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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.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

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 (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.

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-A2 T , shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764 T . The genome of strain TLL-A2 T was sequenced and compared to that of T. mesophilum DSM 13764 T . Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764 T and strain TLL-A2 T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2 T and T. mesophilum DSM 13764 T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2 T (=DSM 106434 T , KCTC 62393 T ) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

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 (LD 50 ) of RA-YMΔCas9 was 5.01 × 10 7  CFU, significantly lower than that of the RA-YM strain, which was 1.58 × 10 5  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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 β-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.

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., Competing Interests: The authors declare that they have no conflict of interests., (Copyright © 2019 Xiaoli Huang et al.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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 bla IND-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.

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 ± 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 IKKβ), 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., (Copyright © 2018. Published by Elsevier Ltd.)

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.

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.

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., (Copyright © 2018 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

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.

Elizabethkingia meningoseptica is an emerging nosocomial pathogen associated with high mortality and inherently resistant to many antimicrobial agents. Levofloxacin has been considered as a therapeutic agent based on in vitro susceptibility. We aim to investigate the risk factors and outcomes for levofloxacin-resistant E. meningoseptica bacteraemia. Adult patients with E. meningoseptica bacteraemia were identified retrospectively in a medical centre in Taiwan from January 2011 to July 2015. These strains were identified by the Vitek2 automated system or matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. We compared clinical features and outcomes of patients with levofloxacin-resistant (MIC >2 μg/mL) and levofloxacin-susceptible (MIC ≤2 μg/mL) E. meningoseptica bacteraemia. A total of 93 patients were identified, including 51 (54.8%) with levofloxacin-resistant E. meningoseptica bacteraemia. The APACHE II score (OR, 1.08; 95% CI, 1.02-1.14; p = 0.008) was the only independent risk factor for levofloxacin-resistant E. meningoseptica bacteraemia. The 14-day mortality for patients with levofloxacin-resistant E. meningoseptica bacteraemia (attributable mortality: 30.7%) was significantly higher than that for patients with the levofloxacin-susceptible strain (56.9% versus 26.2%, p = 0.003). In the multivariate analysis of risk factors for mortality, appropriate definite antibiotic use was the only factor associated with 14-day survival (OR, 0.11; 95% CI, 0.02-0.55, p = 0.007). The levofloxacin-resistant strain was borderline significantly associated with mortality (OR, 3.09; 95% CI, 0.88-10.91, p = 0.079). The early identification of levofloxacin resistance in E. meningoseptica isolates is important to tackle this multi-drug resistance pathogen.

The outbreak of Riemerella anatipestifer (RA) infection has been confirmed in meat-type domestic ducks (Anas platyrhynchos) for the first time in 27 years in Japan. In January 2014, increased mortality in a 14- to 21-day-old duck flock was reported to veterinary officials by the owner. The affected ducks exhibited reduced movement, ataxia and dorsal recumbency with leg paddling. Pathological findings were typical for an RA infection. Fibrinous and heterophilic pericarditis, airsacculitis, perihepatitis, ventriculitis and meningitis were observed. The bacterial isolate from duck organs was identified as RA by PCR-based 16S ribosomal RNA sequencing.

The aim of the investigations was to determine the influence of Ornithobacterium rhinotracheale (ORT) on the development of pathomorphological lesions in the respiratory organs and on the health status of experimentally infected broiler breeders and pheasants from the rearing stage. There was no evidence of clinical signs in infected broiler breeder hens nor in the group of infected pheasants except for one bird in the latter group which exhibited slower movement and gasping. The frequency and intensity of pathomorphological lesions were higher in pheasants. The gross pathology findings were characterized mainly by redness of the mucosa of the upper respiratory tract and accumulation of mucous content in the nasal cavities, infraorbital sinuses, larynx and trachea. Histopathology confirmed the presence of inflammation of the upper respiratory tract. Lesions in the lungs included hyperaemia, granulomatous and fibrinous pneumonia. ORT was reisolated only from the group of infected pheasants. Reisolation was successful from the respiratory organs (trachea, larynx, infraorbital sinuses, and lungs) of eight out of 10 infected birds. The serological response in both species was characterized by rapid production of specific antibodies that reached a maximum level in the blood in the first week after experimental infection. The antibody titres decreased gradually and were maintained at a stable level until the 12th week after inoculation. Fourteen weeks post-inoculation specific antibodies could not be detected by enzyme-linked immunosorbent assay.

