Objective Immunodeficient patients are particularly vulnerable to neuroinvasive infections that can be challenging to diagnose. Metagenomic next-generation sequencing can identify unusual or novel microbes and is therefore well suited for investigating the etiology of chronic meningoencephalitis in immunodeficient patients. Methods We present the case of a 34 year-old man with X-linked agammaglobulinemia from Australia suffering from three years of meningoencephalitis that defied an etiologic diagnosis despite extensive conventional testing, including a brain biopsy. Metagenomic next-generation sequencing of his cerebrospinal fluid and brain biopsy tissue was performed to identify a causative pathogen. Results Sequences aligning to multiple Cache Valley virus genes were identified via metagenomic next-generation sequencing. Reverse transcription polymerase chain reaction and immunohistochemistry subsequently confirmed the presence of Cache Valley virus in the brain biopsy tissue. Interpretation Cache Valley virus, a mosquito-borne orthobunyavirus, has only been identified in three immunocompetent North American patients with acute neuroinvasive disease. The reported severity ranges from a self-limiting meningitis to a rapidly fatal meningoencephalitis with multi-organ failure. The virus has never been known to cause a chronic systemic or neurologic infection in humans. Cache Valley virus has also never previously been detected on the Australian continent. Indeed, our research subject traveled to North and South Carolina and Michigan in the weeks prior to the onset of his illness. This report demonstrates that metagenomic next-generation sequencing allows for unbiased pathogen identification, the early detection of emerging viruses as they spread to new locales, and the discovery of novel disease phenotypes.

BackgroundIntestinal absorption of bile acids is mediated by the apical sodium‐dependent bile acid transporter (ASBT). Fecal bile acid dysmetabolism has been reported in dogs with chronic inflammatory enteropathy (CIE).ObjectiveCharacterization of ASBT distribution along the intestinal tract of control dogs and comparison to dogs with CIE.AnimalsTwenty‐four dogs with CIE and 11 control dogs.MethodsThe ASBT mRNA and protein expression were assessed using RNA in situ hybridization and immunohistochemistry, respectively. The concentrations of fecal bile acids were measured by gas chromatography‐mass spectrometry. The fecal microbiota dysbiosis index was assessed with a quantitative polymerase chain reaction panel.ResultsIn control dogs, ASBT mRNA expression was observed in enterocytes in all analyzed intestinal segments, with highest expression in the ileum. The ASBT protein expression was restricted to enterocytes in the ileum, cecum, and colon. Dogs with CIE had significantly decreased expression of ASBT protein in the ileum (P = .001), which was negatively correlated with histopathological score (ρ = −0.40; P corr = .049). Additionally, dogs with CIE had a significantly increased percentage of primary bile acids in feces compared to controls (P = .04). The fecal dysbiosis index was significantly higher in dogs with CIE than in control dogs (P = .01).Conclusions and Clinical ImportanceThese findings indicate that ileal protein expression of ASBT is downregulated in dogs with CIE. This change may be linked to the inflammatory process, intestinal dysbiosis, and fecal bile acid dysmetabolism observed in these patients.

The massasauga Sistrurus catenatus was historically divided into three subspecies, but this long-standing taxonomy has recently been called into question. Genetic research now recognizes a split of the species into the eastern massasauga S. catenatus and western massasauga S. tergeminus, with the latter split into two subspecies, the desert massasauga S. t. edwardsii and the prairie massasauga S. t. tergeminus. Although the distinction between geographically isolated populations of S. catenatus and S. tergeminus is well-supported genetically, the geographic relationships among populations of S. t. tergeminus and S. t. edwardsii remain unresolved because of incomplete sampling throughout the species’ range. This poses a difficult challenge for conservation and management of this species. Sistrurus t. tergeminus does not have state or federal conservation status, but S. t. edwardsii has been petitioned for listing under the U.S. Endangered Species Act. In this study, we used nuclear and mitochondrial DNA from 52 individuals from 7 states to explore the taxonomic and geographic relationships between S. t. tergeminus and S. t. edwardsii populations. Maximum likelihood and Bayesian inference frameworks for both nuclear and mtDNA genes indicated that S. t. tergeminus and S. t. edwardsii populations were genetically indistinguishable. However, at the species level, we did find eight well-supported mtDNA clades within S. tergeminus, including individuals from five peripheral populations in 1) Arizona and western New Mexico, 2) Colorado and Kansas, 3) Missouri, 4) Oklahoma, and 5) southern Texas. These peripherally isolated populations surrounded a larger population of individuals from north-central Texas, New Mexico, and Oklahoma that was contiguous with three additional genetically distinct populations. We conclude that the putative subspecies S. t. tergeminus and S. t. edwardsii, as currently defined, most likely represent polytypic phenotypes of S. tergeminus rather than discrete taxonomic entities. Instead, we suggest that S. tergeminus existed historically as a large, contiguous collection of populations that only recently became fragmented into several, as opposed to two, potentially discrete taxonomic entities.

Fixation is the first step towards preservation of tissues and can impact downstream histological applications. Historically, formalin has been the fixative of choice in both research and clinical settings due to cost, accessibility, and broad applicability. Here, we describe a method for collection of porcine colon, and compare the usage of Carnoy’s solution (CS) to a 10% neutral buffered formalin (NBF) in tissue fixation. Consecutive colon samples were collected from 24 four-wk-old piglets and fixed in CS for 45 min or NBF for 24 h. We measured the thickness of the inner mucus layer using Alcian Blue stain and found thicker inner mucus layers in porcine colons fixed with CS as compared to NBF (P < 0.0001). Carnoy’s solution-fixed colon exhibited greater bacterial cell counts than NBF-fixed colon (P < 0.0022) after labeling with an eubacterial probe in fluorescent in situ hybridization (FISH). No difference was observed between the mucosal height (P = 0.42) and number of goblet cells (P = 0.66) between the 2 fixatives. From this, we concluded CS is more suitable than NBF for the preservation of the mucus layer and the associated mucosal bacteria in the porcine colon without compromising on overall tissue morphology. This study provides a useful sampling and fixation methodology for histology studies in the porcine gastrointestinal tract, and may be beneficial to microbiota, pathology, and nutrition studies.

Post-weaning enteropathies in swine caused by pathogenic E. coli, such as post-weaning diarrhea (PWD) or edema disease (ED), remain a significant problem for the swine industry. Reduction in the use of antibiotics over concerns of antibiotic resistance and public health concerns, necessitate the evaluation of effective antibiotic alternatives to prevent significant loss of livestock and/or reductions in swine growth performance. For this purpose, an appropriate piglet model of pathogenic E. coli enteropathy is required. In this study, we attempted to induce clinical signs of post-weaning disease in a piglet model using a one-time acute or lower daily chronic dose of a pathogenic E. coli strain containing genes for both heat stable and labile toxins, as well as Shiga toxin. The induced disease state was monitored by determining fecal shedding and colonization of the challenge strain, animal growth performance, cytokine levels, fecal calprotectin, histology, fecal metabolomics, and fecal microbiome shifts. The most informative analyses were colonization and shedding of the pathogen, serum cytokines, metabolomics, and targeted metagenomics to determine dysbiosis. Histopathological changes of the gastrointestinal (GI) tract and tight junction leakage as measured by fecal calprotectin concentrations were not observed. Chronic dosing was similar to the acute regimen suggesting that a high dose of pathogen, as used in many studies, may not be necessary. The piglet disease model presented here can be used to evaluate alternative PWD treatment options.