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Aims: In light of the evidence supporting a significant role of the gut microbiome in Gulf War Illness (GWI) pathology, we sought to examine its contribution to GWI susceptibility in a mouse model. We also aimed to identify bacterial taxa and microbially-derived metabolites associated with disease susceptibility., Main Methods: Male mice receiving pyridostigmine bromide (PB) orally, and controls were evaluated for symptoms of GWI at 8 weeks post-treatment. The Kansas criteria were adapted to assess behaviors associated with the following domains: gastrointestinal alterations, pain, mood, cognitive function, skin and respiratory disturbances. PB-treated subjects were classified into susceptible (GWI-S) or resilient (GWI-R). The status of the gut microbiome was assessed via analyses of the 16S rRNA gene and microbial-derived metabolites were evaluated with metabolomics tools., Key Findings: Our results indicate that application of the Kansas criteria to behavioral outcomes in PB-treated mice resulted in a GWI susceptibility rate of ~35 %, similar to the one reported in humans. The composition and structure of the gut microbiome was different in GWI-S subjects compared to both control and GWI-R mice at 8 weeks but differences in microbial community structure were observed prior to PB treatment between GWI-R and GWI-S mice. GWI-S subjects exhibited a pattern of differentially abundant bacterial taxa and microbial metabolites., Significance: To our knowledge, this is the first preclinical report in which a stratification by susceptibility to GWI and its association with the gut microbiome is described. In light of the research conundrum that vulnerability to GWI represents, the use of tools that could provide further insight into this complex factor should be considered., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mariana Angoa-Perez reports financial support was provided by US Department of Defense. Mariana Angoa-Perez reports financial support was provided by US Department of Veterans Affairs. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Preterm birth (PTB), often preceded by preterm labor, is a major cause of neonatal morbidity and mortality worldwide. Most PTB cases involve intra-amniotic inflammation without detectable microorganisms, termed in utero sterile inflammation, for which there is no established treatment. In this study, we propose homeostatic macrophages to prevent PTB and adverse neonatal outcomes caused by in utero sterile inflammation. Single-cell atlases of the maternal-fetal interface revealed that homeostatic maternal macrophages are reduced with human labor. M2 macrophage treatment prevented PTB and reduced adverse neonatal outcomes in mice with in utero sterile inflammation. Specifically, M2 macrophages halted premature labor by suppressing inflammatory responses in the amniotic cavity, including inflammasome activation, and mitigated placental and offspring lung inflammation. Moreover, M2 macrophages boosted gut inflammation in neonates and improved their ability to fight systemic bacterial infections. Our findings show that M2 macrophages are a promising strategy to mitigate PTB and improve neonatal outcomes resulting from in utero sterile inflammation., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Introduction: It is well known that chronic opioid use disorder is associated with alterations in gastrointestinal (GI) function that include constipation, reduced motility, and increased bacterial translocation due to compromised gut barrier function. These signs of disrupted GI function can be associated with alterations in the gut microbiome. However, it is not known if long-access opioid self-administration has effects on the gut microbiome., Methods: We used 16S rRNA gene sequencing to investigate the gut microbiome in three independent cohorts (N=40 for each) of NIH heterogeneous stock rats before onset of long-access heroin self-administration (i.e., naïve status), at the end of a 15-day period of self-administration, and after post-extinction reinstatement. Measures of microbial α- and β-diversity were evaluated for all phases. High-dimensional class comparisons were carried out with MaAsLin2. PICRUSt2 was used for predicting functional pathways impacted by heroin based on marker gene sequences., Results: Community α-diversity was not altered by heroin at any of the three phases by comparison to saline-yoked controls. Analyses of β-diversity showed that the heroin and saline-yoked groups clustered significantly apart from each other using the Bray-Curtis (community structure) index. Heroin caused significant alterations at the ASV level at the self-administration and extinction phases. At the phylum level, the relative abundance of Firmicutes was increased at the self-administration phase. Deferribacteres was decreased in heroin whereas Patescibacteria was increased in heroin at the extinction phase. Potential biomarkers for heroin emerged from the MaAsLin2 analysis. Bacterial metabolomic pathways relating to degradation of carboxylic acids, nucleotides, nucleosides, carbohydrates, and glycogen were increased by heroin while pathways relating to biosynthesis of vitamins, propionic acid, fatty acids, and lipids were decreased., Discussion: These findings support the view that long access heroin self-administration significantly alters the structure of the gut microbiome by comparison to saline-yoked controls. Inferred metabolic pathway alterations suggest the development of a microbial imbalance favoring gut inflammation and energy expenditure. Potential microbial biomarkers and related functional pathways likely invoked by heroin self-administration could be targets for therapeutic intervention., Competing Interests: 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., (Copyright © 2024 Greenberg, Winters, Zagorac, Kracht, Francescutti, Cannella, Ciccocioppo, Woods, Mackle, Hardiman, Kuhn, Kalivas, Kuhn and Angoa-Perez.)

The olfactory neuroepithelium (OE) is one of the few neuronal tissues where environmental pathogens can gain direct access. Despite this vulnerable arrangement, little is known about the protective mechanisms in the OE to prevent viral infection and its antiviral responses. We systematically investigated acute responses in the olfactory mucosa upon exposure to vesicular stomatitis virus (VSV) via RNA-seq. VSVs were nasally inoculated into C57BL/6 mice. Olfactory mucosae were dissected for gene expression analysis at different time points after viral inoculation. Interferon functions were determined by comparing the viral load in interferon receptor knockout ( Ifnar1 -/- and Ifnlr1 -/- ) with wildtype OE. Antiviral responses were observed as early as 24 h after viral exposure in the olfactory mucosa. The rapidly upregulated transcripts observed included specific type I as well as type III interferons ( Ifn ) and interferon-stimulated genes. Genetic analyses demonstrated that both type I and type III IFN signaling are required for the suppression of viral replication in the olfactory mucosa. Exogenous IFN application effectively blocks viral replication in the OE. These findings reveal that the OE possesses an innate ability to suppress viral infection. Type I and type III IFNs have prominent roles in OE antiviral functions.

Background: Preterm birth preceded by spontaneous preterm labour often occurs in the clinical setting of sterile intra-amniotic inflammation (SIAI), a condition that currently lacks treatment., Methods: Proteomic and scRNA-seq human data were analysed to evaluate the role of IL-6 and IL-1α in SIAI. A C57BL/6 murine model of SIAI-induced preterm birth was developed by the ultrasound-guided intra-amniotic injection of IL-1α. The blockade of IL-6R by using an aIL-6R was tested as prenatal treatment for preterm birth and adverse neonatal outcomes. QUEST-MRI evaluated brain oxidative stress in utero. Targeted transcriptomic profiling assessed maternal, foetal, and neonatal inflammation. Neonatal biometrics and neurodevelopment were tested. The neonatal gut immune-microbiome was evaluated using metagenomic sequencing and immunophenotyping., Findings: IL-6 plays a critical role in the human intra-amniotic inflammatory response, which is associated with elevated concentrations of the alarmin IL-1α. Intra-amniotic injection of IL-1α resembles SIAI, inducing preterm birth (7% vs. 50%, p = 0.03, Fisher's exact test) and neonatal mortality (18% vs. 56%, p = 0.02, Mann-Whitney U-test). QUEST-MRI revealed no foetal brain oxidative stress upon in utero IL-1α exposure (p > 0.05, mixed linear model). Prenatal treatment with aIL-6R abrogated IL-1α-induced preterm birth (50% vs. 7%, p = 0.03, Fisher's exact test) by dampening inflammatory processes associated with the common pathway of labour. Importantly, aIL-6R reduces neonatal mortality (56% vs. 22%, p = 0.03, Mann-Whitney U-test) by crossing from the mother to the amniotic cavity, dampening foetal organ inflammation and improving growth. Beneficial effects of prenatal IL-6R blockade carried over to neonatal life, improving survival, growth, neurodevelopment, and gut immune homeostasis., Interpretation: IL-6R blockade can serve as a strategy to treat SIAI, preventing preterm birth and adverse neonatal outcomes., Funding: NICHD/NIH/DHHS, Contract HHSN275201300006C. WSU Perinatal Initiative in Maternal, Perinatal and Child Health., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

