We also examine the origins of SIVcpz (the predecessor of HIV-1) in greater detail than previous studies, and find that there are still large portions of the genome with unknown origins. Observed patterns of CST are likely driven by a combination of ecological circumstance and innate immune factors.Cross-species transmission (CST) has led to many devastating epidemics, but is still a poorly understood phenomenon. HIV-1 and HIV-2 (human immunodeficiency virus 1 and 2), which have collectively caused over 35 million deaths, are the result of multiple CSTs from chimpanzees, gorillas, and sooty mangabeys. While the immediate history of HIV is known, there are over 45 lentiviruses that infect specific species of primates, and patterns of host switching are not well characterized. We thus took a phylogenetic approach to better understand the natural history of SIV recombination and CST. We modeled host species as a discrete character trait on the viral phylogeny and inferred historical host switches and the pairwise transmission rates between each pair of 24 primate hosts. We identify 14 novel, well-supported, ancient cross-species transmission events. We also find that lentiviral lineages vary widely in their ability to infect new host species: SIV Virus cross-species transmissions (CSTs) can cause devastating epidemics, including EBOV, MERS, and HIV. HIV is actually one of over 45 similar viruses, called SIVs, which infect primates; the emergence of HIV as a human pathogen was the result of multiple CSTs, and yet it is not understood how commonly these viruses switch hosts. We used publicly available genetic sequence data to reconstruct the evolutionary history of these viruses. We found evidence for at least 13 instances of recombination between lineages of SIV, and identified at least 14 ancient cross-species transmission events where an SIV switched hosts. These results provide important ecological context to the HIV pandemic, suggesting that while SIV CST is relatively rare over evolutionary time, it is an important factor that has shaped SIV biology and evolution. As demonstrated by the recent epidemics of EBOV and MERS, and by the global HIV pandemic, viral cross-species transmissions (CST) can be devastating ,2. As susmm  from sooty mangabeys to humans  and frequencies 4 [ESS = 189]) were well over 200.“Pruned” dataset: 65 million steps, sampled every 15,000 states, 10% burnin removed. Posterior distribution of ~3900 trees per segment.“Resampled” dataset: 65 million steps, sampled every 15,000 states, 10% burnin removed. Posterior distribution of ~3900 trees per segment. All ESS values except for the AG substitution rate in segment 2 (ESS = 190) were well above 200.et al. [n host states. We use a non-reversible state transition matrix with n × (n–1) individual transition parameters [sum of the binary indicator variables corresponding to each pairwise rate parameter  was an exponential distribution with mean = 20–23. Convergence was again assessed by visually inspecting the log files in Tracer. MCMC chain parameters and ESS values were as follows.These tree distributions were used to estimate the transmission network. As in Faria et al. , we moderameters . We alsorameters . An expo“Main” dataset: 25 million steps, sampled every 5,000 states, 5 million states discarded as burnin.“Pruned” dataset: decreased phylogenetic noise in the empirical tree distributions for this dataset led to narrow tree distributions and poor mixing (but clear convergence). We thus ran 40 parallel chains of 25–35 million steps each, sampled every 20,000 states . We discarded 10 million steps of each chain as burnin and concatenated the remaining states.“Resampled” dataset: 45 million steps, sampled every 5,000 steps, 20 million steps discarded as burnin.For the main and supplemental datasets, more than 97% of the rate, indicator, and actualRate  parameters, and all other parameters, had an ESS well over 200 (most > 1000). The pruned dataset had a posterior ESS of 677, but the component tree likelihood ESS values ranged from 57–287; 91% of the rate, indicator, and actualRate parameters had an average ESS value > 200.any transmission between a given pair of hosts) [Statistical support for each transmission is summarized as a Bayes factor (BF), calculated by comparing the posterior and prior odds that a given rate is non-zero  . The ancS1 FigFor pairs of biallelic sites (ignoring rare variants), R^2 was used to estimate how strongly the allele in one site predicts the allele in the second site, with values of 0 indicating no linkage and 1 indicating perfect linkage. The mean value of R^2 was 0.044, indicating very low levels of linkage overall.(TIF)Click here for additional data file.S2 FigThe alignment used for GARD analyses (LANL compendium with HIV overrepresentation reduced) was split along the breakpoints identified by GARD to yield the 12 genomic segments, and a maximum likelihood tree was constructed for each. The number of steps required to turn one tree topology into another was assessed for each pair of trees with the Rooted Subtree-Prune-and-Regraft (rSPR) package. Segment pairs with similar topologies have lower scores than segments with less similar topologies.(TIF)Click here for additional data file.S3 FigA: All available high-quality lentivirus sequences were randomly subsampled up to 25 sequences per host for the main dataset. We included the 24 hosts with at least 5 sequences available in this dataset. B: For the supplemental dataset, we randomly subsampled up to 40 sequences per host, and included the 15 hosts with at least 16 sequences available in this dataset. For both datasets, a small number of additional sequences were permitted for the few hosts that are infected by multiple viral lineages in order to represent the full breadth of known genetic diversity of lentiviruses in each host population.(TIF)Click here for additional data file.S4 FigValues for the asymmetric transition rates between hosts, as estimated by the CTMC, were calculated as rate × indicator (element-wise for each state logged). We report the average posterior values above. Bayes factors represent a ratio of the posterior odds / prior odds that a given actual rate is non-zero. Because each of the 12 segments contributes to the likelihood, but they have not evolved independently, we divide all Bayes factors by 12 and report the adjusted values above (and throughout the text).(TIF)Click here for additional data file.S5 FigTips are color coded by known host state; branches and internal nodes are color coded by inferred host state, with color saturation indicating the confidence of these assignments. Monophyletic clades of viruses from the same lineage are collapsed, with the triangle width proportional to the number of represented sequences.(TIF)Click here for additional data file.S6 FigValues for the asymmetric transition rates between hosts, as estimated by the CTMC, were calculated as rate × indicator (element-wise for each state logged). We report the average posterior values above. Bayes factors represent a ratio of the posterior odds / prior odds that a given actual rate is non-zero. Because each of the 12 segments contributes to the likelihood, but they have not evolved independently, we divide all Bayes factors by 12 and report the adjusted values above (and throughout the text).(TIF)Click here for additional data file.S7 FigThe phylogeny of the host species' mitochondrial genomes forms the outer circle. Arrows with filled arrowheads represent transmission events inferred by the model with Bayes' factor (BF) ≥ 3.0; black arrows have BF ≥ 10, with opacity of gray arrows scaled for BF between 3.0 and 10.0. Transmissions with 2.0 ≤ BF < 3.0 have open arrowheads see . Width o(TIF)Click here for additional data file.S8 FigTips are color coded by known host state; branches and internal nodes are color coded by inferred host state, with color saturation indicating the confidence of these assignments. Monophyletic clades of viruses from the same lineage are collapsed, with the triangle width proportional to the number of represented sequences.(TIF)Click here for additional data file.S9 Figy = x; the linear regression is shown in gray.Each datapoint represents on of the 210 possible transmissions between each pair of the 15 hosts present in the supplemental, main, and main/pruned datasets. The main dataset consists of 5–25 sequences from each of 24 host species; the pruned dataset is identical, but with short taxa removed from each segment. The supplemental dataset consists of 16–40 sequences from each of 15 host species. The black dashed line shows (TIF)Click here for additional data file.S10 FigValues for the asymmetric transition rates between hosts, as estimated by the CTMC, were calculated as rate × indicator (element-wise for each state logged). We report the average posterior values above. Bayes factors represent a ratio of the posterior odds / prior odds that a given actual rate is non-zero. Because each of the 12 segments contributes to the likelihood, but they have not evolved independently, we divide all Bayes factors by 12 and report the adjusted values above (and throughout the text).(TIF)Click here for additional data file.S11 FigThe phylogeny of the host species' mitochondrial genomes forms the outer circle. Arrows represent transmission events inferred by the model with Bayes' factor (BF) ≥ 3.0; black arrows have BF ≥ 10, with opacity of gray arrows scaled for BF between 3.0 and 10.0. Width of the arrow indicates the rate of transmission . As illustrated and discussed, pruning short taxa from the topology can introduce unusual model behavior.(TIF)Click here for additional data file.S12 FigTips are color coded by known host state; branches and internal nodes are color coded by inferred host state, with color saturation indicating the confidence of these assignments. Monophyletic clades of viruses from the same lineage are collapsed, with the triangle width proportional to the number of represented sequences.(TIF)Click here for additional data file.


