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Runge M.,Food and Veterinary Institute Braunschweig Hanover | Von Keyserlingk M.,Food and Veterinary Institute Braunschweig Hanover | Braune S.,Food and Veterinary Institute Braunschweig Hanover | Becker D.,Institute for Hygiene and Environment of the Free and Hanseatic City of Hamburg | And 4 more authors.
Pest Management Science | Year: 2013

BACKGROUND: Genetically based resistance to anticoagulants has led to increasing difficulties in the control of rodents over recent decades. The possible impact of rodenticide-resistant rats on the infection risk of humans and livestock by zoonotic pathogens is generally unknown. Hence, in a monitoring programme in the German federal states of Lower Saxony and Hamburg, more than 500 Norway rats were analysed for both Tyr139Cys polymorphisms within the VKORC1 gene and zoonotic agents. RESULTS: Evidence of resistance was almost completely restricted to the known resistance area in southern Lower Saxony. Homozygous mutations were only found in urban areas sampled owing to the occurrence of rat control problems and were missing in bycatches of rats by muskrat trappers in rural areas. In more than 25% of the rats, zoonotic bacteria (Leptospira, Salmonella, Yersinia and Coxiella) were detected. There was no obvious correlation between the occurrence of rats carrying zoonotic pathogens and anticoagulant resistance. CONCLUSION: Zoonotic agents and genetically based resistance conferred by the Tyr139Cys polymorphism are both unevenly distributed in Lower Saxony. The study provides the basis for further studies focusing on districts with high levels of pathogens and resistance to assess the potential health risk of their combined occurrence. © 2012 Society of Chemical Industry.


Schiwy A.,RWTH Aachen | Brinkmann M.,RWTH Aachen | Thiem I.,Food and Veterinary Institute Braunschweig Hanover | Guder G.,Food and Veterinary Institute Braunschweig Hanover | And 12 more authors.
Nature Protocols | Year: 2015

This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.


Geduhn A.,Julius Kuhn Institute | Geduhn A.,University of Munster | Jacob J.,Julius Kuhn Institute | Schenke D.,Julius Kuhn Institute | And 3 more authors.
PLoS ONE | Year: 2015

Anticoagulant rodenticides (ARs) are commonly used to control rodent infestations for biocidal and plant protection purposes. This can lead to AR exposure of non-target small mammals and their predators, which is known from several regions of the world. However, drivers of exposure variation are usually not known. To identify environmental drivers of AR exposure in non-targets we analyzed 331 liver samples of red foxes (Vulpes vulpes) for residues of eight ARs and used local parameters (percentage of urban area and livestock density) to test for associations to residue occurrence. 59.8% of samples collected across Germany contained at least one rodenticide, in 20.2% of cases at levels at which biological effects are suspected. Second generation anticoagulants (mainly brodifacoum and bromadiolone) occurred more often than first generation anticoagulants. Local livestock density and the percentage of urban area were good indicators for AR residue occurrence. There was a positive association between pooled ARs and brodifacoum occurrence with livestock density as well as of pooled ARs, brodifacoum and difenacoum occurrence with the percentage of urban area on administrative district level. Pig holding drove associations of livestock density to AR residue occurrence in foxes. Therefore, risk mitigation strategies should focus on areas of high pig density and on highly urbanized areas to minimize non-target risk. © 2015 Geduhn et al.


PubMed | Food and Veterinary Institute Braunschweig Hanover, Federal Institute of Hydrology BFG and RWTH Aachen
Type: Journal Article | Journal: Nature protocols | Year: 2015

This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.


PubMed | Julius Kuhn Institute and Food and Veterinary Institute Braunschweig Hanover
Type: Journal Article | Journal: PloS one | Year: 2015

Anticoagulant rodenticides (ARs) are commonly used to control rodent infestations for biocidal and plant protection purposes. This can lead to AR exposure of non-target small mammals and their predators, which is known from several regions of the world. However, drivers of exposure variation are usually not known. To identify environmental drivers of AR exposure in non-targets we analyzed 331 liver samples of red foxes (Vulpes vulpes) for residues of eight ARs and used local parameters (percentage of urban area and livestock density) to test for associations to residue occurrence. 59.8% of samples collected across Germany contained at least one rodenticide, in 20.2% of cases at levels at which biological effects are suspected. Second generation anticoagulants (mainly brodifacoum and bromadiolone) occurred more often than first generation anticoagulants. Local livestock density and the percentage of urban area were good indicators for AR residue occurrence. There was a positive association between pooled ARs and brodifacoum occurrence with livestock density as well as of pooled ARs, brodifacoum and difenacoum occurrence with the percentage of urban area on administrative district level. Pig holding drove associations of livestock density to AR residue occurrence in foxes. Therefore, risk mitigation strategies should focus on areas of high pig density and on highly urbanized areas to minimize non-target risk.


