Section for Microbiology

Oslo, Norway

Section for Microbiology

Oslo, Norway

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Bjelland A.M.,Section for Microbiology | Johansen R.,Norwegian Veterinary Institute | Brudal E.,Section for Microbiology | Hansen H.,University of Tromsø | And 2 more authors.
Microbial Pathogenesis | Year: 2012

Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the Gram-negative bacterium Vibrio (Aliivibrio) salmonicida. To study the pathogenesis of this marine pathogen, Atlantic salmon was experimentally infected by immersion challenge with wild type V. salmonicida and the bacterial distribution in different organs was investigated at different time points. V. salmonicida was identified in the blood as early as 2 h after challenge demonstrating a rapid establishment of bacteremia without an initial period of colonization of the host. Two days after immersion challenge, only a few V. salmonicida were identified in the intestines, but the amount increased with time. In prolonged CV cases, V. salmonicida was the dominating bacterium of the gut microbiota causing a release of the pathogen to the water. We hypothesize that V. salmonicida uses the blood volume for proliferation during the infection of the fish and the salmonid intestine as a reservoir that favors survival and transmission. In addition, a motility-deficient V. salmonicida strain led us to investigate the impact of motility in the CV pathogenesis by comparing the virulence properties of the mutant with the wild type LFI1238 strain in both i.p. and immersion challenge experiments. V. salmonicida was shown to be highly dependent on motility to gain access to the fish host. After invasion, motility was no longer required for virulence, but the absence of normal flagellation delayed the disease development. © 2011 Elsevier Ltd.


Bjelland A.M.,Section for Microbiology | Sorum H.,Section for Microbiology | Tegegne D.A.,University of Tromsø | Winther-Larsen H.C.,Section for Microbiology | And 3 more authors.
Infection and Immunity | Year: 2012

Vibrio (Aliivibrio) salmonicida is the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth of V. salmonicida in the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome of V. salmonicida LFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS in V. fischeri, was deleted. Compared to the parental strain, the litR mutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, the litR mutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with the litR mutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host. © 2012, American Society for Microbiology.


Brudal E.,Section for Microbiology | Brudal E.,University of Oslo | Ulanova L.S.,University of Oslo | Lampe E.O.,University of Oslo | And 3 more authors.
Infection and Immunity | Year: 2014

Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granulomalike structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells. © 2014, American Society for Microbiology.


Nilsson C.,Section for Microbiology | Kjellin E.,Sahlgrenska University Hospital | Welinder-Olsson C.,Sahlgrenska University Hospital | Lindmark H.,Section for Microbiology
Virulence | Year: 2011

An evaluation of 22 EHEC genes was carried out for virulence classification of VTEC. The data consisted of 116 patient isolates and 42 beef isolates. The symptoms among patients ranged from mild (diarrhea) to severe (bloody diarrhea and HUS). A cluster of genes-efa1, eae, ecf4, paa and ureC-were more frequent in patient isolates than beef isolates. They also contributed to the classification of high virulence isolates compared with low virulence isolates. These genes may together constitute a general virulence factor, being also associated with well-known virulence serogroups O157, O26, O103, O111, O145, O121 and O118. In a regression model of patient versus beef isolates, the combined presence of efa1 and paa proved a particularly efficient indicator of patient isolates (OR: 32.9). In contrast, a single gene, vtx2 2 (subtype vtx2 of vtx2) was a relatively efficient predictor of high virulence among patient isolates (OR: 41.6), but not of virulence in general. Significant interaction effects observed between genes need to be addressed and clarified in future studies. © 2011 Landes Bioscience.


Abayneh T.,Section for Microbiology | Abayneh T.,Addis Ababa Institute of Technology | Colquhoun D.J.,Norwegian Veterinary Institute | Colquhoun D.J.,University of Bergen | And 2 more authors.
Journal of Fish Diseases | Year: 2014

This study describes a novel multilocus variable number tandem repeat analysis (MLVA) based on six variable number of tandem repeat (VNTR) loci for genotyping of 37 Edwardsiella piscicida (previously Edwardsiella tarda) isolates from multiple sources. The number of alleles identified for each of the six VNTR loci ranged from 3 to 5 with VNTR loci 1 (DI = 0.632) and 3 (DI = 0.644), displaying the highest degrees of polymorphism. MLVA typing of the 37 E. piscicida isolates resulted in the identification of five major clusters consistent with their geographical origins, and were designated as MLVA types I, II, III, IV and V. Types III and V were resolved further into subtypes largely consistent with outbreak source. An MLVA profile comprising a string of integers representing the number of tandem repeats for each allele provided a unique identification for each MLVA type and/or strain. The MLVA protocol described in the current study is robust, relatively simple, has a higher power of resolution than multilocus sequence analysis (MLSA) and is capable of discriminating closely related isolates. © 2013 John Wiley & Sons Ltd.


Larsen A.K.,Section for Arctic Veterinary Medicine | Larsen A.K.,Member of the Fram Center | Nymo I.H.,Section for Arctic Veterinary Medicine | Nymo I.H.,Member of the Fram Center | And 5 more authors.
PLoS ONE | Year: 2013

A high prevalence of Brucella pinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal (Cystophora cristata); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B. pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B. pinnipedialis reference strain (NCTC 12890) from harbor seal (Phoca vitulina), B. ceti reference strain (NCTC 12891) from harbor porpoise (Phocoena phocoena) and a B. ceti Atlantic white-sided dolphin (Lagenorhynchus acutus) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B. pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains tested were able to establish a persistent infection in primary alveolar macrophages from hooded seal. © 2013 Larsen et al.


