The gastrointestinal tract contains a vast community of microbes that to this day remain largely unculturable, making studies in this area challenging. With the newly affordable advanced sequencing technology, important breakthroughs in this exciting field are now possible. However, standardized methods of sample collection, handling, and DNA extraction have yet to be determined. To help address this, we investigated the use of 5 common DNA extraction methods on fecal samples from 5 different species. Our data show that the method of DNA extraction impacts DNA concentration and purity, successful NGS amplification, and influences microbial communities seen in NGS output dependent on the species of fecal sample and the DNA extraction method used. These data highlight the importance of careful consideration of DNA extraction method used when designing and interpreting data from cross species studies. The gastrointestinal (GI) tract contains a vast community of microbes that greatly outnumber host cells  and playSeveral procedures for extraction of DNA from fecal samples have been described. Most of these studies focus on DNA extraction from human samples, with only a few of these methods reported for use on other species –12. FurtIn this study, fresh fecal samples were collected from eight individuals within five species groups  and DNA was extracted using five different DNA extraction methods . The present study evaluates DNA extraction performance of these methods with a focus on DNA yield, purity, NGS output, and cost. Our results demonstrate that certain extraction methods, when performed according to the manufacturer’s instructions, perform preferably in samples from specific host species. Additionally, spectrophotometric assessment of DNA elutions prior to PCR had poor predictive value with regard to successful amplification and sequencing. Lastly, comparison of sequencing results from the same sample subjected to different extraction methods show that, while the detection of a limited number of rare taxa may vary, overall community profiles agreed fairly well in the majority of samples. These findings will enable investigators to choose the optimal DNA extraction method to suit current study needs.All studies were performed in accordance with the recommendations put forth in the Guide for the Care and Use of Laboratory Animals and were approved by the University of Missouri Institutional Animal Care and Use Committee. For public owned animals (dog and cat) prior consent of the owner was obtained before fecal samples were obtained.Danio rerio) were subjected to euthanasia with 0.1% clove oil in aquarium water, and the entire GI tract was removed and placed in a sterile 2 mL cryovial. The tissue and fecal contents were promptly frozen and stored in a -80°C freezer for two weeks prior to DNA extraction. Mouse (Mus musculus) samples: five freshly evacuated fecal pellets per mouse were placed in a 1.5 mL sterile Eppendorf tube. Samples were promptly frozen and stored in a -80°C freezer for two weeks. Prior to each extraction method, one fecal pellet per mouse was removed from the freezer and extraction was performed as described below. Cat (Felis domesticus), dog (Canis familiaris), and horse  samples: freshly evacuated fecal samples were collected from privately owned cats and dogs. Freshly evacuated equine fecal samples were obtained from horses present in the University of Missouri Veterinary Medical Teaching Hospital for reasons unrelated to gastrointestinal conditions. Upon collection, samples were placed in 50 mL conical tubes and stored in a -80°C freezer for two weeks prior to DNA extraction. A small piece of fecal material was promptly removed from the frozen sample immediately prior to performance of each extraction method; the remaining sample was returned to the freezer.Zebrafish samples: Zebrafish (All DNA was extracted and quantified at the University of Missouri Metagenomics Center (MUMC).DNA extraction was performed following the manufacturer’s recommendations with slight modifications to allow for mechanical disruption. Briefly, for cat, dog, and horse samples, 25 mg of feces was placed into a sterile 2 mL round-bottom tube containing 500 μL sterile PBS and a 0.5 cm diameter stainless steel bead. Samples were mechanically disrupted using a TissueLyser II  for 3 minutes at 30 Hz, followed by centrifugation at 200 × g for 5 min. A 200 μL volume of supernatant was removed and placed in a sterile 1.5 mL Eppendorf tube. An equal volume of buffer AL was added and the samples were processed thereafter following the manufacturer’s protocol. For mouse samples, one fecal pellet was placed into a 2 mL sterile round bottom tube containing 500 μL sterile PBS and a 0.5 cm diameter stainless steel bead and processed as described above. For fish samples, the entire GI tract was placed into an autoclaved 2 mL round-bottom tube containing 500 μL sterile PBS and a 0.5 cm diameter stainless steel bead and processed as described above. All samples were eluted in 200 μL AE buffer.DNA extraction was performed following the manufacturer’s recommendations with small adaptions due to equipment availability. Briefly, for cat, dog, and horse samples, 0.25 mg of fecal sample was added to the dry bead tube containing 750 μL of bead solution and gently vortexed. C1 solution was added, the sample briefly vortexed, and incubated at 65°C for 10 minutes following the recommended protocol. Samples were shaken for 10 min. in a TissueLyser II  at 30Hz. Samples were centrifuged at 13,000 × g for 1 min., the supernatant transferred to the provided 2 mL collection tube, and the remainder of the protocol was followed as recommended by the manufacturer. For mouse samples, one fecal pellet was added to the dry bead tube containing 750 μL of bead solution and processed as described above. For fish samples, the entire GI tract was placed in the dry bead tube containing 750 μL of bead solution and processed as described above. All samples were eluted in 100 μL solution C6.DNA extraction was performed following the manufacturer’s recommendations with small adaptions due to equipment availability. Briefly, for cat, dog, and horse samples 25 mg of feces was placed into a 2 mL sterile round-bottom tube containing 500 μL of sterile PBS and a 0.5 cm diameter stainless steel bead. Samples were mechanically disrupted using a TissueLyser II  for 2 minutes at 25 Hz, followed by centrifugation at 14,000 × g for 2 minutes. A 400 μL volume of supernatant was removed and placed in a sterile 2 mL screw cap tube containing 2 mg of sterile glass beads and 100 μL of lysis buffer ATL. Samples were processed on a TissueLyser II  for 10 min. at 50 Hz and the remainder of the protocol was followed as recommended by the manufacturer. For mouse samples, one fecal pellet was placed into a 2 mL sterile round-bottom tube containing 500 μL of sterile PBS and a 0.5 cm diameter stainless steel bead and the sample was processed as described above. For fish samples, the entire GI tract was placed in a 2 mL sterile round-bottom tube containing 500 μL of sterile PBS and a 0.5 cm diameter stainless steel bead and the sample was processed as described above. All samples were eluted in 150 μL AE buffer.DNA extraction was performed following the manufacturer’s recommendations. Briefly, for cat, dog, and horse samples, 200 mg of feces was placed in a sterile, round-bottom 2 mL tube containing 1.4 mL ASL lysis buffer and the remainder of the protocol was followed as described by the manufacturer. For mouse samples, one fecal pellet was placed into a sterile, round-bottom 2 mL tube containing 1.4 mL lysis buffer and the sample was processed as described above. For fish samples, the entire GI tract was placed in a sterile, round bottom 2 mL tube containing 1.4 mL lysis buffer and the sample was processed as described above. All samples were eluted in 200 μL AE buffer.DNA extraction was performed as previously described . BrieflyFor all extraction methods, DNA concentrations were determined fluorometrically  and purity was assessed via 260/280 and 260/230 absorbance ratios, as determined via spectrophotometry . Samples were stored at -20°C until sequencing.(3:00)+[98°C(0:15)+50°C(0:30)+72°C(0:30)] × 25 cycles +72°C(7:00). Amplicons were then pooled for sequencing using the Illumina MiSeq platform and V2 chemistry with 2×250 bp paired-end reads, as previously described [Library construction and sequencing was performed at the University of Missouri DNA Core facility. DNA concentration of samples was determined fluorometrically and all samples were normalized to 3.51 ng/μL for PCR amplification. Bacterial 16S rRNA amplicons were generated via amplification of the V4 hypervariable region of the 16S rRNA gene using single-indexed universal primers (U515F/806R) flanked by Illumina standard adapter sequences and the following parameters: 98°Cescribed . Samplesde novo and reference-based chimera detection and removal, and remaining contiguous sequences were assigned to operational taxonomic units (OTUs) via de novo OTU clustering and a criterion of 97% nucleotide identity. Taxonomy was assigned to selected OTUs using BLAST [http://prime.psc.riken.jp/Metabolomics_Software/StatisticalAnalysisOnMicrosoftExcel/index.html).Assembly, binning, and annotation of DNA sequences was performed at the MU Informatics Research Core Facility. Briefly, contiguous DNA sequences were assembled using FLASH software , and culng BLAST  against ng BLAST  of 16S rp values ≤ 0.05.Statistical analysis was performed using Sigma Plot 12.3 . Differences between DNA extraction methods in total DNA concentration were determined using ANOVA with Student Newman-Keuls post hoc test. Differences in phylum relative abundance (non-rarefied) following successful NGS amplification, i.e., achieving 10,000 sequences, were determined using either ANOVA with Student Newman-Keuls post hoc test or a t-test with Mann-Whitney post hoc test. Receiver operating characteristic (ROC) curves were generated from 260/280 and 260/230 absorbance ratios obtained from samples of each host species using success of amplification as the binary classifier. Statistical differences in microbial diversity were determined at a uniform depth using ANOVA with Student Newman-Keuls post hoc test. Results were considered statistically significant for In addition to traditional “bench-top” techniques for DNA extraction, there are many commercially available kit-based methods. To assess the suitability of several of these methods for extraction of fecal DNA from multiple host species with the intended downstream use of next-generation sequencing, samples were collected from eight individual mice, cats, dogs, horses, or forty zebrafish, and processed according to the manufacturer’s protocol or published methods. For cat, dog, and horse samples, the same fecal bolus was used for all extraction methods. For mice, five fecal samples were collected at the same time and one pellet was used for each method. For zebrafish, forty age-matched fish from the same tank were used, eight per extraction method. Regarding overall DNA yield from a standard mass of starting material, the amount of DNA