Immune Activation and Inflammatory Response Mediated by the NOD/Toll-like Receptor Signaling Pathway-The Potential Mechanism of Bullfrog (Lithobates catesbeiana) Meningitis Caused by Elizabethkingia miricola.

Elizabethkingia miricola is an emerging opportunistic pathogen that is highly pathogenic in both immunocompromised humans and animals. Once the disease occurs, treatment can be very difficult. Therefore, a deep understanding of the pathological mechanism of Elizabethkingia miricola is the key to the prevention and control of the disease. In this study, we isolated the pathogenic bacteria from bullfrogs with dark skin color, weak limbs, wryneck, and cataracts. Via subsequent morphological observations and a 16S rRNA gene sequence analysis, the pathogen was identified as Elizabethkingia miricola. The histopathological and transmission electron microscopy analysis revealed that the brain was the main target organ. Therefore, brain samples from diseased and healthy bullfrogs were used for the RNA-Seq analysis. The comparative transcriptome analysis revealed that the diseased bullfrog brain was characterized by the immune activation and inflammatory response, which were mediated by the "NOD-like receptor signaling pathway" and the "Toll-like receptor signaling pathway". We also performed qRT-PCR to examine the expression profile of inflammation-related genes, which further verified the reliability of our transcriptome data. Based on the above results, it was concluded that the NOD/Toll-like receptor-related networks that dominate the immune activation and inflammatory response were activated in the brain of Elizabethkingia miricola-infected bullfrogs. This study contributes to the search for therapeutic targets for bullfrog meningitis and provides basic information for establishing effective measures to prevent and control bullfrog meningitis.

Comparison of three species of Elizabethkingia genus by whole-genome sequence analysis.

Elizabethkingia are found to cause severe neonatal meningitis, nosocomial pneumonia, endocarditis and bacteremia. However, there are few studies on Elizabethkingia genus by comparative genomic analysis. In this study, three species of Elizabethkingia were found: E. meningoseptica, E. anophelis and E. miricola. Resistance genes and associated proteins of seven classes of antibiotics including beta-lactams, aminoglycosides, macrolides, tetracyclines, quinolones, sulfonamides and glycopeptides, as well as multidrug resistance efflux pumps were identified from 20 clinical isolates of Elizabethkingia by whole-genome sequence. Genotype and phenotype displayed a good consistency in beta-lactams, aminoglycosides and glycopeptides, while contradictions exhibited in tetracyclines, quinolones and sulfonamides. Virulence factors and associated genes such as hsp60 (htpB), exopolysaccharide (EPS) (galE/pgi), Mg2+ transport (mgtB/mgtE) and catalase (katA/katG) existed in all clinical and reference strains. The functional analysis of the clusters of orthologous groups indicated that 'metabolism' occupied the largest part in core genome, 'information storage and processing' was the largest group in both accessory genome and unique genome. Abundant mobile elements were identified in E. meningoseptica and E. anophelis. The most significant finding in our study was that a single clone of E. anophelis had been circulating within diversities of departments in a clinical setting for nearly 18 months.

Investigation of Proteus vulgaris and Elizabethkingia meningoseptica invasion on muscle oxidative stress and autophagy in Chinese soft-shelled turtle (Pelodiscus sinensis).

Muscle is an important structural tissue in aquatic animals and it is susceptible to bacterial and fungal infection, which could affect flesh quality and health. In this study, Chinese soft-shelled turtles were artificially infected with two pathogens, Proteus vulgaris and Elizabethkingia meningoseptica and the effects on muscle nutritional characteristics, oxidative stress and autophagy were assayed. Upon infection, the muscle nutritional composition and muscle fiber structure were notably influenced. Meanwhile, the mRNA expression of Nrf2 was down-regulated and Keap1 up-regulated, thus resulting in a decrease in antioxidant capacity and oxidative stress. However, with N-acetylcysteine treatment, the level of oxidative stress was decreased, accompanied by significant increases in antioxidant enzyme activities and the mRNA levels of SOD, CAT, GSTCD, and GSTO1. Interestingly, there was a significant increase in autophagy in the muscle tissue after the pathogen infection, but this increase could be reduced by N-acetylcysteine treatment. Our findings suggest that muscle nutritional characteristics were dramatically changed after pathogen infection, and oxidative stress and autophagy were induced by pathogen infection. However, N-acetylcysteine treatment could compromise the process perhaps by decreasing the ROS level and regulating Nrf2-antioxidant signaling pathways.