Huang Y.,University of Veterinary Medicine Hannover | Ryll M.,University of Veterinary Medicine Hannover | Walker C.,University of Veterinary Medicine Hannover | Walker C.,Keele University | And 3 more authors.
Berliner und Munchener Tierarztliche Wochenschrift | Year: 2014

Enteric Redmouth Disease (ERM), caused by Yersinia (Y.) ruckeri is one of the most important diseases in salmonid aquaculture. Outbreaks of ERM were controlled by vaccines directed against motile strains of the bacterium, until recently nonmotile vaccine-resistant strains evolved and caused severe outbreaks. Non-motile isolates were found widespread in aquaculture populations in north-western Germany. In the present study, 82 Y. ruckeri isolates were isolated from trout hatcheries in North Rhine Westfalia, Lower Saxony and Hessen and only 20% of them were motile. In order to further characterise the Y. ruckeri isolates from fish aquaculture populations in north-western Germany, the fatty acid compositions of 82 Y. ruckeri field isolates from this area and of the Y. ruckeri reference strain DSM 18506 were analysed by gas chromatography. All Y. ruckeri isolates exhibited 15 major fatty acids, including 12:0, 13:0, 13.957 (equivalent chain length, ECL unknown), 14:0, 14.502 (ECL unknown), 15:0, 16:1ω5c, 16:0, 17:1ω8c, 17:0 CYCLO, 17:0, 16:1 2OH, 18:1ω9c, 18:1ω7c and 18:0. From a dendrogram, all isolates were close to one another, clustering together; while slight differences were detected among the isolates and the reference strain DSM 18506. Compared to their epidemiological and biochemical characteristics, there was no relationship found between the fatty acid profiles, API 20E profiles, motility and geographic distribution. Our results show that the fatty acid composition of Y. ruckeri isolates from north-western Germany is highly homogenous. © 2014 Schlütersche Verlagsgesellschaft mbH & Co. KG.


Huang Y.,University of Veterinary Medicine Hannover | Adamek M.,University of Veterinary Medicine Hannover | Walker C.,University of Veterinary Medicine Hannover | Walker C.,Keele University | And 2 more authors.
Berliner und Munchener Tierarztliche Wochenschrift | Year: 2014

The aim of this study was to investigate differences in presence and expression of virulence factors between biotype 1 and 2 strains of 82 Yersinia (Y.) ruckeri isolates, collected from North West Germany during the period of 2004–2012, and to analyze the cytotoxicity of these strains to different fish cell lines. The common virulence factor genes, such as yhlA and yhlB encoding for hemolysin YhlA, rucC and rupG encoding for ruckerbactin, yrp1 and yrpDEF for ABC exporter protein system, and two flagellar genes, including flgA for flagellar secretion chaperones and flhA for flagellar secretion apparatus, were found present in both biotype 1 and 2 isolates of Y. ruckeri collected from North West Germany using multiplex PCR. mRNA expression of these genes was compared between the two biotypes of Y. ruckeri. There was no significant diversity (p >0.05) in the expression of these genes between biotype 1 and 2 strains. 27 Y. ruckeri isolates from different typing groups were analysed in cytotoxicity tests to common carp brain (CCB), epithelioma papulosum cyprini (EPC), fathead minnow epithelial (FHM) and rainbow trout gonad-2 (RTG-2) cells, respectively. In vitro cytotoxicity of the isolates to CCB, EPC and FHM was higher than that to RTG-2 (p < 0.05). At 15°C the maximum cytotoxicity to FHM and EPC was higher in non-motile strains than in motile stains after an incubation of 24 h (p <0.05), however, after 48 h, there was no significant difference (p >0.05) of cytotoxicity between those two biotypes. Our results suggest that biotype 2 strains from North West Germany are homogenous with biotype 1 strains on the basis of genetic virulence factor genes. At lower temperature non-motile Y. ruckeri isolates were found more active than motile strains, which could explain why in winter non-motile strains were found more often responsible for ERM outbreaks than motile strains. © 2014 Schlütersche Verlagsgesellschaft mbH & Co. KG.


Baumer A.,University of Veterinary Medicine Hannover | Fabian M.,University of Veterinary Medicine Hannover | Wilkens M.R.,University of Veterinary Medicine Hannover | Steinhagen D.,University of Veterinary Medicine Hannover | Runge M.,Food and Veterinary Institute Braunschweig Hanover
Diseases of Aquatic Organisms | Year: 2013

Cyprinid herpesvirus-3 (CyHV-3, koi herpesvirus, KHV) is the causative agent of an economically important disease in carp. The mode of transmission of this virus, especially how the infectious agent is introduced into ponds de novo, is not known in detail. The aim of this study was to investigate the shedding of CyHV-3 from fish with latent infections, under aquaculture conditions. Ponds in Saxony, Germany, with latently infected carp were examined at different times during the production cycle to investigate the influence of fish farming procedures on virus activation and shedding. Carp and water samples were investigated by quantitative real-time PCR. Some of the latently infected carp shed CyHV-3. Virus shedding was induced mainly when the ponds were drained and the carp either harvested or moved to different ponds, and was independent of the water temperature. This indicated that during these times there was a risk that effluent water from the ponds could disseminate the infectious agent. During summer, on-growing carp are infected with low numbers of CyHV-3. These findings are important for disease management strategies in carp aquaculture and for the design of testing protocols for the detection of latent infection in carp populations. © Inter-Research 2013.