Bacterial meningitis is a disease with a high morbidity and mortality. It may be caused by the zoonotic pathogen Capnocytophaga canimorsus, which is part of the commensal oral flora in dogs and cats. We report three cases of C. canimorsus meningitis in a nationwide cohort study of bacterial meningitis patients and performed a review of the literature. Three episodes of C. canimorsus meningitis were identified in three patients included in a nationwide cohort study from 2006 through 2014. The calculated annual incidence was 0.03 per million adults. When combined with the literature, 33 patients were identified of which 28 were male (85%). The median age was 63 years, and 13 (42%) were immunocompromised, which consisted of alcoholism in 7 (21%). Animal contact could be established in 29 of 30 patients (93%) and consisted of dog bites in 22 of 29 (76%). One patient died (3%) and 8 had neurological sequelae upon discharge (25%), most often hearing loss (n = 6, 19%). Capnocytophaga canimorsus meningitis is associated with dog bites. Although mortality is relatively low, survivors often have neurological sequelae., (© 2015 Blackwell Verlag GmbH.)

Flavobacterium columnare is the causative agent of columnaris disease and is responsible for significant economic losses in aquaculture. F. columnare is a Gram-negative bacterium, and 5 genetic types or genomovars have been described based on restriction fragment length polymorphism of the 16S rRNA gene. Previous research has suggested that genomovar II isolates are more virulent than genomovar I isolates to multiple species of fish, including rainbow trout Oncorhynchus mykiss. In addition, improved genotyping methods have shown that some isolates previously classified as genomovar I, and used in challenge experiments, were in fact genomovar III. Our objective was to confirm previous results with respect to genomovar II virulence, and to determine the susceptibility of rainbow trout to other genomovars. The virulence of 8 genomovar I, 4 genomovar II, 3 genomovar II-B, and 5 genomovar III isolates originating from various sources was determined through 3 independent challenges in rainbow trout using an immersion challenge model. Mean cumulative percent mortality (CPM) of ~49% for genomovar I isolates, ~1% for genomovar II, ~5% for the II-B isolates, and ~7% for the III isolates was observed. The inability of genomovar II isolates to produce mortalities in rainbow trout was unanticipated based on previous studies, but may be due to a number of factors including rainbow trout source and water chemistry. The source of fish and/or the presence of sub-optimal environment may influence the susceptibility of rainbow trout to different F. columnare genomovars.

Unlike Elizabethkingia meningoseptica, the clinical importance of E. anophelis is poorly understood. We determined the clinical and molecular epidemiology of bacteremia caused by Elizabethkingia-like species from five regional hospitals in Hong Kong. Among 45 episodes of Elizabethkingia-like bacteremia, 21 were caused by Elizabethkingia, including 17 E. anophelis, three E. meningoseptica and one E. miricola; while 24 were caused by other diverse genera/species, as determined by 16S rRNA gene sequencing. Of the 17 cases of E. anophelis bacteremia, 15 (88%) were clinically significant. The most common diagnosis was pneumonia (n = 5), followed by catheter-related bacteremia (n = 4), neonatal meningitis (n = 3), nosocomial bacteremia (n = 2) and neutropenic fever (n = 1). E. anophelis bacteremia was commonly associated with complications and carried 23.5% mortality. In contrast, of the 24 episodes of bacteremia due to non-Elizabethkingia species, 16 (67%) were clinically insignificant. Compared to non-Elizabethkingia bacteremia, Elizabethkingia bacteremia was associated with more clinically significant infections (P < 0.01) and positive cultures from other sites (P < 0.01), less polymicrobial bacteremia (P < 0.01), and higher complication (P < 0.05) and mortality (P < 0.05) rates. Elizabethkingia bacteremia is predominantly caused by E. anophelis instead of E. meningoseptica. Elizabethkingia bacteremia, especially due to E. anophelis, carries significant morbidity and mortality, and should be considered clinically significant unless proven otherwise.

Cat and dog bites are a common cause of injury in young children. Bergeyella zoohelcum is a rarely reported zoonotic pathogen that is a part of cat and dog oral flora. We present a case of a child with B. zoohelcum abscess and cellulitis after a dog bite and review previously reported cases.

