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.     

Bacterially conditioned thromboembolism in dairy cows--a retrospective study of 31 necropsy cases with special consideration of the causative complex

A study of 340 necropsied dairy cattle from northern Bavaria resulted in 31 animals (9%) showing evidence of pyemic thromboembolism. The most frequent pathomorphologic lesions consisted in endocarditis valvularis thromboticans of the tricuspid and/or mitral valve (21), embolic pneumonia (26), nephritis (13) and renal infarction (12). The most common isolate found in bacteriologic culture was Arcanobacterium pyogenes in 26 of 31 cases, followed by Staphylococcus aureus (2), Mannheimia haemolytica (2) and Streptococcus bovis (1) in rare cases. In 27 of 31 cows a possible cause of pyemic thromboembolism was found; in 23 cases claw diseases and decubital ulcera were probably responsible.     

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.     

A three-year longitudinal study on the effects of a diet containing genetically modified Bt176 maize on the health status and performance of sheep

This study shows that a diet including insect-resistant Bt176 maize, fed to 53 ewes and their progeny for 3 years, did not have adverse effects on their health or performance and that no horizontal gene transfer to ruminal microorganisms or animal tissues was detected. No differences were observed regarding performance, reproductive traits, haematological parameters, antioxidant defences, lymphocyte proliferative capacity, phagocytosis and intracellular killing of macrophages, and ruminal microbial population characteristics between control and genetically modified (GM) maize-fed animals. Immune response to Salmonella abortus ovis vaccination was more efficient in GM maize fed sheep. No modifications of histological features of tissues were found; however, cytochemical analyses of ruminal epithelium by Ki67 staining provided evidence of proliferative activation of basal cells in all GM maize-fed ewes. Preliminary electron microscopy analyses of the liver and pancreas revealed smaller cell nuclei containing increased amounts of heterochromatin and perichromatin granules in GM maize-fed lambs. Meat protein content and water loss by cooking were slightly affected by the dietary treatment. No transgenic DNA was detected in tissues, blood, and ruminal fluid or ruminal bacteria. Longitudinal studies should be included in evaluation of food safety whenever possible and sheep may be a useful animal model for toxicological assessment.     

Efficacy of Flectron-eartags (cypermethrin) for control of midges (Culicoides) as the vectors of bluetongue virus in cattle: field studies and biossays

When in 2006 infection with bluetongue for the first time occurred in Germany the registered and already against flies and tabanids in cattle proofed Flectron ear tags were used against the blood feeding vector midges (Culicoides) also. However, the efficacy against gnats was not yet proofed. The efficacy of 1 and 2 ear tags (1,067 g cypermethrin per ear tag) per animal was investigated in North Germany with 237 heifers and dairy cows. Midges were caught in suction light traps close to the cattle on pasture or became trapped by mouth operated aspirators directly at the skin of the animal bodies. Within 12,051 specimens of midges 12 species of Culicoides could be identified. On grasslands 3 species, C. obsoletus, C. pulicaris and C. dewulfi were found to be dominant. These 3 species are also known to be vectors of BTV. The toxic efficacy was found for 14 days with 1 ear tag and up to 21 days with 2 ear tags. This duration of efficacy was confirmed in the laboratory with hair clippings from the dorsal line and the ventral abdomen (bioassay). In accordance with workers in the U.S.A. it is concluded that insecticide-impregnated ear tags will reduce the number of biting midges, and by this way the risk of infection with BTV in herds of treated cattle will be reduced as well as in other cattle of a particular region. It is concluded that ear tags are of considerable value as part of an integrated control program for BT, e.g. vaccination.     

Effects of glucagon‐like peptides 1 and 2 and epidermal growth factor on the epithelial barrier of the rumen of adult sheep