Schistosomiasis, one of the neglected tropical diseases, is endemic in more than 70 countries. However, the clinical diagnosis of patients with a low degree of infection is an unsolved technical problem. In areas endemic for schistosomiasis japonica, proctoscopy detection of eggs has been one method used for clinical diagnosis. However, it is often a challenge to find typical live eggs and it is difficult to distinguish live eggs from large numbers of partially degraded and/or completely degraded eggs within colon biopsy tissue. To address this problem, we tested six different morphological and biochemical/molecular markers ; AOS (antioxidase); SDHG (succinic dehydrogenase) and SjR2 mRNA (retrotransposons 2 of S.japonicum genome mRNA)), including four new markers , to determine the viability of S. japonicum eggs deposited in human and mouse colon tissues. Our ultimate aim is to obtain a new method that is more sensitive, practical and accurate to clinically diagnose schistosomiasis.S. japonicum infection with or without treatment with praziquantel (PZQ). Four new biochemical or molecular markers were used for the detection of egg viability from mouse liver and intestinal samples: CalS; AOS; SDHG and SjR2 mRNA. Subsequently, all markers were employed for the detection and analysis of eggs deposited in biopsy materials from patients with suspected schistosomiasis japonica for clinical evaluation. Microscopic examination of the egg morphology, worm burden in vivo and ALP  levels were used as a reference standard to evaluate the sensitivity and reliability of four new markers detecting egg viability.Tissue samples were collected from mice at six different time points during S. japonicum eggs deposited in tissues of hosts with schistosomiasis, especially cases with chronic schistosomiasis, is complex and egg viability is difficult to judge morphologically, particularly eggs with a fuzzy structure or partially modified eggs. We found that the majority of the viable schistosome eggs determined by four new markers  were morphologically difficult to identify.The results of the study showed that the morphology of Among the markers, the most sensitive and specific method was the detection of SjR2 mRNA and the most simple, rapid and practical method was the detection of SDHG. Therefore, the detection of SDHG is the most practical for clinical application and its use could improve the accuracy in diagnosing active schistosome infection.The online version of this article (doi:10.1186/s12879-017-2362-4) contains supplementary material, which is available to authorized users. S.japonicum, S. mansoni and S. haematobium are the most common species and schistosomiasis is second only to malaria as a parasitic disease in terms of public health importance. S. japonicum, causing zoonotic schistosomiasis, is found only in Asia, and has 60 million people at risk of infection with the potential for further increases in transmission [Schistosomiasis is a disease mainly caused by schistosome eggs lodging and damaging the organs and tissues of the human host. Six schistosome species are endemic in > 70 subtropical and tropical countries, infecting 230 million people and causing 200,000 deaths annually –3. S.japsmission . In Chinsmission . The allS. japonicum and S. mansoni have a similar life cycle in their definitive mammalian hosts. Adult worms reside in the mesenteric veins and excrete eggs that primarily circulate to, and lodge in, the liver and intestines. The eggs lodged in the colon submucosa and mucous layers, prior to rupturing into the lumen of the bowel and passing out with the feces, can be used for clinical diagnosis. In China, field and clinical diagnosis techniques for S. japonicum are mainly dependent on stool examination of eggs and immunological detection of specific antibodies [tibodies , 7. Methtibodies . Howevertibodies , and fortibodies . In conttibodies , 12. Thetibodies –16, but Detecting schistosome eggs from rectal biopsy specimens, typically employed in the clinic for the diagnosis of chronic and advanced schistosomiasis, partially remedies the drawbacks of the previously mentioned diagnostic techniques. Nevertheless, there are still some prominent problems with this method. One difficulty is the probability of finding randomly scattered eggs within biopsy tissues. The other difficulty is finding live eggs from a large number of partially degraded and/or completely degraded eggs within colon biopsy tissue. In addition, the use of this technique requires the professional skills of a biopsy specialist . FurtherS. mansoni eggs in paraffin tissue sections from the liver and intestine of infected mice has been used by Giboda and Zdarska [S. mansoni eggs were detected in the intestines of mice by endoscopic confocal laser scanning microscopy [S. japonicum eggs from perfused rabbit liver can also be determined by the physical characteristics of the eggs [The viability of schistosome eggs has been studied using TTC ninhydrin, acridine orange and iodine staining. However, these methods are complicated and none are applicable to the clinic –20. The  Zdarska  to detercroscopy . This methe eggs . HoweverS. japonicum challenge infection model and collected samples at different time points pre- and post-infection, and before and after praziquantel (PZQ) treatment. Liver and colon tissues were collected as well as information on pathology and parasitological measures such as liver and colon condition, adult worm and egg numbers and egg morphology. These parameters were then compared with five egg viability markers: calcified substance , antioxidase (AOS), succinic dehydrogenase (SDHG), SjR2 mRNA and alkaline phosphatase (ALP). Subsequently, the egg viability assays were used on proctoscopic samples from schistosomiasis outpatients to evaluate the applicability and practical value of these markers in clinical diagnosis. ALP and CalS used paraffin embedded tissue sections while AOS and SDHG required intact eggs from tissue. Our results indicated that all four new viability indicators, CalS, AOS, SDHG and SjR2 mRNA, could be employed as markers to evaluate the survivability and viability of eggs. Of these, the detection of SjR2 mRNA was the most sensitive, specific and reliable but also technically complex, and the detection of SDHG was the simplest, most practical and amenable to the demands of clinical application.Our study was designed to address the limitations of current diagnostic methods for differentiating between viable and non-viable eggs, particularly for application on rectal biopsy samples. To achieve this we used the mouse/S. japonicum cercariae were released from naturally infected snails collected from the Dongting Lake, Hunan province. The mice were divided into post infection (PI) and post treatment (PT) time course groups. The PI group were percutaneously [n = 6), 90 days  and 180 days  prior to perfusion. The PT group were infected as above and then treated with two consecutive gavage dosages (300 mg/kg/day) of PZQ  at 45 days post-infection. The PT mice were divided into three groups and maintained for 30 days , 90 days  and 180 days  before perfusion. Another two groups of uninfected mice (n = 3) were used as controls.Forty-two 7–8 week old male and female Kunming mice  were used for experimental schistosome infections. aneously  exposed v/v) ethyl alcohol for 15 min. The pleuroperitoneal cavities were opened under aseptic conditions to expose the livers and the gross morphology was recorded [v/v) paraformaldehyde solution for 16 h and stored in PBS at 4 °C. The AOS assay and SDHG staining of intact eggs were carried out within 4 h of sample collection.Mice were euthanized and immersed in 75%  and then pressed between two DEPC-treated glass slides. Images were recorded, the egg morphology noted, and then the slides were stored in liquid nitrogen.All the colorectal mucosa biopsy specimens were obtained from patients with schistosomiasis in the outpatient service department of Xiangyue Hospital, Schistosomiasis Control and Prevention Institution of Hunan Province, Yueyang, At the time of sample collection the following data were collected: basic patient information, past medical history, history of contact with infectious water, anti-schistosomiasis drug treatment, present symptoms, results of clinical examination, and ultrasonic examination of liver. The data were recorded in tables specifically designed for the current study. Two pieces of colon mucosa retrieved through a routine proctoscopic biopsy were smeared between two glass slides for microscopic observation. The tissues containing schistosome eggs were captured and analyzed with an Image Acquisition System (Olympus BX41microscrope fixed with an Olympus DP71 camera). The samples were divided into batches with different processing for the various marker assays. The first batch of 35 egg-containing samples were collected for detection of ALP and were fixed with 4% paraformaldehyde solution, visualized microscopically and paraffin embedded. A further 33 and 30 samples were collected for the AOS and SDHG assays, respectively, following microscopy. Finally, 48 samples were collected for mRNA assay, 38 of which contained The eggs were then further classified from the captured images into mature eggs, immature eggs, partially degraded eggs and completely degraded eggs using a published protocol . Some faS. japonicum eggs were fixed, dehydrated and embedded in paraffin by conventional methods. Serial tissue section slices (10 μm) were cut with a Leica (RM2225) microtome, and 38 successive tissue sections per tissue sample were mounted on glass slides. The prepared