extracted was dependent on the DNA extraction technique and the host species from which the fecal sample was collected. For all species examined, except the dog, the isopropanol extraction method produced the greatest total DNA yields, and in horse samples, the isopropanol method resulted in significantly higher yields when compared to all other extraction methods . In the A more practical measure of performance is the quality of the DNA elutions generated from each method. The presence of myriad undesirable substances in feces is often blamed for samples that result in low coverage  due to PCR inhibition. To assess the quality of the DNA extracted via the methods under evaluation, samples were amplified in a 96-well format using single-indexed primers, and pooled for sequencing on the Illumina MiSeq platform. Based on rarefaction analysis of previously published sequencing data , we defiPrior to NGS, successful PCR-based amplification requires preparation of template DNA containing little to no protein, RNA, or polysaccharides, and purity is often assessed via 260/280 and 260/230 absorbance ratios, determined by spectrophotometry. To compare the predictive power for successful amplification of each absorbance ratio, and also to establish cut-off values for one or both of the ratios, receiver-operating characteristic (ROC) curves were generated for each species, with successful amplification as the binary classifier. ROC curves are frequently used in the development of diagnostic assays to compare positive and negative predictive values of test results, and to determine a threshold value with acceptable sensitivity and specificity based on test results of a group of “known” samples. The area under the curve (AUC) can be used as a measure of overall diagnostic  accuracy; an AUC of 1 denotes 100% sensitivity and specificity while an AUC of 0.5 suggests completely random performance. Unexpectedly, neither high 260/280 or 260/230 absorbance ratios accurately predicted the success of NGS amplification . For exaFusobacteria  was detected in the relative abundance of the rare phylum Cyanobacteria, with QIAamp-processed samples showing greater abundance when compared to isopropanol-extracted samples. No differences were detected in the relative abundance of any other phylum in the mouse samples extracted with those three methods.In human studies, it has been shown that variation in DNA extraction can influence the bacterial communities detected during sequencing . To determine whether the extraction chemistries tested resulted in differential lysis and subsequent skewing of the microbial profile, results of 16S rRNA amplicon sequencing were compared. Annotated to the taxonomic level of phylum, those samples that reached the threshold of 10,000 sequences generally showed good agreement with some exceptions. For example, sequencing of zebrafish samples extracted using the PowerFecal kit or manual isopropanol method detected a higher relative abundance of microbial families within the phylum ria Figs  and 4 whmplified . In the To further evaluate the effect of DNA extraction method on NGS output, principle component analysis (PCA) was performed including only data from methods that resulted in successful amplification in half or more samples for each host species . Data frWith the decrease in costs associated with NGS, characterization of GI microbial communities is becoming increasingly feasible. However, to date, the majority of studies have focused on human fecal samples with far less optimization of techniques for use with samples from other species. In the current study, the performance of five DNA extraction methods was evaluated using samples collected from five diverse host species. Taken as a whole, the data suggest that samples from a particular host species may be amenable to DNA extraction using only certain methods.This phenomenon was first noticed when comparing the concentration of DNA in elutions from each method. While the manual isopropanol precipitation yielded consistently high levels of DNA, the PowerFecal kit resulted in comparable yields from zebrafish and mouse samples, and the DNeasy kit produced comparable yields in feline and canine samples. One possible explanation for the greater yields generated via isopropanol extraction is the fact that the other kit-based methods rely solely on retention of DNA in a column-based matrix. In the isopropanol method, DNA is precipitated via addition of a chaotropic salt and isopropanol on ice, followed by subsequent elution over a column. That said, other differences in the extraction chemistry may also influence efficiency of lysis and removal of PCR inhibitors.We also noted considerable variability in the amount of DNA extracted within several samples, dependent on extraction method used, as evidenced by the large standard deviation among sample groups. This variation could be due to the efficiency of the DNA extraction methods tested. However, this could also be due to variability in the diet that these animals consume or the amount of true microbial biomass loaded with each sample as cat and dog samples were found to contain a large amount of particulate matter such as hair and bone fragments. Similarly, horse fecal samples contained substantial amounts of large particulate fiber matter. Although care was taken during measurement of starting sample mass, these particulates could have an effect on the amount of fecal biomass that was available for DNA extraction.More importantly, the method of DNA extraction was found to strongly influence the number of samples that successfully amplified, but in a species-dependent fashion. For example, the isopropanol method was uniquely well-suited for use with zebrafish samples, resulting in successful amplification in 8/8 samples, while successful amplification and sequencing of equine samples necessitated use of the PowerFecal kits. Taken together, these data suggest that the substances present in the feces responsible for PCR inhibition likely differ between host species.Furthermore, all zebrafish samples had high A260/A280 and A260/A230 absorbance ratios, suggesting relatively “clean” elutions, but three out of the five extraction methods tested completely failed NGS amplification. This may be an artifact of processing as the entire zebrafish GI tract was processed along with fecal contents. Extracted samples likely contained a large amount of host DNA which may have resulted in dilution of bacterial DNA when performing PCR with a standard DNA concentration. However, yields from isopropanol-extracted samples were noted to be the highest and performed well. Conversely, in the cat, dog and horse samples, both high and low A260/A280 and A260/A230 absorbance values were detected, possibly indicating contamination from guanidine residue during DNA extraction or dietary carbohydrate carryover. These samples were thus expected to have variable to unsuccessful amplification, however several such samples successfully amplified. Taken together, these results indicate that DNA purity, as determined via 260/280 or 260/230 absorbance ratio, cannot reliably predict successful amplification.Fusobacteria, while samples extracted via the DNeasy kit showed predominantly Proteobacteria. When overall microbial profiles were compared at the level of OTU via PCA, we noted substantial variation among the samples extracted with the DNeasy kits. Due to the scant amount of digesta present in zebrafish, DNA was extracted from individual fish as compared to serial or identical fecal samples from other species evaluated in this study. Even though all fish used for this study were specific pathogen free and housed in the same aquarium, differences in OTU variation could be due to true differences between individual fish. That said, it is worth noting that the community profiles generated using the isopropanol extraction method are not in agreement with other published reports of zebrafish microbiota [et al. and other groups have identified Proteobacteria as the predominant phylum in the gut of zebrafish, Fusobacteria was the primary phylum detected in the current study. Whether this is a function of extraction method or due to a difference in the populations tested is unclear.Several studies have shown that variation in DNA extraction can impact NGS-based characterizations of bacterial communities. In the present study, only small differences in NGS output were observed for most samples at the phylum level, with the aforementioned exception of zebrafish samples. In zebrafish however, use of the PowerFecal kit or the isopropanol method resulted in detection of a higher relative abundance of the phylum crobiota . While RCollectively, these data highlight the need to match appropriate fecal DNA extraction methods to the host species in question. As the feasibility of NGS increases and with rising pressure from funding agencies to enhance experimental reproducibility , it is p


Intestinal ischemia is a common complication of intestinal obstruction and arises from impaired perfusion. The resultant local and systemic inflammatory response and bacterial translocation come with a significant degree of morbidity and mortality. This study therefore aimed to investigate the predictive value of elevated levels of serum lactate and phosphate as biomarkers of intestinal ischemia among patients with mechanical intestinal obstruction.This was a cross-sectional analytical study done at Mulago Hospital in Uganda. Ethical approval was obtained. All eligible patients had a blood sample drawn for assay analysis. Determination of bowel ischemia status was by physical examination at laparotomy. Analyses were performed using Stata software, version 10.1, and 2 × 2 tables were used to calculate sensitivity and specificity.Serum lactate was predictive of bowel ischemia, while phosphate was not. Of the 81 patients enrolled 70 qualified for analysis; 40/70 (57%) had ischemic bowel, while 30/70 (43%) had normal bowel. Among those with ischemic bowel, 28/40 (70%) had reversible ischemia, and 12/40 (30%) had irreversible ischemia. Serum lactate assay had a sensitivity of 66% and specificity of 53% for bowel ischemia in general and a higher sensitivity of 71% and specificity of 80% for irreversible bowel ischemia.p = 0.011), PPV = 14%, but more significantly predictive of irreversible ischemia (p = 0.009), PPV = 42%. NPV for lactate in both forms of ischemia was 93%. Hernias  were the most common cause of intestinal obstruction.Lactate was predictive of bowel ischemia in general (Serum lactate assay had moderate sensitivity for bowel ischemia due to acute mechanical intestinal obstruction. The assay can be used to aid diagnosis of bowel ischemia in low technology settings. The diagnosis of intestinal ischemia is still a major challenge in sub-Saharan Africa where the acute form of intestinal ischemia caused by intestinal obstruction is most prevalent -4. It isMechanical intestinal obstruction, the most common cause of bowel ischemia in our setting -4, is onClinically useful markers of early gut ischemia would allow for more effective treatment and monitoring, which in turn would reduce the current