Veterinary bacteriology in Denmark from the 1880s to 2022.

This paper gives an account of the history of veterinary bacteriology including clinical veterinary bacteriology as well as the area of veterinary public health in Denmark from the 1880s to 2022. We describe key persons, including B. Bang, C.O. Jensen, K.A. Jensen and others who made important contributions to the development of these areas of microbiological expertise, and we discuss how challenges ranging from bovine tuberculosis to bacterial antimicrobial resistance have been met. Further, we describe progress in research on important bacterial pathogens both with regard to animal clinical aspects and zoonotic food-related aspects. Finally, we describe current issues in relation to One Health and research organization.

Vibrio vulnificus produces quorum sensing signals of the AHL-class.

Vibrio vulnificus is an aquatic pathogenic bacterium that can cause vibriosis in humans and fish. The species is subdivided into three biotypes with the fish-virulent strains belonging to biotype 2. The quorum sensing (QS) phenomenon mediated by furanosyl borate diester or autoinducer 2 (AI-2) has been described in human strains of biotype 1, and here we show that the luxS gene which encodes AI-2 is present in all strains of V. vulnificus regardless of origin, biotype or serovar. In this study, we also demonstrate that V. vulnificus produces QS signals of the acylated homoserine lactone (AHL) class (AI-1). AHLs were detected in strains of biotype 1 and 2 from water, fish and human wound infections but not in strains isolated from human septicaemic cases. The AHL compound was identified as N-butanoyl-homoserine-lactone (C(4)-HL) by both reporter strains and by HPLC-high-resolution MS. C(4)-HL was detected when AHL-positive strains were grown in low-nutrient medium [modified sea water yeast extract (MSWYE)] but not in rich media (tryptic soy broth or brain-heart infusion) and its production was enhanced when blood factors were added to MSWYE. C(4)-HL was detected in vivo, in eels infected with AHL-positive biotype 2 strains. No known AHL-related gene was detected by PCR or Southern blot suggesting that AHL-related genes in V. vulnificus are different from those found in other Gram-negative bacteria.

Distribution of three genes involved in the PJM1 iron-sequestering system in various Vibrio anguillarum serogroups.

Gene sequences with high homologies to the two genes fatA and fatD, associated with the pJM1 virulence plasmid-encoded iron-sequestering system (anguibactin) in Vibrio anguillarum serogroup O1 strains, were found in a range of serogroup O2a strains and one NT 4 strain that either contained no plasmids or only small-sized plasmids. None of these strains contained the angR gene responsible for regulation of the anguibactin synthesis. DNA sequences of presumed fatA and fatD fragments from one O2a strain and one NT4 strain were identical to each other. The fatA sequence of these strains included 35 nucleotide substitutions and 7 amino acid substitutions out of 555 nucleotides when compared with the fatA sequence of the O1 strain 76775. The fatD sequence included 10 nucleotide substitutions and 1 amino acid substitution out of 543 nucleotides when compared with 76775. The fact that the majority of nucleotide substitutes in the fatA and fatD fragments did not result in amino acid substitutes suggest that these sequences may to some degree be preserved by selective pressure. The functional role of these genes in the O2a serogroup is most likely different from their role in the pJM1 system.

Profiling of acylated homoserine lactones of Vibrio anguillarum in vitro and in vivo: Influence of growth conditions and serotype.