Riemerella anatipestifer infection affects intestinal barrier structure and immune reactions in the duck caecum.

Riemerella anatipestifer (RA) infection causes high mortality and poor feed conversion, leading to great economic losses to the duck industry. This study investigated the effects of RA on the intestinal morphology and immune response of ducks. Histological examination showed that RA infection caused intestinal injury, including significantly reduced mucosal thickness on days 2, 3 and 5, significantly reduced villus height on days 1, 2, 3 and 5 (P < 0.05) and significantly reduced villus height to crypt depth ratios on days 2, 3, 5 and 9 of RA infection (P < 0.05). The expression of intestinal mucosal layer construction-associated genes and tight junction genes was significantly altered on at least one time point (day 1, 2, 3, 5, 9 or 14) after RA infection. Quantitative real-time polymerase chain reaction revealed that RA infection affected intestinal mucosal immune function. The genes encoding TLR4 (toll like receptor-4), TRAF6 (TNF receptor-associated factor 6), MYD88 (myeloid differentiation factor 88), IFN-γ (interferon-γ), IL (interleukin)-4 and IL-8 were significantly upregulated on day 2 of RA infection. Taken together, these results indicate that RA infection negatively affects intestinal barrier function in ducks due to impaired mucosal and villus-crypt structure and alters the mRNA expression of mucous layer construction-, intestinal tight junction-, and intestinal mucosal immunity-related genes.

Insights into the microbiome of farmed Asian sea bass (Lates calcarifer) with symptoms of tenacibaculosis and description of Tenacibaculum singaporense sp. nov.

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes), most notably Tenacibaculum maritimum. The impact of tenacibaculosis on the fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissues of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farmed fish. The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One isolated strain, TLL-A2T, shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764T. The genome of strain TLL-A2T was sequenced and compared to that of T. mesophilum DSM 13764T. Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764T and strain TLL-A2T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2T and T. mesophilum DSM 13764T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2T (=DSM 106434T, KCTC 62393T) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

Candidate Animal Disease Model of Elizabethkingia Spp. Infection in Humans, Based on the Systematic Pathology and Oxidative Damage Caused by E. miricola in Pelophylax nigromaculatus.

Most species of the genus Elizabethkingia are pathogenic to humans and animals, most commonly causing meningitis. However, our understanding of the pathogenic mechanisms involved is poor and there have been few pathological studies of Elizabethkingia spp. in animals. To understand the host injury induced by Elizabethkingia spp., we established a model of E. miricola infection in the black-spotted frog (Pelophylax nigromaculatus). The systematic pathology in and oxidative damage in the infection model were investigated. Our results show that recently isolated E. miricola is a bacterium that mainly parasitizes the host brain and that neurogenic organs are the predominant sites of damage. Infection mainly manifested as severe brain abscesses, meningoencephalitis, necrotic spondylitis, and necrotic retinitis. The liver, spleen, kidney, gastrointestinal tract, and lung were also affected to varying degrees, with bacterial necrotic inflammation. P. nigromaculatus also suffered enormous damage to its oxidative system during E. miricola infection, which may have further aggravated its disease state. Our results provide a preliminary reference for the study and treatment of Elizabethkingia spp.-induced neurological diseases in animals.

Cas9 regulated gene expression and pathogenicity in Riemerella anatipestifer.

Riemerellosis, a Riemerella anatipestifer infection, can cause meningitis, pericarditis, parahepatitis, and airsacculitis in ducks, leading to serious economic losses in the duck meat industry. However, the molecular mechanism of the pathogenesis and virulence factors of this infection are poorly understood. In the present study, we created a mutant strain RA-YMΔCas9 using trans-conjugation. Bacterial virulence tests indicated that the median lethal dose (LD50) of RA-YMΔCas9 was 5.01 × 107 CFU, significantly lower than that of the RA-YM strain, which was 1.58 × 105 CFU. The distribution and blood bacterial load from the infection groups showed no significant difference in the brain between the RA-YMΔCas9 mutant and the wild-type RA-YM strains, however, the number of mutant strains were significantly reduced in the liver, heart, and blood. Animal immunization experiments demonstrated that the intranasal administration of RA-YMΔCas9 in ducklings provided 80% protection after challenge with the wild-type strain, showing potential use as a live mucosal vaccine. RNAseq analysis indicated that Cas9 protein played a regulatory role in gene expression. This study is the first to report on the involvement of Cas9 in the regulation and pathogenesis of R. anatipestifer, and provides a theoretical basis for the development of relevant genetic engineering vaccines.