Elizabethkingia meningoseptica is an infrequent cause of hospital-acquired infections. The clinical and microbiological profiles of infections due to E. meningoseptica over a seven-year period at a Level-I trauma centre are reported in this study. Medical records of patients from whose clinical samples E. meningoseptica was isolated on more than one occasion were reviewed. A total of 21 cases were observed during the study, 16 (76.2%) of which exhibited multidrug resistance. The observed in-hospital mortality rate was 47.6%. A high index of clinical suspicion and effective detection of E. meningoseptica in clinical samples are requisite for improved clinical outcome., (Copyright © 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Flavobacterium columnare is an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. The F. johnsoniae ompA promoter (PompA) was fused to sacB to construct a counterselectable marker for F. columnare. F. columnare carrying PompA-sacB failed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacB was constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes, cslA and cslB, were deleted. The ΔcslA and ΔcslB mutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslA ΔcslB) was completely deficient in chondroitin lyase activity. Cells of F. columnare wild-type strain G4 and of the chondroitin lyase-deficient ΔcslA ΔcslB mutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors of F. columnare but may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylum Bacteroidetes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Myroides sp. are gram negative aerobes and known for its oppurtunistic pathogenicity in humans. In the present study, Myroides odoratimimus isolated from the gut of Mugil cephalus showed potential infectivity to the experimental grey mullet and acted as an ultimate pathogen with significant symptoms. Furthermore, the inoculum isolated from the infected fishes were cultured and the selected colonies were reisolated and reinjected into healthy juveniles of M. cephalus. Characterizations of the re-isolated bacteria were the same as those of the isolated M. odoratimimus from naturally infected mullet. The median lethal dose (LD50) of the bacteria was 3 × 10(6) CFU fish(-1). In order to assay the accuracy of infection, the production of reactive oxygen species (ROS), the respiratory burst activity of blood leukocytes of mullet before and after challenge was measured, an indicator of the innate immune system. The mullet infection increased the respiratory burst activity and superoxide dismutase activity. In addition, the innate immune response of TLR 9 expression against M. odoratimimus infection and CpG ODN treatment in disease model, zebrafish confirms the M. odoratimimus infection and pathogenicity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Columnaris disease can be problematic in tilapia (Oreochromis spp.) production. An understanding of the pathogenesis and virulence of Flavobacterium columnare is needed to develop prevention strategies. The objective of this study was to determine the virulence of genetically defined isolates of F. columnare in sex-reversed hybrid tilapia, Oreochromis niloticus (L.)×O. aureus (Steindachner). A series of immersion challenge trials were performed using isolates of the five established genomovars of F. columnare: I, II, II-B, III and I/II. The mean per cent mortality of fish challenged with genomovar I, II and III isolates ranged from 0 to 100, 3.3-78 and 3.3-75%, respectively. The mean per cent mortality of fish challenged with genomovar II-B ranged from 35 to 96.7%, and the only genomovar I/II isolate tested caused no mortality. Contrary to previous work in other fish species, there did not appear to be an association between F. columnare genomovar and virulence in tilapia. The challenge model used resulted in acute mortality. An alternative challenge model was tested by cohabitating healthy fish with dead fish infected with F. columnare. This method resulted in rapid appearance of clinical signs and mortality, suggesting the potential for F. columnare to increase in virulence upon growth on/in a fish host., (© 2014 John Wiley & Sons Ltd.)

Chryseobacterium spp are widely distributed in nature but data of their isolation from clinical samples is scanty. Here, we report the first case of AmpC producing C. gleum causing pyonephrosis in a patient having bilateral nephrolithiasis on double J (DJ) stent. The present isolate was resistant to vancomycin, erythromycin, clindamycin, carbapenems and ciprofloxacin and susceptible to tetracycline and minocycline. The patient was treated with tetracycline and recovered without the need for removal of the DJ stent. The environmental surveillance carried out to trace the nosocomial origin of the isolate was negative. Since antimicrobial susceptibility of this isolate is different from previous reports, we emphasise that in vitro susceptibility testing should be sought to choose optimal antimicrobial agents for these Nonfermentative Gram-Negative Bacilli (NFGNBs) with different susceptibility patterns.