Kashulin A.,University of Tromsø | Sorum H.,Section for Microbiology
Aquaculture | Year: 2014

Cold-Water Vibriosis (CWV) is a well-known disease which significantly influences the aquaculture industry of the North Atlantic coasts (Egidius et al., 1981). Due to wide implementation of apparently effective vaccines in all farmed Atlantic salmon (Salmo salar) the phenomenon of CWV was not a research focus for nearly two decades (Lillehaug, 1990, 1991). Although prevented by vaccination since the 1980s, CWV was again reported in farmed, vaccinated Atlantic salmon in 2012 (Johansen, 2012). Since CWV emerged as a recognized infection in the early 1980s (Egidius et al., 1981; Sørum et al., 1990), several attempts have been undertaken to identify the initial steps of the pathogenesis of CWV. However, no final explanation to how Aliivibrio (Vibrio) salmonicida enters the host has been reported. In this study, we present a novel and simple model for analyzing the initial steps of CWV. Our results demonstrate that initiation of CWV is more complex than was previously thought. In particular, A. (V.) salmonicida enters the host much faster than was anticipated. To identify the initial pathogenic steps in CWV, Atlantic salmon fry were differentially immersed in a suspension of A. (V.) salmonicida and the number of bacteria entering the host was measured. The putative roles of the gills, skin, rectal and oral routes as well as the role of the fin blood vessels as portals of infection were investigated. Bacterial counts were obtained from freshly collected blood samples, thus representing immediate snapshots of the early stages of host invasion. The results clearly indicated that skin was a major route of infection. The experimental design reported in this study provides a new, rapid and cost-effective model for studying CWV. © 2013 Elsevier B.V.


Bjelland A.M.,Section for Microbiology | Fauske A.K.,Section for Microbiology | Nguyen A.,Oslo University College | Orlien I.E.,Oslo University College | And 2 more authors.
Frontiers in Microbiology | Year: 2013

The Gram-negative bacterium Vibrio salmonicida is the causative agent of cold-water vibriosis (CV), a hemorrhagic septicemia that primarily affects farmed Atlantic salmon (Salmo salar L.). The mechanisms of disease development, host specificity and adaptation, as well as the immunogenic properties of V. salmonicida are largely unknown. Therefore, to gain more knowledge on the pathogenesis of CV, 90 Atlantic salmon parr were injected intraperitoneally with 6 × 106 CFU of V. salmonicida LFI1238. Samples from blood and spleen tissue were taken at different time points throughout the challenge for gene expression analysis by two-step reverse transcription (RT) quantitative real-time polymerase chain reaction. Out of a panel of six housekeeping genes, accD, gapA, and 16S rDNA were found to be the most suitable references for expression analysis in Vibrio salmonicida. The bacterial proliferation during challenge was monitored based on the expression of the 16S rRNA encoding gene. Before day 4, the concentrations of V. salmonicida in blood and spleen tissue demonstrated a lag phase. From day 4, the bacterial proliferation was exponential. The expression profiles of eight genes encoding potential virulence factors of V. salmonicida were studied. Surprisingly, all tested virulence genes were generally highest expressed in broth cultures compared to the in vivo samples. We hypothesize that this general muting of gene expression in vivo may be a strategy for V. salmonicida to hide from the host immune system. To further investigate this hypothesis, the expression profiles of eight genes encoding innate immune factors were analyzed. The results demonstrated a strong and rapid, but short-lasting innate immune response against V. salmonicida. These results suggest that the bacterium possesses mechanisms that inhibit and/or resist the salmon innate immune system until the host becomes exhausted of fighting the on-going and eventually overwhelming infection. © 2013 Bjelland, Fauske, Nguyen, Orlien, Østgaard and Sørum.


PubMed | Section for Microbiology and Oslo University College
Type: | Journal: Frontiers in microbiology | Year: 2014

The Gram-negative bacterium Vibrio salmonicida is the causative agent of cold-water vibriosis (CV), a hemorrhagic septicemia that primarily affects farmed Atlantic salmon (Salmo salar L.). The mechanisms of disease development, host specificity and adaptation, as well as the immunogenic properties of V. salmonicida are largely unknown. Therefore, to gain more knowledge on the pathogenesis of CV, 90 Atlantic salmon parr were injected intraperitoneally with 6 10(6) CFU of V. salmonicida LFI1238. Samples from blood and spleen tissue were taken at different time points throughout the challenge for gene expression analysis by two-step reverse transcription (RT) quantitative real-time polymerase chain reaction. Out of a panel of six housekeeping genes, accD, gapA, and 16S rDNA were found to be the most suitable references for expression analysis in Vibrio salmonicida. The bacterial proliferation during challenge was monitored based on the expression of the 16S rRNA encoding gene. Before day 4, the concentrations of V. salmonicida in blood and spleen tissue demonstrated a lag phase. From day 4, the bacterial proliferation was exponential. The expression profiles of eight genes encoding potential virulence factors of V. salmonicida were studied. Surprisingly, all tested virulence genes were generally highest expressed in broth cultures compared to the in vivo samples. We hypothesize that this general muting of gene expression in vivo may be a strategy for V. salmonicida to hide from the host immune system. To further investigate this hypothesis, the expression profiles of eight genes encoding innate immune factors were analyzed. The results demonstrated a strong and rapid, but short-lasting innate immune response against V. salmonicida. These results suggest that the bacterium possesses mechanisms that inhibit and/or resist the salmon innate immune system until the host becomes exhausted of fighting the on-going and eventually overwhelming infection.


Reverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species.Relative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene.Although the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.

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