Using 16S rRNA sequencing, the vaginal microbiota from cervicovaginal fluid samples was characterized into five Community State Types (CST) dominated by Lactobacillus spp.: CSTI (Lactobacillus crispatus), CSTII (Lactobacillus gasseri), CSTIII (Lactobacillus iners), CSTV (Lactobacillus jensenii); and mixed anaerobes—CSTIV. This was then related to the vaginal metabolite profile and pH determined by 1H-Nuclear Magnetic Resonance spectroscopy and pH indicator paper, respectively. At 20–22 weeks, the term-delivered women (TDW) indicated a proportion of CSTI-dominated microbiota >2-fold higher compared to the preterm-delivered women (PTDW) , and a slightly higher proportion at 26–28 weeks . CSTV was >2-fold higher in the PTDW compared to TDW at 20–22  and 26–28 weeks . Furthermore, at 26–28 weeks no PTDW had a CSTII-dominated microbiome, in contrast to 28% of TDW (P < 0.0001). CSTI-dominated samples showed higher lactate levels than CSTV at 20–22 weeks (P < 0.01), and 26–28 weeks (P < 0.05), while CSTII-dominated samples indicated raised succinate levels over CSTV at 26–28 weeks (P < 0.05). These were supported by Principal coordinates analysis, which revealed strong clustering of metabolites according to CST. In addition, the CSTI-dominated samples had an average pH of 3.8, which was lower than those of CSTII—4.4, and CSTV—4.2 (P < 0.05). Elevated vaginal lactate and succinate were associated with predominance of CSTI and II over CSTV in women who delivered at term compared with their preterm counterparts. This suggests that L. jensenii-dominance and decreased lactate and/or succinate could increase the risk of PTB, while L. crispatus/gasseri may confer some protection against inflammation-associated PTB and highlight the need for further study in this area.A major challenge in preventing preterm birth (PTB) is identifying women at greatest risk. This pilot study prospectively examined the differences in vaginal microbiota and metabolite profiles of women who delivered prematurely compared to their term counterparts in a cohort of asymptomatic (studied at 20–22,  Preterm birth (PTB) is the leading contributor to infant mortality and morbidity globally with associated multi-billion dollar health costs  that suppress growth of competing anaerobic species.Thus, in order to understand the mechanisms and prognostic indicators of spontaneous PTB, there is a need to elucidate the role of the vaginal microflora. In recent years, the role of human microbiota in maintaining health and influencing disease has become more prominent , that may indicate a biological mechanism for the microbiota observations  Committee of the National Research Ethics Service (REC Number 13/YH/0167).The study participants were recruited via the antenatal clinics and the labor ward assessment unit of the Jessop Wing Maternity Hospital, Sheffield, UK.n = 121 samples i.e., 20–22, n = 80; and 26–28 weeks, n = 41) and those presenting to the delivery suite with symptoms of threatened but not established preterm labor   Figure . This asFollowing written informed consent, clinical research staff obtained two high vaginal swab (HVS) samples from the posterior vaginal fornix with dry polystyrene Dacron swabs  from each woman after the passage of a sterile vaginal (Cusco's) speculum. To obtain adequate amounts of CVF, each swab was rolled in a clockwise direction and moved across the posterior fornix for a minimum of 1 min, the first swab being employed for microbiome studies and the second for metabolite assays. The collected samples were immediately processed or stored at −20°C for up to 3 days pending analysis: one swab was used for DNA extraction and the other for metabolite analysis. In addition, a third HVS was sent for routine clinical microbiological culture and sensitivity tests for BV and urinary tract infections.During vaginal fluid sample collection, the vaginal pH was also determined from a sample of vaginal discharge obtained by a dry swab, which was then smeared on a narrow range pH paper .Swabs were rinsed in 400 μl of PBS (Sigma) and underwent vortex mixing for 5 min. Two-fifty microliters of swab eluate was transferred to a clean tube and incubated with 1.5 mg/ml lysozyme (sigma) for 1 h at 37°C. DNA was extracted using the QIAamp DNA mini kit column (Qiagen) according to the manufacturer's instructions. Purified DNA was eluted from the QIAamp kit column in 100 μl of ultrapure water and stored at −20°C until further processing.A 500 bp region of the 16S rRNA gene (covering V1–V3) was PCR-amplified from 5 ng of extracted DNA sample using primers comprising universal 16S primers 27F  with hierarchical clustering via a Pearson method according to sample composition.Additionally, sequences were clustered into Operational Taxonomic Units (OTUs) at a sequence dissimilarity distance of 0.03 using an average neighbor algorithm, again using the mothur package. Samples were sub-sampled —an action that in the case of our data did not compromise richness or increase rarefaction) .Given the non-normal and complex nature of vaginal communities , there were many counts of zero. Therefore, the method used for the analysis was to create a binary variable based on whether the species made up at least half of the sample . This method was independently selected by health statistics experts at the Statistical Services Unit, University of Sheffield . The proportion of women for whom the species made up at least half of their microbiota was compared between the women who subsequently delivered preterm and those who delivered at term, i.e., the proportions of the dominant CST defining OTUs . Confidence intervals were obtained for the estimated difference in proportions  and transferred into a 5 mm NMR tube . With new batch of samples and swabs, several unused (sterile) polystyrene Dacron swabs were also prepared and analyzed identically as background signal controls, taking into account any manufacturer batch variation changes—no changes were noted. To compare metabolite levels between term and preterm pregnancy outcomes and between CST groupings we used t-tests (with Welch's correction) and ANOVA (with Tukey's multiple comparisons test), as indicated, since, unlike the OTU abundance data the metabolite data were normally distributed (as tested by a D'Agostino & Pearson omnibus test using the software GraphPad Prism package v7).A swab-end obtained at the same time and anatomical location as the microbiota swab was suspended in 600 μl of PBS  and vortexed for 5 min to wash the CVF into solution. The swab was disposed of safely afterwards. The eluate was then centrifuged at 13,000 rpm for 3 min to separate swab particles and any cellular material from the vaginal fluid solution. The supernatant was carefully aspirated into a separate clean 1.5 ml microfuge tube and stored at −80°C until analysis. Prior to 1H-NMR spectra were acquired using the Watergate water suppression pulse sequence , for each vaginal sample , with 5 mm broadband observe probe, 1H-NMR spectra, 2-D NMR spectra were obtained: 1H-1H watergate-double quantum filtered correlation spectroscopy (DQFCOSY) – NS = 256, D1 = 0.5 s, AQ = 0.832 × 0.022 s, SW = 9.0 × 9.0 ppm, TD = 6,000 × 160; 1H-13C presat-heteronuclear single quantum correlation spectroscopy (HSQC) – NS = 1,024, D1 = 1 s, AQ = 0.078 × 0.006 s, SW = 10.0 × 150 ppm, TD = 624 × 180; 1H-13C presat-heteronuclear multiple bond correlation spectroscopy (HMBC) – NS = 1,024, D1 = 1 s, AQ = 0.128 × 0.005 s, SW = 10.0 × 230 ppm, TD = 1,024 × 200; and 1H-1H presat-clean total correlation spectroscopy (TOCSY) – NS = 16, D1 = 1.5 s, AQ = 0.284 × 0.071 s, SW = 9.0 × 9.0 ppm, TD = 2,048 × 512. All spectra were referenced to the 1H lactate signal at δ = 1.30 ppm. A Representative 1-D 1H-NMR spectrum is shown in Supplementary Figure Data was acquired and processed using Bruker Topspin 2.1.6 software to produce a phase and baseline corrected spectrum. In order to assign metabolites to the 1H-NMR spectra were integrated for peak area . To correct for differences in vaginal fluid concentration (variation in the swab sample volumes), each metabolite integral was divided by the total spectrum integral  to provide a normalized integral (N.I.), which is reported throughout this manuscript  was performed to determine associations between study metabolites and CST subtypes and delivery outcomes.n = 25).The demographic, pregnancy, and delivery outcome details of study participants are summarized in Table From our 158 barcoded 16S rRNA gene amplicon samples, we obtained a final dataset of 1,264,109 high quality sequences  with a mean length of 442 bases after quality filtering, and alignment to the SILVA reference database, screening of the alignment, and removal of chimeras. This provided a final mean yield of 8,000 sequences per sample for further analysis.The number of observed OTUs (at a distance of 0.03) per sample after sub-sampling to 2,000 sequences per sample ranged from 1 to 80 (median = 15). The median estimated coverage of the communities according to Good's non-parametric coverage calculator was 99.5% at this sequencing depth. The diversity (Simpson's inverse diversity index) of the communities is shown in Figure Comparison of the overall bacterial community structure of vaginal swab samples from term and preterm women were performed using the thetaYC metric from which a distance matrix was constructed and visualized using PCoA. As shown in Figure Lactobacillus spp. . Using this division system we observed that among all the samples combined CSTIII  was the most abundant, followed by CSTI , CSTII , CSTV , CSTIII/V , Streptococcus anginosus (1.26%), CST* (Lactobacillus delbrueckii and Lactobacillus helveticus 1.33%), and CSTIV . These assignments were confirmed by repeating the PCoA analyses with samples assigned to CSTs, where, as shown in Figure As has been observed in other studies of the vaginal microbiota, analysis of the OTUs in each sample revealed microbial communities that were dominated by . Figure . In fact. Figure . We then. Figure  with a cLactobacillus, with one exception where the woman's CST assignments changed between 20–22 and 26–28 weeks. In the latter case, the microbiota CST changed from ~40:52 (iners:jensenii) at 20 weeks to ~61:38 (iners:jensenii) at 27 weeks gestation [Supplementary Table Prevotella amnii and Prevotella timonensis, alongside Lachnospiracae BVAB1 and Escherichia coli). Two samples were dominated by S. anginosus (a milleri-group Streptococcus) (1.3%), while only three of the samples (1.9%) did not show dominance by a single species of Lactobacillus, i.e., these samples instead contained approximately equal proportions of L. iners (CSTIII) and L. jensenii (CSTV) and as a result have been annotated as CSTIII/V.CST categorization of the samples confirmed that vaginal microbiota of women at 20–22 weeks who were resampled at 26–28 weeks continued to be dominated by the same species of L. crispatus) dominated microbiota present in the term than the preterm groups at 20–22 weeks  and a slightly higher proportion in the 26–28 weeks group   was more than 2-fold lower in the term than preterm groups