp ladder (100–1000 bp) is run on both sides of sample group. The number of repeat units within each allele is indicated.PCR amplification of (TIFF)Click here for additional data file.S2 FigC. abortus MLST-encoded protein alleles (b)Amino-acid substitutions among  Conserved sites are shaded gray in the protein sequence alignments. C. abortus hemN alleles  (a)A. Nucleotide substitutions among three  The positions in which the C. abortus hemN alleles present substitutions (positions 261 & 343) are shaded by colours. The substitution (point mutation) that occurs within the clonal complex at the position 261  is synonymous, whereas the substitution at the position 343  is non-synonymous (see b). Residues G and A, at the positions 261 & 343, respectively, are unique among all chlamydial hemN alleles. C. abortus hemN-encoded protein alleles (b)Amino-acid substitutions among three  The non-synonymous amino-acid Thr (T) harboured at the position 115 of hemN_21-encoded protein is unique among all chlamydiae; shaded by distinct colour. C. abortus gatA alleles  (a)B. Nucleotide substitutions among three  The positions in which the C. abortus gatA alleles present substitutions (positions 165 & 374) are shaded by colours. The substitution (point mutation) that occurs at the position 165 within the clonal complex  is synonymous (see b). Residue G at the position 374 characteristic for C. abortus clonal complex, is unique among all chlamydial gatA alleles. C. abortus gatA-encoded protein alleles (b)Amino-acid substitutions among three  The amino-acid Gly (G) characterizing the C. abortus clonal complex is unique among all chlamydial gatA-encoded protein alleles at the conserved position 125; shaded by distinct colour. C. abortus fumC alleles  (a)C. Nucleotide substitutions among four  The position in which the C. abortus fumC alleles present substitutions  are shaded by colours. The substitution (point mutation) that occurs at the position 440 within the clonal complex  is synonymous (see b). Residue T at position 243 of fumC_17 harbouring in the LLG singleton is unique among all chlamydial fumC alleles. C. abortus fumC-encoded protein alleles (b)Sequence alignment among  Non-synonymous substitutions were not observed. C. abortus enoA alleles  (a)D. Nucleotide substitutions among three  The position in which the C. abortus enoA alleles present substitutions  are shaded by colours. Residue A at the conserved position 317, is unique among all chlamydial enoA alleles. enoA-encoded protein alleles (b)Amino-acid substitutions among  The non-synonymous amino-acid Glu (E) characterizing the LLG singleton is unique among all chlamydial enoA-encoded protein alleles at the conserved position 106; shaded by distinct colour.Consensus blocks are shaded light-gray; 20% has been taking as a minimal fraction of a specific nucleotide at a defined consensus position. (PDF)Click here for additional data file.S3 FigA. Minimum-spanning-tree analysis of the ruminant C. abortus STs and the avian ST36 previously designated as C. abortus [C. abortus clonal complex. The numbers between the circles define the number of locus variations. The circle colours indicate the corresponding MLVA types (MTs); nt, MT not typeable. B. The individual MLST allelic profile of ST36 is shown in comparison with the other C. abortus profiles. abortus . The gra(TIFF)Click here for additional data file.S4 FigA. Maximum likelihood trees are shown for each MLST locus; in each tree locus-alleles corresponding to known chlamydial STs are included. B. A maximum likelihood tree, highlighted in red border, is shown on the basis of concatenated sequences of all seven loci corresponding to six ruminant  and one avian (ST36) C. abortus STs. STs belonging to the clonal complex or singleton lineages are highlighted white on a black or grey background, respectively.(TIFF)Click here for additional data file.S5 FigA. In the minimum-spanning-tree the MTs are displayed as circles. Circle sizes represent the number of C. abortus strains, isolates or samples; circles are colored by the corresponding STs Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers. Staphylococcus aureus/MRSA .–24.mec (in (PVL) , 13–22]mec (SCCmin (PVL) .S. aureus range from hemolysins, e.g., alpha- and beta-hemolysins  ,,S. aureuLA, HLB) –33. ThesLA, HLB) , 34–37]–37S. aurLA, HLB) –41, and LA, HLB) . Currentdization . These adization –45.S. aureus producing toxins should be treated differently than infections with S. aureus lacking those toxins. The presence of PVL mandates special infection control and eradication measures , or a clinical condition related to PVL or TSST1 might be treated with gamma globulin and/or compounds inhibiting toxin biosynthesis  in addition to the standard regimen . Ba. Ba25]. i.e., most specific and sensitive) combination of capture and detection antibodies for each target ,), sea-320 (GenBank accession number CP001996.1 [1144119…1144898]) and sea-N315 (GenBank accession number BA000018.3 [2011380…2012153]). Several S. aureus isolates with all of these sea alleles were tested. Anti-SEA-antibodies detected SEA corresponding to alleles sea-FRI100 and sea-320, whereas SEA of allele sea-N315 was not identified.For SEA, different alleles can be recognized that will be designated here S. aureus species marker SPA  an anS. aurCurrently, there is a set of genotypic methods in bacterial routine diagnostics for strain identification and determination of relevant virulence and resistance markers. Common techniques are PCR or DNA microarray hybridization. Most of these assays give information about the presence of species-specific virulence or resistance genes, but not about their expression. For protein expression and functionality, molecular phenotypic techniques are well established. ELISAs, lateral flow tests for protein detection in general, or automated micro dilution techniques, with regard to resistance markers, are current methods. A main disadvantage of ELISAs or LFs is that only one, or a low number of targets can be tested in one experiment. The multiplex protein array developed herein allows a rapid, parallel and economic performance to detect several markers within a single experiment. A pure culture harvested from a blood agar plate can be used to perform the assay. These are optimal preconditions for integrating the assay into routine laboratories.S. aureus. All S. aureus isolates tested in this study gave positive signals for SPA. By contrast, the tested coagulase-negative staphylococci (CoNS), S. epidermidis, S. sciuri and S. capitis, as well as another control strain, E. coli, tested negative for this S. aureus species-specific marker. Therefore, these anti-protein A-antibodies act as positive controls for S. aureus isolates in the experiments.The array contains antibodies against epitopes of protein A. This cell wall located protein is used in this assay as species marker for The qualitative detection of proteins using the antibody microarray was verified by checking the presence of corresponding genes using the DNA microarray. Generally, the results described herein showed very good concordance of genotypic and phenotypic results.i.e., in case of a MRSA-positive result), be isolated from other patients to avoid transmissions or even outbreaks. In addition, appropriate treatment can be initiated. Recently, a new mecA homologue, mecC, was discovered. It is located on a novel SCCmec element type XI and also confers resistance to methicillin . Am. AmS. auThe newly developed assay as described herein allows the multiplex detection of staphylococcal proteins, and contains the option to test the influence of external factors on protein expression. This might be a useful test in routine diagnostics as well as for research purposes.S1 File(XLSX)Click here for additional data file.