slides were stored at 4 °C for subsequent ALP staining and detection of calcium compounds.Tissue blocks containing 2O. Finally, the slices were sealed with glycerin jelly. False positive reactions were avoided by including a negative control for each staining experiment. The negative controls were dewaxed tissue sections boiled to inactivation ALP. All stained sections were microscopically examined. Viable eggs were defined as those with a strong blue or purple stain relative to the negative control sample. Stained and unstained eggs in each slice were counted and the images captured. The positive rate of stained eggs in a section was calculated.NBT  when mixed with BCIP  and catalyzed by ALP creates detectable blue or purple deposits in histology tissue sections. ALP detection was completed according to the manufacturer’s protocol and our previous study . Briefly3 and then exposed to a 30 W UV lamp for 1 h.The negative control slide was left unstained. Samples then washed two times with ddH2O to terminate the reaction and re-dyed for 1 min with 1% neutral red. After dehydration of gradient ethanol and sealing slices with neutral gum, slices were observed under a microscope. Eggs partially or completely covered with black calcium granules were considered as negative or dead. Eggs without black calcium granules were identified as positive and therefore viable. The positive and negative eggs were counted and images captured.The standard procedure for the detection of calcium  was foll2O2 substrate reaction system that results in insoluble brown or pale brown granules in the presence of peroxidase; an optimized procedure is available [2O2 solution and incubated at for 30 min at RT in darkness with shaking. The reaction was terminated with ddH2O and the tissue blocks were pressed between two glass slides for microscopic observation. A negative control tissue block was prepared by heat inactivation at 100 °C for 10 min prior to staining. Eggs with brown or deep yellow granules were judged as positive eggs. The positive and negative eggs were counted, images were captured, and the DAB positive rate of each group was calculated.Antioxidase (AOS) in the form of peroxidase was detected with a modified DAB -Hvailable . Fresh tS. japonicum eggs was detected using the MTT  assay. The MTT staining reagent  was prepared according to the manufacturer’s protocol (Sigma). Fresh tissue samples were pressed between two glass slides and egg types recorded prior to dyeing. The mounted slide was covered with the dye reagent in a humidified box for 30 min.The dye reagent was flicked off and the reaction was terminated with distilled water. The mounted slides were examined microscopically. Egg vitality was classified as weakly positive, positive, strongly positive or negative depending on the degree of staining and previously recorded egg type. Eggs were classified using the following criteria: no color change was negative (−), uneven light purple was weakly positive (+), even purple reaction was positive (++), dark violet was strongly positive (+++)  Fig.  and b. TS. japonicum retrotransposon gene, SjR2 [−9/(DNA length × 660), the number of amplicon copies was calculated, which was diluted into seven standards of 1 copies, respectively.The ne, SjR2 , was selne, SjR2 . A 408 bRNA extraction was carried out according to a standardized method . Approxi® green fluorescent dye quantitative method (Vazyme AceQ™ qPCR) [SjR2 mRNA was detected using a real-time PCR assay based on the SYBRQ™ qPCR) . A 20 μlP ≤ 0.05. The Pearson product moment correlation coefficient r was adopted for correlation analysis.SPSS 19 statistical software was used to analyze the data. Differences between groups were tested with the non-parametric Anova Kruskal-Wallis H test and the Nemenyi test for pairwise comparisons. The Chi-square test was used for testing the results of each marker compared with different types of eggs. Statistically significant results were defined as Prior to the commencement of this study, the study protocol was reviewed and approved by the Ethics committee of Hunan Institute of Parasitic Disease (reference number: 2014-S003). Before participating in the study, all subjects were given detailed explanations about the objectives and methodologies of this research. Written informed consents to participate and publish were obtained from all participants who provided colorectal mucosa biopsy samples voluntarily. The study was performed in accordance with the recommendations of the Chinese code of practices for the care and use of animals for scientific purpose. The Ethical Committee of the Center for Parasitology Research (ECCPR) has approved all experimental procedures, including animal handling, under animal license number: syxk (Xiang) 2011–0001 and in accordance with strict ethical standards. Laboratory animal quality conformance license number:43,006,700,002,928.Five tissue fragments of identical colon segments from each mouse, either in infected groups or in treatment groups, were pressed between glass slides for microscopic examination. The eggs were classified using morphology as immature, mature, partially degraded, completely degraded, and unknown type . However, for partially degraded eggs, AOS and SDHG as viability markers, were not significantly different (P = 0.128).As indicated in Table P<0.05) compared with the results of the unknown type, suspected viable and partially degraded eggs by morphology.The results of three biochemical markers testing eggs within rectal mucosa samples from schistosomiasis japonica patients showed that a high ratio of positive eggs appeared in suspected viable unknown type and partially degraded eggs Table . The posS. japonicum eggs belonging to different eggs types deposited in intestinal tissues from mice 45 days post-infection . However, there was no difference between the SjR2 mRNA levels in the 45 dPI, 90 dPI, 180 dPI and 30 dPT groups (P>0.05). In addition, the mRNA levels of S.japonicum eggs in livers were higher than those in intestines at every infection time point (P<0.05).SjR2 mRNA was undetectable in the liver and colon tissue samples from uninfected control mice Table . CompareS. japonicum eggs deposited in intestinal and liver tissues in the 45dPI,90dPI,180dPI and 30dPT groups were significantly higher than that of the normal group, respectively (P < 0.001). The mRNA levels of S. japonicum eggs in livers were more significantly higher than those in the intestines at every post infection time point (P < 0.05).The mRNA levels of S. japonicum egg mRNA in colon tissue without eggs was significantly lower than in tissue containing completely degraded, partially degraded and suspected viable egg types (P < 0.001). Moreover, the level of mRNA in intestinal mucosa samples only containing completely degraded eggs was significantly lower than those containing partially degraded and suspected viable eggs (P < 0.01). The level of mRNA in the samples mainly containing suspected viable eggs was significantly higher than samples with partially degraded eggs (P < 0.01). As a diagnostic method to detect viable eggs the SjR2 PCR assay proved to be highly sensitive. Patient samples containing suspected viable eggs or partially degraded eggs had 100% or 50% positivity rates, respectively. Moreover, the results of detection of the samples either with completely degraded eggs or without any eggs were negative.Our findings from biopsy samples collected from 45 patients complement and confirm the results obtained from the experimental infections Fig. . OverallSchistosome egg viability, from a biological perspective, refers to the survival of eggs after being laid by a female worm till egg death. The main factor affecting viability is the micro environment encountered by the egg. For example, eggs deposited in living host tissues can lead to a short survival time  due to the effect of the host immune system. Eggs trapped in feces outside the mammalian host can survive for 80 days at 1–4 °C  whereas Survival times of schistosome eggs are measured using the miracidium hatching method for eggs in feces and morphological observation of eggs in tissue samples. However, according to our findings, assessing schistosome egg viability using morphological observation of sigmoid-rectal biopsy samples is inadequate. Morphological diagnosis is not ideal for two reasons: microscopic evaluation requires a specialized and experience operator; and it is very difficult to determine the viability of partially degraded or/and completely degraded eggs within colon biopsy tissue, especially atypical eggs with indeterminable viability that are easily misdiagnosed.S. japonicum and S. mansoni and urine or bladder tissues for S. haematobium. However, the sensitivities of the Kato-Katz thick smear technique and miracidium hatching test do not exceed 70% [S. japonicum and S. mansoni [At present, although there are many clinical diagnostic and field monitoring methods for schistosomiasis, including immunological detection of specific antigens or antibodies and molecular biological measurement of specific gene fragments, the gold standard for definitive diagnosis is microscopic observation of eggs or miracidia in stool or intestinal tissues for ceed 70% , 38, and mansoni .We believe that the development of a rapid and simple method for determining the viability of schistosome eggs will help to further improve the effectiveness of rectal examination in the etiological diagnosis of schistosomiasis. In the present study, we evaluated several biomarkers and undertook morphological observations in the mouse model of schistosome infection and with