at 20–22  and 26–28 weeks groups . Also, at 26–28 weeks none of the preterm patient samples were dominated by CSTII (L. gasseri) in contrast to 28% of term patients (P < 0.0001). When the Laud and Dane method was used, these data were supported at 26–28 weeks, with the lack of CSTII in the patients in the preterm group, P = 0.03. Also there was a trend toward a higher proportion of CSTI in the term women compared to their preterm counterparts (P = 0.06), again in support of our observations above. Overall, using both statistical methods, there appears to be a link between CSTI—L. crispatus and L. gasseri and health.When we analyzed CST assignment in relation to birth outcome in our cohorts, we used both a simple Fisher's exact test, as employed by Kindinger et al.  and a muL. crispatus) had an average pH of 3.83, with one-way ANOVA analysis revealing that this was significantly lower than samples assigned as CSTII (L. gasseri) and CSTV (L. jensenii) which had mean pH values of 4.38 and 4.19, respectively (P < 0.05). One-way ANOVA analysis also revealed that the mean pH of CSTII samples (L. gasseri) was significantly higher than that of CSTIII (L. iners), at 4.38 and 4.03, respectively (P < 0.05).It is well-established that vaginal pH varies in relation to health with a pH of 3–4.5 considered a signature of a healthy vaginal environment and variations upwards a sign of an imbalanced vaginal environment, e.g., onset of BV  from these samples, plotting spectra for samples that were then normalized to overall area under the spectrum for each metabolite peak as previously described by our group , we examined the levels of the metabolites in term and preterm women from the same cohorts as for our microbiota studies. Within this data   Figure .L. jensenii and L. crispatus might be associated with differing outcomes in our cohorts, we then analyzed our metabolite data in relation to CST, using the normalized integrals and One-way ANOVA with Tukeys multiple comparison test applied. As shown in Figures L. crispatus)-dominated samples compared to those dominated by CSTV (L. jensenii) (P < 0.01). These differences were replicated for lactate when the groups were analyzed according to gestational age at sampling, with CSTI showing significantly higher lactate than CSTV-dominated samples at both 20–22 weeks (P < 0.01), and 26–28 weeks (P < 0.05) , and although the trend was the same in the subsets categorized by gestational time point of the studies, it did not reach statistical significance. For CSTII (L. gasseri), we observed 3- to 5-fold raised succinate levels over CSTV in the total cohort (P < 0.0001), at 20–22 weeks (ns) and at 26–28 weeks (P < 0.05)  Figure . We also) Figure . These a) Figure . MultivaFor the first time, we highlight important links between microbial community state-types and targeted metabolite profiles in relation to PTB, thereby highlighting the potential functional and clinical significance of combining these determinations to improve our understanding of the mechanisms of inflammation-associated PTB. We show that community composition  seems to differ between women who delivered at term and those who delivered preterm.This study presents data from vaginal fluid samples of a population of asymptomatic and symptomatic pregnant women studied across mid-trimester, many of who had a previous history of PTB but with a low prevalence of BV or any other genital infection. We analyzed the vaginal microbiota using 16S rRNA gene sequencing while collecting data on pH and metabolite expression levels. Our aim was 3-fold, firstly, to identify any changes in vaginal microbiota early in pregnancy in women who subsequently deliver preterm and to establish baseline information about the microbiota of these cohorts. Secondly, we examined whether any identified changes in microbiota related to wider functional changes in the vaginal microenvironment . Thirdly, we determined if insight could be gleaned from vaginal microbial and metabolite profiles of pregnant women in mid-trimester regarding subsequent PTB.Lactobacillus spp.  and CSTI .In relation to the microbiota, it has been established that the vaginal microbiota is relatively simple, i.e., in most cases it is dominated by a single Donders, . As obseDonders, , the samL. crispatus, and potentially L. gasseri, might be associated with pregnancies that progress to term and may be “health” promoting, although we acknowledge that larger sample sizes would ideally be needed based on the data gathered here. This is intriguing given the increasing evidence that L. crispatus, has a number of phenotypic attributes that might predispose to maintenance of a healthy vaginal microbiota able to out-compete BV-associated bacterial (BVAB) species that are considered risk factors for PTB  dominance. We observed that 25% (>2-fold) of women delivering preterm had vaginal microflora dominated by L. jensenii compared to 10% of the women that delivered at term. To our knowledge this is the first report of such an observation. However, previously a longitudinal study suggested that in pregnancy L. jensenii may be indicative of a more unstable microbiota  associated with microbiota that transitioned between CSTs . In addition, we found only a small percentage of samples dominated by mixed anaerobes (0.63%). This could not be fully explained in the current study because while our primers contained a potential mismatch that could reduce detection of Gardnerella spp., they were well suited for detection of a broad variety of Bacteroidetes group and Fusobacterium spp. as highlighted by their extensive use in assessing anaerobe-rich oral and other rich microbiota samples, including vaginal samples , an organism known to produce succinate  and to express metabolic genes for the production of lactate in vivo  and L. gasseri (CSTII) compared to L. jensenii (CSTV). While intriguing, especially given the immunomodulatory effects of succinate  niches in relation to health and disease and to identify potentially associated organisms. However, one drawback of this approach is the inability to examine what the overall functional consequences of the microbiota might be. As a result we utilized a n Figure . Indeed,n Figure , while en Figure ,b. This We also observed a differential expression of metabolites in relation to the dominant CSTs in the overall study population . There were higher lactate and succinate levels in the CSTI-dominated samples compared to those dominated by CSTV. Similarly, CSTI-dominated samples showed higher lactate levels than CSTV at 20–22 and 26–28 weeks. CSTII-dominated microbiota also indicated raised succinate levels over CSTV in the overall cohort and in women studied at 26–28 weeks only. This was supported by PCoA, which revealed strong clustering of metabolites according to CST in the total population. Taken together, the observed differences in metabolite expression levels in relation to the unique microbiota signature suggests that the dominant Lactobacillus spp. (CST) rather than gestational age at sampling seems to influence specific metabolite profiles.L. crispatus may decrease vaginal pH in vivo by producing high amounts of lactate from glycogen deposits in the vaginal ecosystem  over other lactobacilli, particularly the potentially detrimental L. jensenii (CSTV). Also, for the first time, we highlight that L. gasseri may be associated with vaginal health, albeit in a small, high-risk, largely Caucasian population. Furthermore, our data suggest that CSTs may influence the vaginal microenvironment by altering levels of lactate, succinate and acidity.In conclusion, our data present a picture of the vaginal microbiota that tentatively agrees with the potentially beneficial presence of an Though preliminary, our findings do raise the possibility that further study of the CST Lactobacilli spp. is warranted. Importantly, it highlights the need to focus on the influence of specific lactobacilli-dominated CSTs in larger cohorts of pregnant women including those at low risk for prematurity.This study was carried out in accordance with the recommendations of the Yorkshire and Humber (Sheffield) Committee of the National Research Ethics Service (REC Number 13/YH/0167) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Yorkshire and Humber (Sheffield) Committee of the National Research Ethics Service.1H-NMR metabolite data. VS and DA collected the clinical swab material that was initially processed by JP and EA. VS and DA collated clinical patient information. All authors read and approved the final manuscript.DA and GS designed the study. JP, GS, EA, DA, VS, MP, SR, and JK all contributed to writing the manuscript with GS, JP, EA, and DA leading the process. JP, JK, and GS processed and analyzed the microbiota data. GS, SR, EA, and MP processed and analyzed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Paradiplozoon hemiculteri , a member of the Diplozoidae, parasitizes the gills of Asian fish. Not only is the type material unavailable for this species, the original description was poor and somewhat conflicting, and adequate molecular data were not available. What is more, the available morphological and molecular data are inconsistent and fluctuate significantly. Here, we present a redescription of P. hemiculteri based on morphological and molecular data from new isolates collected from the type host, the sharpbelly Hemiculter leucisculus , captured at the neotype locality ; a neotype for P. hemiculteri was designated from this collection. The length and width of the body, buccal suckers, pharynx, attachment clamps, sickle and the central hook handle were all measured and the shape of the anterior and posterior part of the median plate and anterior and posterior joining sclerites accurately documented. Phylogenetic analyses based on the sequences of the second rDNA internal transcribed spacer (ITS2) indicated that all new samples clustered together and differed clearly from sequences attributed to P. hemiculteri, which are deposited in GenBank. Our results confirm that P. hemiculteri is the only diplozoid that has demonstrably been found on the gills of H. leucisculus to date. Members of the class Monogenoidea  can be classified into two subclasses: Polyonchoinea and Heteronchoinea . Within Paradiplozoon homoion , which has been recorded from more than fifteen cyprinid fish species [According to Khotenovsky 1985) , the Dip , the Di species ,18,21.Paradiplozoon, six from Sindiplozoon, two from Inustiatus, one from Diplozoon, and one from Eudiplozoon [Paradiplozoon hemiculteri by Ling (1973); however, the morphological descriptions in these studies have largely been imperfect and/or inaccurate e.g. Chen (1973) [P. hemiculteri should be located, the types of this species are missing and likely lost. Therefore, we collected new material from Shaoguan, Guangdong Province neotype locality and type host, from which a neotype for P. hemiculteri was selected and designated. In this work, we redescribe P. hemiculteri based on the new morphological and molecular data from the neotype and new specimens from the type host in southern China.In China, 