Recent reports suggest an emergence of novel Chryseobacterium spp. associated with aquaculture-reared fish worldwide. Herein, we report on multiple Chryseobacterium spp. infecting Great Lakes fishes that are highly similar to previously detected isolates from Europe, Africa, and Asia but have never before been reported in North America. Polyphasic characterization, which included extensive physiological, morphological, and biochemical analyses, fatty acid profiling, and phylogenetic analyses based upon partial 16S rRNA gene sequences, highlighted the diversity of Great Lakes' fish-associated chryseobacteria and also suggested that at least 2 taxa represent potentially novel Chryseobacterium spp. Screening for the ability of representative chryseobacteria to elicit lesions in experimentally challenged fish showed that they induced varying degrees of pathology, some of which were severe and resulted in host death. Median lethal dose (LD50) experiments for the isolate that elicited the most extensive pathology (Chryseobacterium sp. T28) demonstrated that the LD50 exceeded 4.5 × 108 cfu, thereby suggesting its role as a facultative fish-pathogenic bacterium. Histopathological changes in T28-infected fish included epithelial hyperplasia of the secondary lamellae and interlamellar space that resulted in secondary lamellar fusion, monocytic infiltrate, and mucus cell hyperplasia, all of which are consistent with branchitis, along with monocytic myositis, hemorrhage within the muscle, liver, adipose tissue, and ovaries, spongiosis of white matter of the brain, multifocal edema within the granular cell layer of the cerebellar cortex, and renal tubular degeneration and necrosis. The findings of this study underscore the widespread presence of chryseobacteria infecting Great Lakes fish.

Riemerella anatipestifer, a Gram-negative bacillus, causes septicemia that can result in high mortality for ducklings. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant outer membrane protein A (rOmpA) and plasmid constructs containing CpG oligodeoxynucleotides (ODN). Results showed that CpG ODN enhanced both humoral and cell-mediated immunity elicited by rOmpA as early as two weeks after primary immunization. When compared to ducks immunized with rOmpA, ducks immunized with rOmpA+CpG ODN showed higher levels (p<0.05) of antibody titer, T cell proliferation, and percentages of CD4(+) and CD8(+) T cell in peripheral blood mononuclear cells (PBMCs). The relative fold inductions of mRNA expression of Th1-type (IFN-γ and IL-12), and Th2-type (IL-6) cytokines in PBMCs isolated from ducks immunized with rOmpA+CpG ODN were significantly higher than those of the rOmpA group. Homologous challenge result showed that the rOmpA+CpG ODN vaccine reduced the pathological score by 90% in comparison with the saline control. In conclusion, our study found that CpG ODN can enhance both humoral and cellular immunity elicited by a rOmpA vaccine. The rOmpA+CpG ODN vaccine can be further developed as a subunit vaccine against R. anatipestifer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chryseobacterium indologenes is an environmental organism which is usually an opportunistic pathogen, most usually associated with nosocomial or device-related infections. This case, affecting a fit and well adventure traveler, demonstrates that it may be an agent of severe sepsis in otherwise healthy humans., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Tenacibaculum maritimum is a Gram-negative bacterium that causes large losses in farmed turbot, Psetta maxima (L.). Main lesions included erosive and ulcerative skin injuries in different locations in the body of the fish. This study was set up to gain insights into the immune response of the turbot against this bacterial infection. To determine the variations in the peripheral blood leukocytes a minimum of 200 leukocytes in blood smears were counted. The presence of immunoglobulin positive (Ig(+)) cells in spleen, kidney, intestine, thymus and skin from turbot experimentally infected with T. maritimum were studied using an immunohistochemical method. In fish challenged with the bacterium the percentage of circulating granulocytes, lymphocytes and trombocytes showed significant differences at different points of sampling. Moreover, the number of Ig(+) cells significantly increased in the spleen, kidney, intestine and thymus. In the skin a strong immunoreactivity was observed in the interstitial liquid in damaged areas. The results obtained in this study indicated that the infection by T. maritimum in turbot induced an immune response that involved changes in the number of peripheral blood leukocytes and in the number and distribution of Ig(+) cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Host genetic resistance against disease-causing pathogens can be enhanced through family-based selective breeding. At present, there is an incomplete understanding of how artificial selection of fish alters host physiology and response following pathogen exposure. We previously reported the generation of selectively-bred rainbow trout Oncorhynchus mykiss lines with either increased resistance (ARS-Fp-R) or susceptibility (ARS-Fp-S) to bacterial cold water disease (BCWD). This study (1) determined baseline reference-range intervals for packed cell volume (PCV) and 18 plasma biochemistry analytes, and (2) examined pathophysiological changes following infection between the genetic lines. PCV and biochemistry reference-range intervals did not significantly differ between genetic lines; thus data were pooled into a single reference-range population (n = 85). ARS-Fp-R and ARS-Fp-S line fish were intraperitoneally challenged with Flavobacterium psychrophilum, and plasma was collected on Days 1, 3, 6, and 9 post-challenge. Splenic bacterial load was measured using an F. psychrophilum-specific qPCR assay. In both genetic lines, changes were observed in mean PCV, total protein, albumin, glucose, cholesterol, chloride, and calcium, falling outside the established reference intervals and significantly differing from phosphate-buffered saline challenged fish, on at least 1d post-challenge. Mean PCV, total protein, and calcium significantly differed between ARS-Fp-R and ARS-Fp-S line fish on Day 9 post-infection, with values in the ARS-Fp-S line deviating most from the reference interval. PCV, total protein, cholesterol, and calcium negatively correlated with bacterial load. These findings identify divergent pathophysiological responses between ARS-Fp-R and ARS-Fp-S line fish following laboratory challenge that are likely associated with differential survival.