schistosomiasis patients. Our results showed that eggs that were positive using a biomarker assay could be assumed to be viable. Eggs definitively confirmed by morphology to be viable, including immature and mature eggs, were positive using a biomarker assay; for example, the SDHG assay had a positivity rate of 97–99%. In addition, our biomarker assays generated positive results from difficult to diagnose samples that contained partially denatured, suspected viable or unknown type eggs.In summary, this study had the following findings. Within infected mouse liver and intestinal tissues collected one month post PZQ treatment, few eggs were determined to be viable morphologically compared with the increased number of viable eggs detected with biomarkers. Furthermore, three months post treatment, mouse liver and intestinal tissue had no morphologically viable eggs but SDHG-positive eggs were still detected. These results indicate that a proportion of schistosome eggs can survive for about three months in the mouse model after parasite clearance.Unlike the simple infection pattern in the mouse model, samples from schistosomiasis japonica patients were more complex. Morphological observation of the eggs in biopsied colonic mucosa from 70 cases of suspected schistosomiasis japonica patients indicated that repeated contact with infectious water and PZQ treatment can make definitive diagnosis difficult. In particular, for some cases there was no direct relationship between existing viable eggs and PZQ treatment. Following clinical proctoscopy, all 70 cases were reported to have “recent and long-term degraded eggs” due to the difficulty in identifying egg viability. However, by using biomarkers assays, such as egg-specific mRNA, 50% of cases with suspected viable eggs were found to have viable eggs compared with 10% of cases using morphology alone. In addition, from 30 suspected schistosomiasis cases, 13 were found to have viable eggs when using SDHG as a biomarker. However, the major benefit in using biomarker assays will be determining schistosome egg viability in colon biopsy tissue samples containing unknown type and partially degraded eggs.S. japonicum egg viability than the conventional morphological procedure. The tested biomarkers were effective for samples containing partially degraded, suspected viable and unknown type eggs in the mouse model post-treatment and for suspected schistosomiasis patients. Both SDHG and AOS assays are simple and convenient for detecting egg viability within host tissue but SDHG was more sensitive.Among the tested biomarkers there was no significant difference in mouse tissues before treatment for three biomarkers . However, the sensitivity of AOS and SDHG were lower than that of ALP. Nonetheless, ALP, AOS, SDHG and CalS were significantly higher than the morphological observations. The highest sensitivity was obtained with SDHG detection and the MTT method and SjR2 mRNA detection with RT-qPCR. These two methods may prove to be more effective in the clinic for evaluating S. japonicum eggs. All four biomarkers have value for differentiating between viable and non-viable schistosome eggs. In particular, CalS, SDHG and SjR2 mRNA all offer advantages over morphological observation. However, each method has its advantages and disadvantages in the context of clinical practice. For example, the CalS detection method, similar to ALP detection, has high sensitivity, but requires a paraffin or frozen section of the test sample, adding complexity and possibly influencing the results because of egg loss during the procedure. Applying the real-time fluorescent quantitative PCR method to detect the specific mRNA of S. japonicum eggs is a sensitive, specific and quantitative evaluative method, but the technical requirements will restrict its wider use in the clinic and field. Both DAB detection of AOS and the MTT detection of SDHG have the advantage of a simple, one step operation and rapid results (within 30 min) for detecting viable eggs in colonic biopsy specimens. It should be noted that for the AOS or SDHG assays tissue blocks need to be fully compressed between two slides to effectively expose the eggs before testing. In addition, for correctly assessing weak positive eggs a comparison must be made with the unstained egg as the background color may affect the results.This is the first study reporting the use of four new biomarkers  for measuring the viability of This paper reports on the use of four new markers  for assessing the viability of schistosome eggs. The results of our morphological observations indicated that in schistosomiasis hosts, especially human patients, the morphology of schistosome eggs is varied and complex, and clinical diagnosis is particularly difficult for samples containing partially degraded eggs, suspected viable eggs or unknown type eggs, whereas all four biomarkers we measured were able to differentiate between viable and non-viable schistosome eggs and detect a high proportion of viable eggs in samples with partially degraded eggs, suspected viable or unknown type eggs. Among the four biomarkers, the detection of SjR2 mRNA was the most specific and sensitive method, while the SDHG method is simple, rapid and clinically practical. Our findings suggest that, compared with conventional diagnosis by endoscopic biopsy using egg morphology, SDHG is able to accurately determine the viability of schistosome eggs regardless of morphology type. Therefore, SDHG is the most practical for clinical application and would improve the accuracy of the diagnosis of active schistosome infection.Additional file 1: Figure S1.Categories of eggs in colon tissue from infected mice. A: Immature eggs ; B, D, I: Unknown viability eggs; C and E: Mature eggs (larger in size with miracidium present); F: Partially degraded eggs (miracidium with disordered structure with the the appearance of black particles); G and H: Completely degraded eggs (black particles present in eggs or whole egg appears black). Magnification is 100× for all images. (JPEG 52 kb)Additional file 2: Figure S2.Types of eggs in biopsied colonic mucosa from schistosomiasis patients. Viable eggs with an intact miracidium are present in panels A, B and H (red arrows). Panels C, D and L contain partially degraded eggs that are light in color with disordered structure (green arrows). Panels B, G, I, J, K, M and N reveal completely degraded black eggs (black arrows). Unknown viability eggs containing miracidia with unclear structure are present in panels A, E, F and O (yellow arrows). Images are all at 100× magnification. (JPEG 88 kb)Additional file 3: Table S1.Clinical data and egg viability in proctoscopic tissue samples collected from 76 schistosomiasis patients. (DOCX 12 kb)Additional file 4: Figure S3.S. japonicum eggs in mouse colonic tissue. A: 45dPI group; B: 90dPI group; C: 180dPI group; D:30dPT group; E: 90dPT group; F: 180dPT group. The arrows in Fig. A point to positive eggs, which were stained blue/black by NBT. The staining results show that: all eggs in the 45dPI group were positive and negative eggs began to appear in the 90dPI group; Positive eggs decreased after treatment of mice with PQZ and, notably, eggs in the180dPI group were all negative. The eggs that had no color development were mostly empty or had a fuzzy structure (400× magnification). (JPEG 60 kb)ALP staining results for Additional file 5: Figure S4.S. japonicum eggs in mouse colonic tissue. A: 45dPI group; B: 90dPI group; C: 120dPI group; D: 30dPT group; E: 90dPT group; F: 180dPT group. Eggs indicated by the blue arrows were negative (live) and stained blue with the von Kossa stain. Calcified eggs, were positively stained black? with the von Kossa stain and are highlighted (red arrows). In the Figure, as the tissue background was stained with eosin, the miracidium in the egg was also stained red (400× magnification). (JPEG 62 kb)CalS staining results for Additional file 6: Figure S5.S. japonicum eggs in mouse colonic tissue. A, B and C indicate free eggs isolated from either tissues of infected mice with S. japonicum at 45 days and 180 days post infection or tissues of treated mice at 90 days post treatment. After staining with DAB, eggs in Fig. A developed a positive deep yellow or brown color. Some eggs in Fig. B were positive response and are indicated by blue arrows. In Fig. C, no eggs were positive. Figs AOS staining results for Additional file 7: Figure S6.S. japonicum-specific RNA real-time qPCR. The first 6 standards of the real-time qPCR (copy number was 105 Copies ~ 1 Copy) presented as a complete amplified curve, but the last standard (0.1 copy) did not have a t completely amplified curve. The linear relationship of the standard curve line was optimum, and the amplifying effectiveness was 98.05%; the dissolved melt curve line presented as single peak. (JPEG 50 kb)Amplified, standard and melt curve lines of