high morbidity and mortality 10-40%) [0-40% 1--7,10-14.Serological biomarkers such as serum lactate and phosphate have been found useful and for now seem to offer some hope for solving diagnostic challenges associated with intestinal obstruction and ischemia -14. ThesThis was a cross-sectional analytical study.Mulago National Referral Hospital is the main teaching Hospital for Makerere University College of Health Sciences in Kampala. It is situated in Kampala, the capital city of Uganda, and has a 1,500-bed capacity, admitting up to 50 patients daily with various surgical emergencies to its Accident and Emergency Unit.The study was conducted from January to May 2012 inclusive.All patients admitted to the emergency and general surgical wards with a diagnosis of mechanical intestinal obstruction were included.Patients with potential causes of lactic acidosis, such as diabetic ketoacidosis, severe hypotension, and renal and hepatic failure, were excluded. Patients on antiretroviral drugs, especially stavudine, and those who died or whose symptoms resolved just before surgery, and those younger than 1 year were also excluded.Data were collected using a questionnaire, including patient demographics and bowel status. Bowel status was categorized as normal bowel, reversibly ischemic and irreversibly ischemic bowel, and serum levels of lactate and phosphate were analyzed. Eligible patients were counseled, consented and recruited by the investigators and assistants during the perioperative period.Under aseptic techniques, 5 ml of peripheral venous blood was drawn (avoiding the use of a tourniquet), 2.5 ml delivered to a gray-top fluoride container for lactate and 2.5 ml to a plain red-top container for phosphate. Both containers were delivered within 30 min to the laboratory for analysis or stored in a refrigerator in standing position at 4°C in cases when delay in delivery was anticipated. In the laboratory, samples were routinely centrifuged before analysis for 5 min at 10,000 revolutions per minute to obtain serum. The normal laboratory reference serum level ranges were 0.5–2.2 mmol/l for lactate and 0.9–1.5 mmol/l for phosphate .Intraoperatively, the researcher(s) and attending surgeon physically demonstrated and established the absence or presence of ischemia or its sequel by observing gut appearance, texture and the site involved, using clinical judgment that took into consideration the following criteria:Normal bowel was pink and glistening with normal peristalsis and normal arterial pulsations or mild alteration of one of them.Reversible ischemic demonstrated a red, dull sheen and reduced peristalsis or arterial pulsations that would improve drastically upon relief of the obstruction.Irreversible ischemic was dark or black, with a total loss of sheen and permanent reduction or absence of peristalsis and arterial pulsations.These findings were agreed upon by consensus of the operating surgeon, assistant and investigator(s).Tourniquet use was always avoided when drawing blood samples. Blood was not drawn from the cannulated limb (with an i.v. line/drip). Late night samples were kept at 4°C in standing position. All samples were centrifuged in the laboratory before analysis.Cobas Intergra 400 (Roche), based on an accredited (SANAS) laboratory in Mulago Hospital. Centrifuged samples (serum) were frozen at −20°C in case of the need for sample reruns.Random on-the-spot lactate measurements (with a portable lactate meter) were done before samples were delivered to the laboratory to serve as a control for the laboratory measurements. Sample handling was uniform—i.e., transportation and storage were only carried out by the investigator and one research assistant and the handling and processing by one and the same qualified laboratory technologist using the same well-calibrated machine, p values were ≤0.05. Two-by-two tables were used to calculate the sensitivity and specificity expressed as percentages.Analyses were performed using Stata software, version 10.1. Statistical tests included Mann-Whitney U and Fischer’s exact tests. The Kruskal-Wallis equality-of-population rank test was used for independent analysis of factors across three groups defined by bowel status . Cox regression analysis was used in both univariate and multivariate models. Significant Approval was obtained from the Research and Ethics Committee of Makerere College of Health Sciences, School of Medicine. Informed written consent was obtained from all participants and parental consent for all below 18 years. Those who were incapacitated at the time of data collection were consented later when they were able to consent.In this study, 81 patients who fulfilled the criteria were enrolled. Seventy of them had the outcome of interest, and their data were analyzed. Eleven patients were excluded because they had concomitant non-ischemic conditions such as gut perforations, which were not a result of prior obstruction.Table More males were seen  compared to women , and proportionately more males had both normal and ischemic bowel intraoperatively. The median age group was 38 years (IQR 52-25); however, those with ischemic bowel had a median age of 42 (IQR 64–24), relatively older than those with normal bowel . Patients with bowel ischemia had longer duration of symptoms than those with normal bowel, and the majority of patients with bowel ischemia had most of the signs. There were differences in the distribution of biomarkers. Biomarker levels were higher in patients with bowel ischemia than those with normal bowel as indicated in Figure A sensitivity of 66% and specificity of 53% for bowel ischemia in general were found, with a positive predictive value of 14% and negative predictive value of 93%. For irreversible ischemia, the sensitivity was 71% and specificity 80%, which correspond to a positive predictive value of 42% and negative predictive value of 93%. Participants with irreversible bowel ischemia had higher levels of serum lactate compared to normal and reversible bowel ischemia groups. There were no major differences in phosphate levels across groups, as shown in Table Kruskal-Wallis equality-of-population rank test, factors were compared and analyzed independently among three groups defined by bowel status . Significant factors were gender (0.03), abdominal pain duration (0.04), abdominal swelling duration (0.04) and lactate levels (0.01), as indicated in Table With the ender 0.0, abdominp-value, 0.03), while lactate had a persistently significant p-value (0.001 and 0.009) in both univariate and multivariate models, respectively. Phosphate levels were not significant in both analyses and models, indicating very low sensitivity and specificity , but they were slightly more than the reversible ischemia group, which had 28 (40%). For irreversible ischemia (12/40), gangrene was the most common feature; however, several patients had a mixed pattern of infarction, gangrene and necrosis.The ileum was the main part of the gut involved , followed by the colon , and the omentum  was the least. For all gut parts, there were comparatively more patients with ischemic bowel than normal bowel. Groin hernias were the main cause of intestinal obstruction (47%), followed by gut volvulus (23%), then adhesions (17%) and intussusceptions (14%).There was a weak correlation between lactate level and age and a weak negative correlation between phosphate level and age. There was no difference in the distribution of serum levels of lactate and phosphate by gender. There was no correlation of duration of symptoms and serum levels of both lactate and phosphate.Costs per test were US$ 2 and US$ 3 for lactate and phosphate assays, respectively.We set out to investigate the diagnostic value of the association between serum lactate and phosphate in relation to bowel ischemia. There was an association between serum lactate and bowel ischemia, but not between serum phosphate and bowel ischemia. The population studied was mostly male, with a male:female ratio of 2:1, though the bioassays didn’t vary by gender. The mean age was 39 years; this would be considered young in the context of mechanical intestinal obstruction.The role of serum lactate in predicting bowel ischemia was modest; it was far below the higher values of sensitivity found by several studies ,15. HoweGearhart et al. found 100% sensitivity for intestinal ischemia with a combination of alpha-glutathione S-transferase and lactate . This hiPhosphate had no association with bowel ischemia in this study, which is a departure from the findings of Jamieson , who foup = 0.009). For phosphate, it was insignificant (p = 0.960). The weak correlation with phosphate was further confirmed ; long symptom duration is considered a risk factor for bowel ischemia [The majority of patients had ischemia 40/70 (57%). The median duration of abdominal pain was 72 h (IQR 120-36) (Ileum was the most common part of the gut involved in the obstruction (52%), mainly contained in obstructed/incarcerated hernias, which was also the most prevalent intraoperative diagnosis. These findings correspond rather accurately with those of Kiyengo  where thIn this study the main causes of mechanical intestinal obstruction were hernias (33%). The reason for the prevalence of intestinal obstruction caused by hernias is that many patients live with untreated hernias as opposed to western countries where access to care is early. Other causes included sigmoid volvulus 14%), adhesions and intussusceptions in declining order of proportion. These study findings are similar to those of MacAdam (1960) 4%, adhes, who regIn western countries, mechanical intestinal obstruction  is mainly caused by post-surgical adhesions in over 60% of cases ,22.Though serum lactate is non-specific for bowel ischemia, it may be useful in settings where high-level investigative abilities are limited. The physical assessment of bowel status has limited accuracy (70–89%) ,24. The Patients with mechanical intestinal obstruction are operated on as soon as they are resuscitated, but sometimes logistical limitations lead to delays. Whereas the majority of patients did not wait longer than 6 h, it is possible that during this waiting time significant changes in bowel status and biomarker levels occurred. From the single blood sample drawn, it was not possible to observe the ongoing changes in biomarker levels. Large-scale studies are needed especially in sub-Saharan Africa to further explore the role of serum lactate and phosphate assays in aiding bowel ischemia diagnosis.Serum lactate assay had moderate sensitivity for bowel ischemia caused by acute mechanical intestinal obstruction in a sub-Saharan Africa resource-limited setting. This assay may augment the diagnosis of bowel ischemia in the emergency setting.I.V: Intravenous; IQR: Interquartile ratio; NPV: Negative predictive value; PPV: Positive predictive value.The authors declare that they have no competing interests.GM conceived the concept. KLR collected the data, participated in the analysis and wrote the first draft. GM and OFN contributed to revising the drafts. GM performed critical reviews of the manuscript. All authors approved the final manuscript.