Transcriptomic response of rainbow trout (Oncorhynchus mykiss) skeletal muscle to Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiologic agent of rainbow trout fry syndrome (RTFS). This pathogen infects a wide variety of salmonid species during freshwater stages, causing significant losses in the aquaculture industry. Rainbow trout (Oncorhynchus mykiss) infected with F. psychrophilum, presents as the main external clinical sign ulcerative lesions and necrotic myositis in skeletal muscle. We previously reported the in vitro cytotoxic activity of F. psychrophilum on rainbow trout myoblast, however little is known about the molecular mechanisms underlying the in vivo pathogenesis in skeletal muscle. In this study, we examined the transcriptomic profiles of skeletal muscle tissue of rainbow trout intraperitoneally challenged with low infection dose of F. psychrophilum. Using high-throughput RNA-seq, we found that 233 transcripts were up-regulated, mostly associated to ubiquitin mediated proteolysis and apoptosis. Conversely, 189 transcripts were down-regulated, associated to skeletal muscle contraction. This molecular signature was consistent with creatine kinase activity in plasma and oxidative damage in skeletal muscle. Moreover, the increased caspase activity suggests as a whole skeletal muscle atrophy induced by F. psychrophilum. This study offers an integrative analysis of the skeletal muscle response to F. psychrophilum infection and reveals unknown aspects of its pathogenesis in rainbow trout.

A combined retrospective, prospective and experimental study of Ornithobacterium rhinotracheale infection in chickens.

The aim of this study was to examine the histopathological and immunohistochemical changes caused by natural and experimentally-induced Ornithobacterium rhinotracheale infection in the respiratory system of chickens. To this end, three different studies were carried out. The first was a retrospective study of 82 field cases with respiratory disorders compatible with O. rhinotracheale infection. The bacterium was immunohistochemically detected in the lungs in 48 of 82 field cases, and 50 ß-haemolytic (BH) and non-haemolytic (NH) strains were isolated. In the second study, an experimental model of the disease was created using 3-week-old broiler chickens, to identify possible differences of pathogenicity between the BH and NH isolates by the intravenous (IV) and intratracheal (IT) inoculation routes (IR). The group challenged with the NH isolate showed more severe lung lesions than the group challenged with the BH isolate at 7-days postinoculation (p.i.). The 14-day p.i. groups challenged with either the BH or NH isolates by the IT or IV IR had a higher histologic grade of pulmonary and hepatic lesions and a higher total histologic grade of lesions suggesting more severe pathology with longer time of exposure. A direct association between the inoculation routes and the organs affected was shown. Finally, a slaughterhouse study was carried out from October 2014 to May 2015, in which the histologic grade of lesions was significantly higher in immunohistochemically positive for O. rhinotracheale lungs of dead-on-arrival chickens.

Deletion of the Riemerella anatipestifer type IX secretion system gene sprA results in differential expression of outer membrane proteins and virulence.

Riemerella anatipestifer (RA), the causative agent of infectious serositis that targets ducklings and other poultry, secretes protein via the type IX secretion system (T9SS). The proteins transported by T9SS are located on the bacterial cell surface or secreted into the extracellular milieu. In this study, a sprA deletion mutant was constructed encoding a core protein of T9SS to investigate its influence on outer membrane protein expression and its role in virulence. Compared with the wild-type RA-YM strain, the deletion mutant ΔsprA failed to digest gelatin, showed the same growth rate in the logarithmic phase and exhibited greater sensitivity to the bactericidal activity of duck sera, whereas the complemented strain restored these phenotypes. The outer membrane proteome of RA-YM and the ΔsprA mutant were analyzed by Tandem Mass Tags, which revealed 198 proteins with predicted localization to the cell envelope. Sixty-three of these proteins were differentially expressed in the outer membrane, with 43 up-regulated and 20 down-regulated. Among the twelve outer membrane proteins which were secreted by T9SS, four proteins were up-regulated and one protein was down-regulated. Animal experiments demonstrated that the median lethal dose of the mutant strain ΔsprA was about 500 times higher than that of the wild-type RA-YM strain, and bacterial loads in blood, brain, heart, liver and spleen of the ΔsprA-infected ducks were significantly reduced. Our results indicate that the SprA is a virulence-associated factor of RA, and its absence results in altered abundance of outer membrane proteins, and secretion disorders associated with some of the T9SS effector proteins.