The composition of the vaginal microbiota is heavily influenced by pregnancy and may factor into pregnancy complications, including spontaneous preterm birth. However, results among studies have been inconsistent due, in part, to variation in sample sizes and ethnicity. Thus, an association between the vaginal microbiota and preterm labor continues to be debated. Yet, before assessing associations between the composition of the vaginal microbiota and preterm labor, a robust and in-depth characterization of the vaginal microbiota throughout pregnancy in the specific study population under investigation is required. Here, we report a large longitudinal study ( n  = 474 women, 1,862 vaginal samples) of a predominantly African-American cohort-a population that experiences a relatively high rate of pregnancy complications-evaluating associations between individual identity, gestational age, and other maternal characteristics with the composition of the vaginal microbiota throughout gestation resulting in term delivery. The principal factors influencing the composition of the vaginal microbiota in pregnancy are individual identity and gestational age at sampling. Other factors are maternal age, parity, obesity, and self-reported Cannabis use. The general pattern across gestation is for the vaginal microbiota to remain or transition to a state of Lactobacillus dominance. This pattern can be modified by maternal parity and obesity. Regardless, network analyses reveal dynamic associations among specific bacterial taxa within the vaginal ecosystem, which shift throughout the course of pregnancy. This study provides a robust foundational understanding of the vaginal microbiota in pregnancy and sets the stage for further investigation of this microbiota in obstetrical disease. IMPORTANCE There is debate regarding links between the vaginal microbiota and pregnancy complications, especially spontaneous preterm birth. Inconsistencies in results among studies are likely due to differences in sample sizes and cohort ethnicity. Ethnicity is a complicating factor because, although all bacterial taxa commonly inhabiting the vagina are present among all ethnicities, the frequencies of these taxa vary among ethnicities. Therefore, an in-depth characterization of the vaginal microbiota throughout pregnancy in the specific study population under investigation is required prior to evaluating associations between the vaginal microbiota and obstetrical disease. This initial investigation is a large longitudinal study of the vaginal microbiota throughout gestation resulting in a term delivery in a predominantly African-American cohort, a population that experiences disproportionally negative maternal-fetal health outcomes. It establishes the magnitude of associations between maternal characteristics, such as age, parity, body mass index, and self-reported Cannabis use, on the vaginal microbiota in pregnancy., Competing Interests: The authors declare no conflict of interest.

NF-κB-inducing kinase (NIK), which is essential for the activation of the noncanonical NF-κB pathway, regulates diverse processes in immunity, development, and disease. Although recent studies have elucidated important functions of NIK in adaptive immune cells and cancer cell metabolism, the role of NIK in metabolic-driven inflammatory responses in innate immune cells remains unclear. In this study, we demonstrate that murine NIK-deficient bone marrow-derived macrophages exhibit defects in mitochondrial-dependent metabolism and oxidative phosphorylation, which impair the acquisition of a prorepair, anti-inflammatory phenotype. Subsequently, NIK-deficient mice exhibit skewing of myeloid cells characterized by aberrant eosinophil, monocyte, and macrophage cell populations in the blood, bone marrow, and adipose tissue. Furthermore, NIK-deficient blood monocytes display hyperresponsiveness to bacterial LPS and elevated TNF-α production ex vivo. These findings suggest that NIK governs metabolic rewiring, which is critical for balancing proinflammatory and anti-inflammatory myeloid immune cell function. Overall, our work highlights a previously unrecognized role for NIK as a molecular rheostat that fine-tunes immunometabolism in innate immunity, and suggests that metabolic dysfunction may be an important driver of inflammatory diseases caused by aberrant NIK expression or activity., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The existence of a placental microbiota is debated. The human placenta has historically been considered sterile and microbial colonization was associated with adverse pregnancy outcomes. Yet, recent DNA sequencing investigations reported a microbiota in typical human term placentas. However, this detected microbiota could represent background DNA or delivery-associated contamination. Using fifteen publicly available 16S rRNA gene datasets, existing data were uniformly re-analyzed with DADA2 to maximize comparability. While Amplicon Sequence Variants (ASVs) identified as Lactobacillus, a typical vaginal bacterium, were highly abundant and prevalent across studies, this prevalence disappeared after applying likely  DNA contaminant removal to placentas from term cesarean deliveries. A six-study sub-analysis targeting the 16S rRNA gene V4 hypervariable region demonstrated that bacterial profiles of placental samples and technical controls share principal bacterial ASVs and that placental samples clustered primarily by study origin and mode of delivery. Contemporary DNA-based evidence does not support the existence of a placental microbiota.ImportanceEarly-gestational microbial influences on human development are unclear. By applying DNA sequencing technologies to placental tissue, bacterial DNA signals were observed, leading some to conclude that a live bacterial placental microbiome exists in typical term pregnancy. However, the low-biomass nature of the proposed microbiome and high sensitivity of current DNA sequencing technologies indicate that the signal may alternatively derive from environmental or delivery-associated bacterial DNA contamination. Here we address these alternatives with a re-analysis of 16S rRNA gene sequencing data from 15 publicly available placental datasets. After identical DADA2 pipeline processing of the raw data, subanalyses were performed to control for mode of delivery and environmental DNA contamination. Both environment and mode of delivery profoundly influenced the bacterial DNA signal from term-delivered placentas. Aside from these contamination-associated signals, consistency was lacking across studies. Thus, placentas delivered at term are unlikely to be the original source of observed bacterial DNA signals., (© 2023. The Author(s).)

The lake sturgeon (Acipenser fulvescens; LST) is the only native sturgeon species in the Great Lakes (GL), but due to multiple factors, their current populations are estimated to be <1% of historical abundances. Little is known about infectious diseases affecting GL-LST in hatchery and wild settings. Therefore, a two-year disease surveillance study was undertaken, resulting in the detection and first in vitro isolation of a herpesvirus from grossly apparent cutaneous lesions in wild adult LST inhabiting two GL watersheds (Erie and Huron). Histological and ultrastructural examination of lesions revealed proliferative epidermitis associated with herpesvirus-like virions. A virus with identical ultrastructural characteristics was recovered from cells inoculated with lesion tissues. Partial DNA polymerase gene sequencing placed the virus within the Family Alloherpesviridae, with high similarity to a lake sturgeon herpesvirus (LSHV) from Wisconsin, USA. Genomic comparisons revealed ~84% Average Nucleotide Identity between the two isolates, leading to the proposed classification of LSHV-1 (Wisconsin) and LSHV-2 (Michigan) for the two viruses. When naïve juvenile LST were immersion-exposed to LSHV-2, severe disease and ~33% mortality occurred, with virus re-isolated from representative skin lesions, fulfilling Rivers’ postulates. Results collectively show LSHV-2 is associated with epithelial changes in wild adult LST, disease and mortality in juvenile LST, and is a potential threat to GL-LST conservation.

Mice are frequently used as animal models for mechanistic studies of infection and obstetrical disease, yet characterization of the murine microbiota during pregnancy is lacking. The objective of this study was to characterize the microbiotas of distinct body sites of the pregnant mouse-vagina, oral cavity, intestine, and lung-that harbor microorganisms that could potentially invade the murine amniotic cavity, thus leading to adverse pregnancy outcomes. The microbiotas of these body sites were characterized through anoxic, hypoxic, and oxic culture as well as through 16S rRNA gene sequencing. With the exception of the vagina, the cultured microbiotas of each body site varied by atmosphere, with the greatest diversity in the cultured microbiota appearing under anoxic conditions. Only cultures of the vagina were comprehensively representative of the microbiota observed through direct DNA sequencing of body site samples, primarily due to the predominance of two Rodentibacter strains. Identified as Rodentibacter pneumotropicus and Rodentibacter heylii, these isolates exhibited predominance patterns similar to those of Lactobacillus crispatus and Lactobacillus iners in the human vagina. Whole-genome sequencing of these Rodentibacter strains revealed shared genomic features, including the ability to degrade glycogen, an abundant polysaccharide in the vagina. In summary, we report body site-specific microbiotas in the pregnant mouse with potential ecological parallels to those of humans. Importantly, our findings indicate that the vaginal microbiotas of pregnant mice can be readily cultured, suggesting that mock vaginal microbiotas can be tractably generated and maintained for experimental manipulation in future mechanistic studies of host vaginal-microbiome interactions. IMPORTANCE Mice are widely utilized as animal models of obstetrical complications; however, the characterization of the murine microbiota during pregnancy has been neglected. Microorganisms from the vagina, oral cavity, intestine, and lung have been found in the intra-amniotic space, where their presence threatens the progression of gestation. Here, we characterized the microbiotas of pregnant mice and established the appropriateness of culture in capturing the microbiota at each site. The high relative abundance of Rodentibacter observed in the vagina is similar to that of Lactobacillus in humans, suggesting potential ecological parallels. Importantly, we report that the vaginal microbiota of the pregnant mouse can be readily cultured under hypoxic conditions, demonstrating that mock microbial communities can be utilized to test the potential ecological parallels between microbiotas in human and murine pregnancy and to evaluate the relevance of the structure of these microbiotas for adverse pregnancy outcomes, especially intra-amniotic infection and preterm birth.