33 species have been reported from five diplozoine genera, 23 from iplozoon ,36,39. Sy Ling 193; howeven (1973) –33. MoreP. hemiculteri are now unavailable, the present study is based on morphological and molecular examination of ten adult diplozoid worms collected from the gills of Hemiculter leucisculus , the type host fish species, from the neotype locality in Shaoguan, Guangdong Province, southern China (24.810329°N 113.597547°E) in 2015. Two individuals already fused into the X-shaped adult worm were separated, one haptor being fixed in 96% ethanol for molecular investigation. The second haptor was placed on a slide in a drop of water, covered with the cover slide, while excess water was drawn off with filtration paper until rupture of the parasite’s body appeared, and the sample was infiltrated/fixed with ammonium picrate-glycerine 1:1 (GAP) [As the type specimens of After the fixation of the haptor in GAP, the sclerites of diplozoid clamps laid nearly in one plane, enabling accurate measurement.An Olympus BX51 light microscope  equipped with differential interference contrast and Stream Motion digital image analysis software v. 1.5 was used to measure the length and width of the body, buccal suckers, pharynx, attachment clamps and the central hook sickle and handle . All mea2, 2 μl 10 mM dNTP, 1 μl (5 U/μl) of Taq DNA polymerase (Thermo Scientific), 32 μl PCR H2O, 2 μl 10 μM specific forward primer (DITS2F 5́-GGCTYRYGGNGTCGATGAAGAACGCAG-3́) and reverse primer (DITS2R 5́-GCCGGATCCGAATCCTGGTTAGTTTC TTTTCCT-3́), and 1 μl (50 ng) of DNA template. Amplification took place in a MyCycler thermal cycler system  using the following protocol: 94 °C for 2 min; 30 times 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s and a final 10 min extension at 72 °C. The PCR products obtained were separated on agarose gel (1%), stained with GoldView (Dongsheng Biotech), purified using a High Pure PCR Product Purification Kit (Roche) and cloned onto Escherichiacoli TOP10 using the CloneJET PCR Cloning Kit (Thermo Scientific), according to the manufacturer’s protocol. Colonies were subsequently checked for fragment presence using PCR, with positive plasmids isolated using the High Pure Plasmid Isolation Kit (Roche) and sequenced in both directions using DITS2F and DITS2R PCR primers. The individual sequences obtained were compared with the NCBI database using the Basic Local Alignment Search Tool (BLAST).The DNA from ten samples was isolated according to the protocol published by Zavodna et al., 2008) . Sequenc008 . SeqParadiplozoon spp. sequences from GenBank (Inustiatus inustiatus (DQ098893) was used as the outgroup for both phylogenetic analyses Type host: Site: GillsOriginal type locality: Huanggang, Hubei Province, People’s Republic of China .Original type material: The holotype originally described by Ling, 1973 is now unavailable .Neotype locality: Shaoguan, Guangdong Province, People’s Republic of China .P. hemiculteri should be located: Laboratory of Fish Parasitology, Institute of Hydrobiology, Chinese Academy of Sciences, People’s Republic of China.Type material: Type specimens apparently lost. Official collection where the types of P. hemiculteri neotype and three voucher specimens are deposited at the Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic. Three further voucher specimens are deposited at the Laboratory of Fish Parasitology, School of Life Science, South China Normal University, People’s Republic of China.Neotype material: The IPCAS M-565 Article 75 of International Commission on Zoological Nomenclature was thoroughly followed.Paradiplozoon characteristics [Adult Diplozoidae (Diplozoinae with eristics ) displayTotal body length, including haptor, 3153 μm ; maximum body width 931 μm . Oral opening sub-terminal, pair of oval-shaped buccal suckers of 58 (50-67) x 54 (42-65) μm (n = 16), located ventrally at the anterior part of the body, near to the opening of a muscular pharynx; pharynx 66 (50-83) x 54 (38-62) μm (n = 16). Branched intestinal caeca located in the forebody, though some branches reach the hindbody near the attachment apparatus. Intestinal caeca run in all directions. Vitellaria present in the forebody; ovarium and testis located in the hindbody along with a clearly visible ootype. Eggs were not observed.The opisthaptors in the adult worm each comprise four pairs of clamps organised into two rows with two central hooks between them b, 2a. ThThe clamps are formed by fine sclerites. The anterior end of the median plate is rectangular with rounded corners and a narrow trapeze spur connected to the anterior end of the plate. The anterior joining sclerite, connected to the proximal tip of the anterior jaw, has the typical inverted V-shape. The posterior end of the median plate narrows and terminates with a wide rounded sclerite with an opening. The posterior joining sclerite is the same length as the anterior joining sclerite.The central hooks are situated near the first (anterior-most) pair of clamps. The length of the central hook sickle was 17.6 μm (17.0-18.1) (n = 16) and handle length was 36.6 μm (34-37.9) (n = 16). A barely visible wing covers the blade. Complete drawings of the third clamp and central hook of the neotype are provided in KY124645 − KY124654.DNA sequences isolated and amplified from the ITS2 fragment (762 bp) of ten adult worms were all similar, with less than 1% variability. The ten sequences have now been deposited in GenBank under accession numbers As the results of Bayesian inference and maximum likelihood analysis were almost identical, we pooled the results into one figure. The final tree is based on Bayesian inference phylogenetic tree topology with branch supports stated for both analysis types  clustered with different Paradiplozoon species , respectively.All our samples clustered together and created a well-supported and separated group from those of and 0.3% . InteresP. hemiculteri, a parasite of the gills of H. leucisculus, remains somewhat controversial at this time. Morphological descriptions of diplozoons isolated from H. leucisculus tend to be either inaccurate or inconsistent, with published illustrations of the clamps and central hook often ignoring key characteristic markers [DQ098884, DQ098887, DQ098888, DQ098892, KY290757-61) are probably misnamed as P. hemiculteri. In order to address this, we performed parallel morphological and molecular analyses on ten samples collected from the gills of the original type host H. leucisculus caught at the neotype locality, Shaoguan, Guangdong Province, southern China.In reviewing the available literature and corresponding sequence data deposited in public databases, we observed that taxonomic classification of the Asian diplozoon  markers ,18,39. FP. hemiculteri naturally parasitising the type host H. leucisculus. We evaluated our redescription based on published results of other authors [P. hemiculteri is now unavailable, hence further morphological comparison was impossible.Based on our re-evaluation of the major morphological features , we determined that our specimens represent  authors ,18,39. WP. hemiculteri, we decided to undertake additional molecular analysis based on amplification of the 762 bp ITS2 fragment from DNA templates of ten adult worms (see above). Our sequence data did not match any sequence saved in GenBank, including some sequences already assigned as P. hemiculteri  [Paradiplozoon group cannot be based on the single Sindiplozoon ctenopharyngodoni sequence included in the analysis.Due to the inconsistencies in previously published morphological data on P. hemiculteri displays high species complexity. Similar high species complexity has been shown in marine capsalid monogeneans [Paradiplozoon, as has recently been discovered in other monogenean genera such as Gyrodactylus [Gyrodactylus species/lineages  were difficult to separate, molecular data (complete cytochrome oxidase I and ITS2 genes) clearly indicated different genotypes of cryptic species. The lack of published morphological descriptions for Chinese diplozoons is a major hindrance for this study as it means these hypotheses cannot be reliably confirmed or refuted at this time. This highlights the importance of combining accurate morphological analysis with molecular analysis of markers as the main means of species identification.Based on the high morphological similarity and degree of homology in their genomes, it is possible that ogeneans  and fresogeneans . Both thdactylus ,29. In tP. hemiculteri, thereby addressing the absence of type material and inaccuracies in the original illustrations and sequence clasifications that have been the norm up until now. Our results confirm P. hemiculteri (Diplozoidae) as the only diplozoid demonstrably parasitising the gills of its Asian fish host, H. leucisculus.In conclusion, this study provides an accurate redescription of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Surgery for advanced gastric cancer (AGC) often includes dissection of splenic hilar lymph nodes (SHLNs). This study compared the safety and effectiveness of different approaches to SHLN dissection for upper- and/or middle-third AGC.We retrospectively compared and analyzed clinicopathologic and follow-up data from a prospectively collected database at the Peking University Cancer Hospital. Patients were divided into three groups: in situ spleen-preserved, ex situ spleen-preserved and splenectomy.P = 0.017). Length of postoperative hospital stay was longer in the splenectomy group than in the in situ group (P = 0.002) or the ex situ group (P < 0.001). The splenectomy group also lost more blood volume (P = 0.007) and had a higher postoperative complication rate (P = 0.005) than the ex situ group. Kaplan–Meier (log rank test) analysis showed significant survival differences among the three groups (P = 0.018). Multivariate analysis showed operation duration (P = 0.043), blood loss volume (P = 0.046), neoadjuvant chemotherapy (P = 0.005), and N stage (P < 0.001) were independent prognostic factors for survival.We analyzed 217 patients with upper- and/or middle-third AGC who underwent R0 total or proximal gastrectomy with splenic hilar lymphadenectomy from January 2006 to December 2011, of whom 15.2 % (33/217) had metastatic SHLNs, and from whom 11.4 % (53/466) of the dissected SHLNs were metastatic. The number of harvested SHLNs per patient was higher in the ex situ group than in the in situ group (The ex situ procedure was more effective for SHLN dissection than the in situ procedure without sacrificing safety, whereas splenectomy was not more effective, and was less safe. The SHLN dissection method was not an independent risk factor for survival in this study. The estimated incidence and mortality of gastric cancer in 2013 were 984,000 and 841,000 worldwide, respectively .The incidence of upper- and/or middle-third gastric cancer has steadily increased, especially in Asia . AccordiReportedly, 7.3–26 % of SHLNs in upper- and/or middle-third AGC are metastatic –12. PropTwo main operative procedures for SHLN dissection spare the spleen. Ex situ and in situ dissection are defined depending on