Effect of experimental Ornithobacterium rhinotracheale infection along with live infectious bronchitis vaccination in broiler chickens.

Ornithobacterium rhinotracheale (ORT), a bacterium causing respiratory tract infection, has led to a significant problem in the intensive poultry production in Egypt. Polymerase chain reaction-amplified 784-bp specific ORT DNA fragments were found in 7 ORT isolates from lungs, air sacs, and tracheas of commercial broilers or layers in Egypt in 2015. The objective of this study was to investigate the role of the live variant IBV 4/91 with ORT infection. A total of 120 14-d-old broiler chickens (Cobb 500) were equally divided into 4 groups for experimental infection in a complete randomized design. Group 1 was infected with ORT strain and live infectious bronchitis vaccine (IBV 4/91) simultaneously; group 2 was infected with the bacterial strain alone; group 3 was vaccinated only with IBV 4/91, and group 4 was the non-vaccinated and non-infected control group. The respiratory signs, post-mortem lesions (tracheitis and pneumonia) and histopathological findings of lungs, trachea, and air sacs in the experimentally infected broiler chickens appeared to be more prominent in the chickens of group 1 than group 2. With respect to body weight, weight gain, feed conversion rate, and Ornithobacterium re-isolation, there was a difference (P ≤ 0.05) among the chickens of group 1 and the other groups. This reveals that the use of live infectious bronchitic vaccines, which is a common practice in the local Egyptian field of production, may concomitantly increase the pathogenicity of ORT in broiler chickens.

Impairing of gill health through decreasing immune function and structural integrity of grass carp (Ctenopharyngodon idella) fed graded levels dietary lipids after challenged with Flavobacterium columnare.

The current study conducted to investigate the hypothesis that low or excess levels of lipids increased the gill rot morbidity through impairing the immune function and structural integrity in the gill of grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ±â€¯0.53 g were fed diets containing six graded levels of lipids at 0.59%, 2.14%, 3.60%, 5.02%, 6.66% and 8.01% diets for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that compared with optimal lipids supplementation (2.14%-8.01% lipids diets), low or excess levels of lipids impaired fish immune function through declining the activities of humoral compounds, down-regulated the mRNA levels of anti-inflammatory cytokines, inhibitor of κBα (IκBα) and ribosomal p70S6 kinase (S6K1), and up-regulated pro-inflammatory cytokines, nuclear factor κB p65 (NF-κB p65) (not p52), IκB kinase α (IKKα) (not IKKß), IKKγ and eIF4E-binding protein (4EBP) in the gill of young grass carp. In addition, low or excess levels of lipids decreased young grass carp physical barrier function through down-regulating the mRNA levels of ZO-1 (rather than ZO-2b), Claudin b, c, 3, 12, 15a, 15b, 7b, 7a and Occludin through MAPKK 6/p38 MAPK/MLCK signaling molecules, decreasing antioxidant ability via Kelch-like ECH-associating protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2) signaling molecules, and down-regulating the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) and up-regulating the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), Caspase-3, -8 and -9 and Fas ligand (FasL) in the gill of grass carp. Based on the quadratic regression analysis for the gill rot morbidity, C3 and MDA contents, the dietary lipids requirements for young grass carp have been estimated to be 5.60%, 6.01% and 4.58% diets.

Molecular Characterization of Chryseobacterium indologenes with Multidrug Resistance in the Brazilian Amazon Region.

Chryseobacterium indologenes is an emerging nosocomial pathogen that produces IND-type chromosomal metallo-beta-lactamase. The phenotype and molecular aspects of two multidrug resistant C. indologenes strains and the analysis of the tertiary structure of the IND enzyme were studied. Identification of species and susceptibility tests were performed using the Vitek-2 compact. Chromosomal and plasmid DNA were extracted using PureLink™ Genomic DNA Mini Kit and PureLink Quick Plasmid Miniprep Kit, and the sequencing was performed using ABI 3130 genetic analyzer. Two strains were isolated and are registered as P-23 and P-113. Of the two, P-113 was sensitive to ciprofloxacin and cefepime only, whereas the P-23 showed reduced sensitivity to ceftazidime, ciprofloxacin, and tigecycline. The genetic analysis of both isolates identified the presence of the blaIND-like gene, with similarity to IND-3 and IND-8 alleles. The IND-3 identified in the P-133 sample presented a single mutation at position T355G, which corresponds to a nonsynonymous substitution of the amino acid at position 119 (Ser→Ala). The phylogenetic analysis of INDs showed lineages that are circulating in Asian and European countries. These results emphasize the need for effective preventive actions to avoid the dissemination of this type of pathogen in the hospital environment.