Avena sativa) and the possible involvement of hydrogen peroxide (H2O2) in the dysfunctions observed. Four oat cultivars with differential resistance responses  to powdery mildew  were used. Results demonstrated that stomatal dysfunctions were genotype but not response-type dependent since genotypes with similar resistance responses when assessed histologically showed very different locking patterns. Maximum quantum yield (Fv/Fm) of photosystem II were compromised in most Bga–oat interactions and photoinhibition increased. However, the extent of the photosynthetic alterations was not directly related to the extent of HR. H2O2 generation is triggered during the execution of resistance responses and can influence stomatal function. Artificially increasing H2O2 by exposing plants to increased light intensity further reduced Fv/Fm ratios and augmented the patterns of stomatal dysfunctions previously observed. The latter results suggest that the observed dysfunctions and hence a cost of resistance may be linked with oxidative stress occurring during defense induced photosynthetic disruption.Stomatal dysfunction known as “locking” has been linked to the elicitation of a hypersensitive response (HR) following attack of fungal pathogens in cereals. We here assess how spatial and temporal patterns of different resistance mechanisms, such as HR and penetration resistance influence stomatal and photosynthetic parameters in oat ( Avena sativa L. caused by the biotrophic fungus Blumeria graminis (DC.) E.O. Speer f. sp. avenae Em. Marchal. This disease is common in humid temperate climates widespread in north west Europe and in south east United States of America causing yield losses up to 40%  which exhibited a lower grain yield, smaller grains and less grain protein compared to uninoculated controls (Arabidopsis lines and attributed a metabolic cost to the presence of one resistance (R) gene – RPM1  which would be evolutionarily prohibitive in the absence of persistent disease pressure  cultivars (cvs) with different resistance responses to powdery mildew (Blumeria graminis f. sp. avenae (hereafter Bga) to (1) assess the extent to which stomatal dysfunction is directly related to the extent of HR and/or the different resistance responses (2) to explored the role of H2O2 and overall oxidative stress in the resistance-associated physiological dysfunctions (3) to identify oat cultivars with resistance response to powdery mildew displaying minimum physiological alterations upon pathogen attack.In this work, we used a genotypically diverse series of oats  , previously identified as resistant to  (Selma) , were us (Selma)  previous-2 s-1 photon flux density  but for the experiments of increased light intensity, after inoculation plants were subjected to a moderate light intensity regime of 450 μmol m-2 s-1 photon flux density supplied by high-output white fluorescent tubes .Plants were grown individually in 30 × 110 mm plastic centrifuge tubes (with two 5 mm drainage holes) filled with peat: sand (3:1). Tubes were stood in trays filled to a ∼50 mm depth in compost which was watered freely throughout. Plants were grown in a room with 20°C, 65% relative humidity and under 12 h dark/12 h light. Plants grew usually under a light intensity regime of 250 μmol mBga or Bgh, respectively, using a settling tower (-2 (checked by counts made from glass slides laid adjacent to leaves).For powdery mildew inoculation, the first fully expanded leaf (12 days seedlings), of the oat or barley plants was inoculated with ng tower  to give For histological studies, plants were maintained in the above mentioned growth chamber. Then at 36 h after inoculation (h.a.i) the central 30 mm leaf segment was excised and fixed on pads moistened with 3:1 ethanol:glacial acetic acid (v/v), and cleared with lactoglycerol , as described before  to avoidPercentages of germlings hampered in the infection process before or at time of cell penetration (penetration resistance), percentage of germlings inducing early or late cell death and percentage of established colonies were determined from 100 germinated urediniospores per leaf segment. Death of attacked epidermal cells was recognized by whole-cell autofluorescence , 2012.gl) was measured in ten plants per genotype with an AP4 cycling porometer that allows a non-destructive and rapid method for stomatal conductance measurement  as described in gl is the sum of cuticular and stomatal conductance, but as cuticular conductance of oat is low , of fully expanded second leaves twice a day, 3 h after the onset of the light period and 2 h before the end of the dark period. In light, a single measurement took <20 s, and in darkness slightly longer as the g1 was lower. Consequently, 10 plants were measured under 5 min. In each experiment, sets of 10 healthy and 10 inoculated plants of the chosen genotypes were measured, each set being held in adjacent trays on the growth room bench. The porometer was wiped clean after measuring inoculated leaves to avoid transferring the pathogen. Measurements in the powdery mildew highly susceptible cv. Selma were stopped earlier than in the other cvs in order to avoid experimental interference with sporulation.Leaf water conductance  of treated plants with respect to the control curve of non-stressed plants was calculated using the following formulai is the conductance at assessment date i, ti is the number of days after the first observation on assessment date i and k is the number of successive observations.where SFluorescence quenching analyses were measured using modulated fluorescence on second leaves of dark-adapted plants with a PAM 2100 Fluorometer . Measurements were made in four different replications according to .-2 s-1). Then, a 1-s saturating light pulse  value. When fluorescence returned again to the Fo level, plants were illuminated by a non-saturating continuous red “AL” (655nm) for 5 min to drive photosynthesis and gives F’ values. During the AL induced fluorescence kinetics, saturating pulses were applied with 20-s intervals in order to keep track of the fluorescence parameters Fm’ (the maximum fluorescence level in the light-adapted state). After several minutes under AL, and when the F value remained low and constant, AL was turned off and a far-red radiation (735 nm) that excited preferentially PS I was switched on during 4 s to determine the value of Fo′. During the induction kinetic induced by AL the ML is automatically switched to a higher frequency of 100 kHz in order to achieve a better signal to noise ratio and time resolution. In ML, while measuring F0, the frequency of 100 k Hz is replaced by 1.6 k Hz to avoid any chlorophyll fluorescence induction kinetics. Based on these basic parameters obtained, other parameters with significant physiological relevance such as i) Maximum Quantum Yield of PSII photochemistry (Fv/Fm) and ii) PSII operating efficiency  were derived.The Fo was determined after dark adaptation (at least 30 min) with a pulsed low red measuring light (ML)  and hence qN (NF/Fv). The qI, was calculated as NF20/Fv.  and 150 μL of KI was added to a microtiter well. The absorbance was measured at 390 nm 10 min later and was stable at least 30 min afterward. A calibration curve was performed with H2O2 standards at different concentrations in a similar way.HCell membrane stability (CMS) was measured in five plants per accession according to where T and C refer to the treated and control samples, respectively; the subscripts 1 and 2 refer to the initial and final conductance readings, respectively.All experiments were performed according to completely randomized designs. For ease of understanding, means of raw percentage data are presented in tables and figures. However, for statistical analysis, data recorded as percentages were transformed to arcsine square roots ]) to normalize data and stabilize variances throughout the data range. Data were subjected to analysis of variance (ANOVA) using SPSS software for comparison of treatments and analysis of interactions between factors, after which residual plots were inspected to confirm data conformed to normality. Significance of differences between means was determined by contrast analysis (Scheffe’s). Pearson correlations were calculated to detect statistical correlations between traits measurements. In addition, least significant difference (LSD) values were added to tables for comparison.Bga inoculation in several oat cultivars with different genetic backgrounds (Table 1).To further test the correlation between the execution of different resistance responses and stomatal lock-up , 2010, wBga with more than 65% of attack sites forming colonies with no sign of visible HR. The remaining sites exhibited penetration resistance due to papilla formation. All other tested oat cvs were significantly more resistant than the susceptible Selma (Table 1). Cory, Charming and Orblanche showed similar levels of HR to Bga, being observed at nearly 50% of attack sites, with most of the remaining exhibiting penetration resistance (∼45%). These proportions were similar to that elicited by Bgh in the barley cv. P01 which was included to allow comparison with our previously published results  but complete data along the monitored time course can be accessed in Supplementary Figure -2s-1. Following inoculation, a significant reduction on stomatal conductance (P < 0.001) was observed in barley P01; confirming previous results. Of all the oat cultivars screened, only Cory exhibited a reduction that was similar to that observed in P01; with ∼15% reduction in the AUCPC values on inoculated plants compared to the non-inoculated control . Oat cvs. Charming and Orblanche and the susceptible cv. Selma exhibited no significant reduction in stomatal conductance during daytime. Measurements on Selma were stopped from 5 days after inoculation (d.a.i) as fungal sporulation could influence the measurements of gl .Having characterized the responses of our panel of oat genotypes to Figure 1B), most cvs showed increased conductance following powdery mildew inoculation. Thus, Cory, and Orblanche showed increased stomatal conductance from 2 d.a.i. (P < 0.001) . These increases were nevertheless far below the ∼142% increase in stomatal conductance observed in the barley P01 . In the case of Charming, there were no significant differences in stomatal conductance in control and inoculated