Abstract A challenge to selectively breeding fish populations for improved disease resistance in aquaculture is an incomplete understanding of how artificial selection affects innate immunity at the host-pathogen level. The objective of this study was to determine whether Rainbow Trout Oncorhynchus mykiss bred for differential susceptibility to bacterial cold-water disease exhibited altered tissue damage and cellular inflammatory response following experimental challenge with Flavobacterium psychrophilum. Fish from disease-resistant (ARS-Fp-R) and disease-susceptible (ARS-Fp-S) lines were experimentally challenged as juveniles, and mortalities, as well as survivors, were sampled for histopathology during the acute phase of the disease. Microscopic lesions were quantified or semiquantified and statistically compared for changes over time and between genetic lines. Significant progression in the degree of perisplenitis, splenic necrosis, splenic inflammatory infiltrates, average splenic ellipsoid area, total splenic ellipsoid area, and peritonitis was present over time in both genetic lines on at least one postinfection time point. No differences were found between renal inflammatory infiltrates and renal hematopoietic cell depletion over time. Perisplenitis was significantly lower in fish from the ARS-Fp-R line on day 9 postinfection than in fish from the ARS-Fp-S line. The ARS-Fp-R line demonstrated a trend towards reduced splenic necrosis compared with the ARS-Fp-S line that approached significance, and fish from the ARS-Fp-S line were 3.6 times more likely than fish from the ARS-Fp-R line to have a higher splenic necrosis lesion score after day 3 postinfection. These findings support the hypothesis that differential survival is a result of divergence in disease magnitude and not altered disease course between genetic lines. Characterization of histopathologic changes between genetic lines and over time helps elucidate mechanisms of disease resistance and contributes to our understanding of disease pathogenesis in fish infected with F. psychrophilum. Received January 7, 2014; accepted March 10, 2014.

Myroides spp and Chryseobacterium spp are uncommon clinical isolates, though more frequently reported to cause infections than other pigmented non-fermentors. Two cases of Myroides odoratus and Chryseobacterium indologenes infection in a diabetic with pulmonary tuberculosis and a patient with de-compensated alcoholic liver disease, respectively, are reported here. Anti-microbial susceptibility testing of the isolates was performed by determining the minimum inhibitory concentration. The clinical picture, characteristic features of the isolates and the antibiotic susceptibility pattern are discussed briefly.

Background: Flavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. The bacterium forms three colony morphotypes (Rhizoid, Rough and Soft), but the differences of the morphotypes are poorly known. We studied the virulence of the morphotypes produced by F. columnare strain B067 in rainbow trout (Onconrhynchus mykiss) and used high-resolution scanning electron microscopy to identify the fine structures of the cells grown in liquid and on agar. We also analysed the proteins secreted extracellularly and in membrane vesicles to identify possible virulence factors., Results: Only the Rhizoid morphotype was virulent in rainbow trout. Under electron microscopy, the cells of Rhizoid and Soft morphotypes were observed to display an organised structure within the colony, whereas in the Rough type this internal organisation was absent. Planktonic cells of the Rhizoid and Rough morphotypes produced large membrane vesicles that were not seen on the cells of the Soft morphotype. The vesicles were purified and analysed. Two proteins with predicted functions were identified, OmpA and SprF. Furthermore, the Rhizoid morphotype secreted a notable amount of a small, unidentified 13 kDa protein absent in the Rough and Soft morphotypes, indicating an association with bacterial virulence., Conclusions: Our results suggest three factors that are associated with the virulence of F. columnare: the coordinated organisation of cells, a secreted protein and outer membrane vesicles. The internal organisation of the cells within a colony may be associated with bacterial gliding motility, which has been suggested to be connected with virulence in F. columnare. The function of the secreted 13 kDa protein by the cells of the virulent morphotype cells remains unknown. The membrane vesicles might be connected with the adhesion of cells to the surfaces and could also carry potential virulence factors. Indeed, OmpA is a virulence factor in several bacterial pathogens, often linked with adhesion and invasion, and SprF is a protein connected with gliding motility and the protein secretion of flavobacteria.