Vibrio anguillarum produces several interlinked acylated homoserine lactone (AHL) signal molecules which may influence expression of its virulence factors such as exoprotease production and biofilm formation. Using both thin layer chromatography and HPLC-high resolution mass spectrometry (HPLC-HRMS), we demonstrate in this study that the same types of AHLs are produced by many serotypes of V. anguillarum and that altering in vitro growth conditions (salinity, temperature and iron concentration) has little influence on the AHL-profile. Most strains produced N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-hydroxy-C6-HSL) as the dominant molecules. Also, two spots with AHL activity appeared on TLC plates, which could not be identified as AHL structures. Trace amounts of N-(3-hydroxy-octanoyl)-l-homoserine lactone, N-(3-hydroxy-decanoyl)-l-homoserine lactone and N-(3-hydroxy-dodecanoyl)-l-homoserine lactone (3-hydroxy-C8-HSL, 3-hydroxy-C10-HSL and 3-oxo-C12-HSL, respectively) were also detected by HPLC-HRMS analysis from in vitro cultures. Most studies of quorum sensing (QS) systems have been conducted in vitro, the purpose of our study was to determine if the same acylated homoserine lactones were produced in vivo during infection. Extracts from infected fish were purified using several solid phase extraction strategies to allow chromatographic detection and separation by both TLC and HLPC-HRMS. 3-oxo-C10-HSL and 3-hydroxy-C6-HSL were detected in organs from fish dying from vibriosis, however, compared to in vitro culturing where 3-oxo-C10-HSL is the dominant molecule, 3-hydroxy-C6-HSL was prominent in the infected fish tissues. Hence, the balance between the QS systems may be different during infection compared to in vitro cultures. For future studies of QS systems and the possible specific interference with expression of virulence factors, in vitro cultures should be optimised to reflect the in vivo situation.

An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum).

The fish pathogen Vibrio anguillarum produces quorum sensing signal molecules, N-acyl homoserine lactones (AHLs), which in several Gram-negative human and plant pathogenic bacteria regulate virulence factors. Expression of these factors can be blocked using specific quorum-sensing inhibitors (QSIs). The purpose of this study was to investigate the effect of a QSI, furanone C-30, on mortality of rainbow trout during challenge with V. anguillarum. Addition of 0.01 or 0.1 microM furanone C-30 to rainbow trout infected by cohabitation caused a significant reduction in accumulated mortality from 80-100% in challenge controls to 4-40% in treated groups. Furanone C-30 had no effect in an immersion challenge system, probably due to a very high water exchange and a rapid dilution of furanone C-30. Growth and survival of V. anguillarum were not affected by the concentrations of furanone C-30 used in the challenge experiments, thus avoiding selection for resistance. To elucidate the mechanism of disease control by furanone C-30, we determined its effect on the bacterial proteome, motility, and respiration. No effects were seen of furanone C-30 in any of these experiments. Although no cytotoxic effect on HeLa cells were observed, exposure to 1 microM (or higher) concentrations of furanone C-30 had detrimental effects on the rainbow trout. Our results indicate that QSIs can be used in non-antibiotic based control of fish diseases. However, they also underline the need for development of novel, less toxic QSI compounds and the need for understanding the exact mechanism(s) of action.

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout.

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum.

Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.

Application of Lime ( Citrus aurantifolia ) Juice to Drinking Water and Food as a Cholera-Preventive Measure.

The protective activity of lime juice against cholera was suggested in a recent case-control study from Guinea-Bissau, On the basis of these findings, we investigated the vibriocidal properties of juice from lime ( Citrus aurantifolia ) fruits added to well water, tap water, and food samples. Well-water samples showed initial pH values from 3.5 to 4,9 whereas water samples from municipal or private taps showed initial pH values between 7.5 and 8.3, A 5-log reduction in Vibrio cholerae O1 counts to <100 CFU/ml of well water was shown for two test strains after exposure to 0.5% lime juice (pH 3.3) for 12 min. In tap water, no significant reduction in CFU of V. cholerae was found after exposure to 0% (pH 8.3) and 0.5% (pH 5.6) lime juice whereas exposure to 1.0% lime juice (pH 4.4) for 120 min caused a 5-log reduction to <100 CFU/ml of tap water for the test strains. A 3-log reduction of V. cholerae was found in food samples containing 3.5% and 5.0% lime juice after 120 min exposure. Our results show that during epidemics of cholera in areas without safe sources of drinking water, juice from citrus fruits added to water and food in palatable concentrations may be appropriate measures in reducing the transmission of cholera. However, local characteristics of the water, in particular its alkalinity, should be considered before applying this measure.