Suitability of dehydrogenase activity assay as an index of soil biological activity

Summary Dehydrogenase activity was studied in typical soils of the Northwest German Lower Plains in order to test some criticisms raised by nannipieri et al. (1990) about methodology. We found that in the same soils with the sampe crop, dehydrogenase activity varies significantly. In the field dehydrogenase activity was dependent on the soil type and not on the cropping system. There is no adequate explanation of these findings. Dehydrogenase activity is a parameter of soil microbiological activity and is influenced by many factors. Either purely accidental the results are or dehydrogenase activity is affected by unknown ecological interactions and by the composition of soil microflora. We conclude that without simultaneous investigations of other microbiological parameters (microbial biomass, ATP levels, enzyme activities, etc.) the measurement of dehydrogenase activity creates confusion and may impede valid ecological comparisons of soils.Dedicated to Prof. H.-P. Blume, Kiel, on the occasion of his 60th birthday     

Microbial decomposition of fuel oil after compost addition to soil

The effects of adding straw and bark compost at different nitrogen levels to fuel oil contaminated soil was investigated using the CHCl₃ fumigation-extraction method to measure the soil microbial biomass, The addition of these substrates decreased the percentage of extractable fuel oil, increased the percentage of fuel oil mineralized to CO₂, but also increased the fraction of unrecovered fuel oil residues. The different N levels had no significant effects on fuel oil decomposition in the presence of the compost substrates. The addition of the compost substrates increased markedly the content of microbial biomass in contrast to the fuel oil addition which had no effect on the biomass level indicating toxic properties.     

Structural investigations of flavonol glycosides from sea buckthorn (Hippophaë rhamnoides) pomace by NMR spectroscopy and HPLC-ESI-MS(n)

Four flavonol glycosides were isolated from an extract of sea buckthorn pomace (Hippopha? rhamnoides) by Sephadex LH-20 gel chromatography and semipreparative HPLC. Their structures were elucidated by hydrolysis studies, ESI-MS(n), UV, and (1)H and (13)C NMR spectroscopy. The occurrence of the major flavonol glycoside kaempferol 3-O-beta-sophoroside-7-O-alpha-rhamnoside in sea buckthorn is described here for the first time. A further 21 flavonol glycosides of Sephadex LH-20 fractions of sea buckthorn pomace were characterized by HPLC-DAD-ESI-MS. The characteristic MS-MS and MS(3) fragmentation pattern of flavonol glycosides previously identified in sea buckthorn juice and of flavonol glycosides identified by NMR spectroscopy gave valuable indications for their identification. The results demonstrate that loss of the sugar moiety from C-7 of the aglycon is more favored than fission of the glycosidic linkage at the C-3 position. Thus, most of the compounds identified were 7-rhamnosides of isorhamnetin, kaempferol, and quercetin, which exhibit different substitution patterns at the C-3 position, mainly glucosides, rutinosides, and sophorosides. In addition, numerous flavonol glycosides were detected lacking a sugar moiety at C-7. Finally, eight flavonol derivatives were identified that are acylated by hydroxybenzoic or hydoxycinnamic acids.     

Application of Campylobacter molecular classification and typing techniques in veterinary medicine: old-established methods and new perspectives

In this review the application and usefulness of Campylobacter genotypical classification and typing in veterinary medicine will be discussed.While there is a large area of overlapping applications between the veterinary and the medical field, several differences exist, as the spectrum of veterinary pathogens is different from the human and contaminated food of healthy animal origin may cause disease in man. In general, genotyping in the veterinary field can be applied in three different areas: (a) purely diagnostic purposes for classification of Campylobacter species and subspecies, (b) typing methods useful for monitoring or surveillance of animals as well as food products of animal origin, and (c) typing methods that can be applied during outbreaks and for source tracing. In addition, typing methods applied in areas (b) and (c) should be distinguished in regard to local short-term and global long-term epidemiology, respectively. While a whole plethora of discriminative typing methods are available, classification tools of certain species and subspecies are still missing. Perspectively, as the genomes of many relevant Campylobacter species have now been sequenced, this will help to identify several species specific loci, the products of which should be available to develop easy and fast applicable diagnostic tools. Global cooperation, sharing of strains and databases should close the currently existing gaps in Campylobacter identification tools.     

Susceptibility of bacterial pathogens against lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) as determined in the BfT-GermVet monitoring program 2004-2006

A total of 368 bacterial pathogens, including 72 coagulase-positive and coagulase-variable staphylococci, 97 beta-haemolytic streptococci, 51 Escherichia coli, 75 Pasteurella multocida, 25 Mannheimia haemolytica, 25 Pseudomonas aeruginosa, and 23 Arcanobacterium pyogenes, were investigated for their susceptibility to the three combinations of antimicrobial agents lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) in comparison to their susceptibility to the corresponding single substances. When comparing the minimum inhibitory concentrations (MICs) determined for any of the three combinations with those for the single substances, the lowest MIC of one of the two substances usually determined the MIC of the combination.This observation was made for all three combinations and all bacterial pathogens tested.Thus, it is assumed that the combination of lincomycin with spectinomycin as well as that of penicillin with either neomycin or dihydrostreptomycin resulted in an extended spectrum of target bacterial pathogens rather than in an increase in antimicrobial efficacy.     