whether the pancreas and spleen are treated within the peritoneal cavity or not. The in situ dissection approach is more difficult as the SHLN dissection is implemented in a narrow and small space, and can thus lead to bleeding; however, it avoids moving the pancreas and spleen and shortens surgical time. In contrast, ex situ dissection is performed under direct vision, which provides a better exposure, and is thus less difficult.To our knowledge, no previous study has directly compared the effectiveness and safety of these three approaches. We therefore investigated which of these three dissection approaches was better for patients with upper- and/or middle-third AGC.This study was performed after approval by the Ethics Committee of Peking University Cancer Hospital. Informed consent was obtained from each patient. We retrospectively collected clinical and pathological data from a prospectively collected database at the Peking University Cancer Hospital. We included 217 patients with upper- and/or middle-third AGC who had undergone R0 total or proximal gastrectomy with SHLN dissection from January 2006 to December 2011. Their primary diagnoses were confirmed by endoscopic biopsies analysis. Clinical staging was mainly confirmed by ultrasound endoscopy, chest, abdominal and pelvic computed tomography scans, and laparoscopic exploration. Patients with other types of tumors, such as gastrointestinal stromal tumor or lymphoma, were excluded.All the enrolled patients underwent laparoscopic exploration to exclude distant metastatic disease. After that, all the patients received R0 resection with total or proximal gastrectomy and SHLN dissection. The lymph node dissection scope was mainly D2/D2+, according to the definition in the Japanese gastric cancer treatment guidelines . The appIn the splenectomy group, splenectomy was performed with full mobilization of the distal pancreas and spleen. Lymph nodes along the splenic artery were completely dissected. The splenic artery was usually ligated and divided 5–6 cm away from its origin. The spleen and lymph nodes at the hilum of the spleen were removed, with the pancreas preserved.In the in situ spleen-preserved group, the spleen and the pancreas were not mobilized from the retroperitoneum. Lymph nodes along the splenic artery were dissected. All the soft tissues at the splenic hilum were removed as cautiously as possible.In the ex situ spleen-preserved group, splenic hilar lymphadenectomy was performed with full mobilization of the distal pancreas and spleen. The spleen was moved outside the peritoneal cavity. Lymph nodes along the splenic artery and at the splenic hilum were completely dissected, with the pancreas and spleen preserved, and then replaced into the peritoneal cavity.After the surgery, the patients stayed in hospital to get recovery. Before they left the hospital, the discharge criteria must be all fulfilled. The discharge criteria included: absence of subjective complaints, tolerance of solid oral intake, return of bowel function, absence of intravenous fluids/medications, adequate mobility of daily living and self-care , adequate pain control on oral analgesia only, adequate wound condition, removal of drainage tube, absence of infectious complications, absence of postoperative complications, absence of abnormal physical signs or laboratory test , acceptance of discharge, adequate home/social condition.The clinicopathological data collected from the database included age, sex, body mass index (BMI), neoadjuvant chemotherapy (NACT) regimens, tumor location, tumor size, presence of multi-tumor, range of gastrectomy, degree of lymph node dissection (LND), SHLN dissection procedure, tumor differentiation, lymphovascular invasion (LVI), depth of tumor invasion, number of harvested and metastatic lymph nodes, postoperative complications, mortality, length of postoperative hospital stay, operation duration, blood loss volume, and survival outcomes. Terminology used to describe the clinicopathologic parameters was based on the Japanese Gastric Cancer Association classification of gastric carcinoma .Follow-up was conducted mainly through telephone interviews, E-mail communication, or outpatient reviews. As of April 26, 2016, the percentage of follow-up was 96.7 % (210/217).P < 0.05 (two-sided) was considered significant in the statistical analysis.All statistical analysis was performed through IBM SPSS Statistics 20.0 software . For quantitative variables, normal distribution was tested first. Variables of normal distribution were expressed as means ± standard deviation, and tested by analysis of variance among the three groups. If not, the variables were expressed as medians with ranges, and compared by Kruskal–Wallis non-parametric test. For categorical data, the chi-squared test or Fisher’s exact test was performed. Kaplan–Meier estimation and the log-rank tests were used to calculate survival. In the pairwise comparisons, the original calculated P value and the Bonferroni-corrected threshold were listed. If the P value was less than this Bonferroni-corrected threshold, then the comparison was considered to be statistically significant. Cox proportional hazards regression model was used to confirm independent prognostic factors through univariate and multivariate analysis. Except in the pairwise comparison, n = 68), ex situ (n = 118), and splenectomy (n = 31). Some of the patients in the splenectomy group had intended to undergo in situ or ex situ approach after abdominal exploration, but encountered unintended splenic injury resulting in splenectomy. Of all the thirty-one patients in the splenectomy group, two patients underwent conversion from in situ approach to splenectomy, and three patients underwent conversion from ex situ approach to splenectomy. The rates of conversion from in situ and ex situ procedures to splenectomy were 2.86 % (2/70) and 2.48 % (3/121), respectively. All of their clinicopathologic factors except the number of patients who received NACT and the range of gastrectomy were comparable among the three groups; however, lower percentages of the in situ group underwent NACT and total gastrectomies than the ex situ and splenectomy groups . Of 466 harvested SHLNs, 11.4 % (53/466) were metastatic, including 8.3 % (10/121) in the in situ group, 11.8 % (32/271) in the ex situ group, and 14.9 % (11/74) in the splenectomy group (P = 0.349).All 217 patients in our study underwent SHLN dissection, and all of the dissected lymph nodes were confirmed by pathological examination. Of the 217 patients, 33 (15.2 %) were found to have metastatic SHLNs, including 8.8 % (6/68) of the in situ group, 14.4 % (17/118) of the ex situ group, and 32.3 % (10/31) of the splenectomy group (P = 0.047), length of postoperative hospital stay (P = 0.001), and blood loss volume (P = 0.027). Further paired comparisons revealed that the number of harvested SHLNs per patient was higher in the ex situ group than in the in situ group (P = 0.015). The length of postoperative hospital stay was significantly longer in the splenectomy group than in the other two groups . The splenectomy group also had significantly greater blood loss volume than did the ex situ group (P = 0.007). The three groups did not significantly differ in total harvested lymph nodes per patient (P = 0.313) or operation duration (P = 0.695).Surgery-related parameters were compared among the three groups Tables  and 4, aP = 0.011); and were notably higher in the splenectomy group than in the ex situ group . The three groups did not significantly differ in reoperation rate (P = 0.359) or postoperative mortality rate (P = 0.363).Postoperative complication rates were: in situ group: 17.6 % (12/68); ex situ group: 12.7 % (15/118); and splenectomy group: 35.5 % . Median survival times were: in situ group: 34.5 months, ex situ group: 71.1 months, and splenectomy group: 21.1 months; 5-year overall survival rates were: in situ group: 46 %, ex situ group: 50 %, and splenectomy group: 23 %. The three groups were found to significantly differ by Kaplan–Meier survival analysis , blood loss volume (P = 0.046), use of NACT (P = 0.005), and N stage (P < 0.001) to be independent prognostic factors for survival . Surgeons were inclined to use ex situ or splenectomy procedures for SHLNs suspected of having metastases, to perform dissections more effectively.Although the incidence of gastric cancer has decreased worldwide, upper- and/or middle-third AGC has shown an increasing trend. As far as we know, the only way to cure gastric cancer is radical surgery, which includes gastrectomy and lymph node dissection. The currently recommended surgical procedure for advanced upper- and/or middle-third gastric cancer is total gastrectomy with D2 lymph node dissection . SHLNs aOptimal procedure for SHLN dissection has long been debated. Many previous studies have reported that splenectomy in this situation did not lead to longer survival , 18, 19,We found ex situ procedure was more effective for SHLN dissection than in situ splenic-preserving procedure and did not sacrifice surgical safety. Ex situ spleen-preserving procedure might improve the integrity of lymphadenectomy at the splenic hilum . In the Interestingly, although Kaplan–Meier and log-rank analysis showed significant differences in survival among the three groups, Cox regression analysis of proportional hazards did not show SHLN dissection procedure to be an independent risk factor for survival. The significant difference shown in the Kaplan–Meier method might be caused by some other factors such as the imbalance of grouping in our study. The higher postoperative complication rate in the splenectomy group probably had adverse effects on survival, which is supported by earlier studies , 24.Our study also had some limitations. First, it was a retrospective study, and selection bias was difficult to avoid. For instance, the percentages of patients who received NACT were much higher in the splenectomy and ex situ groups, probably because patients with later-stage disease were more likely to receive NACT. The choice of lymphadenectomy procedure was decided by surgeons, who usually chose patients with later-stage disease for ex situ or splenectomy procedures, as these methods seem to be more effective means to dissect the SHLNs. Similarly, more patients in the splenectomy or ex situ groups underwent total gastrectomies, which are more suitable for patients with later-stage disease. The three groups did not significantly differ with regard to other clinicopathologic parameters. Second, as the sample size in the splenectomy group was much smaller than that in the other two groups, a type II error might have occurred.Ex situ SHLN dissections were safer than splenectomies. Compared with in situ procedures, ex situ procedures apparently dissected SHLNs more effectively. Although the survival in these three groups significantly differed in Kaplan–Meier analysis, SHLN dissection method was not an independent risk factor for survival. Multicenter, large-scaled, randomized controlled trials are needed to clarify the optimal splenic hilar lymphadenectomy procedure.


