Proof of Concept for Prevention of Natural Colonization by Oral Needle-Free Administration of a Microparticle Vaccine.

There has been substantial interest in the development of needle-free vaccine administration that has led to a variety of approaches for delivery through the skin for induction of a systemic immune response. The mucosal administration of vaccines has inherently been needle-free, but the simple application of vaccines on the mucosal surface by itself does not lead to mucosal immunity. Since many important bacterial infections develop after initial colonization of the upper respiratory tract of the host, prevention of colonization could not only prevent infection but also eliminate the reservoir of pathogens that reside exclusively in that ecologic niche. This study was designed to provide proof of concept for a needle-free immunization approach that would reduce or eliminate colonization and prevent infection. In order to accomplish this a microparticle vaccine preparation was delivered just below the oral mucosal epithelial cell layer where it would lead to a robust immune response. A vaccine antigen (mutant transferrin binding protein B) shown to be capable of preventing infection in pigs was incorporated into a polyphosphazene microparticle preparation and delivered by a needle-free device to the oral sub-epithelial space of pigs. This vaccination regimen not only provided complete protection from infection after intranasal challenge by Glaesserella parasuis but also eliminated natural colonization by this bacterium. Notably, the complete prevention of natural colonization was dependent upon delivery of the microparticle preparation below the epithelial layer in the oral mucosa as intradermal or intramuscular delivery was not as effective at preventing natural colonization. This study also demonstrated that a primary immunization in the presence of maternal antibody limited the resulting antibody response but a robust antibody response after the second immunization indicated that maternal antibody did not prevent induction of B-cell memory.

Some Gammaproteobacteria are enriched within CD14+ macrophages from intestinal lamina propria of Crohn's disease patients versus mucus.

Crohn's disease causes chronic inflammation in the gastrointestinal tract and its pathogenesis remains unclear. In the intestine of Crohn's disease patients, CD14+CD11+CD163low macrophages contribute to inflammation through the induction of Th17 cells and production of inflammatory cytokines; the CD14+CD11c+163high fraction is anti-inflammatory through the production of IL-10 in normal cases. In this report, the 16S rRNA gene amplicon sequencing method was used to identify bacteria that are specifically present in intestinal CD14+CD11c+ macrophages of Crohn's disease patients. Bacteria present in intestinal CD14+CD11c+ macrophages and mucus of Crohn's disease patients were separated into different clusters in principal coordinates analysis. There was a statistically significant increase in the relative composition of CD14+CD11c+ macrophages from mucus in two phyla (Proteobacteria [p = 0.01] and Actinobacteria [p = 0.02]) and two families (Moraxellaceae [p < 0.001] and Pseudomonadaceae [p = 0.01]). In addition, OTU-1: Acinetobacter and OTU-8: Pseudomonadaceae tended to concentrate in the CD14+CD11c+CD163low subset, whereas OTU-10: Proteus, OTU-15: Collinsella tended to concentrate more in the CD14+CD11c+CD163high subset than the other subset and mucus.

Specific Chicken Egg Yolk Antibody Improves the Protective Response against Gallibacterium anatis Infection.

Gallibacterium anatis is a pathogen associated with peritonitis and salpingitis in chickens and other avian species. Novel safety prevention strategies are urgently needed because of widespread multidrug resistance and antigenic diversity. The objective of this study was to produce a specific chicken egg yolk antibody and evaluate its protective response against a G. anatis infection model in 4-week-old chicks. Enzyme-linked immunosorbent assays showed that hens immunized with the recombinant N terminus of Gallibacterium toxin A (GtxA-N) had significantly increased antibody titers against GtxA-N in serum and egg yolk IgY. Western blotting showed that IgY antibody had specificity against GtxA-N in the egg yolks of immunized hens. The growth of G. anatis in brain heart infusion (BHI) broth and agar was significantly inhibited by the GtxA-N-specific IgY antibody. The protective effects of the specific IgY antibody were evaluated in G. anatis-infected chicks after intramuscular injection (10 mg/ml). The anti-GtxA-N antibody titers in the sera of G. anatis-challenged chicks following an injection of specific IgY antibody were significantly higher than those of the control and the nonspecific IgY groups, but lower lesion scores for the peritoneum, liver, and duodenum were found after specific IgY antibody treatment. The results from this study suggest that the GtxA-N-specific IgY antibody could potentially improve the protective response against G. anatis infection in chicks.