plants during the experimental time course of 8 days . No increases in the AUCPC of night-time stomatal gl were observed in the susceptible Selma, albeit it started to show a significant higher night-time gl from 5 d.a.i. . Similarly than for stomatal data, the results are given using the values corresponding to the area under the progress curve of the maximum quantum efficiency (Fv/Fm) of the complete time course. Bga inoculation significantly reduced Fv/Fm ratios in all oat cvs . The highest reduction in Fv/Fm was observed in Cory while Charming and Orblanche exhibited a slightly lower reduction. However, overall, the highest reduction was observed in the barley cv. P01 challenged with Bgh with a reduction up to more than 50% . In all cases, the highest reduction in Fv/Fm was observed around 7–8 d.a.i.To relate pathogen impacts on stomata with overall plant physiology effects, the status of the photosynthetic electron transport was estimated through chlorophyll fluorescence parameters . From the resistant cultivars assessed only Charming showed no increased photoinhibition following pathogen attack. Overall, and as happened with stomatal dysfunction, Charming cv. showed the smallest alteration in chlorophyll fluorescence parameters whereas Cory showed the highest alteration, similar or slightly lower than in the barley genotype P01.Dark relaxation measurements on inoculated oat and barley leaves showed that photoinhibition significantly increased in Cory, Orblanche and also in the barley P01, being this increase of more than 50% in P01 . In some cvs this could reflect a diversion of reductants  from a generalized whole tissue pattern of H2O2 to a more focused infection site specific oxidative burst. In this context, infection linked H2O2 content at 5 d.a.i. was higher than at 2 d.a.i. with no significant interaction between time and cultivars.It is widely known that a transient oxidative burst is generated during the execution of the penetration resistance and HR i.e., . In addiovements . We ther-2 s-1. Fungal development and leaf epidermal cell responses under this light regime were assessed to check for any possible change of the resistance responses (Table 2). Overall, mildew development and plant cell responses to the fungi under the moderate high light intensity did not differ from that observed under the normal regime  the overall H2O2 content but this was again reduced (P < 0.001) in response to pathogen attack in resistant cultivars, with the exception of Charming . The susceptible cv. Selma did not exhibit the clear response observed in the resistant cultivars. There were interactions between all factors indicating that the different genotypes responded in a different manner to the light and inoculation treatment. However, overall, under the moderate high light regime we observed a slightly higher reduction of the H2O2 content in inoculated respect to control plants . Assessment of CMS showed no cell damage consequence of oxidative stress under any of the light regimes used and after pathogen attack .The increase of light intensity increased also significantly . Thus, whereas Charming and Selma still did not show any differences in daytime conductance following inoculation, Cory, Orblanche and P01, showed higher decreases in diurnal g1 after pathogen attack than those observed under the normal light regime . A similar effect was observed regarding night-time g1. Thus, there was a significantly (P < 0.001) higher stomatal lock-up in all inoculated resistant cvs under the increased light intensity compared with the normal intensity . Interestingly there was a strong and significant interaction (P < 0.001) between cultivars, light intensity and inoculation indicating that not all genotypes responded in the same way. Thus, whereas under the normal light intensity the increases of night-time g1 were approximately of 131.7 and 82.7% for Cory and Orblanche, under the increased light intensity the night-time g1 increased, respectively, up to 203.1 and 110.8% with respect to non-inoculated plants. In addition Charming, that previously did not increase night-time g1, showed a slight stomatal lock-up when superimposing pathogen inoculation and higher light intensity. Overall, P01 barley did not respond to the increased light intensity with further nocturnal g1 increases, although it indeed increased g1 at the later time points of the time course  decrease in the Fv/Fm values of the area under the progress curve from 5.73 to 5.58  but no significant effect on photoinhibition . Confirming the previous results, under the moderate high light, pathogen challenge reduced significantly (P < 0.001) Fv/Fm ratios in all cultivars tested. However, there was no significant interaction between inoculation and light intensity, indicating that the overlapping of the increased light with the pathogen challenge did not change further the proportion of the Fv/Fm reduction previously observed. However, there were interactions between cultivars and inoculation and between cultivars and light intensity treatment. Thus, for instance, the resistant cv. Charming, that under the normal light assayed did not show photoinhibition following pathogen attack, showed it under the higher light intensity .Regarding the effect of the moderate light increase in chlorophyll fluorescence parameters, there was an overall slight but significant . However, when overlapping the inoculation treatment correlation turned to be negative .2 for photosynthesis while preventing water losses , formation of lipids microdomains, plasma membrane-cell wall adhesion, reorganization of the cytoskeleton, polarization of the cytoplasm and the endomembrane system, activation of transcription for synthesis of antifungal peptides, for secondary metabolites and for inhibitors of cell wall-degrading enzymes, synthesis of phytoalexins and monolignols, secretion of callose and other papilla components, phenolic and protein cross-linking, accumulation of H2O2 and other antifungal compounds  such as hydrogen peroxide or superoxide anion is one of the first responses of a plant to pathogen attack . The ROSablished . Using to leaves . Indeed,o leaves , 2010. T closure . The str2O2 through increasing light intensity lead to overall elevated H2O2 levels both, in control and inoculated plants. However, it did not lead to direct cell damage, as indicated by CMS assessment. This suggests that observed effects on stomata might be more directly related to H2O2 signaling than with direct damage of the cell components. Interestingly, the H2O2 reduction of inoculated respect to control plants was still observed when overlapping pathogen inoculation under the higher light intensity. However, the proportional reduction of H2O2 in inoculated respect to controls slightly increased under the highest light intensity, Thus, not H2O2 levels per se but the proportional higher reduction respect to control levels might explain the higher lock-up observed when overlapping higher light intensity and pathogen inoculation. On the other hand, increased photosynthesis, which can be promoted by higher light intensity, has been reported to increase night-time stomatal openings (2O2 levels are reduced following effective resistance for relatively so long also need to be elucidated. It is known that at time of attempt penetration, Blumeria graminis up-regulate a gene encoding a catalase CatB, which has been demonstrated to be secreted at the host-pathogen interface (2O2 was visualized around attack sites. However, we found genotype-dependent differences with Charming cv. showing fewer changes in H2O2 content and physiological dysfunctions than other resistant cvs. Thus, some of the mechanisms reducing the H2O2 are expected to relay on the host oxidant/antioxidant machinery.As expected, manipulation of Hopenings  and mighnterface . Further2O2 levels and chlorophyll fluorescence parameters. However, chlorophyll fluorescence parameters were strongly correlated with both daytime and night-time conductance. This suggests that the disruption of photosynthetic electron transport is not a direct consequence of oxidative damage but it is a feature of pathogen/resistance-induced stomatal dysfunctions. It is worth mentioning that the resistant cv. Charming, similar to Cory or Orblanche in term of resistance, showed the lowest fluctuations on H2O2 levels after pathogen attack and also the lowest stomatal or photosynthetic dysfunctions. Although for several of the assessed parameters it could seem that Charming behave physiologically similarly to the susceptible cv. Selma following pathogen attack it is worth to note that, as stated previously, measurements in Selma finished earlier to avoid experimental interference with sporulation. Late effects of the powdery mildew disease development on susceptible cultivars involve impaired stomatal opening in the light and disease-induced senescence (reviewed by Data showed no direct correlation between H2O2, rather than cell death per se are major determining factors, offer the possibility for such consequences to be avoided or reduced.Although further work is needed to dissect the molecular bases governing the physiological dysfunctions derived from the resistance responses, our data, suggesting that background genotypic effects, involving a role for HJS-M and GM-B carried out most of the experimental work and data analysis. DR and LM contributed to critical reading and writing and discussion of the results. EP designed experiments, and contributed to the interpretation and discussion of results and writing of the manuscript.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.