The existence of an amniotic fluid microbiota (i.e., a viable microbial community) in mammals is controversial. Its existence would require a fundamental reconsideration of fetal in utero exposure to and colonization by microorganisms and the role of intra-amniotic microorganisms in fetal immune development as well as in pregnancy outcomes. In this study, we determined whether the amniotic fluid of mice harbors a microbiota in late gestation. The profiles of the amniotic fluids of pups located proximally or distally to the cervix were characterized through quantitative real-time PCR, 16S rRNA gene sequencing, and culture (N = 21 dams). These profiles were compared to those of technical controls for bacterial and DNA contamination. The load of 16S rRNA genes in the amniotic fluid exceeded that in controls. Additionally, the 16S rRNA gene profiles of the amniotic fluid differed from those of controls, with Corynebacterium tuberculostearicum being differentially more abundant in amniotic fluid profiles; however, this bacterium was not cultured from amniotic fluid. Of the 42 attempted bacterial cultures of amniotic fluids, only one yielded bacterial growth - Lactobacillus murinus . The 16S rRNA gene of this common murine-associated bacterium was not detected in any amniotic fluid sample, suggesting it did not originate from the amniotic fluid. No differences in the 16S rRNA gene load, 16S rRNA gene profile, or bacterial culture were observed between the amniotic fluids located Proximally and distally to the cervix. Collectively, these data indicate that, although there is a modest DNA signal of bacteria in murine amniotic fluid, there is no evidence that this signal represents a viable microbiota. While this means that amniotic fluid is not a source of microorganisms for in utero colonization in mice, it may nevertheless contribute to fetal exposure to microbial components. The developmental consequences of this observation warrant further investigation., Competing Interests: 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., (Copyright © 2022 Winters, Romero, Greenberg, Galaz, Shaffer, Garcia-Flores, Kracht, Gomez-Lopez and Theis.)

Microbial invasion of the amniotic cavity (MIAC) leading to infection is strongly associated with adverse pregnancy and neonatal outcomes. Limitations of current diagnostic assays to detect MIAC rapidly and accurately have hindered the ability of obstetricians to identify and treat intra-amniotic infections. We developed, optimized, and validated two multiplex quantitative polymerase chain reaction (qPCR) assays for the simultaneous detection and quantification of microbial taxa commonly associated with MIAC. The first assay allows for the quantification of general bacterial and fungal loads in amniotic fluid and includes a human reference gene to allow for assessing the integrity of clinical samples and the DNA extraction process. The second assay allows for the detection and quantification of four specific bacterial taxa commonly associated with MIAC: Ureaplasma spp., Mycoplasma hominis, Streptococcus agalactiae, and Fusobacterium nucleatum. The qPCR assays were validated by using both microbial isolates and clinical amniotic fluid samples. The assays were further validated by comparing qPCR amplification results to those from the microbial culture and bacterial 16S rRNA gene sequencing of amniotic fluid. Both assays demonstrated high reproducibility and are sensitive and specific to their intended targets. Therefore, these assays represent promising molecular diagnostic tools for the detection of MIAC. Most importantly, these assays may allow for administration of timely and targeted antibiotic interventions to reduce adverse perinatal outcomes attributed to intra-amniotic infections., (Copyright © 2021. Published by Elsevier B.V.)

Mitochondria have emerged as key drivers of mammalian innate immune responses, functioning as signaling hubs to trigger inflammation and orchestrating metabolic switches required for phagocyte activation. Mitochondria also contain damage-associated molecular patterns (DAMPs), molecules that share similarity with pathogen-associated molecular patterns (PAMPs) and can engage innate immune sensors to drive inflammation. The aberrant release of mitochondrial DAMPs during cellular stress and injury is an increasingly recognized trigger of inflammatory responses in human diseases. Mitochondrial DNA (mtDNA) is a particularly potent DAMP that engages multiple innate immune sensors, although mounting evidence suggests that cytosolic mtDNA is primarily detected via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. cGAS and STING are widely expressed in mammalian cells and serve as key regulators of type I interferon and cytokine expression in both infectious and inflammatory diseases. Despite growing roles for the mtDNA-cGAS-STING axis in human disease, assays to quantify mtDNA release into the cytosol and approaches to link mtDNA to cGAS-STING signaling are not standardized, which increases the possibility for experimental artifacts and misinterpretation of data. Here, we present a series of protocols for assaying the release of mtDNA into the cytosol and subsequent activation of innate immune signaling in mammalian cells. We highlight genetic and pharmacological approaches to induce and inhibit mtDNA release from mitochondria. We also describe immunofluorescence microscopy and cellular fractionation assays to visualize morphological changes in mtDNA and quantify mtDNA accumulation in the cytosol. Finally, we include protocols to examine mtDNA-dependent cGAS-STING activation by RT-qPCR and western blotting. These methods can be performed with standard laboratory equipment and are highly adaptable to a wide range of mammalian cell types. They will permit researchers working across the spectrum of biological and biomedical sciences to accurately and reproducibly measure cytosolic mtDNA release and resulting innate immune responses. © 2022 Wiley Periodicals LLC. Basic Protocol 1: siRNA-mediated knockdown of TFAM to induce mtDNA instability, cytosolic release, and activation of the cGAS-STING pathway Alternate Protocol: Pharmacological induction of mtDNA release and cGAS-STING activation using ABT-737 and Q-VD-OPH Basic Protocol 2: Isolation and quantitation of DNA from cytosolic, mitochondrial, and nuclear fractions Basic Protocol 3: Pharmacological inhibition of mtDNA replication and release., (© 2022 Wiley Periodicals LLC.)

Zebrafish (Danio rerio) is an attractive model organism to use for an array of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria . Furthermore, this core intestinal microbiome is plastic and can be significantly altered due to external factors. Zebrafish are particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. Members of the resident intestinal microbial community likely must be reduced or eliminated by V. cholerae for colonization, and subsequent disease, to occur. Many studies have explored a variety of aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms but few have researched how a V. cholerae infection changes the resident intestinal microbiome. In this study, 16S rRNA gene sequencing was used to examine how five genetically diverse V. cholerae strains alter the intestinal microbiome following an infection. We found that V. cholerae colonization induced significant changes in the zebrafish intestinal microbiome. Notably, changes in the microbial profile were significantly different from each other, based on the particular strain of V. cholerae used to infect zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiota to enable colonization and that specific microbes that are targeted depend on the V. cholerae genotype.

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae, an intestinal pathogen. V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal microbiome and the role of the type six secretion system (T6SS) in that process. In this study, 16S rRNA gene sequencing was utilized to investigate how strains of V. cholerae both with and without the T6SS alter the aforementioned microbial profiles following an infection. V. cholerae infection induced significant changes in the zebrafish intestinal microbiome, and while not necessary for colonization, the T6SS was important for inducing mucin secretion, a marker for diarrhea. Additional salient differences to the microbiome were observed based on the presence or absence of the T6SS in the V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiome to enable colonization and that the T6SS is important for pathogenesis induced by the examined V. cholerae strains. Furthermore, the presence or absence of T6SS differentially and significantly affected the composition and structure of the intestinal microbiome, with an increased abundance of other Vibrio bacteria observed in the absence of V. cholerae T6SS.