Isolation and characterization of Avibacterium paragallinarum with different nicotinamide adenine dinucleotide requirements.

Twenty field isolates of Avibacterium paragallinarum were obtained from chickens in South Korea during 2011-2015. The isolates were identified by a HPG-2 PCR assay specific for A. paragallinarum and by biochemical tests. Growth requirements, Page serovars, carbohydrate fermentation patterns, and antimicrobial susceptibility were also examined. Most isolates (16/20) showed the typical requirement for nicotinamide adenine dinucleotide (NAD) and an enriched CO2 atmosphere for growth. One isolate needed increased levels of NAD and serum for good growth. Three isolates showed NAD-independent growth on blood agar under aerobic conditions. In terms of carbohydrate fermentation patterns, three biochemical biovars were recognized; these varied with respect to acid production from maltose and D-xylose. The 16 typical NAD-dependent isolates were serovar A while the variants, both NAD-independent isolates and the isolate with increased NAD dependency were non-typeable. All isolates were sensitive to amoxicillin-clavulanic acid, ceftiofur, gentamicin, and spectinomycin. High rates of resistance, including intermediate resistance, to lincomycin (100%), cloxacillin (75%), and erythromycin (70%) were observed. The four variant strains (the three NAD-independent isolates and the isolate showing unusual growth requirements) were more resistant to antibiotics than the typical NAD-dependent strains. The finding of NAD-independent forms of A. paragallinarum extends the known distribution of this form, previously only reported in South Africa, Mexico and Peru. There is clearly a need for increased caution in the diagnosis and, possibly, the control of infectious coryza.

The respiratory tract microbiome and lung inflammation: a two-way street.

The lungs are not sterile or free from bacteria; rather, they harbor a distinct microbiome whose composition is driven by different ecological rules than for the gastrointestinal tract. During disease, there is often a shift in community composition towards Gammaproteobacteria, the bacterial class that contains many common lung-associated gram-negative "pathogens." Numerous byproducts of host inflammation are growth factors for these bacteria. The extracellular nutrient supply for bacteria in the lungs, which is severely limited during health, markedly increases due to the presence of mucus and vascular permeability. While Gammaproteobacteria benefit from airway inflammation, they also encode molecular components that promote inflammation, potentially creating a cyclical inflammatory mechanism. In contrast, Prevotella species that are routinely acquired via microaspiration from the oral cavity may participate in immunologic homeostasis of the airways.vAreas of future research include determining for specific lung diseases (1) whether an altered lung microbiome initiates disease pathogenesis, promotes chronic inflammation, or is merely a marker of injury and inflammation, (2) whether the lung microbiome can be manipulated therapeutically to change disease progression, (3) what molecules (metabolites) generated during an inflammatory response promote cross-kingdom signaling, and (4) how the lung "ecosystem" collapses during pneumonia, to be dominated by a single pathogen.

Development of a lateral flow immunoassay for rapid diagnosis of potato blackleg caused by Dickeya species.

Early detection of potato infections is essential for effective disease management. The aim of this study was to develop a lateral flow immunoassay (LFIA) for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani. Polyclonal antibodies specific to different strains of Dickeya were obtained from rabbits after immunization with bacterial cells of D. dianthicola and D. solani. Enzyme-linked immunosorbent assay testing with use of a wide range of bacterial species showed that the polyclonal antibodies detect closely related strains of D. dianthicola and D. solani. Cross-reactivity with widespread pathogenic bacteria (nine species) and saprophytes of healthy potato plants was not detected. The LFIA based on the obtained antibodies and gold nanoparticles with average diameter of 20 nm was developed. Under optimized conditions, the LFIA method enabled the analysis of potato extracts within 10 min, with a visual limit of detection of 1 × 105 CFU/ml for leaves and 4 × 105 CFU/ml for tubers. The assay was tested on potato stem and tuber extracts, and the results of the LFIA were confirmed in 92.1% of samples using the real-time polymerase chain reaction. The findings confirmed that the developed LFIA could be used for monitoring blackleg infection without the need for special equipment or skills. Graphical Abstract The developed lateral flow immunoassay is an efficient tool for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani.

Gestational diabetes is associated with changes in placental microbiota and microbiome.