Serological Salmonella monitoring in German pig herds: results of the years 2003-2008

The implementation of the European regulations regarding zoonoses demands objective and scientific statements regarding the status of the Salmonella prevalence in the national pig herds of all EU member states. Since 2002, the "QS Qualit?t und Sicherheit GmbH" has carried out serological Salmonella monitoring in German finishing pig herds. All data generated within the monitoring system are entered into the central database Qualiproof(?) (Qualitype AG, Dresden). The dataset investigated included 5,324,532 samples taken between January 1, 2003 and December 31, 2008, originating from 22,490 herds. Blood sera or meat juices were sampled following a standardized sampling scheme (up to 60 samples per year and herd, depending on herd size) using one of four evaluated ELISA-tests. Herds were classified into three categories I (0-20%), II (>20-40%), or III (>40%) by their percentage of yearly positive samples, which was re-calculated quarterly. The number of participating herds increased continuously since the start of the monitoring programme, with regional differences in the degree of participation. In 2008, 10.8% of all samples were positive. Adjusted for the distribution of samples in the German districts, the Salmonella prevalence in Germany was 7.9%. In general the distribution of herds in the categories was relatively stable over time. In the fourth quarter of 2008, 81.9% of the herds were allocated to category I, 14.0% to category II, and 4.0% to category III. However, the prevalence of Salmonella tended to decrease in herds that participated over a longer period. Differences could be found between German geographical regions. Seroprevalences were higher in the Northwest and the Northeast than in the South. This might be due to the relationship between Salmonella seroprevalences and farm densities per district, which were both higher in Northern than in Southern Germany. The Salmonella monitoring system will contribute to the reduction of the Salmonella prevalence in German pork production, when it is supplemented by control measures.     

Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.     

Avian pathogenic Escherichia coli (APEC)

Infections with avian pathogenic Escherichia coli (APEC) cause colibacillosis, an acute and mostly systemic disease resulting in significant economic losses in poultry industry worldwide. Avian colibacillosis is a complex syndrome characterized by multiple organ lesions with airsacculitis and associated pericarditis, perihepatitis and peritonitis being most typical. Environmental factors as well as the constitution of poultry or initial viral infections influence the outcome of APEC-infections. However, several challenge experiments in chickens proofed the role of virulent APEC strains as the single aetiological agent. Currently serotypes O1:K1, O2:K1 and O78:K80 are recognized as the most prevalent, however the number of published serotypes is increasing. In addition, single APEC isolates vary profoundly in virulence, and knowledge about the molecular basis of this variability is still scarce. Known virulence factors of APEC are adhesins (F1- and P-fimbriae), iron acquisition systems (aerobactin and yersiniabactin), hemolysins (hemolysinE and temperaturesensitive hemagglutinin), resistance to the bactericidal effects of serum and phagocytosis (outer membrane protein, iss protein, lipopolysaccharide, K/1)-capsule and colilcin production) as well as toxins and cytotoxins (heat stable toxin, cyto-/verotoxin and flagella toxin). Esperimental studies have shown that the respiratory tract, principally the gas-exchange region of the lung and the interstitium of the air sacs are the most important sites of entry for avian pathogenic E. coli. APEC strains adhere to the epithelial cells of air sacs presumably through F1-fimbriae. After colonization and multiplication the bacteria enter the bloodstream, and the temperature-sensitive hemagglutinin (tsh) seems to be important int his step. After invading the bloodstream APEC cause a septicemia resulting in massive lesins in multiple internal organs and in sudden death of the birds. The ability of the bacteria to acquire iron and the resistance to the bactericidal effects of serum, predominantly conferred by the increased serum survival (iss)--protein, enables APEC to multiply quickly in their hosts. Iss is regarded a specific genetic marker for avian pathogenic E. colistrains. A critical review of the literature published so far on APEC reveals, that these pathotypes are not defined appropriately. This findings urge investigations on the population structure of APEC, enabling the establishment of appropriate diagnostic tools and avoiding the obsolete use of serotyping for APEC diagnosis. So far more than 20 APEC strains have been investigated in animal experiments, explaining contrary published results. Thus, the lack of knowledge in pathogenicity and in immunity of APEC infections urges further experimental studies. As APEC share not only identical serotypes with human pathogens but also specific virulence factors, their zoonotic potential is under consideration.