Sneathia is an emerging pathogen implicated in adverse reproductive and perinatal outcomes. Although scarce, recent data suggest that vaginally residing Sneathia becomes pathogenic following its ascension into the upper urogenital tract, amniotic fluid, placenta, and foetal membranes. The role of Sneathia in women's health and disease is generally underappreciated because the cultivation of these bacteria is limited by their complex nutritional requirements, slow growth patterns, and anaerobic nature. For this reason, molecular methods are typically required for the detection and differential diagnosis of Sneathia infections. Here, we review the laboratory methods used for the diagnosis of Sneathia infections, the molecular mechanisms underlying its virulence, and its sensitivity to antibiotics. We further review the evidence of Sneathia 's contributions to the pathogenesis of bacterial vaginosis, chorioamnionitis, preterm prelabour rupture of membranes, spontaneous preterm labour, stillbirth, maternal and neonatal sepsis, HIV infection, and cervical cancer. Collectively, growing evidence indicates that Sneathia represents an important yet underappreciated pathogen affecting the development and progression of several adverse clinical conditions diagnosed in pregnant women and their neonates, as well as in non-pregnant women.

Objectives: Intra-amniotic infection, defined by the presence of microorganisms in the amniotic cavity, is often accompanied by intra-amniotic inflammation. Occasionally, laboratories report the growth of bacteria or the presence of microbial nucleic acids in amniotic fluid in the absence of intra-amniotic inflammation. This study was conducted to determine the clinical significance of the presence of bacteria in amniotic fluid samples in the absence of intra-amniotic inflammation., Methods: A retrospective cross-sectional study included 360 patients with preterm labor and intact membranes who underwent transabdominal amniocentesis for evaluation of the microbial state of the amniotic cavity as well as intra-amniotic inflammation. Cultivation techniques were used to isolate microorganisms, and broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was utilized to detect the nucleic acids of bacteria, viruses, and fungi., Results: Patients whose amniotic fluid samples evinced microorganisms but did not indicate inflammation had a similar perinatal outcome to those without microorganisms or inflammation [amniocentesis-to-delivery interval (p=0.31), spontaneous preterm birth before 34 weeks (p=0.83), acute placental inflammatory lesions (p=1), and composite neonatal morbidity (p=0.8)]., Conclusions: The isolation of microorganisms from a sample of amniotic fluid in the absence of intra-amniotic inflammation is indicative of a benign condition, which most likely represents contamination of the specimen during the collection procedure or laboratory processing rather than early colonization or infection., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Intra-amniotic infection is strongly associated with adverse pregnancy and neonatal outcomes. Most intra-amniotic infections are due to Ureaplasma species; however, the pathogenic potency of these genital mycoplasmas to induce preterm birth is still controversial. Here, we first laid out a taxonomic characterization of Ureaplasma isolates from women with intra-amniotic infection, which revealed that Ureaplasma parvum is the most common bacterium found in this clinical condition. Next, using animal models, we provided a causal link between intra-amniotic inoculation with Ureaplasma species and preterm birth. Importantly, the intra-amniotic inoculation of Ureaplasma species induced high rates of mortality in both preterm and term neonates. The in vivo potency of U. parvum to induce preterm birth was not associated with known virulence factors. However, term-derived and preterm-derived U. parvum isolates were capable of inducing an intra-amniotic inflammatory response. Both U. parvum isolates invaded several fetal tissues, primarily the fetal lung, and caused fetal inflammatory response syndrome. This bacterium was also detected in the placenta, reproductive tissues, and most severely in the fetal membranes, inducing a local inflammatory response that was replicated in an in vitro model. Importantly, treatment with clarithromycin, a recently recommended yet not widely utilized antibiotic, prevented the adverse pregnancy and neonatal outcomes induced by U. parvum These findings shed light on the maternal-fetal immunobiology of intra-amniotic infection. IMPORTANCE Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Multiple etiologies are associated with preterm birth; however, 25% of preterm infants are born to a mother with intra-amniotic infection, most commonly due to invasion of the amniotic cavity by Ureaplasma species. Much research has focused on establishing a link between Ureaplasma species and adverse pregnancy/neonatal outcomes; however, little is known about the taxonomy of and host response against Ureaplasma species. Here, we applied a multifaceted approach, including human samples, in vivo models, and in vitro manipulations, to study the maternal-fetal immunobiology of Ureaplasma infection during pregnancy. Furthermore, we investigated the use of clarithromycin as a treatment for this infection. Our research provides translational knowledge that bolsters scientific understanding of Ureaplasma species as a cause of adverse pregnancy/neonatal outcomes and gives strong evidence for the use of clarithromycin as the recommended treatment for women intra-amniotically infected with Ureaplasma species., (Copyright © 2020 Motomura et al.)

Gulf War Illness (GWI) is a chronic health condition that appeared in Veterans after returning home from the Gulf War. The primary symptoms linked to deployment are posttraumatic stress disorder, mood disorders, GI problems and chronic fatigue. At first glance, these symptoms are difficult to ascribe to a single pathological mechanism. However, it is now clear that each symptom can be linked individually to alterations in the gut microbiome. The primary objective of the present study was to determine if gut microbiome dysbiosis was evident in a mouse model of GWl. Because the majority of Gulf War Veterans are overweight, a second objective was to determine if a high fat diet (HF) would alter GWI outcomes. We found that the taxonomic structure of the gut microbiome was significantly altered in the GWI model and after HF exposure. Their combined effects were significantly different from either treatment alone. Most treatment-induced changes occurred at the level of phylum in Firmicutes and Bacteroidetes. If mice fed HF were returned to a normal diet, the gut microbiome recovered toward normal levels in both controls and GWI agent-treated mice. These results add support to the hypotheses that dysbiosis in the gut microbiome plays a role in GWI and that life-style risk factors such as an unhealthy diet can accentuate the effects of GWI by impacting the gut microbiome. The reversibility of the effect of HF on the gut microbiome suggests new avenues for treating GWI through dietary intervention.

Traumatic brain injury (TBI) is often accompanied by gastrointestinal and metabolic disruptions. These systemic manifestations suggest possible involvement of the gut microbiota in head injury outcomes. Although gut dysbiosis after single, severe TBI has been documented, the majority of head injuries are mild, such as those that occur in athletes and military personnel exposed to repetitive head impacts. Therefore, it is important to determine if repetitive, mild TBI (rmTBI) will also disrupt the gut microbiota. Male mice were exposed to mild head impacts daily for 20 days and assessed for cognitive behavior, neuropathology and disruptions in the gut microbiota at 0, 45 or 90 days after injury. Deficits in recognition memory were evident at the late post-injury points. Brains show an early increase in microglial activation at the 0-day time point that persisted until 90 days post-injury. This was compounded by substantial increases in astrocyte reactivity and phosphorylated tau at the 90-day time point. In contrast, changes in the microbial community were minor and transient, and very few differences were observed in mice exposed to rmTBI compared to controls. While the progressive emergence of white matter damage and cognitive alterations after rmTBI resembles the alterations observed in athletes and military personnel exposed to rmTBI, these changes could not be linked to systematic modifications in the gut microbiota.

The prevailing paradigm in obstetrics has been the sterile womb hypothesis. However, some are asserting that the placenta, intra-amniotic environment, and fetus harbor microbial communities. The objective of this study was to determine whether the fetal and placental tissues of rhesus macaques harbor bacterial communities. Fetal, placental, and uterine wall samples were obtained from cesarean deliveries without labor (∼130/166 days gestation). The presence of bacteria in the fetal intestine and placenta was investigated through culture. The bacterial burden and profiles of the placenta, umbilical cord, and fetal brain, heart, liver, and colon were determined through quantitative real-time PCR and DNA sequencing. These data were compared with those of the uterine wall as well as to negative and positive technical controls. Bacterial cultures of fetal and placental tissues yielded only a single colony of Cutibacterium acnes This bacterium was detected at a low relative abundance (0.02%) in the 16S rRNA gene profile of the villous tree sample from which it was cultured, yet it was also identified in 12/29 background technical controls. The bacterial burden and profiles of fetal and placental tissues did not exceed or differ from those of background technical controls. By contrast, the bacterial burden and profiles of positive controls exceeded and differed from those of background controls. Among the macaque samples, distinct microbial signals were limited to the uterine wall. Therefore, using multiple modes of microbiologic inquiry, there was not consistent evidence of bacterial communities in the fetal and placental tissues of rhesus macaques. IMPORTANCE Microbial invasion of the amniotic cavity (i.e., intra-amniotic infection) has been causally linked to pregnancy complications, especially preterm birth. Therefore, if the placenta and the fetus are typically populated by low-biomass microbial communities, current understanding of the role of microbes in reproduction and pregnancy outcomes will need to be fundamentally reconsidered. Could these communities be of benefit by competitively excluding potential pathogens or priming the fetal immune system for the microbial bombardment it will experience upon delivery? If so, what properties (e.g., microbial load and community membership) of these microbial communities preclude versus promote intra-amniotic infection? Given the ramifications of the in utero colonization hypothesis, critical evaluation is required. In this study, using multiple modes of microbiologic inquiry (i.e., culture, quantitative real-time PCR [qPCR], and DNA sequencing) and controlling for potential background DNA contamination, we did not find consistent evidence for microbial communities in the placental and fetal tissues of rhesus macaques.

