Evaluation of techniques for the recovery of live intestinal Trichinella spiralis worms from experimentally infected foxes

Previously described methods for the recovery of intestinal Trichinella worms from rodents are not feasible when applied in larger experimental animals such as foxes. In this study,worm recovery by standard technique of simple incubation of the intestine in saline was compared to embedment of the intestine in an agar gel. The small intestines of Trichinella spiralis infected foxes (4-5 days post inoculation) were slit lengthwise and the two corresponding halves were processed with one of the two incubation methods.Worms were recovered from all samples, and the total worm recovery ranged from 0.2-4.4% of the infection dose. The samples from the standard incubation were very unclear and time consuming to count compared with samples from the agar gel embedment,in which the intestinal debris were kept inside the agar. As the agar gel technique generally yielded higher numbers of worms than the corresponding standard incubation sample, it is with some optimisation, recommended for recovery of intestinal Trichinella worms from foxes.     

Comparison of conventional coproscopical methods and commercial coproantigen ELISA kits for the detection of Giardia and Cryptosporidium infections in dogs and cats

Faecal samples of 270 dogs and 100 cats from 3 animal shelters in Germany were comparatively examined using conventional coproscopical methods and commercial coproantigen ELISA kits for the detection of Giardia and Cryptosporidium infections. Giardia cysts were found in 9.5% and 0% of the faecal samples in dogs and cats, respectively, examined once using the ZnCl2-NaCl flotation. However, the Giardia coproantigen ELISA (ProSpecT Giardia Microplate Assay) was positive in 29.5% and 22.4% of the samples from dogs and cats, respectively. Direct faecal smears stained with carbol fuchsin showed Cryptosporidium oocysts in one dog (0.4%) and one cat (1%). In contrast, the Cryptosporidium coproantigen ELISA (ProSpecT Cryptosporidium Microplate Assay) reacted positively in 23% of the samples from dogs and 30% of the samples from cats. Both coproantigen ELISAs were more often positive in coproscopically Giardia-negative canine faecal samples that contained Isospora burrowsi/ohioensis oocysts than in faecal samples without any parasite stage. Possible reasons for these observations are discussed.     

Antimicrobial susceptibility testing of bacteria isolated from animals: considerations concerning the predefinition of breakpoints from the clinical pharmacological viewpoint

In vitro susceptibility tests are performed to receive information for selecting the most suitable antibacterial agent. As result of in vitro susceptibility tests, the minimum inhibitory concentration (MIC) indicates bacteria as resistant or sensitive. To determine MIC, therapeutically relevant breakpoints have to be defined. Microbiological criteria, chemical and physical characteristics as well as pharmacokinetic and toxicological (tolerance) properties of the antimicrobial compounds have to be considered in the selection of the therapeutic agent in addition to clinical experiences. Using some concentration and time dependent antibiotics as examples, it is demonstrated that the above mentioned criteria are not sufficiently considered in currently defined breakpoints.     

Results from a German interlaboratory test to establish the broth microdilution method for the determination of the minimum inhibitory concentration of bacteria from animals

In accordance with NCCLS guideline M31-A2, the DVG working group "antimicrobial resistance" developed a standard operating procedure (SOP) for the determination of the minimal inhibitory concentration (MIC) of antimicrobial agents by broth microdilution. This SOP was evaluated for its fitness for use in a national interlaboratory test. A total of 32 participating laboratories tested five strains (including two internationally accepted reference strains and three field strains representing in total three different bacterial species) three times at a one week interval each, using uniform microtitre plates. In 31 of the 32 laboratories more than 80% of MIC determinations performed yielded values in the expected range. In total 94.0% of the results were reproducible, with a lesser deviation of 4.0% from the expected values for laboratories performing MIC determination as a matter of routine (46.9%), compared to 7.9% for laboratories without such routine (53.1%). Comparing the consistency of results on the basis of the tested strains, a higher reproducibility of the results was observed for reference strains (96.1%) than for field strains (92.6%). In particular results obtained for the Streptococcus uberis field strain were afflicted with a higher error ratio (98 deviations from the expected values). Among the tested antimicrobial agents, a higher variability of results was recorded only for gentamicin with 16.7% divergent MIC determinations (mean value 6.0%). The high reproducibility of the results confirmed by this interlaboratory study underlines the robustness of the developed SOP as well as broth microdilutions as the method of choice for MIC determina tion.     

Cross-resistance between antimicrobial agents used in veterinary medicine: molecular background and practical consequences for susceptibility testing

Phenotypic resistance of veterinary pathogens to more than one antimicrobial agent (multi-resistance) may be caused by intrinsic resistance to the antimicrobial agents, acquired cross-resistance, or acquired co-resistance. Known cross-resistances allow to select so-called "representative substances" which are tested and the results of which can also be regarded as being valid for other members of the same class of antimicrobial agents. In general, a limitation in the number of antimicrobial agents to be tested in routine diagnostics is necessary because of capacity and cost efficiency. This is of particular relevance when the broth microdilution method - recommended as the method of choice - with 96-well microtiter plates is used. The knowledge about the relationship between different resistance phenotypes and the corresponding resistance mechanisms is of major value for both, the laboratory personnel and the veterinary practitioner. This review explains how "representative substances" for the most relevant classes of antimicrobial agents used in veterinary medicine are chosen on the basis of known cross-resistances.