Based on functional classification via Mapman, ~50% of the identified proteins fall into six categories, including protein metabolism, transport, and lipid metabolism. Of note, elaioplasts contained ATP synthase and ADP, ATP carrier proteins at high abundance, indicating important roles for ATP generation and transport in elaioplast biogenesis. Additionally, a comparison of proteins between citrus chromoplast and elaioplast proteomes suggest a high level of functional conservation. However, some distinctive protein profiles were also observed in both types of plastids notably for isoprene biosynthesis in elaioplasts, and carotenoid metabolism in chromoplasts. In conclusion, this comprehensive proteomic study provides new insights into the major metabolic pathways and unique characteristics of elaioplasts and chromoplasts in citrus fruit.Elaioplasts of citrus peel are colorless plastids which accumulate significant amounts of terpenes. However, other functions of elaioplasts have not been fully characterized to date. Here, a LC–MS/MS shotgun technology was applied to identify the proteins from elaioplasts that were highly purified from young fruit peel of kumquat. A total of 655 putative plastid proteins were identified from elaioplasts according to sequence homology  A study of the proteins in bodies called elaioplasts in citrus fruit cells will help to clarify elaioplast formation and functions. Plant cells contain several types of membrane-bound bodies called plastids, with elaioplasts being an incompletely characterized type. They are known to store oils in the cells, especially molecules called terpenes that influence the fragrances and flavors of citrus fruits. Researchers in China, led by Yunliu Zeng at Huazhong Agricultural University, analyzed the protein molecules in elaioplasts of kumquat peel to investigate possible roles for elaioplasts other than oil storage. They identified more than 600 proteins and assigned likely functions to many. The results suggest the proteins are involved in chemical energy transactions and the manufacture of various categories of biomolecules. This will guide research to fully characterize the activities and significance of elaioplasts. These plastids are reported as the sites for several essential processes, including carbon fixation, nitrogen assimilation, and the biosynthesis of isoprenoids, starches, fatty acids (FA), and amino acids. Although plastids are a family of organelles possessing the same plastid genome (encoding approximately 100 proteins), the majority (>95%) of the 2000–3500 different proteins are imported from the surrounding cytoplasm rather than being synthesized in situ3. It is the imported proteins that define the developmental fate of plastids, leading to the different morphologies and special functions within specific plastids4. Recently, large-scale proteomics analysis has become an efficient approach for the investigating subcellular organelles at the protein level2. Special attention has been given to chloroplasts as well as chromoplasts in several species, including tomato, citrus, pepper, papaya, watermelon, carrot, and orange curd cauliflower11. In contrast, relatively few proteomics studies have focused on other types of plastid.Plants contain several kinds of plastids, such as chloroplasts in photosynthetic tissues , chromoplasts in pigment-containing fruits, as well as colorless leucoplasts in starch-storing or lipid-storing seeds12 and terpenes in the outer peel of citrus14. Plastid isolation is a prerequisite not only for assessing proteome composition, but also for understanding metabolic processes, enzyme localization, metabolite transporters, and biogenesis. Although protocols for the isolation of elaioplasts are available for some plant tissues, including petals of snapdragon flowers (Antirrhinum majus)15, developing seeds of mustard (Brassica campestris)17, and maize (Zea mays)18, as well as the outer peel of citrus (Citrofortunella mitis)14, the proteome profile of elaioplasts in these tissues is still unknown.Leucoplasts are colorless plastids that include amyloplasts, elaioplasts, and proteoplasts that store starches, lipids, or proteins, respectively. Amyloplasts have been well studied and have been shown to be enriched in many storage tissues, including seeds, tubers/root vegetables, and fruits. In contrast, elaioplasts are much less studied in plants. Besides their role in storage, elaioplasts were also reported to possess crucial biosynthetic functions, for example in the biosynthesis of long-chain FAs in developing castor seeds19. As the major component of essential oil, terpenes have been employed for manufacturing of perfumes and flavorings, and they are also used as antiseptic preservative agents to improve the safety and shelf-life of fruit during storage21. Although significant progress has been made in understanding chromoplast differentiation in the peel of citrus fruits23, little information is available about the regulatory mechanisms underlying elaioplast biogenesis.Citrus are one of the most important horticultural crops around the world, and have a unique anatomical structure, which consists of the pericarp (peel) and an edible endocarp (flesh). The endocarp accumulates large amounts of carotenoids within chromoplasts, which not only bring the red, yellow, and orange colors of fruit flesh, but also provide several important groups of nutrients. The pigmented region of pericarp contains active secretory cells that are characterized by numerous elaioplasts. Elaioplasts have been demonstrated to be involved in the biosynthesis of terpenes in essential oil, which are then exported into the secretory pocket, greatly affecting the aroma and taste of the fruitIn the present study, a comprehensive proteomic analysis using nLC-MS/MS-Q Exactive technology was used to identify proteins in elaioplasts isolated from the peel of kumquat by density gradient centrifugation. A total of 655 plastid protein candidates were detected that were predicted to participate in the general metabolism or unique metabolic pathways in elaioplasts, including flavor synthesis and fruit maturation. Overall, the present study provides an important insight into elaioplast biogenesis and formation of essential flavors.Fortunella margarita Swingle) was used for elaioplast isolation. Fresh mature pulp of ‘Hong Anliu’ orange (C. sinensis) was used for chromoplast isolation. Both citrus cultivars were collected from Guangxi Institute of Citrus Research, located at Guilin city, Guangxi, China.The outer peel of ~150 earlier-stage green fruits (0.5–0.8 cm in diameter) of ‘Rong An’ kumquat  soluble polyvinylpyrrolidone 360, and 0.1% (w/v) bovine serum albumin). The homogenized sample was passed through two layers of Micracloth . The pooled filtrate was centrifuged for 8 min at 5000 × g. The pellet was carefully re-suspended in SPBTK buffer at pH 7 and then centrifuged for 90 s at 800 × g. The resulting supernatant was centrifuged at 5000 × g for 10 min. The suspension containing most of the plastids was gently layered onto the top of a discontinuous gradient of 0.75, 0.92, 1.20, 1.50 M sucrose in 50 mM Tricine-NaOH (pH 6.5) and then centrifuged at 100,000 × g for 90 min in a Hitachi P40ST rotor. Four zones, designated as bands 1, 2, 3, 4 from the top to the bottom of the gradient, were clearly separated by sucrose gradient centrifugation. Each band was carefully recovered and examined under an optical microscope at ×100 magnification . The purity of each fraction was tested by western blot analysis (described below). The elaioplast layer (band 1) was recovered. Chromoplast isolation from sweet orange was performed as described previously10. The final pellets containing elaioplast or chromoplast fractions were collected and flash-frozen in liquid nitrogen and kept at −80 °C. The plastid samples were prepared in two biological replicates.Elaioplasts were purified according to the procedure described by Michel et al.®. A total of 30 μg of proteins were used for immunoblot analysis as previously described10. At least two independent replicate experiments were performed for each immunoblot with similar result.Polyclonal antibodies were diluted as appropriate against plastid Rubisco large subunit , cytosolic UDP-glucose pyrophosphorylase , vacuolar ATPase , and mitochondrial voltage-dependent anion-selective channel protein 1 , as well as two photosynthesis-related antibodies (light-harvesting complex (Lhca1 and Lhca4), 1:5000) from Agrisera9. The filter-assisted sample preparation method was adapted for protein digestion. Briefly, 200 μg of proteins were mixed with 100 μl of 100 mM dithiothreitol (DTT), 150 mM Tris–HCl pH 8, 4% (w/v) SDS, incubated at 100 °C for 10 min, and then cooled to 25 °C. Next, UA buffer  was used to remove detergent, DTT, and other low-molecular-mass substances by repeated ultrafiltrate , and then centrifuged for 30 min at 14,000 × g. The samples were washed using 100 μl of UA buffer followed by centrifugation for 30 min at 14,000 × g, and then incubated under darkness for 30 min by adding 100 μl of UAI buffer . Further washes (three times) with 100 μl of UA buffer followed by two washes using 100 μl of 50 mM NH4HCO3 (pH 8.5) were performed. Finally, the resulting proteins were digested with 4 μg of trypsin (Promega) overnight (16–18 h) at 37 °C. The concentration of peptide mix was estimated by UV absorbance at 280 nm as described previously9.Protein extraction was performed as previously described9nLC-MS/MS analysis was performed on an Easy-nLC system equipped with a Thermo Scientific Q Exactive mass spectrometer. 5 μg of peptide mixture was injected onto an Acclaim PepMap100 trap column  fitted with a C18-reversed phase analytical column  in mobile phase A (0.1% formic acid in water). Peptides were eluted with a linear 120-min gradient of mobile phase B (0.1% formic acid in 84% acetonitrile) at a flow rate of 0.3 μl/min. The separated MS data were obtained as described in ref. Citrus sinensis (http://www.pytozome.com or http://citrus.hzau.edu.cn), and the complete chloroplast genome sequence of C. sinensis (accession no. NC_008334). The search parameters were performed as described in ref. 9 Only the proteins identified by two or more peptides in two bio-replicates were retained for further analysis.MS/MS spectra were searched with the MaxQuant software package (version 1.1.0.36) against non-redundant protein databases of https://mapman.gabipd.org/mapman). Additional curation was performed according to classifications in PPDB (http://ppdb.tc.cornell.edu). The proteins identified in citrus plastids were queried against three comprehensive plastid proteome databanks ; Plprot ; downloaded in October 2016); and two plastidial protein subset databanks . There were 2816 plastid proteins in plprot, 1265 AT-CHLORO, 2292 in PPDB, 2977 in SUBA and 2038 in Uniprot.Detected proteins were categorized based on Mapman functional bins (https://urgi.versailles.inra.fr/predotar/), Target P (http://www.cbs.dtu.dk/services/TargetP/), and WoLF PSORT (http://www.genscript.com/wolf-psort.html). Proteins were tentatively assigned as plastidial (i) if predicted to be plastid targeted by at least one of the three programs, and/or (ii) if homologous to plastid proteins annotated in at least two plastidial databanks. Additionally, sequence information was manually curated if they were available in the literature. The putative plastid proteins of elaioplast and chromoplast were listed in Supplementary Table Subcellular localization analysis was carried out using three software programs: Predotar /2 with ImageJ (http://rsbweb.nih.gov/ij/).Isolated plastids were carefully layered onto microscope slides and visualized using a DP70 camera coupled to an Olympus BX61 microscope. Plastid samples for transmission electron microscopy (TEM) analysis were prepared and examined as described previouslyCitrus genus. Ripened fruits can be eaten as a whole , cytosol (UDPase), vacuole (V-ATPase), and mitochondrion (VDAC1), as well as chloroplast (Lhca1 and Lhca4). As shown in Fig. We conducted a comprehensive proteomic analysis to characterize the proteins found in elaioplasts. A total of 1239 proteins with at least two detected peptides in two bio-replicates, were identified from purified elaioplasts. The raw data obtained from the elaioplast proteins were refined by searches against five plastid databanks  and by three targeting predictors . Based on the chosen criteria for plastid-localized proteins , an inventory of 655 putative plastid proteins was constructed, accounting for 52% of all identified proteins  serve as a major source of reducing power for various metabolic pathways. As expected, we identified several proteins involved in the Calvin cycle within elaioplasts, such as fructose-bisphosphate aldolase, phosphoglycerate kinase, and transketolase. Furthermore, proteins participated in oxidative metabolism were well presented, including glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase. Of note, transketolase and transaldolase (key enzymes of the non-oxidative part of OPPP), which are reported to feed the processes of anabolism or glycolysis30, were also identified in the present study. Glycolysis also occurs in plastids to generate ATP and pyruvate in plants31. Several glycolysis enzymes including aldolase, phosphoglycerate kinase, enolase, and pyruvate kinase were observed. Consistent with previous findings in non-photosynthetic plastids, a substantial number of proteins responsible for the generation of ATP and reducing power were observed8, indicating their high conservation among different plastid types. Taken together, the observation of many proteins involved in essential biosynthetic activities suggests an active metabolism in elaioplasts to produce reducing power and energy, as well as the supply of precursors for the biosynthesis of metabolites.Elaioplasts are a type of heterotrophic plastid, wherein all the metabolic precursors are either generated via oxidative metabolism