The existence of a placental microbiota and in utero colonization of the fetus have been the subjects of recent debate. The objective of this study was to determine whether the placental and fetal tissues of mice harbor bacterial communities. Bacterial profiles of the placenta and fetal brain, lung, liver, and intestine samples were characterized through culture, quantitative real-time PCR (qPCR), and 16S rRNA gene sequencing. These profiles were compared to those of the maternal mouth, lung, liver, uterus, cervix, vagina, and intestine, as well as to background technical controls. Positive bacterial cultures from placental and fetal tissue samples were rare; of the 165 total bacterial cultures of placental tissue samples from the 11 mice included in this study, only nine yielded at least a single colony, and five of those nine positive cultures came from a single mouse. Cultures of fetal intestinal tissue samples yielded just a single bacterial isolate, Staphylococcus hominis , a common skin bacterium. Bacterial loads of placental and fetal brain, lung, liver, and intestinal tissues were not higher than those of DNA contamination controls and did not yield substantive 16S rRNA gene sequencing libraries. From all placental or fetal tissue samples ( n  = 51), there was only a single bacterial isolate that came from a fetal brain sample having a bacterial load higher than that of contamination controls and that was identified in sequence-based surveys of at least one of its corresponding maternal samples. Therefore, using multiple modes of microbiological inquiry, there was not consistent evidence of bacterial communities in the placental and fetal tissues of mice. IMPORTANCE The prevailing paradigm in obstetrics has been the sterile womb hypothesis, which posits that fetuses are first colonized by microorganisms during the delivery process. However, some are now suggesting that fetuses are consistently colonized in utero by microorganisms from microbial communities that inhabit the placenta and intra-amniotic environment. Given the established causal role of microbial invasion of the amniotic cavity (i.e., intra-amniotic infection) in pregnancy complications, especially preterm birth, if the in utero colonization hypothesis were true, there are several aspects of current understanding that will need to be reconsidered; these aspects include the magnitude of intra-amniotic microbial load required to cause disease and its potential influence on the ontogeny of the immune system. However, acceptance of the in utero colonization hypothesis is premature. Herein, we do not find consistent evidence for placental and fetal microbiota in mice using culture, qPCR, and DNA sequencing., (Copyright © 2020 Theis et al.)

Background Intra-amniotic inflammation, which is associated with adverse pregnancy outcomes, can occur in the presence or absence of detectable microorganisms, and involves activation of the inflammasome. Intra-amniotic inflammasome activation has been reported in clinical chorioamnionitis at term and preterm labor with intact membranes, but it has not yet been investigated in women with preterm prelabor rupture of membranes (preterm PROM) in the presence/absence of detectable microorganisms. The aim of this study was to determine whether, among women with preterm PROM, there is an association between detectable microorganisms in amniotic fluid and intra-amniotic inflammation, and whether intra-amniotic inflammasome activation correlates with microbial burden. Methods Amniotic fluids from 59 cases of preterm PROM were examined for the presence/absence of microorganisms through culture and 16S ribosomal RNA (rRNA) gene quantitative real-time polymerase chain reaction (qPCR), and concentrations of interleukin-6 (IL-6) and ASC [apoptosis-associated spec-like protein containing a caspase recruitment domain (CARD)], an indicator of inflammasome activation, were determined. Results qPCR identified more microbe-positive amniotic fluids than culture. Greater than 50% of patients with a negative culture and high IL-6 concentration in amniotic fluid yielded a positive qPCR signal. ASC concentrations were greatest in patients with high qPCR signals and elevated IL-6 concentrations in amniotic fluid (i.e. intra-amniotic infection). ASC concentrations tended to increase in patients without detectable microorganisms but yet with elevated IL-6 concentrations (i.e. sterile intra-amniotic inflammation) compared to those without intra-amniotic inflammation. Conclusion qPCR is a valuable complement to microbiological culture for the detection of microorganisms in the amniotic cavity in women with preterm PROM, and microbial burden is associated with the severity of intra-amniotic inflammatory response, including inflammasome activation.

Host-associated microbial communities, henceforth 'microbiota', can affect the physiology and behavior of their hosts. In mammals, host ecological, social and environmental variables are associated with variation in microbial communities. Within individuals in a given mammalian species, the microbiota also partitions by body site. Here, we build on this work and sequence the bacterial 16S rRNA gene to profile the microbiota at six distinct body sites (ear, nasal and oral cavities, prepuce, rectum and anal scent gland) in a population of wild spotted hyenas (Crocuta crocuta), which are highly social, large African carnivores. We inquired whether microbiota at these body sites vary with host sex or social rank among juvenile hyenas, and whether they differ between juvenile females and adult females. We found that the scent gland microbiota differed between juvenile males and juvenile females, whereas the prepuce and rectal microbiota differed between adult females and juvenile females. Social rank, however, was not a significant predictor of microbiota profiles. Additionally, the microbiota varied considerably among the six sampled body sites and exhibited strong specificity among individual hyenas. Thus, our findings suggest that site-specific niche selection is a primary driver of microbiota structure in mammals, but endogenous host factors may also be influential., (© FEMS 2020.)

The list of pharmacological agents that can modify the gut microbiome or be modified by it continues to grow at a high rate. The greatest amount of attention on drug-gut microbiome interactions has been directed primarily at pharmaceuticals used to treat infection, diabetes, cardiovascular conditions and cancer. By comparison, drugs of abuse and addiction, which can powerfully and chronically worsen human health, have received relatively little attention in this regard. Therefore, the main objective of this study was to characterize how selected synthetic psychoactive cathinones (aka "Bath Salts") and amphetamine stimulants modify the gut microbiome. Mice were treated with mephedrone (40 mg/kg), methcathinone (80 mg/kg), methamphetamine (5 mg/kg) or 4-methyl-methamphetamine (40 mg/kg), following a binge regimen consisting of 4 injections at 2h intervals. These drugs were selected for study because they are structural analogs that contain a β-keto substituent (methcathinone), a 4-methyl group (4-methyl-methamphetamine), both substituents (mephedrone) or neither (methamphetamine). Mice were sacrificed 1, 2 or 7 days after treatment and DNA from caecum contents was subjected to 16S rRNA sequencing. We found that all drugs caused significant time- and structure-dependent alterations in the diversity and taxonomic structure of the gut microbiome. The two phyla most changed by drug treatments were Firmicutes (methcathinone, 4-methyl-methamphetamine) and Bacteriodetes (methcathinone, 4-methyl-methamphetamine, methamphetamine, mephedrone). Across time, broad microbiome changes from the phylum to genus levels were characteristic of all drugs. The present results signify that these selected psychoactive drugs, which are thought to exert their primary effects within the CNS, can have profound effects on the gut microbiome. They also suggest new avenues of investigation into the possibility that gut-derived signals could modulate drug abuse and addiction via altered communication along the gut-brain axis., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The existence of a placental microbiome would require a non-antagonistic relationship between potentially colonizing bacteria and trophoblasts., Objective: The immunologic response of trophoblasts to specific potentially invading bacteria needs further analysis., Methodology: Immortalized first trimester human trophoblasts Swan 71 (Sw.71) were coincubated with Escherichia coli, Lactobacillus jensenii, Lactobacillus crispatus, and incubated alone (i.e., control group; 4 conditions with n = 6 for each condition). Chemokines and cytokines were measured. ANOVA with post hoc pairwise analysis was used to compare cytokines/chemokines concentrations in the 4 culture media., Results: Sw.71 co-incubated with E. coli, L. jensenii or L. crispatus resulted in differential secretion of 11 of the 26 assayed cytokines/chemokines. Sw.71 co-incubated with any of the 3 bacteria responded with significant increased secretion of interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor. All bacteria elicited the secretion of IL-6 and interferon (IFN) α2, 2 proinflammatory cytokines. In addition, Lactobacillus species resulted in increased secretion of IL-12p40 and IFNγ. While E. coli did not modify secretion of anti-inflammatory cytokines, Sw.71 cells responded to co-incubation with Lactobacillus species by secreting increased levels of IL-10 and IL-1ra. Both Lactobacillus species led to a decreased secretion of IL-4., Conclusion: All 3 bacterial species triggered significant release of chemokines and inflammatory cytokines, suggesting that a commensal relationship with trophoblasts may not be feasible., (© 2020 S. Karger AG, Basel.)