Pigs living in intensive husbandry systems may experience both acute and chronic stress through standard management procedures and limitations in their physical and social environment, which may have implications for their immune status. Here, the effect of a new breeding method where pigs were selected on their heritable influence on their pen mates' growth, and environmental enrichment on the immune status of pigs was investigated. Hereto, 240 pigs with a relatively positive genetic effect on the growth of their pen mates (+SBV) and 240 pigs with a relatively negative genetic effect on the growth of their pen mates (−SBV) were housed in barren or straw-enriched pens from 4 to 23 weeks of age . A blood sample was taken from the pigs before, three days after a 24 h regrouping test, and at week 22. In addition, effects of coping style, as assessed in a backtest, and gender were also investigated. Mainly, +SBV were found to have lower leukocyte, lymphocyte and haptoglobin concentrations than -SBV pigs. Enriched housed pigs had a lower neutrophil to lymphocyte (N:L) ratio and lower haptoglobin concentrations, but had higher antibody titers specific for Keyhole Limpet Hemocyanin (KLH) than barren housed pigs. No interactions were found between SBV class and housing. Furthermore, pigs with a proactive coping style had higher alternative complement activity and, in the enriched pens, higher antibody titers specific for KLH than pigs with a reactive coping style. Lastly, females tended to have lower leukocyte, but higher haptoglobin concentrations than castrated males. Overall, these results suggest that +SBV pigs and enriched housed pigs were less affected by stress than -SBV and barren housed pigs, respectively. Moreover, immune activation might be differently organized in individuals with different coping styles and to a lesser extent in individuals of opposite genders. In response to stressful situations, the HPA-axis and sympathetic nervous system are activated which subsequently results in the release of glucocorticoids and catecholamines which prepare the body to fight or flight Pigs in intensive farming systems experience acute stress during standard management procedures such as castration, tail docking, abrupt weaning, regrouping and transport One solution to diminish these negative side effects of stress may be provided by genetics The aim of this study was therefore to investigate both the separate and interacting effects of this new breeding method and housing on the immune status of pigs. Furthermore, the coping style of the pigs was also taken into account, because pigs with different coping styles do not only respond differently to acute and chronic stress The experiment described in this study was approved by the Animal Care and Use Committee of Wageningen University (Protocol Number: 2010055f). Blood sampling was carried out by trained assistants and done as quickly as possible to minimize suffering.2/pig) with a partially slatted and partially concrete solid floor. Barren housed pigs received two hands of wood shavings each day from 6 weeks of age onwards. The other half of the pigs were housed in pens (∼1 m2/pig) enriched with 1.5 kg of straw and 12 kg of wood shavings. All pens were cleaned daily and afterwards 3 kg of fresh wood shavings and fresh straw  were added to the enriched pens. In all pens, a metal chain with a ball and, from 8 weeks of age, a jute sack were attached to the wall of each pen. The jute sack was replaced when needed.The pigs in this study −480 in total, equally divided over five batches - were the same pigs as described in Reimert et al. Each group of pigs consisted of three gilts and three barrows and at least two high-resisting (HR) and two low-resisting (LR) pigs (see section 2.2.1.) and groups diverged in indirect genetic effects for growth, i.e. the heritable effect on the growth of their group members. All pigs in a pen had either an estimated relatively positive indirect genetic effect  or an estimated relatively negative indirect genetic effect on the growth of their pen mates  during the finishing period (from app. 25 to 110 kg). During this period, the growth of a pig is theoretically affected by each of its pen mates which means that the total estimated indirect genetic effect on a pig's growth in this experiment was 10 g/day ((6−1) * 2.00) for the +SBV pigs and −8.1 g/day ((6−1) * −1.62) for the −SBV pigs. Pigs were obtained by mating Topigs-20 sows and Tempo boars with the most extreme positive or negative estimated indirect genetic effects for growth that were available for each batch. Direct breeding values for growth were kept as similar as possible for both SBV classes  breeding value estimations see Camerlink et al., 2013). The study was approved by the Animal Care and Use Committee of Wageningen University.Pigs were subjected to the backtest at approximately two weeks of age At 9 weeks of age, pigs were exposed to a regrouping test Blood was collected from the pigs in the week before the regrouping test at 8 weeks of age, three days after the regrouping test at 9 weeks of age, and at 22 weeks of age . Hereto,In the laboratory, the serum separating tubes were incubated for one hour at 37°C after which they were centrifuged at 5251 g for 12 min at 20°C. Obtained sera were stored at −80°C until further analysis . Blood from the EDTA tubes was used directly (section 2.3.3).The hemolytic activity of both CPW and APW complement was measured using the hemolytic complement assay of Demey et al. 2CO3 × 10 H2O, pH 9.6). After washing with tap water containing 0.05% Tween 20, serial dilutions of serum were added and incubated for one hour at RT. After washing, plates were incubated for one hour at RT with a 1:20000 diluted peroxidase (PO)-conjugated goat antibody directed to swine IgGFC  to detect binding of IgG and with 1:20000 diluted peroxidase (PO)-conjugated goat antibody directed to swine IgMFC  to detect binding of IgM, respectively. After washing, tetramethylbenzidine and 0.05% H2O2 were added as a substrate and incubated for 10 min at RT. The reaction was stopped with 2.5N H2SO4 and the absorbance was measured at 450 nm with a Multiskan . Each absorbance was expressed relatively to the absorbance of a standard positive control serum and antibody titers were determined as described in Schrama et al. Antibody titers of IgG and IgM specific for Keyhole Limpet Hemocyanin (KLH) 9/l) was determined with a Sysmex F-820  and with 10 µl, a smear was made on a microscope plate. After the smears had dried, they were fixed with a methanol solution and thereafter stained using a rapid staining kit . Surplus staining solution was washed away with PBS and then smears were dried. The percentages of lymphocytes, neutrophils, monocytes and eosinophils ) were determined by microscopic examination of the smears and counting 100 leukocytes in total using an Assistant-Counter AC-8. From these counts, the neutrophil:lymphocyte ratio (N:L ratio) was determined. Furthermore, the concentration of lymphocytes (109/l) was determined by multiplying the percentage of lymphocytes with the leukocyte concentration.With 150 µl of the blood in the EDTA tubes, the concentration of leukocytes  which has been validated for pigs . Briefly, 100 µl of hemoglobin was added to 7.5 µl serum and solutions were gently mixed. Thereafter, 140 µl of chromogen was added and incubated for 5 min at RT. The absorbance was read immediately at 600 nm in a microplate reader. Haptoglobin concentrations (mg/ml) were calculated by using a standard linear curve with known concentrations of haptoglobin.SAS  was used for all statistical analyses. Variables could not be obtained from all 480 pigs at each sampling period . This waTo investigate the effect of SBV class, housing, backtest classification and gender on the variables after acute stress, the delta between week 9 and week 8 was calculated and subsequently analyzed with a linear mixed model with SBV class, housing, backtest classification, their two-way interactions, gender, and batch as fixed effects and pen, nested within SBV class, housing, and batch, as random effect. Prior to this analysis, CPW complement activity was log transformed to obtain normality of residuals.For brevity, only significant interactions are reported. Significant interactions were further investigated with post hoc pairwise comparisons using the differences of the least square means. Results are presented as means ± SEM.CPW complement activity was not affected by SBV class, housing or backtest classification (P ≥ 0.29). There was, however, an effect of week on CPW complement activity (P <0.001) , with hiAPW complement activity was affected by backtest classification (P <0.01) and week (P <0.001) . HR pigsKLH-IgG titers were affected by housing (P <0.001) and week (P <0.001) and by the interaction between housing and backtest classification (P <0.05) . Post hoKLH-IgM titers were affected by week (P <0.001) and the interaction between housing, backtest classification and week (P <0.001) . Post hoLeukocytes, lymphocytes and the ratio of neutrophils to lymphocytes (N:L ratio).9/l). The concentration of leukocytes was also affected by week (P <0.001) and by the interaction between housing and week (P <0.05) , but was not affected by housing or backtest classification (P ≥ 0.19).The concentration of leukocytes was affected by SBV class (P <0.05). Pigs with a +SBV had, overall, lower concentrations than −SBV pigs (17.8 ± 0.2 vs. 18.6 ± 0.2 10P <0.05) . Post hoP <0.05) . Leukocy9/l). Furthermore, lymphocyte concentrations were lower in week 22 compared to weeks 8 and 9. Lymphocyte concentrations were not affected by housing or backtest classification (P ≥ 0.22). The delta in the lymphocyte concentration between weeks 8 and 9 was also not affected by SBV class, housing or backtest classification (P ≥ 0.20).The concentration of lymphocytes was also affected by SBV class (P <0.05) and week (P <0.01) . Pigs wiThe N:L ratio was affected by housing (P <0.05) and week (P <0.001) . EnricheHaptoglobin concentrations tended to be affected by SBV class (P <0.1) . Concent9/l, P <0.10) and higher haptoglobin concentrations .Gender did not affect any of the immune variables, except that over the three sampling points, gilts tended to have lower leukocyte concentrations than barrows  and environmental enrichment on the immune status of pigs.In response to acute stress or inflammation, the acute phase response is activated which results, amongst others, in an increase of acute phase proteins, such as haptoglobin, and in complement activation The other effects of week on the variables measured could point to an effect of age. For instance, the increase in KLH-IgG and KLH-IgM natural antibody titers from week 8 to 9 to 22 is in line with other studies reporting rising natural antibody titers with age Even though the pigs in this study were all relatively healthy and were not deliberately immunologically challenged, SBV class, housing and coping style did have clear effects on the immune variables measured. These effects were mainly found for levels of the immune variables per se and only few effects on their change due to regrouping stress. It should be stated that the amount of fresh lesions counted after regrouping did not differ between enriched and barren housing and between the two SBV classes Effects of housing were found on all variables measured except for complement activity and lymphocyte concentrations. Enriched housed pigs had, partly in line with other studies, overall a higher KLH-IgG titer Most notably, +SBV pigs had overall lower concentrations of leukocytes and lymphocytes and tended to have overall lower haptoglobin concentrations than −SBV pigs. In addition, the decrease in the concentration of leukocytes from week 8 to 9 tended to be larger for +SBV pigs than for −SBV pigs, suggesting that they responded differently to the 24 h regrouping test at 9 weeks of age.Although no differences were found in the number of skin lesions after regrouping, indicating no major differences in aggression, after reunion −SBV pigs tended to act more aggressively towards their own original group members, indicating that they differently coped with the stress of regrouping. This seems to be in line with their response to other stressful situations. Previously, it was found that these +SBV pigs behaved somewhat less fearful than the −SBV pigs in several novelty tests No interactions were found between SBV class and coping style which is in agreement with earlier findings on the behavior of these pigs Interestingly, the differences between the +SBV and −SBV pigs are comparable with the differences found between enriched and barren housing: pigs housed in a better physical environment (i.e. enriched pens) may experience less chronic stress and pigs housed in a better social environment (i.e. +SBV pens) also seem to experience less chronic stress. In addition, no interactions were found between SBV class and housing condition which is in line with behavioral results of these pigs In many different animal species two extremes in coping style or personality have been described: proactive and reactive In this study we also found immunological differences between HR and LR pigs, even though we simply divided the population of pigs into HR or LR and did not use the extremes of the population as was done in earlier pig studies Gilts were found to have lower concentrations of leukocytes, but higher haptoglobin concentrations than barrows. The barrows were surgically castrated at 3 days of age which could explain their higher concentrations of leukocytes, because Prunier et al. Environmental enrichment is known to alleviate stress in animals. In this study, enriched housed pigs were found to have a lower N:L ratio and lower haptoglobin concentrations than the barren housed pigs which indeed suggests that enrichment has stress-reducing effects. Stress-reducing effects were also seen in pigs selected for a relatively positive genetic effect on the growth of their pen mates (i.e. +SBV pigs), because these pigs had lower leukocytes, lymphocyte and haptoglobin concentrations compared to pigs that were selected for a relatively negative genetic effect of the growth of their pen mates. Together these results indicate that both genetics and environmental enrichment can be used to improve the welfare of pigs and that the use of both together likely yields the best results.Two effects of gender were found, but these effects were rather subtle. On the other hand, clear differences were found between pigs with a proactive or reactive coping style. Pigs with a proactive coping style seemed to have a more active innate immune status compared to pigs with a reactive coping style, pointing to a difference in dealing with immune challenges. The biological relevance of the results and their implications for health merit further research.


Gokushovirinae were common in both environmental datasets. Analysis of haloarchaeal virus contigs revealed the presence of three contigs distantly related with His1, indicating a possible new lineage of salterproviruses in the Hosabes playa. Based on viral richness and read mapping analyses, the salt pan metaviromes were novel and most closely related to each other while showing a low degree of overlap with other environmental viromes.Viral communities of two different salt pans located in the Namib Desert, Hosabes and Eisfeld, were investigated using a combination of multiple displacement amplification of metaviromic DNA and deep sequencing, and provided comprehensive sequence data on both ssDNA and dsDNA viral community structures. Read and contig annotations through online pipelines showed that the salt pans harbored largely unknown viral communities. Through network analysis, we were able to assign a large portion of the unknown reads to a diverse group of ssDNA viruses. Contigs belonging to the subfamily  Research into the viral community structure of saline environments has focused mainly on marine environments with a limited number of studies looking at moderate or hypersaline lakes and solar salterns ,4,5,6,7.Haloquadratum walsbyi and Salinibacter ruber [H. walsbyi was identified and named EHP1, but host assignment was very speculative [H. walsbyi [Isolation studies of hypersaline environments have revealed four main types of virus morphologies; spindle shaped or fusiform, head-tail, icosahedral and pleomorphic, while filamentous and rod-shaped viruses have also been observed by transmission electron microscopy (TEM) ,17,18,19er ruber . Using cculative . Fosmid- walsbyi . The div walsbyi , limited walsbyi  and one  walsbyi . Thus, m4·2H2O), with the Hosabes playa also containing the rare nitrate mineral Humberstonite (K3Na7Mg2(SO4)6(NO3)2·H2O) [In this study, we have sequenced metaviromes of two different salt pans (playas) in the Namib Desert, namely the Hosabes playa and another near the town of Swakopmund (Eisfeld). Playas found in the Namib Desert are moist, salt-covered, sediment-filled depressions which form in drainage channels whose surface and groundwater flow is obstructed by linear bedrock outcrops, in particular dolerite dykes, and their mineralogy and geochemistry has been well-studied by others ,32,33,343)2·H2O) ,31,32,333)2·H2O) , and theThe Hosabes and Eisfeld playas were identified as sites of interest for bioprospecting for novel enzymes, because of their salt content and their unexplored nature. The use of metaviromics for the exploration of new enzymes was suggested previously, taking into account the high density of coding sequences in viral genomes and the absence of a cultivation step . This waThe two sample sites were located in the Namib Desert approximately 124 km apart, one near the Gobabeb Training and