The cause is usually genetic and various genes are implicated.The 46,XY female is characterised by a male karyotype and female phenotype arising due to any interruption in the sexual development pathways SRY, DHH, NR5A1, NR0B1, AR, AMH, and AMHR2. To study candidate genes simultaneously, we performed whole genome sequencing. This revealed a novel and likely pathogenic missense variant  in SRY, one of the major genes implicated in complete gonadal dysgenesis, hence securing this condition over androgen insensitivity syndrome as the cause of the patient’s disorder of sexual development.Herein we describe a 46,XY woman who was first diagnosed with androgen insensitivity syndrome (testicular feminisation) at 18 years; however, this was later questioned due to the presence of intact Müllerian structures. The clinical phenotype suggested several susceptibility genes including This case highlights the emerging clinical utility of whole genome sequencing as a tool in differentiating disorders of sexual development. DSDs may arise due to environmental factors such as maternal androgen exposure, or they may arise due to mutations in any of the genes involved in sexual development in utero. The leading nosology of the unvirilised 46,XY female are complete androgen insensitivity syndrome (CAIS) and complete gonadal dysgenesis (CGD). Whereas CAIS is due to mutations in the androgen receptor (AR) gene abolishing the actions of testosterone, CGD may be caused by various genetic aberrations leading to failure of testis development and thus testosterone production and aromatisation of testosterone to oestradiol. In both CAIS and CGD, effective testosterone deficiency results in female external genitalia, female body habitus and tall stature. Primary amenorrhoea is the commonest presentation in both conditions – due to Müllerian regression in CAIS and due to gonadal insufficiency in CGD [Disorders of sexual development (DSD) are categorised as 46,XY DSD, 46,XX DSD and chromosomal DSD Table  with abay in CGD . Notablyy in CGD , 2.TableAR gene positioned on the X chromosome and, with an incidence of up to 1/40,000 births, complete AIS (CAIS) is the commonest cause of the XY female. The molecular severity of the mutation dictates phenotypic severity, ranging from mild AIS with male phenotype and gynecomastia and/or infertility to CAIS with an unambiguously female external phenotype [in utero results in variable failure of testicular descent, which may manifest as bilateral inguinal herniae or vulval masses. Sertoli cell function is normal in utero and therefore anti-Müllerian hormone (AMH) production typically leads to Müllerian tract regression [AIS is due to loss-of-function mutations in the henotype . Androgegression . Breast SRY) gene which account for 15 % of cases [in utero onwards. Absence of AMH in utero results in Müllerian tract persistence and development [CGD is characterised by streak gonads due to various genetic defects specific to testicular development Table , includiof cases . The defelopment . EndogenOne of the major utilities of determining an accurate diagnosis in 46,XY DSD is in guiding gonadectomy since the risk of gonadal malignancy differs among XY females. As the tumour risk in CAIS is age-dependent and only amounts to 16 % over the long term in women with CAIS and intact gonads, there is an argument to consider gonadectomy postpubertally after spontaneous breast development through aromatisation of gonadal androgens and at an age where girls may be active participants in their healthcare . In contThe precise cause of 46,XY DSD may also influence decisions regarding hormone replacement as unopposed oestrogen therapy in CGD carries risks of endometrial hyperplasia and carcinoma due to stimulatory effects on the endometrium . As hormAR mutation are used. Nonetheless, women with CAIS cited gamete preservation as a reason for declining gonadectomy in a survey of XY females, therefore this possibility may at least arise in patient-physician discussions [A diagnosis of CGD in 46,XY DSD may offer reproductive opportunities as successful pregnancies using donated ova have been achieved in women with CGD . This recussions .There may be other clinical differences between CAIS and CGD which may only be unmasked by studying XY women with genetically-confirmed diagnoses as ambiguity in the clinical diagnosis is not uncommon. Of relevance to our patient who had type 2 diabetes mellitus, endogenous oestrogen deficiency in childhood and beyond appears to contribute to glucose intolerance. This might account for the disproportionately high rates of diabetes mellitus in gonadal dysgenesis and aromatase deficiency compared to the background population though how this compares to women with CAIS remains to be determined , 10.SRY gene which is otherwise a straightforward cause of CGD. Subsequent investigations have traditionally involved single or staged gene analysis via Sanger sequencing guided by the clinical phenotype.The genetic evaluation of suspected DSD should begin with a karyotype. Fluorescence in situ hybridisation (FISH) may be performed simultaneously in the 46,XY female to confirm an intact An 18-year-old woman first presented for medical attention due to primary amenorrhoea. She was then a tall, thin woman with female external genitalia and no vulval or inguinal masses, however breast development and secondary sexual hair were conspicuously absent. The karyotype was 46,XY leading to a presumptive diagnosis of CAIS. Because of the suspicion of cryptorchid, dysgenetic gonads with high malignant potential, the patient underwent open laparotomy revealing streak gonads which were removed, and an infantile uterus which was left in situ. Histopathology demonstrated bilateral gonadoblastoma, and dysgerminoma in the right gonad. She received abdominopelvic radiotherapy with no tumour recurrence. After gonadectomy, she was commenced on the oral contraceptive pill and later changed to lower dose hormone replacement therapy.2 and her proportions were eunuchoid with height 181 cm, arm span 182 cm and lower segment length 102 cm. There was minimal pubic hair and no axillary hair, but breast development was now normal. She had female external genitalia and a retroverted uterus.At age 46 years, the patient migrated to Australia and sought review at our institution. Her active medical issues included type 2 diabetes mellitus, dyslipidemia, obstructive defecation syndrome and colonic polyps. She had a tall, slim build with a body-mass index of 20.4 kg/mResults of hormonal studies and haematocrit were most consistent with hypogonadal female ranges Table . Dual-enSRY gene, excluding SRY deletion as the cause of the patient’s atypical DSD. Array comparative genomic hybridisation showed no copy number variation at an average effective resolution of 0.2 MB.It was now, in the patient’s fifth decade of life, that her original diagnosis of CAIS was questioned. Whilst the external female phenotype, XY karyotype and absence of secondary sexual hair were consistent with CAIS, the presence of streak gonads and functioning Müllerian structures along with the absence of spontaneous breast development favoured a diagnosis of CGD. Though Müllerian remnants have been observed in women with CAIS, there are no reports of the functioning uterine structures as found in our patient. We performed various genetic studies locally, beginning with a digital karyotype confirming the original 46,XY result. FISH demonstrated an intact DHH, SRY, NR5A1 and NR0B1 [AR and AMH or AMHR2 were considered possible, as this would theoretically result in an unvirilised XY female phenotype with intact Müllerian structures. Whole exome sequencing was contemplated as the next step in our genetic evaluation, however this was deemed less suitable due to the non-contiguous capture and amplification steps which may hinder identification of copy number variations which are a significant cause of CGD [The major genetic possibilities at this stage were sequence variants and copy number variations in known CGD genes such as nd NR0B1 . In keepe of CGD . Single e of CGD , howevere of CGD . We inste of CGD .WGS was performed using the Illumina HiSeq X Ten Sequencing system at the Garvan Institute of Medical Research . BioinfoAKR1C4, DMRT1, MAP2K1, MAP3K1, NR0B1, NR5A1, POR and SRY. All but the NR0B1 and SRY variants were excluded based on variant population frequencies >1 % in the 1000 Genomes (1 K), Exome Sequencing Project (ESP) and Exome Aggregation Consortium (ExAC) databases. The NR0B1 variant was then excluded because of a population frequency >1 % amongst individuals in the ExAC database of the same ethnicity as the patient.Variants found on WGS were filtered based on mutational consequence (snpEff impact high or moderate) and phenotypic correlation. WGS revealed variants in eight genes associated with disorders of sexual development, namely: SRY gene  was confirmed to be present on Sanger sequencing of SRY. This variant has not been previously reported in the 1K, ESP, ExAC, or dbSNP databases, although there is an enrichment of pathogenic missense mutations at other locations of SRY reported in the ClinVar database , and more specifically CGD, thus revising the patient’s diagnosis from CAIS to CGD 30 years after her original presentation. The patient’s Müllerian structures were now considered a consistent feature of her DSD, although the reason for the absence of secondary sexual hair remains unclear as we excluded a concomitant mutation in the AR gene on WGS.The evidence collectively demonstrated this novel SRY mutation thus securing the diagnosis of CGD over CAIS. If we were to have performed single gene analysis based on the patient’s original diagnosis, the AR gene would have been sequenced with no yield. In current clinical practice, the cost of WGS is approximately US$2600 with the exact price depending on laboratory throughput and the technologies employed. The cost is generally less in research settings and further decreases are expected with improving efficiency in the sequencing and bioinformatic processes. With the increasing availability, cost-effectiveness, speed and understanding of WGS, we support its use in the clinical evaluation of the XY female due to the presence of genetic heterogeneity and oftentimes atypical clinical features in the DSD spectrum.In the presented case, the patient was phenotypically female with no evidence of virilisation and we confirmed a 46,XY karyotype. The leading differentials were therefore CAIS and CGD. Whilst the absence of secondary sexual hair was consistent with CAIS, the presence of streak gonads and functioning Müllerian structures along with the absence of spontaneous breast development were more suggestive of CGD contrary to her original diagnosis. To overcome the issues of multiple susceptibility genes and the possibility of either point mutations or copy number variations, we performed WGS which identified a novel


Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV) in faecal samples of recovered goats. Ninety-six (96) faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area. Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants caused by PPR virus (PPRV) of family Paramyxoviridae . The disOptimizations of advanced methods for the detection of PPRV are important for large scale surveillance studies especially to investigate carrier state of the animals. So far, very few reports have been documented in this regard. However, following the acute phase of PPR in small ruminants some animals may experience a long asymptomatic persistent stage. PPR viral RNA detection has been reported for 11 weeks after recovery and in some animals even up to 12 weeks after recovery using haemagglutination (HA) test . FurtherA suspected outbreak of PPR was investigated in an organised goat farm in suburban area of Lahore district, Pakistan, on 20 November 2012. The affected animals were examined for the presence of clinical signs specific to PPR virus infection. The temperature and clinical signs were recorded on prescribed proforma. The outbreak control measures were implemented and symptomatic therapeutic interventions were advised to the farmer. Structured epidemiological investigations were conducted to determine the most likely source of PPR virus transmission. Information regarding previous history of the disease at the farm, vaccination status of the flock, flock size, number of affected animals, number of dead animals, and history of a PPR outbreak in the nearby farm/area was recorded on a prescribed proforma.Necropsy of recently dead animals was performed. The carcasses were examined for the evidence of discharges , diarrhea, and pneumonia. Gross pathological lesions in GIT and respiratory tract were recorded.Ocular, nasal, and oral swabs and faecal samples were collected from live animals. Ocular swabs were collected by inserting a sterile swab (BD sterile swab) beneath the conjunctiva and swirling it so that the ocular secretions may adhere to the swab. The oral and nasal swabs were collected by inserting a sterile swab (BD sterile swab) deep into the oral and nasal cavity. Faecal samples were collected directly from rectum of clinically affected goats in polyethylene zipper bags using sterile gloves. The gloves were changed after collecting each sample.The tissue samples including lungs, liver, spleen, lymph nodes , kidneys, and intestine were collected from dead animals. Each sample from donor animal was given a unique identification number with date. All the samples were transferred to laboratory in cold conditions. The swabs, tissues, and fecal and sera samples were stored at −70°C till further analysis.Oral, ocular, and nasal swabs were processed for analysis. The cotton area of the swab was separated out gently from swab stick with the help of sterile forceps and scissor. Swab was put into a sterile Eppendorf tube containing 1.5 mL of sterile phosphate buffer saline . The swab was completely squeezed in 1.5 mL Eppendorf tube and centrifuged at 10,000 RPM for 3–5 minutes at 4°C. Supernatant was collected and stored at −70°C till further analysis.The tissue samples were processed by making approximately 10% homogenate of infected tissues in sterile PBS (0.01 M pH 7.4). The homogenate was centrifuged at 10,000 RPM for 3–5 minutes at 4°C in a 1.5 mL Eppendorf tube. The supernatant was collected and stored at −70°C till further analysis.μL of lysis buffer and 700 μL of 70% ethanol have been added to 140 μL of tested sample and allowed to spin in spin filtered column for one minute. For washing, wash buffer and buffer RPE are added and allowed to spin. To elute the extracted RNA, 40 μL of RNA's free water was used. The extracted RNA was placed at −20°C until further use. The quantity and purity of extracted RNA were determined using NanoDrop  [RNA was extracted from all the samples using RNeasy kit . A negative control was also included for detection of possible contamination during extraction. The extraction of RNA was performed according to manufacturer instructions. Briefly, 560 μL of TaqMan EZ buffer, 2.5 μL of 25 mM Mn (OAc)2, 03 μL of dNTPs, 01 μL of forward primer, 01 μL of reverse primer and 01 μL of probe, 01 μL of rTh DNA polymerase, and 10.5 μL of nuclease free water were added to make the volume 25 μL. Master mix (22.5 μL) and 2.5 μL of RNA template were added to the wells of optical 96-well reaction plate (MicroAmp™ N801-0560). The plate was covered with adhesive film (MicroAmp) and spun in refrigerated centrifuge at 2500 rpm for 1 minute.Each sample was analysed for the presence of PPR virus specific genome using reverse transcriptase real time polymerase chain reaction (rRT-PCR) . rRT-PCRThe rRT-PCR was performed using ABI7500 real time PCR system (Applied Biosystems) and ABI prism SDS software, an initial reverse transcription temperature at 45°C for 30 min, followed by reverse transcriptase inactivation and DNA polymerase activation at 95°C for 5 min, and then 50 cycles of 15 s at 94°C and 30 s at 60°C. The reporter dye (FAM) signal was measured against the internal reference dye (ROX) signal to normalize the signals for non-PCR-related fluorescence fluctuations that occur from well to well. The data were collected at the annealing step of each cycle and the threshold cycle (Ct) for each sample was calculated by determining the point at which the fluorescence exceeded the threshold limit.The animals positive for PPR were followed for the period of four months from 20 November 2012 to 20 March 2013. Sera and fecal samples were collected from recovered animals at 4, 8, 12, and 16 weeks after outbreak.The sera samples were analysed for the presence of PPRV specific antibodies using anti- nucleocapsid (N) monoclonal antibody (MAb) based competitive ELISA (c-ELISA) . The faeThe animals were raised on a semiextensive system where the animals were taken out for grazing in the morning and supplemented with a concentrate stall feeding in the evening for fattening. The flock consisted of 140 goats with age ranging between 10 and 18 months. The flock had history of introduction of five new animals from a nearby livestock market. None of the animals had a history of vaccination against PPR. There was no outbreak of PPR in the nearby area/village. The morbidity rate was 100%. However, 44 out of 140 animals died during the outbreak with a mortality rate of 31.42%.The clinical examination of the affected animals revealed high fever ranging between 39°C and 42°C, conjunctivitis, mucopurulent nasal and discharges along with depression, anorexia, swollen lips, cough, and diarrhoea. The affected animals showed signs of severe dehydration and their hind quarters were soiled with diarrhoea material. The mouth lesions were found in all the affected animals with red raw areas on inner side of the lips, lower gums, and necrosis on the dorsal surface of the tongue.n = 3 recently dead animals was conducted. On external appearance the carcasses were found to have evidence of dehydration with sunken eyes, rough/dry skin, and hind quarter soiled with diarrhoea material. The internal examination revealed pneumonic lungs with haemorrhages on mucosal surfaces of rumen, abomasum, and large intestine (caecum and colon). Haemorrhages were also observed on liver and kidneys of one animal. The body lymph nodes were inflamed and swollen particularly the mesenteric lymph nodes.The postmortem examination of For laboratory confirmation of PPR a total of 29 swabs and 33 faecal and 12 tissue  samples were collected. Sixteen (55.17%) swabs and 28 (84%) faecal and 10 (83%) tissue samples were found positive for PPRV genome using rRT-PCR. These results confirmed the outbreak of PPR in the flock.n = 96 faecal samples were collected. RNA extraction and rRT-PCR were performed as mentioned earlier. At each sampling n = 96 faecal samples were collected from PPR recovered goats, out of which 46, 37, 29, and 25 samples were found positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively. These results indicated that small ruminants shed PPR viral genome up to four months (16 weeks) after clinical recovery from the disease (Figures The samples were collected according to the method as described earlier. At each sampling A PPR outbreak causing high morbidity and low mortality in a goat herd in Punjab was confirmed on the basis of clinical signs and various lab diagnostic tests. The animals recovered from the outbreak were monitored and their faecal samples were collected for a period of 16 weeks to determine the persistence of PPRV in faecal samples of recovered animals.The results of the study indicated that introduction of new animals into the flock from weekly livestock markets resulted in the initiation of an outbreak in herd of goats under investigation. Similar mode of disease transmission has been reported previously by Asmar et al.  in SaudiDuring investigation typical clinical signs and symptoms of PPR virus infection were observed in infected animals. These include high fever up to 42°C, lesions in mouth, oral and nasal congestion, respiratory signs, and diarrhea leading to death of the animals. Similar findings were made in previous reports , 15, 16.The results of the study indicated detection of PPRV in goats recovered from PPR. However, the number of animals giving positive results decreased from 46 on 4th week after vaccination to 25 on 16th week after vaccination. Previous studies also reported the persistence of PPRV antigen in faecal samples of PPR recovered goats , 18. HowVarious samples have been analysed using different diagnostic tests to understand the persistence of PPRV in animals recovered from PPR . For exaWe used rRT-PCR for detection of PPRV in faecal samples of goats whereas previous studies used HA for detection of PPRV in faecal samples , 18. rRTIn conclusion, this study reported the detection of PPRV in faecal samples of PPR recovered goats using rRT-PCR up to 16th weeks after recovery indicating the possible role of PPR recovered goats in transmission of disease to in-contact healthy animals. However, a study should be carried out to isolate the virus from faecal samples positive for PPRV RNA to further elucidate the role of recovered animals in transmission of disease to healthy animals. Therefore, these results may help in studying the future epidemiology of PPR virus shedding and transmission of PPR virus by fecal material and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area.