Background Microbial invasion of the amniotic cavity resulting in intra-amniotic infection is associated with obstetrical complications such as preterm labor with intact or ruptured membranes, cervical insufficiency, as well as clinical and histological chorioamnionitis. The most widely accepted pathway for intra-amniotic infection is the ascension of microorganisms from the lower genital tract. However, hematogenous dissemination of microorganisms from the oral cavity or intestine, retrograde seeding from the peritoneal cavity through the fallopian tubes, and introduction through invasive medical procedures have also been suggested as potential pathways for intra-amniotic infection. The primary reason that an ascending pathway is viewed as most common is that the microorganisms most often detected in the amniotic fluid are those that are typical inhabitants of the vagina. However, thus far, no studies have shown that microorganisms in the amniotic cavity are simultaneously present in the vagina of the woman from which they were isolated. The objective of the study was to determine the frequency with which microorganisms isolated from women with intra-amniotic infection are also present in the lower genital tract. Methods This was a cross-sectional study of women with intra-amniotic infection with intact membranes. Intra-amniotic infection was defined as a positive culture and elevated concentrations of interleukin-6 (IL-6) (>2.6 ng/mL) in amniotic fluid and/or acute histologic chorioamnionitis and funisitis. Microorganisms isolated from bacterial cultures of amniotic fluid were taxonomically identified through matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and 16S ribosomal RNA (rRNA) gene sequencing. Vaginal swabs were obtained at the time of amniocentesis for the identification of microorganisms in the lower genital tract. The overall bacterial profiles of amniotic fluids and vaginal swabs were characterized through 16S rRNA gene sequencing. The bacterial profiles of vaginal swabs were interrogated for the presence of bacteria cultured from amniotic fluid and for the presence of prominent (>1% average relative abundance) operational taxonomic units (OTUs) within the overall 16S rRNA gene bacterial profiles of amniotic fluid. Results (1) A total of 75% (6/8) of women had bacteria cultured from their amniotic fluid that are typical residents of the vaginal ecosystem. (2) A total of 62.5% (5/8) of women with bacteria cultured from their amniotic fluid also had these bacteria present in their vagina. (3) The microorganisms cultured from amniotic fluid and also detected in the vagina were Ureaplasma urealyticum, Escherichia coli, and Streptococcus agalactiae. (4) 16S rRNA gene sequencing revealed that the amniotic fluid of women with intra-amniotic infection had bacterial profiles dominated by Sneathia, Ureaplasma, Prevotella, Lactobacillus, Escherichia, Gardnerella, Peptostreptococcus, Peptoniphilus, and Streptococcus, many of which had not been cultured from the amniotic fluid samples. (5) Seventy percent (7/10) of the prominent (>1% average relative abundance) OTUs found in amniotic fluid were also prominent in the vagina. Conclusion The majority of women with intra-amniotic infection had bacteria cultured from their amniotic fluid that were typical vaginal commensals, and these bacteria were detected within the vagina at the time of amniocentesis. Molecular microbiological interrogation of amniotic fluid from women with intra-amniotic infection revealed that the bacterial profiles of amniotic fluid were largely consistent with those of the vagina. These findings indicate that ascension from the lower genital tract is the primary pathway for intra-amniotic infection.

Symbiotic microbes that inhabit animal scent glands can produce volatile compounds used as chemical signals by the host animal. Though several studies have demonstrated correlations between scent gland bacterial community structure and host animal odour profiles, none have systematically demonstrated a causal relationship. In birds, volatile compounds in preen oil secreted by the uropygial gland serve as chemical cues and signals. Here, we tested whether manipulating the uropygial gland microbial community affects chemical profiles in the dark-eyed junco ( Junco hyemalis ). We found an effect of antibiotic treatment targeting the uropygial gland on both bacterial and volatile profiles. In a second experiment, we cultured bacteria from junco preen oil, and found that all of the cultivars produced at least one volatile compound common in junco preen oil, and that most cultivars produced multiple preen oil volatiles. In both experiments, we identified experimentally generated patterns in specific volatile compounds previously shown to predict junco reproductive success. Together, our data provide experimental support for the hypothesis that symbiotic bacteria produce behaviourally relevant volatile compounds within avian chemical cues and signals., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Zebrafish ( Danio rerio ) are an attractive model organism for scientific studies, including host-microbe interactions. The organism is particularly useful for studying aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae . Previous studies have established the presence of a core zebrafish intestinal microbiome, and tracked the development of the zebrafish intestinal microbiome from the larval stage to adulthood. An unexplored matter in this host-microbe relationship is the effect of the housing system on the zebrafish intestinal and tank water microbiomes. In this study, we used 16S rRNA gene sequencing to investigate the response of zebrafish intestinal and tank water microbiomes to a change in housing conditions. Zebrafish in the separated fish tanks showed no initial differences in the structures of their intestinal microbial profiles; the same prominent bacteria were present and abundant across tanks. Immediately after the housing switch, the zebrafish intestinal microbial profiles changed in composition and structure. Within 5 days of the housing switch, the intestinal microbiome had stabilized, and changed significantly from the prehousing switch profile. This study demonstrates that although external factors can significantly perturb and alter the zebrafish intestinal microbiome, the microbiome displays a large level of selective resilience whose primary members (namely Vibrio ) persist.

Recent molecular studies concluded that the endometrium has a resident microbiota dominated by Lactobacillus spp. and is therefore similar to that of the vagina. These findings were largely derived from endometrial samples obtained through a transcervical catheter and thus prone to contamination. Herein, we investigated the molecular microbial profiles of mid-endometrial samples obtained through hysterectomy and compared them with those of the cervix, vagina, rectum, oral cavity, and controls for background DNA contamination. Microbial profiles were examined through 16S rRNA gene qPCR and sequencing. Universal bacterial qPCR of total 16S rDNA revealed a bacterial load exceeding that of background DNA controls in the endometrium of 60% (15/25) of the study subjects. Bacterial profiles of the endometrium differed from those of the oral cavity, rectum, vagina, and background DNA controls, but not of the cervix. The bacterial profiles of the endometrium and cervix were dominated by Acinetobacter, Pseudomonas, Cloacibacterium, and Comamonadaceae. Both 16S rRNA gene sequencing and Lactobacillus species-specific (L. iners & L crispatus) qPCR showed that Lactobacillus was rare in the endometrium. In conclusion, if there is a microbiota in the middle endometrium, it is not dominated by Lactobacillus as was previously concluded, yet further investigation using culture and microscopy is necessary.

Background: The human placenta has been traditionally viewed as sterile, and microbial invasion of this organ has been associated with adverse pregnancy outcomes. Yet, recent studies that utilized sequencing techniques reported that the human placenta at term contains a unique microbiota. These conclusions are largely based on the results derived from the sequencing of placental samples. However, such an approach carries the risk of capturing background-contaminating DNA (from DNA extraction kits, polymerase chain reaction reagents, and laboratory environments) when low microbial biomass samples are studied., Objective: To determine whether the human placenta delivered at term in patients without labor who undergo cesarean delivery harbors a resident microbiota ("the assemblage of microorganisms present in a defined niche or environment")., Study Design: This cross-sectional study included placentas from 29 women who had a cesarean delivery without labor at term. The study also included technical controls to account for potential background-contaminating DNA, inclusive in DNA extraction kits, polymerase chain reaction reagents, and laboratory environments. Bacterial profiles of placental tissues and background technical controls were characterized and compared with the use of bacterial culture, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and metagenomic surveys., Results: (1) Twenty-eight of 29 placental tissues had a negative culture for microorganisms. The microorganisms retrieved by culture from the remaining sample were likely contaminants because corresponding 16S ribosomal RNA genes were not detected in the same sample. (2) Quantitative real-time polymerase chain reaction did not indicate greater abundances of bacterial 16S ribosomal RNA genes in placental tissues than in technical controls. Therefore, there was no evidence of the presence of microorganisms above background contamination from reagents in the placentas. (3) 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and background technical controls. (4) Most of the bacterial sequences obtained from metagenomic surveys of placental tissues were from cyanobacteria, aquatic bacteria, or plant pathogens, which are microbes unlikely to populate the human placenta. Coprobacillus, which constituted 30.5% of the bacterial sequences obtained through metagenomic sequencing of placental samples, was not identified in any of the 16S ribosomal RNA gene surveys of these samples. These observations cast doubt as to whether this organism is really present in the placenta of patients at term not in labor., Conclusion: With the use of multiple modes of microbiologic inquiry, a resident microbiota could not be identified in human placentas delivered at term from women without labor. A consistently significant difference in the abundance and/or presence of a microbiota between placental tissue and background technical controls could not be found. All cultures of placental tissue, except 1, did not yield bacteria. Incorporating technical controls for potential sources of background-contaminating DNA for studies of low microbial biomass samples, such as the placenta, is necessary to derive reliable conclusions., (Published by Elsevier Inc.)