BACKGROUND: The human microbiota is a modulator of the immune system. Variations in the placental microbiota could be related with pregnancy disorders. We profiled the placental microbiota and microbiome in women with gestational diabetes (GDM) and studied its relation to maternal metabolism and placental expression of anti-inflammatory cytokines. METHODS: Placental microbiota and microbiome and expression of anti-inflammatory cytokines (IL10, TIMP3, ITGAX, and MRC1MR) were analyzed in placentas from women with GDM and from control women. Fasting insulin, glucose, O'Sullivan glucose, lipids, and blood cell counts were assessed at second and third trimester of pregnancy. RESULTS: Bacteria belonging to the Pseudomonadales order and Acinetobacter genus showed lower relative abundance in women with GDM compared to control (P < 0.05). In GDM, lower abundance of placental Acinetobacter associated with a more adverse metabolic (higher O'Sullivan glucose) and inflammatory phenotype (lower blood eosinophil count and lower placental expression of IL10 and TIMP3) (P < 0.05 to P = 0.001). Calcium signaling pathway was increased in GDM placental microbiome. CONCLUSION: A distinct microbiota profile and microbiome is present in GDM. Acinetobacter has been recently shown to induce IL-10 in mice. GDM could constitute a state of placental microbiota-driven altered immunologic tolerance, making placental microbiota a new target for therapy in GDM.

The Bacterium Frischella perrara Causes Scab Formation in the Gut of its Honeybee Host.

UNLABELLED: Honeybees harbor well-defined bacterial communities in their guts. The major members of these communities appear to benefit the host, but little is known about how they interact with the host and specifically how they interface with the host immune system. In the pylorus, a short region between the midgut and hindgut, honeybees frequently exhibit scab-like structures on the epithelial gut surface. These structures are reminiscent of a melanization response of the insect immune system. Despite the wide distribution of this phenotype in honeybee populations, its cause has remained elusive. Here, we show that the presence of a common member of the bee gut microbiota, the gammaproteobacterium Frischella perrara, correlates with the appearance of the scab phenotype. Bacterial colonization precedes scab formation, and F. perrara specifically localizes to the melanized regions of the host epithelium. Under controlled laboratory conditions, we demonstrate that exposure of microbiota-free bees to F. perrara but not to other bacteria results in scab formation. This shows that F. perrara can become established in a spatially restricted niche in the gut and triggers a morphological change of the epithelial surface, potentially due to a host immune response. As an intermittent colonizer, this bacterium holds promise for addressing questions of community invasion in a simple yet relevant model system. Moreover, our results show that gut symbionts of bees engage in differential host interactions that are likely to affect gut homeostasis. Future studies should focus on how these different gut bacteria impact honeybee health. IMPORTANCE: As pollinators, honeybees are key species for agricultural and natural ecosystems. Their guts harbor simple communities composed of characteristic bacterial species. Because of these features, bees are ideal systems for studying fundamental aspects of gut microbiota-host interactions. However, little is known about how these bacteria interact with their host. Here, we show that a common member of the bee gut microbiota causes the formation of a scab-like structure on the gut epithelium of its host. This phenotype was first described in 1946, but since then it has not been much further characterized, despite being found in bee populations worldwide. The scab phenotype is reminiscent of melanization, a conserved innate immune response of insects. Our results show that high abundance of one member of the bee gut microbiota triggers this specific phenotype, suggesting that the gut microbiota composition can affect the immune status of this key pollinator species.

CRISPRs extending their reach: prokaryotic RNAi protein Cas9 recruited for gene regulation.

A Cas protein from the CRISPR defence system against foreign DNA, also functions in endogenous gene regulation. Sampson et al (2013) have revealed that in pathogenic Francisella bacteria, the Cas9 protein guided by small RNAs represses the mRNA of a lipoprotein. This novel mechanism of post-transcriptional regulation enables the infecting bacteria to evade the TLR2-based innate immune response of its host. Thus, reminiscent of eukaryotic RNAi where some proteins facilitate both genome defence and gene regulation, a central prokaryotic RNAi protein not only destroys invading DNA but also controls mRNA expression.

A CRISPR/Cas system mediates bacterial innate immune evasion and virulence.

CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.

Environmental biodiversity, human microbiota, and allergy are interrelated.

