Effect of chronic and acute enterotoxigenic E. coli challenge on growth performance, intestinal inflammation, microbiome, and metabolome of weaned piglets.

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.

Sheep in wolves' clothing: Temperate T7-like bacteriophages and the origins of the Autographiviridae.

Bacteriophage T7 is an extensively studied virulent phage, and its taxonomic family, the Autographiviridae, is broadly synonymous with a strictly virulent lifestyle. It is difficult to imagine how a T7-like phage could function in a "domesticated" temperate lifestyle, in which it is incorporated into the host's genome. Here we describe two temperate T7-like bacteriophages: ProddE, a Desulfovibrio phage, and Pasto, an Agrobacterium phage. Each contains recognizable T7-like proteins in the canonical T7-like gene order, but with the addition of lysogeny gene modules. While ProddE contains a phage-like repressor, Pasto lysogeny appears to be controlled by a novel MarR-like transcriptional regulator. In addition, we identify similar T7-like prophage elements in a wide variety of Gram-negative bacterial genomes and a small number of Gram-positive genomes. Identification of these elements in diverse bacterial species raises interesting evolutionary questions about the origins of T7-like phages and which lifestyle, temperate or virulent, is the ancestral form.

Comparison of 2 fixatives in the porcine colon for in situ microbiota studies.

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.

Complete Genome Sequence of Escherichia coli Myophage Mangalitsa.

Enteropathogenic Escherichia coli is a prevalent Gram-negative bacterium that can lead to fatal complications from infection in humans. Here, we present the isolation and complete annotation of the 52,329-bp genome of enteropathogenic E. coli ATCC 23545 myophage Mangalitsa. Predicted terminal repeats and temperature sensitivity for plaque formation place Mangalitsa with similar unclassified myoviruses.

Complete Genome Sequence of the Novel Klebsiella pneumoniae Phage Marfa.

Here, we describe the complete genome sequence of the T4-like Klebsiella pneumoniae myophage Marfa. In its 168,532-bp genome, Marfa has 289 genes, for which 122 gene functions were predicted. Many similar proteins are shared between Marfa and phage T4, as well as its closest phage relatives.

Complete Genome Sequence of Klebsiella pneumoniae Myophage Mineola.

Klebsiella pneumoniae is an important human pathogen due to the wide range of infections it can cause and its emerging drug resistance. Isolation and characterization of phage infecting K. pneumoniae could be important for future therapeutic applications. Here, we report the complete genome sequence of the T4-like Klebisella pneumoniae myophage Mineola.

A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain.

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.