Research Station  at 23°30′25.75′′ S, 15°4′16.50′′ E, about 100 km inland from the town of Walvis Bay, while the Eisfeld site is located 21.5 km North East of Swakopmund  at 22°29′5.02′′ S, 14°34′18.06′′ E [Further sample processing occurred at the Institute for Microbial Biotechnology and Metagenomics (IMBM) at the University of the Western Cape (South Africa), where the concentrates were stored for two weeks at 4 °C until processing.On site, metadata was collected (from 2010 to 2015). The pH was measured with narrow range strips . The temperature and conductivity were measured using probes  which were either submersed in the stream or in water collected with a syringe from the stream and measured in a falcon tube. The conductivity meter was calibrated at 25 °C and 12.88 mS/cm buffer solution. Total dissolved solids and total salinity were calculated based on the conductivity at 25 °C.w/v solution of polyethylene glycol (PEG) 10,000. The dialyzed samples were inspected using fluorescence microscopy and no noticeable decrease in viral particles was observed. Virus particles were collected by centrifugation in a Beckman JA20 rotor at 43,667× g for 6 h in autoclaved 30-mL Nalgene polypropylene copolymer (PPCO) tubes, and the individual pellets were resuspended in 1 mL Tris buffer. The suspension was treated with DNase I  and RNase A  at a final concentration of 0.1 μg/mL at 37 °C for 2 h. The efficacy of DNAse treatment was assessed in two ways. The first was to remove an aliquot of the sample to which DNAse had been added and combining it with bacterial genomic DNA which was incubated at 37 °C alongside the sample. This digest was analyzed by agarose gel to determine if the bacterial genomic DNA was degraded. A portion of the DNAse treated sample was then used as template for PCR (35 cycles) to amplify bacterial 16S rRNA genes, including controls spiked with bacterial genomic DNA to check for PCR inhibitors from the sample. If no product was detected when loading the entire PCR reaction (50 μL) on an agarose gel, we proceeded with DNA extraction. Proteinase K (Fermentas) was added to a final concentration of 1 μg/mL and the suspension incubated at 55 °C for 2 h. SDS was added to a final concentration of 0.6% (w/v) and incubated at 37 °C for 1 h. Nucleic acids were purified by performing two rounds of phenol-chloroform-isoamyl alcohol (25:24:1) phase separation followed by one round of chloroform-isoamyl alcohol (24:1) phase separation. DNA was precipitated by the addition of 1/10 volume of sodium acetate  and 2 volumes of 100% ethanol at 4 °C overnight. Samples were centrifuged at 29,000× g for 10 min to pellet the DNA, which was resuspended in 30 μL of TE (Tris-EDTA) buffer. The two separate DNA extractions per site were pooled for the whole genome amplification. Ten nanograms of DNA from each sample was used to perform Phi29 amplification  using the manufacturer’s recommendations. The DNA was purified using the Qiagen gel extraction kit  for use in preparation of sequencing libraries. Library preparation included a 10% phiX V3 spike as per the manufacturer’s instructions (Illumina Nextera XT guide) with the Illumina Nextera XT library prep kit/MiSeq reagent kit V2 . Three libraries were constructed for DNA from each site, to capture a wider variety of DNA species for sequencing. The amplified DNA was sequenced 2 × (forward and reverse sequencing) 250-bp reads, on the Illumina MiSeq platform located at the University of the Western Cape, Cape Town, South Africa.Two independent DNA extractions were performed per site (GS and SS), using 25 mL of TFF  sample per extraction, to limit any bias arising from the DNA extraction protocol. To ensure that DNAse and RNAse treatments performed later in the protocol were successful, the sample was first dialyzed against 5 L of 100 mM Tris buffer (pH8.0) using Snakeskin™ dialysis tubing  to remove excess salt. The suspension was concentrated down to the original 25 mL by dialysis against a 10% The quality of the raw read files was first checked with FastQC  getting The post-QC reads were assembled using CLC Genomics Workbench as paired files (3 × 2 read files per site), using the default settings, and any residual contigs showing more than 99% sequence identity with phiX were removed. Additional contamination screens were performed using the whole genome shotgun (WGS) submission pipeline of the National Center for Biotechnology Information (NCBI) and any contigs showing contamination were removed. For GS, the assembly resulted in 11,304 contigs with a minimum length of 200 bases at an N50 of 530 and a maximum of 74,962 bases. The assembly of the SS reads gave 22,352 contigs of minimum 200 bases at an N50 of 457 and a maximum contig size of 23,854 bases. These contig files were also submitted to MetaVir and VIROME for analysis ,40,41. Te values <0.001 or BLASTp against the RefSeq virus database including conserved domain search , the latter performed by MetaVir. When multiple taxonomic assignments were found, the assignment was made at a higher taxon. For example, a contig with genes showing similarity to the Podoviridae family and another with genes similar to those of the family Siphoviridae within the same network resulted in an assignment of the order Caudovirales for the network.Contig networks for the Namib Saline sites were generated by first mapping the post-QC reads to their respective contigs using bowtie2 . The reaPhylogenetic trees were generated with MEGA6  using clFor the read mapping analysis of GS and SS, the read files were merged into one file per metavirome. An additional phiX removal step was included as well as a removal of potential human contamination (hg18 bowtie2 reference index) using bowtie2 at setting very-fast, resulting in 15,207,796 reads for GS and 15,465,794 reads for SS. A reference set of metaviromes was downloaded from MetaVir and GenBank as fasta files and used to build a reference with bowtie2-build at the default settings. Alternatively, the GS and SS contigs were used as a reference. Mapping was performed at the very-fast setting using unpaired reads, which means that the mapping algorithm used only 5 iterations before assigning a mapped read which needed to align over its entire length with no mismatches allowed. Some metavirome files could not be converted into reference index files due to their large sizes. To assess the contigs to which the reads were mapped, the samtools package was used to extract all the mapped reads (samtools view—F 4) .To cluster metaviromes based on the known virus fraction, the taxonomic composition of all dsDNA contig datasets available from MetaVir was downloaded (January 2015), including those of the Namib saline sites, GS contigs and SS contigs. The table was expanded to the species level, loaded into R and transformed into a presence/absence table. Bray-Curtis dissimilarities were calculated using the Vegan package, clustered using hclust, and subsequently plotted in R.GS and SS contigs were annotated using the MetaVir pipeline, using MetaGeneAnnotator software  and BLAS2+, Cl−, Mg2+, SO42−, and CO32− present within the spring water. The salinity range based on the measured conductivity was 4.1%–7.5%. For the Eisfeld playa, where the Swakopmund Saline (SS) metavirome was sampled, the pH ranged from 6.5 at the source to 8.5 at the sink. The conductivities for this site reached a higher maximum, ranging from 66–180 mS/cm, giving a TDS range of 42,000–115,000 ppm and a salinity range of 4.5%–8.6%. These values agree well with those reported previously [The Hosabes spring, source of the Gobabeb Saline (GS) metavirome, had a variation in pH from 7.0–7.5 at the source and 9.0–9.5 at the sink, measured over multiple years (2010–2015). The electrical conductivity also displayed a range 61–103 mS/cm) with the lowest conductivities measured at the source and the highest at the sink. This translated into a total dissolved solids (TDS) range of approximately 39,000–66,000 ppm, giving a measure of the number of unbound inorganic solutes such as Naeviously ,33. Theseviously . However mS/cm wieviously ,57.The taxonomic breakdown of the Gobabeb (GS) and Swakopmund Saline (SS) metaviromes is described in To provide a general taxonomic assignment to the unknown reads and contigs, we performed a network analysis. The networks in Inoviridae (network 2), Microviridae , or Geminiviridae (network 17). While the majority of contigs belonging to the families Inoviridae and Microviridae most likely represent bacteriophages, the Geminiviridae family contains plant viruses of which members of the genera Mastrevirus and Begomovirus have been known to infect tropical plants/crops in Sub-Saharan Africa [Myoviridae family while the remaining 11 networks were completely unaffiliated. For the 24 networks, 96 of the 108 contigs were classified as unknown environmental sample by MetaVir. As a result of the network