Background: Mortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events., Results: Juvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures < 23 °C. While co-infection of smallmouth bass by both A. salmonicida and largemouth bass virus can be devastating to juvenile smallmouth bass, the optimal temperatures of each pathogen are 7-10 °C apart, making their synergistic effects highly unlikely under field conditions., Conclusions: The sum of our data generated in this study suggests that largemouth bass virus can be the causative agent of young-of-year smallmouth bass mortality episodes observed at relatively high water temperature.

Flavobacterium columnare, the aetiological agent of columnaris disease, causes significant losses in fish worldwide. In this study, the prevalence of F. columnare infection was assessed in representative Great Lakes fish species. Over 2000 wild, feral and hatchery-propagated salmonids, percids, centrarchids, esocids and cyprinids were examined for systemic F. columnare infections. Logistic regression analyses showed that the prevalence of F. columnare infection varied temporally and by the sex of the fish, whereby females had significantly higher prevalence of infection. A total of 305 isolates of F. columnare were recovered. Amplification of the near complete 16S rRNA gene from 34 representative isolates and subsequent restriction fragment length polymorphism analyses demonstrated that all belonged to F. columnare genomovar I. Phylogenetic analysis of near complete 16S rRNA gene sequences also placed the isolates in genomovar I, but revealed some intragenomovar heterogeneity. Together, these results suggest that F. columnare genomovar I is widespread in the Great Lakes Basin, where its presence may lead to mortality., (© 2016 John Wiley & Sons Ltd.)

In 2011, the Fathead Minnow nidovirus (FHMNV; Genus Bafinivirus, Family Coronaviridae, Order Nidovirales) was isolated from pond-raised juvenile Muskellunge Esox masquinongy suffering from lingering mortality at the Wild Rose Hatchery in Wild Rose, Wisconsin. Moribund Muskellunge exhibited tubular necrosis in the kidneys as well as multifocal coalescing necrotizing hepatitis. The FHMNV was also isolated from apparently healthy juvenile Muskellunge at the Wolf Lake State Fish Hatchery in Mattawan, Michigan. The identity of the two syncytia-forming viruses (designated MUS-WR and MUS-WL from Wild Rose Hatchery and Wolf Lake State Fish Hatchery, respectively) as strains of FHMNV was determined based on multiple-gene sequencing and phylogenetic analyses. The pathogenicity of the MUS-WL FHMNV strain was determined by experimentally infecting naive juvenile Muskellunge through intraperitoneal injection with two viral concentrations (63 and 6.3 × 10(3) TCID50/fish). Both doses resulted in 100% mortality in experimentally infected fish, which exhibited severely pale gills and petechial hemorrhaging in eyes, fins, and skin. Histopathological alterations in experimentally infected fish were observed mainly in the hematopoietic tissues in the form of focal areas of necrosis. Phylogenetic analysis of concatenated partial spike glycoprotein and helicase gene sequences revealed differences between the MUS-WL FHMNV, MUS-WR FHMNV, and two other FHMNV originally isolated from moribund Fathead Minnows Pimephales promelas including the index FHMNV strain (GU002364). Based on a partial helicase gene sequence, a reverse transcriptase PCR assay was developed that is specific to FHMNV. These results give evidence that the risks posed to Muskellunge by FHMNV should be taken seriously. Received May 1, 2015; accepted February 8, 2016.

A microsporidian infecting the skeletal muscle of hybrid jundiara (Leiarius marmoratus × Pseudoplatystoma reticulatum) in a commercial aquaculture facility in Brazil is described. Affected fish exhibited massive infections in the skeletal muscle that were characterized by large opaque foci throughout the affected fillets. Histologically, skeletal muscle was replaced by inflammatory cells and masses of microsporidial developmental stages. Generally pyriform spores had a wrinkled bi-layer spore wall and measured 4·0 × 6·0 µm. Multinucleate meronts surrounded by a simple plasma membrane were observed. The polar filament had an external membrane and a central electron dense mass. The development of sporoblasts within a sporophorous vesicle appeared synchronized. Ultrastructural observations and molecular analysis of 16S rDNA sequences revealed that the microsporidian was Pleistophora hyphessobryconis. This study is the first report of a P. hyphessobryconis infection in a non-ornamental fish.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen. Analysis of its genomic characteristics is required to determine the worldwide distribution of the various populations of this bacterium. Genomic alignments between the 01-B526 pathogenic strain and the A449 reference strain have revealed a 51-kb chromosomal insertion in 01-B526. This insertion (AsaGEI1a) has been identified as a new genomic island (GEI) bearing prophage genes. PCR assays were used to detect this GEI in a collection of 139 A. salmonicida subsp. salmonicida isolates. Three forms of this GEI (AsaGEI1a, AsaGEI1b, AsaGEI2a) are now known based on this analysis and the sequencing of the genomes of seven additional isolates. A new prophage (prophage 3) associated with AsaGEI2a was also discovered. Each GEI appeared to be strongly associated with a specific geographic region. AsaGEI1a and AsaGEI2a were exclusively found in North American isolates, except for one European isolate bearing AsaGEI2a. The majority of the isolates bearing AsaGEI1b or no GEI were from Europe. Prophage 3 has also a particular geographic distribution and was found only in North American isolates. We demonstrated that A. salmonicida subsp. salmonicida possesses unsuspected elements of genomic heterogeneity that could be used as indicators to determine the geographic origins of isolates of this bacterium., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bacterial communities play important roles in the biological functioning of crustaceans, yet little is known about their diversity, structure, and dynamics. This study was conducted to investigate the bacterial communities associated with the benthic amphipod Diporeia, an important component in the Great Lakes foodweb that has been declining over the past 3 decades. In this study, the combination of 16S rRNA gene sequencing and terminal restriction fragment length polymorphism revealed a total of 175 and 138 terminal restriction fragments (T-RFs) in Diporeia samples following treatment with the endonucleases HhaI and MspI, respectively. Relatively abundant and prevalent T-RFs were affiliated with the genera Flavobacterium and Pseudomonas and the class Betaproteobacteria. T-RFs affiliated with the order Rickettsiales were also detected. A significant difference in T-RF presence and abundance (P = 0.035) was detected among profiles generated for Diporeia collected from 4 sites in Lake Michigan. Comparison of profiles generated for Diporeia samples collected in 2 years from lakes Superior and Michigan showed a significant change in diversity for Lake Superior Diporeia but not Lake Michigan Diporeia. Profiles from one Lake Michigan site contained multiple unique T-RFs compared with other Lake Michigan Diporeia profiles, most notably one that represents the genus Methylotenera. This study generated the most extensive list of bacteria associated with Diporeia and sheds useful insights on the microbiome of Great Lakes Diporeia that may help to reveal potential causes of the decline of Diporeia populations.

