Intervet Innovation GmbH

Schwabenheim, Germany

Intervet Innovation GmbH

Schwabenheim, Germany

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Silley P.,MB Consult Ltd. | Silley P.,University of Bradford | De Jong A.,Bayer AG | Simjee S.,Elanco Animal Health | Thomas V.,Intervet Innovation GmbH
International Journal of Antimicrobial Agents | Year: 2011

Antimicrobial surveillance systems in Denmark (DANMAP), The Netherlands (MARAN), Spain (VAV) and Sweden (SVARM) as well as the European Antimicrobial Susceptibility Surveillance in Animals (EASSA) were reviewed. Data have been considered for extended-spectrum cephalosporins, fluoroquinolones and macrolides against food-borne and commensal bacteria. The greatest challenge arises from the lack of agreement between programmes on what is meant by resistance through the use of different interpretive criteria. Indeed, it is shown here that the extent of the differences depends on the antibacterial compound being investigated, the methodology and the interpretive criteria used. This emphasises a need to agree a definition for resistance and for epidemiological cut-off values and to consider harmonising the antimicrobials used in surveillance. This analysis of the data highlights the usefulness of using both epidemiological cut-off values and clinical resistance breakpoints for the purpose of detection of decreased susceptibility and development of clinical resistance, respectively. It is concluded that harmonisation in resistance monitoring programmes is needed since there is potential for data to be appropriately used within risk analysis, providing the opportunity to implement appropriate risk management steps as a response to the public health issues arising from changes in antibiotic resistance in food-borne pathogens and commensal organisms. © 2011 Elsevier B.V. and the International Society of Chemotherapy.


Spehr V.,Intervet Innovation GmbH | Warrass R.,Intervet Innovation GmbH | Hocherl K.,University of Regensburg | Ilg T.,Intervet Innovation GmbH
Applied Biochemistry and Biotechnology | Year: 2011

(3′-5′)-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1β, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages. © 2011 Springer Science+Business Media, LLC.


Wischke C.,Research Center Geesthacht GmbH | Wischke C.,Berlin Brandenburg Center for Regenerative Therapies | Neffe A.T.,Research Center Geesthacht GmbH | Steuer S.,Intervet Innovation GmbH | And 2 more authors.
European Journal of Pharmaceutical Sciences | Year: 2010

A family of oligo[(e{open}-caprolactone)-co-glycolide]dimethacrylate (oCG-DMA) derived networks of different glycolide contents as well as precursor molecular weights has been synthesized by crosslinking oCG-DMA, providing matrices of different hydrophilicity, network density, and morphology at body temperature. Such networks were loaded with a hydrophilic model drug, ethacridine lactate, either before crosslinking or afterwards by swelling in drug solution. Disadvantageous alterations of the shape-memory functionality and degradation characteristics were observed only in few loaded materials. Loading by swelling generally resulted in low payloads, which slightly increased for more hydrophilic polymer networks, and a substantial burst and fast subsequent release for all investigated materials. Loading before crosslinking gave almost no burst and higher subsequent release rates over longer periods of time. Overall, depending on the needs of a specific application, a material from this polymer family with the desired mechanical properties, shape-memory functionality, and degradation pattern can be selected and combined with drugs when considering that (i) loading by swelling is best suited for applications that require high initial doses and (ii) loading before crosslinking allows easy variation of payloads and low burst release for therapeutics that are non-sensitive to chemical alterations during crosslinking. © 2010 Elsevier B.V.


Koch O.,Intervet Innovation GmbH | Koch O.,Molisa GmbH
Molecular Informatics | Year: 2012

Turns are essential for protein structure as they allow the polypeptide chain to fold backup on itself. They also occur within protein binding sites, at protein - protein interfaces and in small bioactive peptides, where they can play a crucial role for molecular recognition. Turn structures are an important class of protein secondary structure, although relatively little attention is paid to them with respect to helices and β-sheets. Protein structure prediction, functional analysis of proteins and peptides, and computer-aided drug design could all benefit from making use of accurately predicted turn structures from amino acid sequence. Here, recent advances of turn structure prediction and the underlying turn classification will be discussed together with their applications. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Rose S.,University of Southern Denmark | Desmolaize B.,University of Southern Denmark | Jaju P.,University of Southern Denmark | Wilhelm C.,Intervet Innovation GmbH | And 2 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2012