or actively imported from the cytosol to sustain anabolic reactions10 and amyloplasts26.Several proteins involved in photosynthesis were found in elaioplasts although the presence of chloroplasts was not observed using light microscopy and there was no signal obtained by immunoblotting using two photosynthesis-related antibodies Fig. . This su28. We detected several enzymes for the synthesis of FA as well as glycolipids and phospholipids in elaioplasts, such as acetyl E1 beta pyruvate dehydrogenase complex, E2-dihydrolipoamide acetyltransferase, E3-dihydrolipoamide dehydrogenase 1, long-chain acyl-CoA synthetase 7/8/9, and plastidial ketoacyl-ACP reductase , which are associated with generation of polyunsaturated FA hydroperoxides, could contribute to the biosynthesis of jasmonic acid (JA)35 and FA-derived flavor compounds36. In the present study, several enzymes involved in the LOX pathway  and JA metabolism  were detected. Therefore, it is possible that the presence of lipoxygenases in citrus elaioplasts and chromoplasts10 may play a key role in generating odor or flavor through production of LOX-derived volatiles in the outer peel and flesh of a fruit, respectively. A similar role for lipoxygenases in chromoplasts of tomato has been proposed6.FA are known to be synthesized in plastids26. Taken together, our data suggest that these proteins involved in the biosynthesis of several types of amino acids, including aromatic amino acids have important functions in the active synthesis of amino acids within the elaioplasts.Elaioplasts are also important sites for amino acids synthesis. A large number of proteins responsible for amino acid metabolism were identified in elaioplasts, such as threonine synthase, aspartate aminotransferase, alanine transaminase, argininosuccinate synthase, cystathionine beta-lyase, 3-dehydroquinate synthase, isopropylmalate isomerase, isopropylmalate dehydrogenase, and ketol-acid reductoisomerase  cycle and antioxidant system of plastids, such as glutathione peroxidases, peroxiredoxins (PRXs), superoxide dismutases (SODs), and thioredoxin. Additionally, several key enzymes involved in second active oxygen species scavenging system were identified in elaioplasts, including the AsA-GSH cycle catalyzed by ascorbate peroxidases (APXs), as well as the AsA-dependent generation systems catalyzed by (momo)dehydroascorbate reductase38. Like in photosynthetic chloroplasts, APXs encountered in elaioplasts may indicate the presence of the AsA-dependent system involves the reduction of H2O2 using AsA as reductant39. The activation of plastid SOD expression was detected to be dependent on a copper chaperone for SOD40. Thus, as expected, Fe-SOD and Mn-SOD were detected and their overexpression induced an enhancement of plant tolerance in response to environmental stresses42. The presence of a large number of enzymes involved in oxidative stress response indicates a functionally active redox system in citrus elaioplasts and suggests the roles of these proteins in ROS metabolism, allowing the plastid to adapt to various environmental signals and processes during elaioplast development as fruit maturation proceeds.Active redox system is a key regulator for plant development and fruit senescence and has a wide range of functions in gene expression, enzyme activities, and protein import in plastids43, which is important for the biogenesis of either chloroplasts44 or chromoplasts1. However, the import machinery remains unclear in elaioplasts. In this study, numerous translocons including TOC33/75/159 and TIC62/55/40/22/110 in the protein import machinery were identified in elaioplast proteome  Thirty ribosomal proteins of translation machinery were identified in citrus elaioplasts. These proteins are represented by plastid-encoded 30S and 50S subunit, together with nucleus-encoded 40S and 60S subunit. These findings suggest the presence of translation machinery in elaioplast biogenesis. (3) Maintaining a balance between protein synthesis, import, turnover, and processing is an important factor determining plastid fate and maintaining their homeostasis46. As expected, we identified numerous caseinolytic peptidase (Clp) proteases in the study , which have been described as critical for plastid development48. The extensive presence of Clp protease family in elaioplasts might contribute to elaioplast biogenesis by processing and/or cleavage of transit peptides of proteins in the plastids.Protein synthesis, degradation, and translocation regulate the biogenesis of plastids. In this study, proteins associated with import, ribosome assembly and proteases account for 16.9% of the total plastid proteins Fig. . (1) Impd-erythritol 4-phosphate (MEP) pathway, which provides precursors to synthesize monoterpenes within plastids. The fourth (orange1.1t03905) was a putative terpene synthase, which might be responsible for the accumulation of d-limonene (accounting for up to 90% among monoterpene compounds) in outer peel of citrus50. The importance of DXS in regulating MEP substrate flux in kiwifruit and grape has previously been reported52 and the identification of MEP pathway proteins and TPS genes in citrus elaioplasts indicates the key role that elaioplasts play in basic metabolism.The protein abundance for all proteins detected in kumquat elaioplasts was determined based on intensity-based absolute quantification (iBAQ). The 30 most abundant proteins of kumquat elaioplasts are listed in Table 53. ATP synthases are involved in energy production and proton transport. The identification of these proteins at high abundance suggests they may play an important role in active energy production and transport during elaioplast development. The presence of an active ATP generation system that is also necessary for the formation of membranes in plastids54. Elevated energy production has also be observed in other heterotrophic plastids, such as chromoplasts6 and proplastids55.Eight proteins involved in transport or electron transport categories, such as ADP, ATP carrier protein, ATP-binding cassette transporter, ATPase, as well as ATP synthase beta/epsilon chain were also detected in kumquat elaioplasts. ATPase acts as an essential regulator of membrane trafficking, which couples free energy generated from ATP hydrolysis to transport of substances across the membranes of many organellesTo determine whether the ultrastructure was different between elaioplasts and chromoplasts, we purified chromoplasts from citrus flesh for comparison with elaioplasts. TEM analysis showed that the size of elaioplasts was 2.15 ± 0.70 μm in diameter, which was significantly smaller than that of chromoplast (2.59 ± 0.60 μm) Fig. . The num57. Consistently, CCD1 was detected in elaioplasts and chromoplasts.Given the structural differences between elaioplasts and chromoplasts, it was not surprising that differences in protein distribution patterns on SDS-PAGE gels were also observed Fig. . We furt9, which is confirmed by the present study (Fig. 58, and in our previous chromoplast proteome analysis we identified citrus homologs for 24 of these 30 proteins9. In this study, we identified five additional plastoglobule-localized proteins (Supplementary Table 58. In pepper, it is reported to be important for the formation of carotenoid-lipoprotein sequestration substructures and contributes to elevated levels of carotenoid accumulation59. Fibrillin and its homologs were identified at high abundance in elaioplasts (Table 60, they might possess a unique plastoglobule proteome. Therefore, the further determination of plastoglobule proteomes of this colorless, non-photosynthetic plastid is necessary and meaningful.The extensive formation of plastoglobules within chromoplasts of citrus flesh is the most representative feature during chromoplast biogenesisudy Fig. . Thirty ts Table , suggestin silico homology analyses, and the majority of these proteins share similar distribution patterns in Mapman functional classes between elaioplast and chromoplast. The identification of the abundant proteins associated with the MEP pathway and terpene biosynthesis in elaioplasts suggest that this biochemical process may define a basic feature of elaioplasts. In conclusion, this proteomic analysis provides a comprehensive understanding of citrus elaioplasts as a key step towards uncovering the biogenesis of the elaioplast and its terpene metabolism.The present study reveals several important characteristics of the elaioplasts of kumquat peel. An inventory of 655 candidate proteins predicted to be localized in citrus elaioplasts was constructed based on Supplemental Figures 1-3Supplementary Table S1: Characteristics of the identified citrus plastid proteinsSupplementary Table S2: The proteome profiles of ‘secondary metabolism’ class proteins in elaioplasts and chromoplastsSupplementary Table S3: Comparative protein profiling involved in secondary metabolites in plastidsSupplementary Table S4: The core proteome of plastoglobules of elaioplasts and chromoplasts


Mycobacterium leprae, is a major public health problem in poor and developing countries of the Americas, Africa, and Asia. MicroRNAs (miRNAs), which are small non-coding RNAs (18–24 nucleotides), play an important role in regulating cell and tissue homeostasis through translational downregulation of messenger RNAs (mRNAs). Deregulation of miRNA expression is important for the pathogenesis of various neoplastic and non-neoplastic diseases and has been the focus of many publications; however, studies on the expression of miRNAs in leprosy are rare. Herein, an extensive evaluation of differentially expressed miRNAs was performed on leprosy skin lesions using microarrays. Leprosy patients, classified according to Ridley and Jopling’s classification or reactional states (R1 and R2), and healthy controls (HCs) were included. Punch biopsies were collected from the borders of leprosy lesions  and from 9 HCs. miRNA expression profiles were obtained using the Agilent Microarray platform with miRBase, which consists of 1,368 Homo sapiens (hsa)-miRNA candidates. TaqMan quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to validate differentially expressed miRNAs. Sixty-four differentially expressed miRNAs, including 50 upregulated and 14 downregulated  were identified after comparing samples from patients to those of controls. Twenty differentially expressed miRNAs were identified exclusively in the reactional samples (14 type 1 and 6 type 2). Eight miRNAs were validated by RT-PCR, including seven upregulated  and one downregulated (hsa-miR-1290). These miRNAs were differentially expressed in leprosy and several other diseases, especially those related to the immune response. Moreover, the integration of analysis of validated mi/mRNAs obtained from the same samples allowed target pairs opposite expression pattern of hsa-miRNA-142-3p and AKR1B10, hsa-miRNA-342-3p and FAM180b, and hsa-miRNA-484 and FASN. This study identified several miRNAs that might play an important role in the molecular pathogenesis of the disease. Moreover, these deregulated miRNAs and their respective signaling pathways might be useful as therapeutic markers, therapeutic targets, which could help in the development of drugs to treat leprosy.Leprosy, a chronic infectious disease caused by  Mycobacterium leprae, is a major public health problem in poor and developing countries in the Americas, Africa, and Asia  occurs with poor preservation or absence of cellular immunity. R1 is an immunocellular response that occurs mainly in patients with TT and the borderline forms of the disease . The clinical signs of R1 include swelling and erythema of preexisting lesions, and when very intense, there can be necrosis and ulceration. Histopathological examination typically reveals more extensive, confluent, and poorly delimited granulomas associated with interstitial and intracellular edema, fibrin deposition, focal or confluent necrosis, and varying degrees of epithelial hyperplasia  occurs with relative preservation of specific cellular immunity against erplasia . R2 is cerplasia .There is no specific treatment to prevent the occurrence of these reactional phenomena and no blood marker that identifies reactional episodes or their intensity. In addition, there is no any effective treatment regimen for all cases. During episodes, neurological lesions often worsen, typically resulting from direct aggravation of the neural branches and other tissues, which can lead to permanent functional disabilities.M. leprae is able to subvert host immune mechanisms by modifying the expression of genes in parasitized cells, making these cells an environment conducive to the survival of the bacilli; in addition, it is speculated that a similar strategy is used for their spread to adjacent tissues and distant organs.The pathophysiological mechanisms of leprosy progression and reactional episode onset are virtually unknown . It is aThere are a large number of studies showing that non-coding RNAs are important for the maintenance of cell and tissue homeostasis and that their dysregulation is involved in the development of diseases . Among tIt is well known that altered expression of miRNAs occurs in various types of diseases, but most often this has been described in neoplasms. Studies have provided a better understanding of the pathophysiological mechanisms of different diseases at the molecular level, some of which have displayed peculiar miRNA expression patterns allowing for their molecular classification . The miRIn a recent study on the expression of mRNAs in skin samples using microarrays, hundreds of differentially expressed mRNAs were identified in the spectrum of leprosy and its reactional states suggesting their participation in the pathophysiology of these conditions . Some ofLeprosy is a disease that is difficult to treat, and this is especially true of the reactional episodes. Drugs currently used, including corticosteroids and thalidomide, significantly disrupt homeostasis, leading to difficult-to-control