High prevalence of leg disorders in fattening meat turkey farm was observed. Four birds as well as tracheal and joint swabs were submitted to the Bavarian Health and Food Safety Authority in Oberschleissheim and to the Institute of Poultry Diseases, Faculty of Veterinary Medicine, Free University of Berlin. At the post-mortem, all birds showed an inflammation of the hock joints (intertarsal joint). The histopatholical investigations revealed a chronic inflammation of the joint and amyloid deposits in the joints in two cases as well as in different tissues (liver, spleen and kidneys) in another two cases. Using polymerase chain reaction, Ornithobacterium rhinotracheale-DNA could be detected in the examined tracheal and joint swabs. On the other hand, Mycoplasma gallisepticum- and Mycoplasma synoviae-DNA could not be detected. A causal correlation between the detected infectious agent and amyloidosis in relation to the leg disorders were discussed.

Elizabethkingia meningoseptica has been deemed a potentially important threat to patients in critical care areas because of its multidrug-resistant phenotype and its ability to adapt to various environments. This review considers the incidence, factors which predispose to, and clinical features of, E. meningoseptica sepsis, along with antimicrobial susceptibility patterns of clinical E. meningoseptica isolates and reportedly successful measures for the prevention and control of infections caused by this bacterium. The English-language literature from the PubMed database was reviewed. The incidence of E. meningoseptica bacteraemia has increased over the last decade. Patients at high risk of E. meningoseptica infection include preterm children, the immunocompromised and those exposed to antibiotics in critical care units. Vancomycin, rifampicin, newer fluoroquinolones, piperacillin-tazobactam, minocycline and possibly tigecycline are preferred empirical choices for E. meningoseptica infection according to in-vitro susceptibility data. Combination therapy has been used for infections not responding to single agents. Saline, lipid, and chlorhexidine gluconate solutions as well as contaminated sinks have been implicated as sources of infection following outbreak investigations. In addition to reinforcement of standard infection control measures, actions that have successfully terminated E. meningoseptica outbreaks include pre-emptive contact isolation, systematic investigations to identify the source of the bacterium and thorough cleaning of equipment and environmental surfaces. As the clinical complexity and incidence of E. meningoseptica infections increase, there is a need for heightened awareness of the potential for this bacterium to cause outbreaks. This will permit timely initiation of active surveillance for infected/colonized patients as well as investigations to identify the likely source of the bacterium, which will, in turn, allow implementation of appropriate infection control measures., (Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Ornithobacterium rhinotracheale (ORT) is a nonhemolytic, gram-negative, pleomorphic, rod-shaped bacterium that causes upper and lower respiratory tract disease in poultry. Recently, hemolytic strains of ORT have been isolated with increasing frequency from field outbreaks. A study was conducted to determine whether the hemolytic phenotype is associated with any change in virulence. Briefly, 225 turkey poults, vaccinated against hemorrhagic enteritis at 4 wk of age, were randomly divided into nine replicates housed in separate rooms: three sham treatment controls (25 poults/replicate), three challenged with a nonhemolytic (NH) field isolate (24 poults/replicate), and three challenged with a hemolytic (H) field isolate (24 poults/replicate). Nine days postvaccination, poults were inoculated intratracheally with either 0.2 ml sterile phosphate-buffered saline (PBS), 2 x 10(8) colony-forming units (CFU) of the NH isolate in 0.2 ml PBS, or 2 x 10(8) CFU of the H isolate in 0.2 ml PBS. Serum and body weights were obtained at 0, 7, 14, and 21 days postinoculation (dpi). Tissues were taken for culture and histopathology from five randomly selected poults/replicates at 7, 14, and 21 dpi. When compared with poults inoculated with the H isolate or controls, those inoculated with the NH isolate showed a highly significant depression in weight gain at 7 dpi. NH poults also had significantly higher levels of antibody against ORT at 14 and 21 dpi. Reisolations decreased over time and, by 21 dpi, only the NH phenotype could be found. Based on a Likert-type scale, poults inoculated with the NH isolate had significantly higher histopathologic lesion scores in lung tissue at 7, 14, and 21 dpi. Results suggest that nonhemolytic field isolates are more virulent then hemolytic ones. These findings are unusual because hemolytic phenotypes are often more virulent in other bacterial species.