Antimicrobial susceptibility testing of bacteria isolated from animals: methods for in-vitro susceptibility testing and their suitability with regard to the generation of the most useful data for therapeutic applications

In-vitro susceptibility testing provides valuable informations for choosing the most suitable antimicrobial agent for the control of bacterial infections in animals. Different diffusion and dilution methods, as conducted according to various approved performance standards, can be used to determine the in-vitro susceptibility of bacterial pathogens. In the present article, problems are discussed which arise from the use of different methods and the difficulty to interpret such results. While most approved performance standards were designed for testing of bacteria from human sources, the NCCLS document M31-A2 exclusively focusses on susceptibility testing of bacteria isolated from animals and--in contrast to all other standards--includes veterinary specific breakpoints for a number of antimicrobial agents used in veterinary medicine. Therefore, performance of in-vitro susceptibility testing of veterinary pathogens should follow the recommendations given in the NCCLS document M31-A2. The microdilution method is recommended as the method of choice for susceptibility testing. The result of a microdilution test is given as the minimum inhibitory concentration (MIC). This value provides a quantitative result which precisely indicates the degree of susceptibility of the tested bacterial strain and in return gives the veterinarian a clear guidance whether therapeutic intervention with the antibiotic in question will be successful.     

Identification of two serological flagellar types (H1 and H2) inPseudomonas syringae pathovars

Flagellar antigen specificity was studied for the speciesPseudomonas syringae, P. viridiflava andP. cichorii. After checking their motility, bacteria were reacted against six polyclonal antisera containing anti-O (LPS) and anti-H (flagellar) antibodies by indirect immunofluorescent staining. Two distinct flagellar serotypes (H1 and H2) were described. The distribution of H1 and H2 serotypes was then determined for a collection of 88 phytopathogenicPseudomonas strains. Serotype H1 was possessed byP. syringae pv.aptata (12 strains),P. s. pv.helianthi (2),P. s. pv.pisi (11), andP. s. pv.syringae (13). Serotype H2 was possessed byP. cichorii (2),P. s. pv.delphinii (1),P. s. pv.glycinea (4),P. s. pv.lacrymans (1),P. s. pv.mori (1),P. s. pv.morsprunorum (10),P. s. pv.persicae (1),P. s. pv.phaseolicola (8),P. s. pv.tabaci (10) andP. s. pv.tomato (1).P. viridiflava (5) revealed HI, H2 and untyped flagella. The following isolates were untypable by the H1/H2 system:P. corrugata (3),P. fluorescens (2),P. tolaasii (1). H1/H2 serotypes distribution is not linked toP. syringae O-serogroups. On the other hand, H1/H2 distribution seems remarkably linked to the new genospecies of theP. syringae group.Abbreviations CFBP French Collection of Phytopathogenic Bacteria, Angers, France - ICMP International Collection of Micro-organisms from Plants, Auckland, New-Zealand - NCPPB National Collection of Plant Pathogenic Bacteria, Harpenden, Great Britain     

Layout proposal for a microtitre plate to be used in routine antimicrobial susceptibility testing of bacteria from infections of dogs and cats

The determination of minimum inhibitory concentrations by broth microdilution is recommended as method of choice for susceptibility testing of veterinary bacterial pathogens. Accordingly, broth microdilution is used in veterinary routine diagnostic laboratories at a progressive rate. To reduce the costs of susceptibility testing, it is reasonable to develop widely accepted uniform microtitre plate layouts that are produced in large quantities. Such microtitre plate layouts have already been developed and published for the susceptibility testing of pathogens from food-producing animals. However, a microtitre plate layout, especially designed for the testing of bacteria from dogs and cats, should be available, too. The choice of the antimicrobial agents or combinations of antimicrobial agents to be included in a suitable layout should be based on the following criteria: (1) the approval and availability of an antimicrobial agent or combination of agents, (2) known cross-resistances, and (3) availability of approved clinical breakpoints. The latter point is of particular importance for the choice of the numbers of concentrations per antimicrobial agent tested and the range of test concentrations. Taking into account these aspects, a science-based layout proposal for microtitre plates, which are suitable for routine testing of bacteria from dogs and cats, is presented and discussed.     

Lack of detectable West Nile virus RNA in brains and kidneys of dogs and cats with immunohistological precipitates using virus-specific antibodies

Five dogs and four cats from Germany suffering from encephalitis revealed positive immunoreactivity using two West Nile virus (WNV) specific monoclonal antibodies in brain and in kidney. However, WNV-infection could not be confirmed by additional PCR analyses. This study indicated that positive immunoreactivity for WNV in dogs and cats must be interpreted cautiously and should be confirmed by a second virus-specific technique.     