disorders such as obesity, diabetes, immunodeficiency, and teratogenesis, among others . Thus, tThis study utilized microarrays to identify differentially expressed miRNAs that might be involved in the pathophysiology of leprosy. These candidate miRNAs were hypothesized to represent novel markers and therapeutic targets for leprosy and its reactional states, which will be the focus of future research.The events in this study occurred in the following order: patients who were consulted at the leprosy Outpatient Clinics of the Lauro de Souza Lima Institute  and Rondonópolis (Mato Grosso) were examined by leprologists and underwent two skin biopsy procedures. One biopsy was processed by histopathological analysis and bacilloscopy. The other was stored immediately after collection in RNAlater solution for future extraction of RNA. This study was performed using the same RNA material extracted from samples used in a recent publication reporting mRNA expression in leprosy .After clinical and histopathological assessments and bacilloscopy, the patients were classified according to Ridley and Jopling’s criteria of disease and reactions  , 5. SixtThe data for all patients, including age, gender, and ethnicity, as well as the identification of each sample, are listed in Table S1 in Supplementary Material. It is important to mention that ethnic factors were not considered since previous studies demonstrated the high individual ancestral variability was observed in Brazilian population  which reflects a singular proportion of Amerindian, European, and African ancestries in its mosaic genome, and that in this population it is not possible to predict the color of persons from their genomic ancestry nor the opposite. The classic skin color stratification for additional analysis is not useful in the population investigated . This stThe following comparisons were used to identify differentially expressed miRNAs in terms of the leprosy spectrum and different reactional states: (1) disease (TT + BT + BB + BL + LL + R1 + R2) vs. HC; (2) clinical forms (TT + BT + BB + BL + LL) vs. HC; (3) between polar forms (TT vs. LL); (4) tuberculoid (TT + BT) vs. HC; (5) lepromatous (BL + LL) vs. HC; (6) TT vs. HC; (7) LL vs. HC; (8) borderline leprosy (BT + BB + BL) vs. HC; (9) reactional type 1 (R1) vs. its respective clinical forms (TT + BT + BB + BL); (10) R1 vs. HC; (11) reactional type 2 (R2) vs. its respective clinical forms (BL + LL); (12) R2 vs. HC.Skin biopsy samples (stored in RNAlater) were individually fragmented using a scalpel and transferred to a ceramic bead tube (CK28-Bertin Technologies). QIAzol reagent  was added, and the samples were processed (homogenization and lysis) in a Precellys 24 homogenizer (Bertin Technologies) with three cycles of 10-s pulses, and further incubated at 4°C for 5 min. Total RNA (including miRNA) was extracted using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions in a QIAcube apparatus (Qiagen). The RNA was recovered in 30 µL of RNase-free water. RNA quantification was performed using a Nanodrop 2000 (ThermoScientific), and integrity was evaluated using a Bioanalyzer 2100 electrophoresis system . Samples with low quality or insufficient RNA for analysis were excluded.®).To evaluate the expression of miRNA, 300 ng of each total RNA sample was subjected to RNA (Cy3) labeling, performed according to the manufacturer’s instructions . The slides used in these assays were miRNA—human miRNA microarray (G4872A-031181—8 × 60 K) . The oligoarrays were hybridized with fluorescent targets at 55°C for 20 h in a hybridization oven. After hybridization, the slides were processed using buffers provided by the manufacturer (Agilent Technologies) to eliminate non-specific targets and background interference, and then were subjected to a drying process that included washing in acetonitrile for 1 min and in a washing and stabilization solution for microarrays for 1 min. The arrays were digitized using the Agilent Bundle Scanner  with a resolution of 3 µm and Feature Extraction software (Version 11.0). The digitized images of each array were submitted for data quality analysis using Agilent Genespring software version 11.0  validation; some were highly upregulated or downregulated based on most comparisons, whereas others were expressed in either type 1 or type 2 reactional states. An unpaired asymptotic Complementary DNA (cDNA) was synthesized from total RNA by adding 0.5 µg of oligo-dT15 to 2 µg of total RNA in a final volume of 5 µL, and incubating the sample at 70°C for 10 min and cooling on ice for oligo annealing. Reverse transcription was performed using SuperScript III enzyme (Invitrogen) according to the manufacturer’s instructions in a final volume of 20 µL. The sample was incubated at 50°C for 1 h, followed by 15 min at 70°C. Aliquots of the obtained cDNA were diluted 10-fold and stored at −20°C.After selecting genes, custom PCR plates were ordered in a 96 × 1 format (Qiagen—miRNA #CMIHS02125). Twenty-four miRNA samples, representing the entire spectrum of disease, the reactional forms, and controls, were validated.Complementary DNA synthesis from miRNA samples was performed using the miScript II RT Kit and HiSpec Buffer (Qiagen). The obtained cDNA was subjected to the RT-PCR protocol of the miScript miRNA PCR Array with the miScript SYBR Green PCR Kit (Qiagen), using an ABI VIIA 7 device (Applied Biosystems).After completing the reaction, the quality of data was analyzed using SDS 2.3 software (Applied Biosystems). The dissociation curves were analyzed for amplification of genomic DNA, primer dimers, and splicing variants. Using amplification plots, the fluorescence intensity threshold was adjusted in the exponential phase of the graph in which the cycle threshold values of each reaction were considered. Duplicates with SDs of less than 0.5 were considered acceptable.The relative expression in each group was compared using a non-parametric Mann–Whitney test.We identify from our list of differentially expressed miRNAs and mRNAs those pairs that (i) were previously described as a validated interaction partners and (ii) show opposite expression pattern in our data. For this analysis, we consider only miRNA/mRNAs found as differentially expressed in each of the following group comparisons . The mRNAs were selected from a previous already published study by the same authors, in which the same set of samples used in this study were analyzed . Each grThe genes selected from the miRNA/mRNA analysis were submitted to pathway enrichment analysis by using the ReactomeFIViz plugin from the Cytoscape softwareThe microarray data set has been submitted to the Gene Expression Omnibus database at NCBIComparing disease (TT + BT + BB + BL + LL + R1 + R2) vs. HC groups, 64 miRNAs (50 upregulated and 14 downregulated) were differentially expressed . The 10 most upregulated or downregulated miRNAs differentially expressed in type 1 reaction and HC are shown in Table In general, miRNAs were heterogeneously expressed. The majority of these miRNAs were differentially expressed in different disease forms or reactional states compared to expression in the HC group, but with higher or lower magnitude . However, some were expressed only in specific groups .Of the 51 miRNAs subjected to RT-PCR validation, 8 were validated . All miRNAs subjected to validation and the associated values for the comparisons are shown in Table MicroRNA/mRNAs found as differentially expressed in each of the following group comparisons  are detailed in Table S14 in Supplementary Material. In summary, in the comparison Clinical forms vs. HC, six miRNAs  are associated with 39 mRNAs. In the comparison TT vs. HC, 6 miRNAs  are associated with 27 mRNAs. In the comparison LL vs. HC, 4 miRNAs  are associated with 44 mRNAs. In the comparison R1 vs. HC, 9 miRNAs  are associated with 62 mRNAs. In the comparison R2 vs. HC, 6 miRNAs  are associated with 57 mRNAs. After comparing the miRNAs validated in this study with the mRNAs validated by Belone et al.’s study, the following mi/mRNAs were identified: hsa-miRNA-142-3p and AKR1B10, hsa-miRNA-342-3p and FAM180b, and hsa-miRNA-484 and FASN , regulation of cholesterol biosynthesis by SREBP (SREBF), synthesis of very long-chain fatty acyl-CoAs, linoleic acid metabolism.Analysis of the human genome has indicated that many genomic sequences encode RNA that does not encode protein. Many longer non-coding RNAs, small nucleolar RNAs, miRNAs, and other small regulatory RNAs are included among the non-coding RNAs. miRNAs (20–24 nucleotides) have been extensively studied. They consist of a group of small non-coding RNA molecules related to small interfering RNAs. miRNAs play important roles in key biological processes such as gene regulation, cell growth, apoptosis, and hematopoietic lineage differentiation. As such, miRNAs are involved in various human diseases such as cancer, vascular disease, immune disease, and infections . RegardiIn this study, a large number of differentially expressed miRNAs were identified mainly due to the varied and polymorphous composition of the cells that contributed to the inflammatory processes in leprosy skin lesions , 5, 14. M. leprae for peripheral neural branches, is a predominantly neural infectious disease. Initially, in its indeterminate phase, before the development of lesions within the R&J spectrum, the peripheral neural branches of the skin or subcutaneous tissue are involved, and this is characterized by minimal inflammatory infiltration, which is predominantly lymphocytic, without well-formed granulomas. Subsequently, with complete disease establishment, granulomas involving neural branches become a histological feature present in all forms and both reactional states of leprosy. Therefore, there is constant interaction between neural branches and the inflammatory process during the disease.Many miRNAs described in the literature are associated with the regulation of the immune response in other diseases; for example, hsa-miR-34, hsa-miR-142-3p, hsa-miR-146a, hsa-miR-150, hsa-miR-155, hsa-miR-214, hsa-miR-223, and hsa-miR-424 were also present in leprosy tissues , probably by regulation of the immune response of the hosts. Functional profiles and experiments generated evidence suggesting that regulation of specific miRNAs during infection might stimulate the immune response or facilitate immune evasion by the pathogens  infection leads to the downregulation of miRNA17 and consequently the upregulation of its targets Mcl-1 and STAT3 -induced cytochrome p450 27B1 and IL-1β, and upregulation of IL-10. Thus, the ability of M. leprae to upregulate hsa-mir-21 could result in the regulation of multiple genes associated with the LL disease form, providing an effective mechanism to escape from vitamin D-mediated antimicrobial pathway  in skin samples of patients with the polar leprosy forms (TT and LL) and HC, the authors identified four validated miRNA  that can be used to discriminate HC from leprosy patients with 80% sensitivity and 91% specificity, respectively. In addition, the same miRNAs can discriminate, with 83% sensitivity and 80% specificity, LL from TT patients . In thisM. leprae-infected patients. It also identified Cbl-b overexpression and loss of miR-181 expression as important characteristics for the progression of leprosy , glioma cell proliferation, and germ cell tumor growth in the testis , 38, 39.N-acetyltransferase 1, is correlated with increased survival in patients with these tumor subtypes , there are no references in the literature regarding their expression in leprosy skin lesions. hsa-miR-1290, validated as downregulated in type 1 reactions compared to that in HCs, is associated with the suppression of proliferation and invasion in NSCLC and is significantly downregulated in luminal-A breast tumors (subtypes . hsa-miRsubtypes , 44. hsasubtypes , 46. hsasubtypes . hsa-miRsubtypes . hsa-miRsubtypes . The funsubtypes . Interessubtypes .MicroRNAs are increasingly described as important players in the regulation of cellular functions. However, the differential expression of miRNAs and target mRNAs in tissues, fluids, or cultured cells does not necessarily mean that there is a related disorder in cell metabolism. Most miRNAs were validated only experimentally with studies directed at the target mRNA. As a single miRNA species can regulate hundreds of mRNAs and because several miRNAs can be regulated by a single mRNA, the broad analysis of multiple mRNA/miRNA interactions becomes very complex, and often these interactions remain unknown as a result. Recently, we published a study on the expression of mRNAs in leprosy using the same samples and types of comparisons used in this study . CompariIn summary, this study has uncovered miRNAs that are deferentially expressed in leprosy skin lesions, several of which were described for the first time in this disease. miRNA expression profiles as they relate to the spectrum of leprosy and the reactional states might provide a solid basis for the understanding of the pathophysiological mechanisms of leprosy. These miRNAs will require validation and functional analyses to evaluate their role in the pathogenesis of leprosy. Thus, this study represents the initial stages of an important field of research to identify molecular markers of this disease or its reactional states. Furthermore, these deregulated miRNAs and their respective signaling pathways could be used as therapeutic targets, consequently enabling the development of novel drugs for the treatment of leprosy.CS, AB, DC, and FS conceived the project. CG and CS performed clinical evaluation of patients and performed biopsy procedures. CS performed histopathological and immunohistochemical analysis. AB, AC, MP, LF, and AT were involved with RNA sample preparation, hybridization, and PCR. CS, MP, AC, AB, AT, LF, RR, and PR were responsible for data analysis. CS and PR wrote the manuscript. All the authors critically revised the paper for intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.