Serosurvey of Avian metapneumovirus, Orithobacterium rhinotracheale, and Chlamydia psittaci and Their Potential Association with Avian Airsacculitis.

Seasonal outbreaks of airsacculitis in China's poultry cause great economic losses annually. This study tried to unveil the potential role of Avian metapneumovirus (AMPV), Ornithobacterium rhinotracheale (ORT) and Chlamydia psittaci (CPS) in avian airsacculitis. A serological investigation of 673 breeder chickens and a case-controlled study of 430 birds were undertaken. Results showed that infection with AMPV, ORT, and CPS was highly associated with the disease. The correlation between AMPV and CPS were positively robust in both layers and broilers. Finally, we determined the co-infection with AMPV, ORT, and CPS was prevalent in the sampled poultry farms suffering from respiratory diseases and the outbreak of airsacculitis was closely related to simultaneous exposure to all three agents.

Clinical manifestations, molecular characteristics, antimicrobial susceptibility patterns and contributions of target gene mutation to fluoroquinolone resistance in Elizabethkingia anophelis.

Objectives: Elizabethkingia anophelis has recently emerged as a cause of life-threatening infections in humans. We aimed to investigate the clinical and molecular characteristics of E. anophelis. Methods: A clinical microbiology laboratory database was searched to identify patients with Elizabethkingia infections between 2005 and 2016. Isolates were re-identified and their species were confirmed using 16S rRNA gene sequencing. Patients with E. anophelis infections were included in this study. Clinical information, antimicrobial susceptibility and mutations in DNA gyrase and topoisomerase IV were analysed. Results: A total of 67 patients were identified to have E. anophelis infections, including 47 men and 20 women, with a median age of 61 years. Comorbidity was identified in 85.1% of the patients. Among the 67 E. anophelis isolates, 40 (59.7%) were isolated from blood. The case fatality rate was 28.4%. Inappropriate empirical antimicrobial therapy was an independent risk factor for mortality (adjusted OR = 10.01; 95% CI = 1.20-83.76; P = 0.034). The isolates were 'not susceptible' to multiple antibiotics. All the isolates were susceptible to minocycline. Susceptibilities to ciprofloxacin and levofloxacin were 4.5% and 58.2%, respectively. Mutations in DNA gyrase subunit A were identified in 11 isolates that exhibited high-level fluoroquinolone resistance. Conclusions: Minocycline has the potential to be the drug of choice in patients with E. anophelis infections. Additional investigations are needed to determine the optimal antimicrobial agents to treat this life-threatening infection.

The Role of the Regulator Fur in Gene Regulation and Virulence of Riemerella anatipestifer Assessed Using an Unmarked Gene Deletion System.

Riemerella anatipestifer, an avian pathogen, has resulted in enormous economic losses to the duck industry globally. Notwithstanding, little is known regarding the physiological, pathogenic and virulence mechanisms of Riemerella anatipestifer (RA) infection. However, the role of Ferric uptake regulator (Fur) in the virulence of R. anatipestifer has not, to date, been demonstrated. Using a genetic approach, unmarked gene deletion system, we evaluated the function of fur gene in the virulence of R. anatipestifer. For this purpose, we constructed a suicide vector containing pheS as a counter selectable marker for unmarked deletion of fur gene to investigate its role in the virulence. After successful transformation of the newly constructed vector, a mutant strain was characterized for genes regulated by iron and Fur using RNA-sequencing and a comparison was made between wild type and mutant strains in both iron restricted and enriched conditions. RNA-seq analysis of the mutant strain in a restricted iron environment showed the downregulation and upregulation of genes which were involved in either important metabolic pathways, transport processes, growth or cell membrane synthesis. Electrophoretic mobility shift assay was performed to identify the putative sequences recognized by Fur. The putative Fur-box sequence was 5'-GATAATGATAATCATTATC-3'. Lastly, the median lethal dose and histopathological investigations of animal tissues also illustrated mild pathological lesions produced by the mutant strain as compared to the wild type RA strain, hence showing declined virulence. Conclusively, an unmarked gene deletion system was successfully developed for RA and the role of the fur gene in virulence was explored comprehensively.