Huang Y.,University of Veterinary Medicine Hannover | Michael G.B.,Institute of Farm Animal Genetics | Becker R.,Institute of Farm Animal Genetics | Kaspar H.,Federal Office of Consumer Protection and Food safety BVL | And 4 more authors.
Veterinary Microbiology | Year: 2014

Enteric red-mouth disease, caused by Yersinia ruckeri, is an important disease in rainbow trout aquaculture. Antimicrobial agents are frequently used in aquaculture, thereby causing a selective pressure on bacteria from aquatic organisms under which they may develop resistance to antimicrobial agents. In this study, the distribution of minimal inhibitory concentrations (MICs) of antimicrobial agents for 83 clinical and non-clinical epidemiologically unrelated Y. ruckeri isolates from north west Germany was determined. Antimicrobial susceptibility was conducted by broth microdilution at 22 ± 2. °C for 24, 28 and 48. h. Incubation for 24. h at 22 ± 2. °C appeared to be suitable for susceptibility testing of Y. ruckeri. In contrast to other antimicrobial agents tested, enrofloxacin and nalidixic acid showed a bimodal distribution of MICs, with one subpopulation showing lower MICs for enrofloxacin (0.008-0.015. μg/mL) and nalidixic acid (0.25-0.5. μg/mL) and another subpopulation exhibiting elevated MICs of 0.06-0.25 and 8-64. μg/mL, respectively. Isolates showing elevated MICs revealed single amino acid substitutions in the quinolone resistance-determining region (QRDR) of the GyrA protein at positions 83 (Ser83-Arg or -Ile) or 87 (Asn87-Tyr), which raised the MIC values 8- to 32-fold for enrofloxacin or 32- to 128-fold for nalidixic acid. An isolate showing elevated MICs for sulfonamides and trimethoprim harbored a ~8.9. kb plasmid, which carried the genes sul2, strB and a dfrA14 gene cassette integrated into the strA gene. These observations showed that Y. ruckeri isolates were able to develop mutations that reduce their susceptibility to (fluoro)quinolones and to acquire plasmid-borne resistance genes. © 2013 Elsevier B.V.


PubMed | Food and Veterinary Institute Braunschweig Hanover, University of Veterinary Medicine Hannover, Institute of Farm Animal Genetics and Federal Office of Consumer Protection and Food safety BVL
Type: Journal Article | Journal: Veterinary microbiology | Year: 2014

Enteric red-mouth disease, caused by Yersinia ruckeri, is an important disease in rainbow trout aquaculture. Antimicrobial agents are frequently used in aquaculture, thereby causing a selective pressure on bacteria from aquatic organisms under which they may develop resistance to antimicrobial agents. In this study, the distribution of minimal inhibitory concentrations (MICs) of antimicrobial agents for 83 clinical and non-clinical epidemiologically unrelated Y. ruckeri isolates from north west Germany was determined. Antimicrobial susceptibility was conducted by broth microdilution at 22 2C for 24, 28 and 48 h. Incubation for 24h at 22 2C appeared to be suitable for susceptibility testing of Y. ruckeri. In contrast to other antimicrobial agents tested, enrofloxacin and nalidixic acid showed a bimodal distribution of MICs, with one subpopulation showing lower MICs for enrofloxacin (0.008-0.015 g/mL) and nalidixic acid (0.25-0.5 g/mL) and another subpopulation exhibiting elevated MICs of 0.06-0.25 and 8-64 g/mL, respectively. Isolates showing elevated MICs revealed single amino acid substitutions in the quinolone resistance-determining region (QRDR) of the GyrA protein at positions 83 (Ser83-Arg or -Ile) or 87 (Asn87-Tyr), which raised the MIC values 8- to 32-fold for enrofloxacin or 32- to 128-fold for nalidixic acid. An isolate showing elevated MICs for sulfonamides and trimethoprim harbored a 8.9 kb plasmid, which carried the genes sul2, strB and a dfrA14 gene cassette integrated into the strA gene. These observations showed that Y. ruckeri isolates were able to develop mutations that reduce their susceptibility to (fluoro)quinolones and to acquire plasmid-borne resistance genes.

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