Rapidly declining biodiversity may be a contributing factor to another global megatrend--the rapidly increasing prevalence of allergies and other chronic inflammatory diseases among urban populations worldwide. According to the "biodiversity hypothesis," reduced contact of people with natural environmental features and biodiversity may adversely affect the human commensal microbiota and its immunomodulatory capacity. Analyzing atopic sensitization (i.e., allergic disposition) in a random sample of adolescents living in a heterogeneous region of 100 × 150 km, we show that environmental biodiversity in the surroundings of the study subjects' homes influenced the composition of the bacterial classes on their skin. Compared with healthy individuals, atopic individuals had lower environmental biodiversity in the surroundings of their homes and significantly lower generic diversity of gammaproteobacteria on their skin. The functional role of the gram-negative gammaproteobacteria is supported by in vitro measurements of expression of IL-10, a key anti-inflammatory cytokine in immunologic tolerance, in peripheral blood mononuclear cells. In healthy, but not in atopic, individuals, IL-10 expression was positively correlated with the abundance of the gammaproteobacterial genus Acinetobacter on the skin. These results raise fundamental questions about the consequences of biodiversity loss for both allergic conditions and public health in general.

Dietary administration of Zooshikella sp. enhance the innate immune response and disease resistance of Paralichthys olivaceus against Sreptococcus iniae.

We report the growth, innate immune response, and disease resistance in olive flounder (Paralichthys olivaceus) challenged with Streptococcus iniae after feeding with diet enriched with Zooshikella sp. strain JE-34 three different concentration i.e. Low (3.4 x 10(4), n = 50), medium (3.5 x 10(6), n = 50), and high (3.4 x 10(8), n = 50) cfu ml(-1) supplemented diets, the changes were monitored on weeks 1, 2, 4, 8, 12, and 16. With all diets the innate immune parameters, such as superoxide anion production, phagocytic and lysozyme activity were not enhanced on week 1 and 4. On the other hand, all tested immune parameters in the treated groups significantly enhanced after 8th week; the weight gain significantly increased after 4th week in fish fed with enriched diets. The mortality in olive flounder after administration with high concentration diet showed 25%. With low and medium enriched diets the mortality was 40% and 35%, respectively. In the infected untreated group mortality was 85% while there was no mortality in the control group. The results suggested that Zooshikella sp. strain JE-34 enriched diets could be used to enhance the innate immune response and disease resistance of P. olivaceus against S. iniae.

Rickettsial infection in farmed Atlantic salmon in eastern Canada.

The cause of death in a postsmolt, Atlantic salmon population with elevated levels of mortalities was investigated. Diagnosis of a rickettsia-like organism was based on gross pathology, histopathology, differential staining, electron microscopy and fluorescent antibody tests. The course of the infection and response to treatment are discussed. This is the first reported occurrence of salmon rickettsias in the Atlantic coast of North or South America.

An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia, an economically devastating rickettsial disease of farmed salmonids. Infected salmonids respond poorly to antibiotic treatment and no effective vaccine is available for the control of P. salmonis. Bacterin preparations of P. salmonis were found to elicit a dose-dependent response in coho salmon (Oncorhynchus kisutch), which varied from inadequate protection to exacerbation of the disease. However, an outer surface lipoprotein of P. salmonis, OspA, recombinantly produced in Escherichia coli elicited a high level of protection in vaccinated coho salmon with a relative percent survival as high as 59% for this single antigen. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein, that are universally immunogenic in mammalian immune systems, were incorporated tandemly into an OspA fusion protein. Addition of these TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a three-fold increase in vaccine efficacy. These results represent a highly effective monovalent recombinant subunit vaccine for a rickettsia-like pathogen, P. salmonis, and for the first time demonstrate the immunostimulatory effect of mammalian TCE's in the salmonid immune model. These results may also be particularly pertinent to salmonid aquaculture in which the various subspecies are outbred and of heterologous haplotypes.

Application of an amperometric immunosensor for the enumeration of Nitrobacter in activated sludge.

A competitive immunosensor using a monoclonal antibody has been developed for the enumeration of Nitrobacter in activated sludge and other environmental samples. Its cross-reactivity was tested against a number of bacterial strains and isolates. All strains of the nitrite-oxidising genera Nitrobacter and Nitrococcus reacted strongly with the monoclonal antibody. The nitrite-oxidising Nitrospira moscoviensis, as well as the ammonia oxidising bacteria and the heterotrophic bacteria tested, did not show any affinity towards the antibody in the immunosensor. The numbers of Nitrobacter were analysed in sludge samples from several wastewater treatment plants in Sweden. Detectable amounts were found in all samples. This study shows the adequacy of using this immunosensor for the enumeration of Nitrobacter in natural environments.