EVALUATION OF CANINE LUMBOSACRAL STENOSIS USING INTRAVENOUS CONTRAST-ENHANCED COMPUTED TOMOGRAPHY

The objective of this study was to evaluate intravenous contrast-enhanced computed tomography as a technique for predicting the within-level location(s) of compressive soft tissues in the canine lumbosacral spine. Pre-operative intravenous contrast-enhanced computed tomography of the L5-S3 vertebral levels was performed in 12 consecutive large breed dogs with lumbosacral stenosis. The images were evaluated for enhancement of soft tissues by two radiologists who were unaware of the surgical findings. For each within-level location (dorsal canal, ventral canal, right lateral recess, left lateral recess) enhancement was classified as present, absent or equivocal. The results were compared with the results of surgical exploration and histopathology of excised tissues. The positive predictive values of intravenous contrast-enhanced computed tomography for compressive soft tissues involving the dorsal canal, ventral canal and lateral recesses were 83%, 100%, and 81% respectively. Negative predictive values for compressive soft tissues involving these locations were 29%, 50%, and 40% respectively.     

Optimisation of gene expression analysis in Atlantic salmon lenses by refining sampling strategy and tissue storage

Analysis of gene expression in the lens is one of the analytical tools employed to investigate cataract formation in Atlantic salmon (Salmo salar L.). High quality RNA preparations are an essential prerequisite for gene expression analysis. The first aim of the present study was to investigate the possible effects of two methods of tissue preservation on the quality of RNA extracted from Atlantic salmon lenses. RNA was extracted from lenses either stored in RNAlater or flash-frozen in liquid nitrogen. Both tissue preservation methods yielded RNA of similarly high quality. The second aim was to examine if stress related to fish handling and the choice of anaesthesia during the sampling procedure affected gene expression in the lens. Six sampling procedures were tested on groups of sea water adapted Atlantic salmon smolt. Fish were either killed instantaneously (control group) or sampled after 30 min anaesthetised with isoeugenol, after 30 min without anaesthesia, after 120 min anaesthetised with isoeugenol, after 120 min with 15 min anaesthesia with metacaine or after 120 min without anaesthesia. The expression levels of specific genes, of special interest in the study of molecular mechanisms of cataractogenesis, were analysed in lenses by real-time RT–PCR. Fish not anaesthetised had significantly increased levels of heat shock protein 70 (HSP70) mRNA after 30 min compared to the control group. Glutathione reductase (GR) and manganese superoxide dismutase (Mn-SOD) were expressed at significantly lower levels in groups of Atlantic salmon sampled after 120 min anaesthetised with isoeugenol or metacaine, and anaesthetised with isoeugenol, metacaine or without anaesthesia, respectively. The same expression patterns were found in corresponding gill tissues for these two antioxidant genes. In conclusion, preservation in liquid nitrogen instead of RNAlater is recommended due to practical conditions in RNA extraction. A quick sampling protocol with the use of anaesthetics and not exceeding 30 min should be preferred to avoid effects of the sampling procedure on lens gene expression in Atlantic salmon.     

Corneal innervation in mesocephalic and brachycephalic dogs and cats: assessment using in vivo confocal microscopy

Objective To determine the density of the canine and feline corneal neural network in healthy dogs and cats using in vivo confocal microscopy (IVCM). Animals examined A total of 16 adult dogs (9 Mesocephalic breeds, 7 Brachycephalic breeds) and 15 cats (9 Domestic Short-haired cats (DSH), 6 Persian cats) underwent IVCM. Procedure Animals were examined with a confocal corneal microscope (HRTII/RCM; Heidelberg Retina Tomograph II/Rostock Cornea Module^®, Heidelberg Engineering, Dossenheim, Germany). The investigations focused on the distribution of the corneal nerves and quantification of central subepithelial and subbasal nerve plexus. Results The corneal stromal nerve trunks, subepithelial and subbasal nerve plexus were observed. The nerve fiber density (NFD) quantified in nerve fiber length in mesocephalic dogs were 12.39 ± 5.25 mm/mm² in the subepithelial nerve plexus and 14.87 ± 3.08 mm/mm² in the subbasal nerve plexus. The NFD of the subepithelial nerve plexus in DSH cats was 15.49 ± 2.7 and 18.4 ± 3.84 mm/mm² in the subbasal nerve plexus. The subbasal NFD of DSH cats was significantly higher than in mesocephalic dogs (P = 0.037). The subepithelial NFD in brachycephalic dogs, and Persian cats were 10.34 ± 4.71 and 9.50 ± 2.3 mm/mm², respectively. The subbasal NFD measured 11.80 ± 3.73 mm/mm² in brachycephalic dogs, and 12.28 ± 4.3 mm/mm² NFD in Persian cats, respectively. The subepithelial and subbasal NFD in Persian cats were significantly lower than in DSH cats (P = 0.028, respectively, P = 0.031), in contrast to brachycephalic vs. mesocephalic dogs. Conclusion The noninvasive IVCM accurately detects corneal innervation and provides a reliable quantification of central corneal nerves.