The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm(42), which encodes a monomethyltransferase and confers a type I MLSB (macrolide, lincosamide, and streptogramin B) phenotype; msr(E), which encodes a macrolide efflux pump; and mph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm(42), msr(E), and mph(E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA (rrl) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm(42), msr(E), and mph(E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Dederer H.,Intervet Innovation GmbH | Werr M.,Intervet Innovation GmbH | Ilg T.,Intervet Innovation GmbH
Insect Biochemistry and Molecular Biology | Year: 2011

Nicotinic acetylcholine receptors (nAChRs) are the binding sites for nicotinoid drugs, such as nicotine and epibatidine, and are the molecular targets of the selectively insecticidal neonicotinoids. In this study we report the full length cDNA cloning of the three Ctenocephalides (C.) felis (cat flea) nAChR α subunits Cfα1, Cfα2, and Cfα3. When expressed in Xenopus oocytes as hybrid receptors with the Gallus gallus (chicken) β2 (Ggβ2) subunit, these cat flea α subunits formed acetylcholine-responsive ion channels. Acetylcholine-evoked currents of Cfα2/Ggβ2 were resistant to α-bungarotoxin, while those of Cfα1/Ggβ2 were sensitive to this snake toxin. The pharmacological profiles of Cfα1/Ggβ2, Cfα2/Ggβ2 and the chicken neuronal receptor Ggα4/Ggβ2 for acetylcholine, two nicotinoids and 6 insecticidal neonicotinoids were determined and compared. Particularly remarkable was the finding that Cfα1/Ggβ2 was far more sensitive to acetylcholine, nicotine and neonicotinoid agonists than either Cfα2/Ggβ2 or Ggα4/Ggβ2: for the anti flea neonicotinoid market compound imidacloprid the respective EC50s were 0.02μM, 1.31μM and 10μM. These results were confirmed for another insect species, Drosophila melanogaster, where the pharmacological profile of the Dmα1 and Dmα2 subunits as hybrid receptors with Ggβ2 in Xenopus oocyte expressions resulted in a similar sensitivity pattern as those identified for the C. felis orthologs. Our results show that at least in a Ggβ2 hybrid receptor setting, insect α1 subunits confer higher sensitivity to neonicotinoids than α2 subunits, which may contribute in vivo to the insect-selective action of this pesticide class. © 2010 Elsevier Ltd.


Koch O.,The Cambridge Crystallographic Data Center | Koch O.,Intervet Innovation GmbH | Cole J.,The Cambridge Crystallographic Data Center
Proteins: Structure, Function and Bioinformatics | Year: 2011

A new automated helix assignment method is presented that leads to a more consistent definition of the helix termini, especially of the helix C-terminus. The method assigns a helix to segments of protein chain where adjacent helical turn structures are observed, capped by specific distorted turn types (e.g., open helical turns without a hydrogen bond) or capping motifs (e.g., the Schellman motif). Helix termini are detected by observing the behavior of the NH group in N-termini and the CO group in C-termini; in each case, the respective group must be free to interact with hydrogen bonding partners outside of the putative helix for a helix terminus to be assigned. The presented assignment method and SHAFT-assigned helices are part of Secbase and are made available with Relibase+ 3.0 and the free web version of Relibase 3.0. The method can also be used for the helix assignments of additional protein structures. © 2011 Wiley-Liss, Inc.


Desmolaize B.,University of Southern Denmark | Rose S.,University of Southern Denmark | Wilhelm C.,Intervet Innovation GmbH | Warrass R.,Intervet Innovation GmbH | Douthwaite S.,University of Southern Denmark
Antimicrobial Agents and Chemotherapy | Year: 2011