assignments, we managed to increase the number of annotations and now only 39 out of 108 contigs remain completely unknown.For the Gobabeb saline spring, 24 networks contained at least one contig with over 1000-fold coverage . Eight nn Africa . Two of n Africa . The autCircoviridae , Microviridae, specifically the subfamily Gokushovirinae , Inoviridae (network 20), or to unassigned ssDNA viruses . The increased presence of circoviruses, the majority of which infect avian species [In the Swakopmund Saline dataset, 29 networks had at least one contig with over 1000-fold coverage, for a total of 154 contigs . The sam species , in the  species . Many ofIn both GS and SS datasets, several contigs with phage integrase genes were identified see . In the The high prevalence of ssDNA genomes in these datasets is most likely caused by the multiple displacement DNA amplification method (MDA), using the phi29 polymerase. Recent research has indicated that MDA preferentially amplifies circular ssDNA viruses when present ,64,65.Microviridae members in general, were identified. To investigate the presence and diversity of potential genomes belonging to this family, all presumed circular contigs with lengths over 4000 bp were extracted from the datasets through MetaVir, and the major capsid protein amino acid sequences were compared to those of selected isolates of the family Microviridae and the major capsid proteins (VP1) of a set of 81 assembled genomes from metavirome datasets [Gokushovirinae and “Pichovirinae”. The gokushovirus group was the most diverse, containing the majority of the genomes of the Namib saline metaviromes, as well as genomes of marine, freshwater and human virome origins. With these additional capsid protein sequences, there seems to be a mostly environmental clade and two human-associated clades within the subfamily Gokushovirinae, with two GS capsid proteins clustering with a human-associated clade. It is possible that the two GS genes belong to phage genomes associated with bacteria present in animal feces . A division into two groups was also witnessed after a PCR amplification study, giving rise to a eukaryote-associated clade and an environmentally-dominated clade [Gokushovirinae subfamily is ubiquitously distributed and therefore probably of importance in general microbial ecology [Chlamydia phage isolates which were used for primer design [In the network analysis, many contigs with similarity to marine gokushoviruses, or datasets . The resed clade . Additio ecology ,68,69. A ecology . Additio ecology ,70. Hower design .Microvirus. Alpaviruses have so far only been found in human-associated viromes [, and the lack of sequences clustering with this group could indicate an absence of human contamination. It is interesting to note that in this analysis, the phiX174-related viruses of the Microvirus genus form a clade which is nested within the “Alpavirinae” clade, albeit with low bootstrap support. Two distinct clades of alpaviruses are also found in the phylogenetic analysis of Roux and colleagues, indicating that this subfamily could be split as more microviruses are sequenced [No capsid proteins of the saline metaviromes in this study were clustered with the “Alpavirinae” or members of the genus  viromes ,71, and equenced .Haloarcula hispanica and has been isolated from Lake Victoria, Australia [Salterprovirus. Rather, taking into account the conservation of the structural proteins, these genomes could be the first representatives of a candidate “Salterproviridae” family.The saline metavirome datasets were mined for contigs showing resemblance to known haloarchaeal viruses. This analysis revealed 152 contigs for GS and 35 contigs for SS which showed varying degrees of similarity. The majority of these signatures belonged to viruses informally categorized as Haloviruses, comprising a group of head-tailed archaeal viruses with myovirus or siphovirus morphologies . To inveustralia ,73. GS custralia , had couSulfolobus spindle-shaped virus 1 (SSV1) and Acidianus spindle-shaped virus 1 (ASV1) which share morphological characteristics with His1, and the newly classified gammapleolipovirus His2  which infects the same host as His1. The three metaviromic putative capsid proteins clustered with that of His1 virus, and were also distantly related to the fuselloviruses, while His2 was practically unrelated. All putative capsid proteins contained two hydrophobic transmembrane domains (data not shown) which were found to be characteristic for fusello-like archaeal viruses (or tailless spindle-shaped viruses) but are not present in members of the family Bicaudaviridae (tailed spindle-shaped viruses) [Salterprovirus and the metavirome contigs, shares an evolutionary relationship with fuselloviruses. Alternatively, as previously suggested by Krupovic and colleagues, all tailless spindle-shaped viruses could be grouped into the family Fuselloviridae and be recognized as different genera, with the genus Salterprovirus becoming “Epsilonfusellovirus” [As the capsid gene is widely used as a phylogenetic marker, the evolutionary relationship between VP1 of the three putative genomes and isolated archaeal viruses was investigated in an amino acid-based phylogenetic tree . We inclviruses) ,75. BaseTo investigate the degree of similarity between GS, SS and other metaviromes, two strategies of read mapping were used. In a first strategy, reads from a set of 44 publically available metaviromes, including ssDNA only and hypersaline metaviromes, were mapped to the GS or SS contigs . In the Of the Gobabeb Saline reads, 92.7% mapped to their own contig dataset, illustrating that the assembly represented almost the entire read dataset. For the Swakopmund data, this was slightly higher at 93.2%. When mapping the two metaviromes against each other, 57.7% of the GS reads mapped to the SS contigs and 55.4% of the SS reads mapped to the GS contigs. This very high degree of similarity between the two saline metaviromes is indicative of a high similarity in the viral community structures, supporting the hypothesis that these similar environments  select for a similar community composition despite the geographic distance (approximately 124 km apart).Since MDA preferentially amplifies ssDNA, we compared the saline reads and contigs with three ssDNA metavirome datasets collected from different ocean regions . This yiComparing the GS and SS reads and contigs with other saline metaviromes, we found that the Namib sites were markedly novel. For the solar saltern metaviromes , no simFor the Gobabeb saline metavirome, over 1000 reads mapped to the reads of freshwater Lake Michigan metaviromes , and to a seawater metavirome read dataset, Indian Ocean virome sample GS108 . The diCellulophaga phage phi47:1, a myovirus isolated from seawater [Circoviridae capsid protein had the majority of mapped SS reads . For the AOS contigs, the SS reads mapped to a range of contigs described as environmental samples in MetaVir, with some hits to the virus families Myoviridae, Mimiviridae and Nudiviridae. We therefore conclude that the seemingly high similarity between the Antarctic soil and the Namib saline metaviromes is caused by a limited number of shared genes possibly belonging to a small fraction of globally ubiquitous viruses.The results of the read mapping of GS and SS reads against metavirome contigs revealed a high degree of similarity between both GS and SS, and the Antarctic hypolith (AH) and open soil (AOS) metaviromes . Given seawater . This exseawater . This waFuselloviridae and Rudiviridae which specifically infect extremely thermophilic and/or acidophilic hosts.Since MDA of the metaviromic DNA is likely to introduce a bias towards ssDNA virus taxa, we conducted the comparison of the known viral groups solely based on presence/absence of viral species in the contig datasets. Hierarchical clustering of the Bray-Curtis dissimilarity matrix confirmed the result from the read mapping data, that the Gobabeb and Swakopmund saline metaviromes were most closely related to each other . All theWe also noted the absence of the recently discovered crAssphage, a genome which was cross-assembled from human gut metaviromes and which has been shown to be ubiquitously distributed in public metagenomes . ShortlyThe Namib saline metaviromes share a high degree of similarity in their sequence space and are distinct from other published metaviromes. MDA of the virome DNA has revealed a high richness of ssDNA viruses, from potential phages to insect, plant and bird virus signatures. Almost all of the known haloarchaeal virus genomes have homologs in these metaviromes, suggesting that these salt pans are ideally suited for the discovery of new archaea-infecting viruses. Furthermore, the presence of many tailed phages makes these sites useful targets to mine for phage enzymes such as DNA polymerases for industrial applications.