Mycobacterium bovis vaccine via the oral route has been suggested to overcome this issue. In this study, the main goal was to assess the interference of the HI vaccine by different routes of administration using a previous vaccination and re-vaccination (boosting) protocol. TB-free kid goats were divided into three groups: oral (n = 16), intramuscular , and control (n = 16). Results showed that there was a significant difference in the percentage of animals positive to the single intradermal test (SIT) and blood based interferon-gamma release assay (IGRA) caused by vaccination when performed in the IM group compared to the oral group (p < 0.001). Nevertheless, no positivity to the SIT or IGRA test was observed in orally vaccinated goats regardless of the different interpretation criteria applied. None of the groups presented positive antibody titers using an in-house ELISA and samples collected 2 months after the boost. These results suggest the potential usefulness of the HI vaccine by the oral route in goats to minimize the interference on diagnostic tests (skin and IGRA tests) and reducing the necessity of defined antigens to replace the traditional purified protein derivatives for diagnosis. Finally, the results pave the way to future efficacy studies in goats using different routes of HI vaccination.Vaccination against tuberculosis (TB) is prohibited in cattle or other species subjected to specific TB eradication campaigns, due to the interference that it may cause with the official diagnostic tests. However, immunization with a heat-inactivated (HI)  Mycobacterium bovis and Mycobacterium caprae, both of which have also been isolated in Spain from other domestic and wildlife species (Tuberculosis (TB) in goats is mostly caused by  species –3. Accor species . These p species . Vaccina species . Bacillu species –12. Howe species , complic species . Therefo species , 16 as i species . However species , 18, 19.M. bovis vaccine has been recently developed, conferring similar protection to BCG against TB in wild boar  ild boar  and imprild boar . Both paild boar , 22. Thee scores . In thesIM route , 24. AnoIM route . MoreoveIM route . NeverthIM route –29.The aim of the present experiment was to assess the interference caused by the HI vaccine using a vaccination and boosting re-vaccination protocol by two different routes of administration  on the cellular and humoral TB diagnostic tests in kid goats. These results will be useful for the design of potential immunization protocols applied in goats with the aim of reducing the high prevalence of caprine TB in regions of Spain where caprine production is uncoupled to the cattle industry and where the TB prevalence is high.Forty-eight female kid goats (2–3 weeks old) were selected from a flock without a previous history of TB. Animals were tested in the farm of origin using a commercial IGRA kit  and an indirect in-house ELISA that was carried out with a readjusted procedure used by Che-Amat et al.  to checkn = 16) non-vaccinated (control); Group 2 (n = 16) intramuscularly vaccinated (IM); and Group 3 (n = 16) orally vaccinated.M. bovis strain Neiker 1403 (spoligotype SB0339) from a naturally infected wild boar was used for the preparation of vaccines as previously described  and M. avium (avian PPD)  according to the R.D. 2611/1996 and European Commission Council Directive 64/432/EEC. The skin fold thickness was measured after 72 h. Bovine and avian PPDs were inoculated on the left and right side of the neck, respectively, with a volume of 0.1 mL using a Dermojet® syringe . Applying the standard interpretation of the SIT test, animals were considered as positive reactors if presenting a skin fold thickness increase of 4 or more mm or the presence of clinical signs . Positive animals to the SCIT test were those showing a positive bovine reaction greater than the avian reaction in more than 4 mm, or the presence of clinical signs at the bovine PPD inoculation site.Both SIT and SCIT tests were carried out 2 months after booster vaccination. SIT and SCIT tests were performed in the neck , using PPD of ® TB Kit, Thermo Fischer Scientific, USA). According to the interpretation criteria prescribed in the Bovine Tuberculosis Spanish Eradication Program, animals were considered positive if the optical density (OD) in the bovine PPD stimulated sample minus the OD of nil antigen sample (PBS) was equal or greater than 0.05 and greater than the avian PPD-stimulated sample. Moreover, a less stringent threshold of 0.1 was also applied for comparison purposes.Blood samples were collected just prior to the vaccination to check the negative status of the goats, and 2 months after re-vaccination. Heparinized whole blood samples were stimulated with bovine and avian PPD  at a final concentration of 20 µg/mL and nil antigen PBS as previously described –33. Afte2SO4 (3 N), and the OD was measured at 490 nm with an ELISA reader. Goat negative and positive control sera were included in every plate in quadruplicate. Negative control serum was obtained from a TB-free goat previously described as M. bovis culture negative from a bTB-free area. Positive control serum was obtained from a goat previously infected with M. bovis in an experimental study. Sample results were expressed as an ELISA percentage (E%), according to the following formula: [sample E% = (mean sample OD/(2 × mean of negative control OD)) × 100]. The cutoff value was calculated using a ROC analysis and was defined as the ratio of the mean sample OD to the double of the mean OD of the negative control. Serum samples with E% values greater than 100 were considered as positive.Goats were tested before vaccination and 2 months after booster vaccination in order to evaluate the humoral response against different antigens using an in-house ELISA with some modifications to that previously described by Che-Amat et al. . BrieflyE%) among groups were analyzed by Kruskal–Wallis and Mann–Whitney U tests. Agreement between tests was measured with the kappa statistic (k) and interpreted as follows: <0.00 poor, 0.00–0.20 slight, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, and 0.81–1.00 almost perfect agreement  were calculated for the percentage of reactors to the different techniques. Comparison of proportions of test reactors between groups was performed using Fisher’s exact test. Skin fold thickness, IFN-γ OD, and ELISA percentage ], three in the IM vaccinated group [18.75% (95% CI 6.59–43.01%)] and two belonging to the control group [12.50% (95% CI 3.50–36.02%)], presented antibody titers against avian PPD above the cutoff point selected  and to control goats   and 12.50% (95% CI 3.50–36.02%) of the animals were positive reactors to both tests in the IM vaccinated group, respectively. None of the animals in the control and oral vaccinated groups showed a positive reaction to SIT and SICT tests (Table p < 0.001 for both comparisons). According to these results, significant differences  were found between the optical densities against bovine PPD in the IM vaccinated and the other groups, 2 months after booster vaccination . However, in the IM vaccinated group, there were 10 [62.50% (95% CI 38.64–81.52%)] and 7 [43.75% (95% CI 23.10–66.82%)] positive reactors out of the 16 animals, employing the two cutoff points 0.05 and 0.1, respectively. The percentage of reactors in the IM vaccinated group was significantly higher compared to the other two groups  were found in the bovine PPD (E%) values among groups 2 months after booster vaccination .Humoral response was measured by an indirect in-house ELISA, detecting only one positive animal to the bovine PPD in the IM vaccinated group 2 months after revaccination. Regarding the oral and control groups, all animals were negative to the bovine PPD, avian PPD, and PPA3. No significant differences . Nevertheless, no agreement was observed between the ELISA and the cell-based diagnostic techniques  in all groups was substantial  suggests that animals have not developed a humoral response yet. Long-term immunogenicity studies would be required to evaluate the humoral response elicited by the HI vaccine, administered orally and intramuscularly. Goats vaccinated with subcutaneous BCG and boosting with a recombinant adenoviral vaccine 8 weeks later induce a peak in the antibody response 2 weeks after the boost  and Spanish Legislation (RD 53/2013), and also approved by the Ethics Committee (Complutense University of Madrid) and the Regional Agriculture Authority .Participated in experimental design: AR, MR, CC, BR, LJ, AD-G, RJ, CG, LD, and JB. Prepared the inactivated vaccines: JG and IS. Conducted field and laboratory work: AR, MR, CC, ASM, AD-G, and JB. Performed data analysis: AR, LD, CG, and JB. Drafted the manuscript: AR and JB. Revised the manuscript: MR, RJ, LD, CG, and JB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.