Respiratory tract infections in cattle are commonly associated with the bacterial pathogens Mannheimia haemolytica and Pasteurella multocida. These infections can generally be successfully treated in the field with one of several groups of antibiotics, including macrolides. A few recent isolates of these species exhibit resistance to veterinary macrolides with phenotypes that fall into three distinct classes. The first class has type I macrolide, lincosamide, and streptogramin B antibiotic resistance and, consistent with this, the 23S rRNA nucleotide A2058 is monomethylated by the enzyme product of the erm(42) gene. The second class shows no lincosamide resistance and lacks erm(42) and concomitant 23S rRNA methylation. Sequencing of the genome of a representative strain from this class, P. multocida 3361, revealed macrolide efflux and phosphotransferase genes [respectively termed msr(E) and mph(E)] that are arranged in tandem and presumably expressed from the same promoter. The third class exhibits the most marked drug phenotype, with high resistance to all of the macrolides tested, and possesses all three resistance determinants. The combinations of erm(42), msr(E), and mph(E) are chromosomally encoded and intermingled with other exogenous genes, many of which appear to have been transferred from other members of the Pasteurellaceae. The presence of some of the exogenous genes explains recent reports of resistance to additional drug classes. We have expressed recombinant versions of the erm(42), msr(E), and mph(E) genes within an isogenic Escherichia coli background to assess their individually contributions to resistance. Our findings indicate what types of compounds might have driven the selection for these resistance determinants. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


Ilg T.,Intervet Innovation GmbH | Schmalz S.,Intervet Innovation GmbH | Werr M.,Intervet Innovation GmbH | Cramer J.,Intervet Innovation GmbH
Insect Biochemistry and Molecular Biology | Year: 2010

Acetylcholinesterase (AChE, EC3.1.1.7.) is a prime target for insecticides and is the site of action of carbamate and organophosphate drugs used to combat the cat flea Ctenocephalides felis. In this paper we report the identification and cDNA cloning of two AChE-encoding genes from the cat flea, cface1 and cface2. Functional heterologous expression of the catalytic domains in Pichia pastoris shows that both genes encode functional enzymes, CfAChE1 and CfAChE2. Bioinformatical analysis of the predicted translation products and heterologous expression of the full length cDNAs in the human cell line HEK293 demonstrate, that CfAChE1 and CfAChE2 possess glycosylphosphatidylinositol membrane anchors and are transported to the cell surface. Recombinant CfAChE1 and CfAChE2 share high sensitivity towards the anti-flea carbamates propoxur and carbaryl, but can be distinguished by their specificity for different acylthiocholine AChE substrates and, particularly, by their differential sensitivity to the non-covalent inhibitor galanthamine. Comparison of substrate specificities and inhibitor sensitivities of both recombinant enzymes with those of AChE activities extracted from adult fleas suggest that CfAChE1, and not CfAChE2, is the dominant activity in C. felis imagoes. Three-dimensional structure models of CfAChE1 and CfAChE2 reveal similarities, but also differences, and compound docking experiments on these models provide potential rationales for the differential substrate and the inhibitor specificities observed experimentally. © 2010 Elsevier Ltd. All rights reserved.


Desmolaize B.,University of Southern Denmark | Rose S.,University of Southern Denmark | Warrass R.,Intervet Innovation GmbH | Douthwaite S.,University of Southern Denmark
Molecular Microbiology | Year: 2011

Mannheimia haemolytica and Pasteurella multocida are aetiological agents commonly associated with respiratory tract infections in cattle. Recent isolates of these pathogens have been shown to be resistant to macrolides and other ribosome-targeting antibiotics. Direct analysis of the 23S rRNAs by mass spectrometry revealed that nucleotide A2058 is monomethylated, consistent with a Type I erm phenotype conferring macrolide-lincosamide resistance. The erm resistance determinant was identified by full genome sequencing of isolates. The sequence of this resistance determinant, now termed erm(42), has diverged greatly from all previously characterized erm genes, explaining why it has remained undetected in PCR screening surveys. The sequence of erm(42) is, however, completely conserved in six independent M. haemolytica and P. multocida isolates, suggesting relatively recent gene transfer between these species. Furthermore, the composition of neighbouring chromosomal sequences indicates that erm(42) was acquired from other members of the Pasteurellaceae. Expression of recombinant erm(42) in Escherichia coli demonstrated that the enzyme retains its properties as a monomethyltransferase without any dimethyltransferase activity. Erm(42) is a novel addition to the Erm family: it is phylogenetically distant from the other Erm family members and it is unique in being a bona fide monomethyltransferase that is disseminated between bacterial pathogens. © 2011 Blackwell Publishing Ltd.

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