Early life adversity increases anxiety in adult rodents and primates, and increases the risk for developing post-traumatic disorder (PTSD) in humans. We hypothesized that early adversity impairs the use of learning signals -negative, aversive prediction errors–to reduce fear in uncertainty. To test this hypothesis, we gave adolescent rats a battery of adverse experiences then assessed adult performance in probabilistic Pavlovian fear conditioning and fear extinction. Rats were confronted with three cues associated with different probabilities of foot shock: one cue never predicted shock, another cue predicted shock with uncertainty, and a final cue always predicted shock. Control rats initially acquired fear to all cues, but rapidly reduced fear to the non-predictive and uncertain cues. Early adversity rats were slower to reduce fear to the non-predictive cue and never fully reduced fear to the uncertain cue. In extinction, all cues were presented in the absence of shock. Fear to the uncertain cue in discrimination, but not early adversity itself, predicted the reduction of fear in extinction. These results demonstrate early adversity impairs the use of negative aversive prediction errors to reduce fear, especially in situations of uncertainty. Early adversity increases anxiety in adult rodents . The +APE is then broadcast to relevant brain regions to strengthen the cue-shock association and increase fear to the cue. When the same cue is encountered again, the prediction will be higher (0.5). If no shock occurs on this trial, the result is surprisingly better than expected. As a result there will be a negative discrepancy between the actual shock and the prediction (0.0 − 0.5 = −0.5). The result is a negative APE (−APE) that will weaken the cue-shock association and reduce fear to the cue. The use of APEs allows subjects to appropriately adjust levels of fear to cues that predict aversive events with uncertainty.During probabilistic reinforcement, a single cue predicts aversive foot shock on 25% of trials so the outcome of any particular trial is uncertain. Because this cue is initially neutral, there is no prediction the shock will occur. The first time the cue is followed by a shock, assigned an arbitrary value of 1.0, the result is surprising and aversive. The discrepancy between the actual shock and the prediction (1.0 − 0.0 = +1.0) results in a We hypothesize that early adversity disrupts the use of −APEs to reduce fear in uncertainty. To test this hypothesis, we gave adolescent rats a 10-day battery of adverse experiences. In adulthood, we assessed their ability to reduce fear via −APEs using a Pavlovian fear conditioning procedure. Three initially neutral cues were then associated with different probabilities of shock: 100, 25, and 0%. We have found that rats initially acquire high levels of fear to all cues, but come to reduce their fear to the 25 and 0% cues  rats were slower to reduce their fear to the 0% cue and never fully reduced fear to the uncertain cue. The level of fear demonstrated to the uncertain cue during discrimination strongly and significantly predicted fear during extinction in all rats. Thus, increased adult anxiety, by way of Early Adversity, may specifically reflect an inability to use APEs to reduce fear. These results have implications for early adversity experiences and the long term, adult risk for anxiety disorders such as PTSD.Subjects were 22 male Long Evans rats approximately 21 days old on arrival, obtained from Charles River Laboratories, and maintained on a 12-h light cycle (lights off at 6:00 PM). Rats were individually housed with food and water freely available. From arrival through postnatal day 56, rats were weighed three times per week: Monday, Wednesday, and Friday. Starting on P56, rats were maintained at 85% of their free-feeding body weight for the duration of Pavlovian fear conditioning. Water was always freely available. All protocols were approved by the Boston College Animal Care and Use Committee and all experiments were carried out in accordance with the NIH guidelines regarding the care and use of rats for experimental procedures.Forced swim took place in a clear, 10 L plastic cylinder. Tail pinch and cat hair exposure took place in a clear, plastic mouse cage free of bedding. Restraint occurred in a clear plastic restraint. The apparatus for Pavlovian fear conditioning consisted of eight individual chambers with aluminum front and back walls, clear acrylic sides and top, and a metal grid floor. Each grid floor bar was electrically connected to an aversive shock generator . A single external food cup and central nose poke opening equipped with infrared photocells, were present on one wall. Auditory stimuli were presented through two speakers mounted on the ceiling of each chamber.Rats arrived to the animal facility on postnatal (P) 21 Figure . From P2EA Rats were placed in 10 L of 10°C water for 5 min. The water level was such that rats could neither reach the bottom nor the top of the container. When 5 min expired, the rat was dried vigorously with a towel and returned to his home cage.Tail pinch took place in a clear, plastic mouse cage free of bedding. A half-inch mini binder clip was placed on the base of each rat's tail for 5 min. When 5 min expired, the binder clip was removed and the rat was returned to his home cage.A ball of hair obtained from a cat certified to be disease-free by a veterinarian was placed in a hair net and suspended from the wire ceiling of a clear, plastic mouse cage free of bedding. Each rat was placed in the cage containing cat hair for 5 min, and was returned to his home cage immediately following the exposure.Each rat was placed into a plastic restraint  for 30 min. When 30 min expired, the rat was promptly removed from the restraint, and returned to his home cage.n = 11) were randomly selected to undergo the early adverse procedure. The remaining rats  arrived at the animal facility on the same day and were handled and weighed as EA rats, but they did not receive any adverse experiences. During the procedure, EA and Control rats were housed in separate rooms. Two weeks following the end of the adverse experience procedure, EA rats joined the colony room in which the Control rats were housed.Half of the rats  cues to be used in Pavlovian discrimination. These 42-min sessions consisted of four presentations of each cue  with a mean inter-trial interval (ITI) of 3.5 min. The order of trial type presentation was randomly determined by the behavioral program, and differed for each rat during each session.Starting on P85, and for the next sixteen sessions, each rat underwent Pavlovian discrimination training. Each of the 16, 54-min sessions consisted of 16 trials, with a mean ITI of 3.5 min. Each auditory cue (A1-3) was 10-s in duration, and was associated with a different probability of foot shock . Cue A1 always predicted shock [p(shock) = 1.00], cue A2 probabilistically predicted shock [p(shock) = 0.25], and cue A3 never predicted shock [p(shock) = 0.00]. For reinforced trials, the foot shock was administered 1 s following the termination of the auditory cue. A single session consisted of four A1 trials, six A2 no shock trials, two A2 shock trials, and four A3 trials. The order of trial type presentation was randomly determined by the behavioral program, and differed for each rat during each session.Six extinction sessions were given, one session per day, following the final discrimination session. The composition of these sessions was exactly like those for pre-exposure. Each 42-min session consisted of four presentations of each cue  with a mean inter-trial interval (ITI) of 3.5 min. The order of trial type presentation was randomly determined by the behavioral program, and differed for each rat during each session.post-hoc comparisons were made with two-tailed t-tests.Body weights were analyzed for group differences using analysis of variance (ANOVA). Behavioral data from Pavlovian fear conditioning were acquired using Med Associates Med-PC IV software. The time stamp for every nose poke and event onset (cues and shocks) during each session was recorded automatically. Raw data were processed in Matlab to extract nose poke rates during two periods: the baseline, which was 20 s prior to cue onset, and the entire 10 s cue. A suppression ratio was calculated as follows: (baseline − cue)/(baseline + cue). A ratio of “1” indicated complete suppression of nose poking during the cue relative to baseline, indicative of a high level of fear. A ratio of “0” indicated no suppression of nose poking, indicating little or no fear  and also performed a one-tailed, independent sample t-test because we were testing a directional hypothesis. The sliding window was moved ahead one session at a time until all 15 windows had been analyzed . A significance level of p < 0.05 was used throughout.A sliding-window analysis was performed to identify differences in suppression ratios to each of the three cues by each Control and EA rat throughout the two pre-exposure, and sixteen discrimination sessions. Starting with the first pre-exposure session, and for each cue separately, we took the mean suppression ratio from trial n → F = 1880.1, p < 0.01] and the day × group interaction , but no main effect of group .All rats had free access to water and food from the day they arrived until they began the Pavlovian fear conditioning procedure. While Control and EA rats arrived at the same weight, EA rats gained weight more slowly than Controls following the early adversity procedure. In adulthood, Control and EA rats did not differ significantly in weight. These descriptions are supported by ANOVA  for body weight that found a significant effect of day , but no effect of group or session × group interaction . By the final extinction session, mean ± SEM baseline nose poke rates for each group were Control: 53.1 ± 9.9, and EA: 52.5 ± 6.0.During Pavlovian fear conditioning, Control and EA rats showed equivalent levels of baseline nose poking throughout pre-exposure, discrimination, and extinction. ANOVA  for baseline nose poke rates revealed a significant effect of session . The significant interaction was driven by higher suppression ratios to the A2 cue by EA rats in the last half of the discrimination sessions  = 5.63, p < 0.05]. Significance was not found when contrasts examined differences in suppression ratios to the A1 or A3 cues . Finally, consistent with both the ANCOVA result and planned comparisons, post-hoc tests of suppression ratios to the A2 cue over the final half of discrimination revealed significant differences between Control and EA rats .In order to be certain any differences in body weight following early adversity did not drive our effects, the 85% body weight for each rat was used as a covariate in ANCOVA. In support of our description above, ANCOVA for suppression ratios  found significant effects of body weight, group, cue, and the session × cue interaction , but most critically a significant session × cue × group interaction  and a covariate × trial interaction . The interaction was the result of nose poke suppression recall on trial 1 being equivalent in all rats ; between factor: group (Control vs. EA)] revealed a significant effect of the covariate  and of covariate . No effect of or interaction with Group was found . To visualize this statistical pattern, we separately plotted suppression ratios in extinction for Control and EA rats showing the highest (gray outlines) and lowest (black outlines) nose poke suppression to the probabilistic cue in discrimination  in the reduction of fear through −APEs. Mirroring EA rats in the current study, rats with neurotoxic DRN lesions were specifically impaired in reducing fear to a probabilistic cue (Berg et al., if Early Adversity impaired the ability to reduce fear to cue predicting shock probabilistically. The answer to this is unequivocally “yes.” Now that this has been demonstrated, the next logical question is how does this impairment come about? One hypothesis we propose is that Early Adversity impaired the ability to generate or utilize −APEs to reduce fear. However, other explanations are possible. Early adversity may generally impair sensory discrimination or probability detection. This could be tested by employing a procedure similar to that here, only using rewards instead of aversive foot shock. Early Adversity could also generally elevate anxiety, increasing fear to all cues present. It's possible this drove the results of this study, only observing an increase in fear to the 100% cue was not possible because fear levels were near ceiling. Along these lines, it would be informative to see if Early Adversity impaired various forms of safety learning (Christianson et al., Here, we asked Early Adversity undoubtedly results in many behavioral and neural changes that increase the risk for adult PTSD. The current results are significant because they demonstrate one specific change may be to impair the use of learning signals—negative, aversive prediction errors—to weaken fearful associations and ultimately reduce fear. Future studies identifying the neural origins of APEs, and their corruption by early adversity, will provide novel targets and more effective pharmacotherapies for PTSD and related anxiety disorders.MM conceived the work, MM/KW/AD acquired, analyzed, interpreted the work, MM/KW/AD drafted and critically revised the manuscript. MM/KW/AD approved the final manuscript. MM/KW/AD are accountable for all aspects of the work.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.