Since the 1990s, populations of the benthic amphipod Diporeia spp. (Diporeia) have sharply declined across much of the Laurentian Great Lakes. This study was undertaken to identify contemporary and historical community composition, structure, and dynamics of parasites infecting Diporeia collected from nine sites in the southern basin of Lake Michigan, where declines of the amphipod have been well documented over the past 20years. An additional aim of this study was to assess whether infection dynamics and dreissenid densities could explain the declines in Diporeia densities that have occurred. We found that Diporeia were host to eight groups of uni- and multicellular pathogens. Of the 3082 amphipods analyzed, 1624 individuals (52.7%) were infected with at least one type of parasite. Ciliophora was the most prevalent parasite (50.08% prevalence of infection), followed by Gregarinasina (2.79%), Microsporidia (0.68%), Cestoda (0.45%), Acanthocephala (0.36%), Haplosporidia (0.23%), Yeast (0.32%), and filamentous Fungi (0.10%). Considerable spatial and temporal variability were observed in parasite prevalences, with prevalences frequently appearing to cycle between low and high values. Parasite species belonging to Microsporidia and Haplosporidia were associated with tissue alteration and host inflammatory response; however, parasite prevalences explained very little in terms of Diporeia density declines at assessed sites. Despite these findings, we do not discount the possibility that parasitic infections may have played a role in declining Diporeia densities in the Great Lakes, as the cyclical prevalences that were observed are possibly suggestive of parasitic outbreaks that are followed by die-offs at affected sites. This study suggests that if parasites have affected Diporeia densities in the Laurentian Great Lakes, then the relationship may be a complicated one., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The phylum Haplosporidia contains coelozoic and histozoic, spore-forming, obligate protozoan endoparasites that infect a number of freshwater and marine invertebrates including bivalves, crustaceans, and polychaetes. In amphipods, haplosporidians cause systemic infection resulting in a range of pathologies. While amphipods belonging to the genus Diporeia (Gammarideae) have been shown to host haplosporidians, the taxonomic relationship of the Diporeia haplosporidian(s) is largely unknown due to the lack of phylogenetic and detailed ultrastructural studies., Methods: The infection characteristics and taxonomic identity of a haplosporidian infecting Diporeia spp. (Diporeia) were based on microscopical investigation, electron microscopy, and Bayesian phylogenetic inference using haplosporidian 16S rRNA gene sequences., Results: In stained sections, the haplosporidian was observed to cause systemic infections in Diporeia that were often accompanied with host tissue degeneration. The haplosporidian appeared as binucleate plasmodia and sporocysts containing different spore maturation stages in the coelom, connective tissue, digestive tissue, and muscle. All of the observed systemic infections progressed to sporogenesis. Transmission electron microscopy revealed that fixed mature spores were slightly ellipsoidal and had a mean spore length X width of 5.34 ± 0.17 × 4.09 ± 0.15 μm. A hinged opercular lid with a length of 3.1 ± 0.17 μm was observed for a number of developing spores. The average thickness of the cell wall was 90.0 ± 8.33 nm. Thin filaments (70 nm) composed of spore wall material were observed projecting from an abopercular thickening of the spore wall. Phylogenetic analysis showed that the haplosporidian is novel bearing some similarities with the oyster pathogen Haplosporidium nelsoni, yet distinctly different., Conclusions: Based on its morphology, genetic sequence, and host, it became evident that the Diporeia haplosporidian is taxonomically novel and we propose its nomenclature as Haplosporidium diporeiae. This is the first report of a haplosporidian infecting Diporeia in Lake Superior.

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg(2+) with an incubation time of 40 min at 63 °C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2)=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The invasion of the Laurentian Great Lakes by the fish-parasitic sea lamprey has led to catastrophic consequences, including the potential introduction of fish pathogens. Aeromonas salmonicida is a bacterial fish pathogen that causes devastating losses worldwide. Currently, there are five accepted subspecies of Aeromonas salmonicida: A. salmonicida subsp. salmonicida, masoucida, smithia, achromogenes, and pectinolytica. We discuss the discovery of an isolate of A. salmonicida that is pathogenic to rainbow trout (Oncorhynchus mykiss) and exhibits unique phenotypic and molecular characteristics. We examined 181 adult sea lamprey (Petromyzon marinus) from the Humber River (Lake Ontario watershed) and 162 adult sea lamprey from Duffins Creek (Lake Ontario watershed) during the spring seasons of 2005-11. Among those, 4/343 (1.2%) sea lamprey were culture positive for A. salmonicida, whereby biochemical and molecular studies identified three of the isolates as A. salmonicida subsp. salmonicida. The remaining isolate (As-SL1) recovered from Humber River sea lamprey was phenotypically more similar to A. salmonicida subsp. salmonicida than to the four other A. salmonicida subspecies. However, unlike A. salmonicida subsp. salmonicida, As-SL1 was sucrose positive, produced an acid-over-acid reaction on triple-sugar iron medium and did not amplify with A. salmonicida subsp. salmonicida specific primers. Phylogenetic analysis based on partial stretches of the 16S rRNA and DNA gyrase subunit B genes further confirmed that the As-SL1 isolate was not A. salmonicida subsp. masoucida, smithia, achromogenes, or pectinolytica. Based on our analyses, the As-SL1 isolate is either an unusual strain of A. salmonicida subsp. salmonicida or a novel A. salmonicida subspecies. The four A. salmonicida isolates that were recovered from sea lamprey were pathogenic to rainbow trout in experimental challenge studies. Our study also underscores the potential role of sea lamprey in the ecology of infectious fish diseases.

Dictyocoela diporeiae n. sp. is described from Diporeia spp. (Amphipoda, Gammaridea) collected from Lake Superior (USA), and its morphology and taxonomic affiliation are discussed. In hematoxylin- and eosin-stained sections of infected amphipods, the microsporidian was observed to infect muscle tissue surrounding the ovaries. Melanized hemocytic encapsulations were often observed in or near masses of microsporidians. The microsporidians appeared as spores measuring 1.99 ± 0.09 μm long by 1.19 ± 0.05 μm wide. Each spore contained eight coils of isofilar polar filaments that were arranged in single ranks. Polar filaments measured 71 ± 3 nm in diameter. A prominent lamellar polaroplast composed of ordered concentric membranes was found at the apical end of the spore surrounding the polar filament. A distinct posterior vacuole was observed at the distal end of the spore. Phylogenetic analysis based on 16s RNA sequences showed that the microsporidian belongs to the genus Dictyocoela, and is most similar to D. berillonum, yet distinctly different. The species is new, based on its morphology, genetic sequence, host, and location within the host., (© A.D. Winters and M. Faisal , published by EDP Sciences, 2014.)

In 2003, viral hemorrhagic septicemia virus (VHSV) emerged in the Laurentian Great Lakes causing serious losses in a number of ecologically and recreationally important fish species. Within six years, despite concerted managerial preventive measures, the virus spread into the five Great Lakes and to a number of inland waterbodies. In response to this emerging threat, cooperative efforts between the Michigan Department of Natural Resources (MI DNR), the Michigan State University Aquatic Animal Health Laboratory (MSU-AAHL), and the United States Department of Agriculture-Animal and Plant Health Inspection Services (USDA-APHIS) were focused on performing a series of general and VHSV-targeted surveillances to determine the extent of virus trafficking in the State of Michigan. Herein we describe six years (2005-2010) of testing, covering hundreds of sites throughout Michigan's Upper and Lower Peninsulas. A total of 96,228 fish representing 73 species were checked for lesions suggestive of VHSV and their internal organs tested for the presence of VHSV using susceptible cell lines. Of the 1,823 cases tested, 30 cases from 19 fish species tested positive for VHSV by tissue culture and were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Gene sequence analyses of all VHSV isolates retrieved in Michigan demonstrated that they belong to the emerging sublineage "b" of the North American VHSV genotype IV. These findings underscore the complexity of VHSV ecology in the Great Lakes basin and the critical need for rigorous legislation and regulatory guidelines in order to reduce the virus spread within and outside of the Laurentian Great Lakes watershed.

The mode of viral hemorrhagic septicemia virus (VHSV) transmission in the Great Lakes basin is largely unknown. In order to assess the potential role of macroinvertebrates in VHSV transmission, Diporeia spp., a group of amphipods that are preyed upon by a number of susceptible Great Lakes fishes, were collected from seven locations in four of the Great Lakes and analyzed for the presence of VHSV. It was demonstrated that VHSV is present in some Diporeia spp. samples collected from lakes Ontario, Huron, and Michigan, but not from Lake Superior. Phylogenetic comparison of partial nucleoprotein (N) gene sequences (737 base pairs) of the five isolates to sequences of 13 other VHSV strains showed the clustering of Diporeia spp. isolates with the VHSV genotype IVb. This study reports the first incidence of a fish-pathogenic rhabdovirus being isolated from Diporeia, or any other crustacean and underscores the role macroinvertebrates may play in VHSV ecology.