Epidermal growth factor (EGF) and glucagon‐like peptides (GLP) modulate the tight junctions (TJ) of the intestinal epithelial barrier (EB) of monogastric animals. This work tried to elucidate whether GLP‐1, GLP‐2 and EGF can affect the EB of the rumen. Ovine ruminal epithelia were incubated in Ussing chambers for 7 hr with 25 or 250 nM of either GLP‐1 or GLP‐2 on the serosal side, with 2.5 nM of EGF on the serosal side or with 0.25 or 2.5 nM EGF on the mucosal side. No treatment affected tissue conductance. Short‐circuit current (I_sc) was affected by time and treatment and their interactions. Only 250 nM of either GLP‐1 or GLP‐2 decreased I_sc in certain periods compared with 25 nM GLP‐1 or 0.25 nM mucosally applied EGF; however, not when compared to control epithelia. Fluorescein flux rates (J_fluor) of ruminal epithelia were affected by treatment, time and time × treatment interaction. The time × treatment interaction was based on an increase in J_fluor between the first and last hour in epithelia incubated with 25 nM GLP‐1 or GLP‐2 and in epithelia incubated with EGF. After 7 hr incubation, claudin‐7 mRNA expression was downregulated in all treatments. Claudin‐1 mRNA was upregulated after incubation with 2.5 nM EGF on the serosal side, claudin‐4 mRNA was downregulated by 2.5 nM EGF on the mucosal side, and occludin mRNA was increased after incubation with 250 nM GLP‐2. The protein abundance of all tested TJ proteins was not influenced by treatment. We conclude that GLP‐1, GLP‐2, and EGF have no obvious acute effects on the EB of ruminal epithelia under simulated physiological conditions ex vivo. However, by decreasing the mRNA expression of claudin‐7 and partly affecting other TJ proteins, they may modulate EB in the longer term or under certain conditions.     

Sequestering Ability of Phytate toward Biologically and Environmentally Relevant Trivalent Metal Cations

Quantitative parameters for the interactions between phytate (Phy) and Al(3+), Fe(3+), and Cr(3+) were determined potentiometrically in NaNO(3) aqueous solutions at I = 0.10 mol L(-1) and T = 298.15 K. Different complex species were found in a wide pH range. The various species are partially protonated, depending on the pH in which they are formed, and are indicated with the general formula MH(q)Phy (with 0 ≤ q ≤ 6). In all cases, the stability of the FeH(q)Phy species is several log K units higher than that of the analogous AlH(q)Phy and CrH(q)Phy species. For example, for the MH(2)Phy species, the stability trend is log K(2) = 15.81, 20.61, and 16.70 for Al(3+), Fe(3+), and Cr(3+), respectively. The sequestering ability of phytate toward the considered metal cations was evaluated by calculating the pL(0.5) values (i.e., the total ligand concentration necessary to bind 50% of the cation present in trace in solution) at different pH values. In general, phytate results in a quite good sequestering agent toward all three cations in the whole investigated pH range, but the order of pL(0.5) depends on it. For example, at pH 5.0 it is pL(0.5) = 5.33, 5.44, and 5.75 for Fe(3+), Cr(3+), and Al(3+), respectively (Fe(3+) < Cr(3+) < Al(3+)); at pH 7.4 it is pL(0.5) = 9.94, 9.23, and 8.71 (Al(3+) < Cr(3+) < Fe(3+)), whereas at pH 9.0 it is pL(0.5) = 10.42, 10.87, and 8.34 (Al(3+) < Fe(3+) < Cr(3+)). All of the pL(0.5) values, and therefore the sequestering ability, regularly increase with increasing pH, and the dependence of pL(0.5) on pH was modeled using some empirical equations.     

Lucerne management in an organic farming system under dry site conditions

Biological nitrogen fixation (BNF) as a result of the legumes–rhizobia symbioses is the main source of nitrogen in organic farming systems. Lucerne (Medicago sativa L.), used as green manure or as forage legume, is important on arable farms under dry site conditions. In a field experiment on organically managed agricultural fields, we examined the impacts of the utilisation system (harvested = forage production versus mulched = green manure) and the crop composition (pure lucerne crops versus lucerne–grass mixtures) on yield, biological nitrogen fixation (BNF), soil inorganic N content, N balance and water consumption of autumn-cultivated lucerne crops. The study was conducted at the University of Natural Resources and Applied Life Sciences, Vienna, in eastern Austria—a region characterized by pannonian site conditions (9.8 °C mean annual temperature, 545 mm average total precipitation) and stockless farming systems. Our results indicate that the utilisation system and the crop composition had no marked influence on above- and below-ground dry matter (DM) and N yield, soil inorganic N contents, BNF, or water use efficiency of lucerne. The level of symbiotically fixed N₂ in harvested lucerne was 89–125 kg N ha⁻¹ (27–33% Ndfa = nitrogen derived from atmosphere) in the first year and 161–175 kg N ha⁻¹ (47–49% Ndfa) in the second year of the study. The high soil inorganic N supply in the first year increased the N uptake from soil by lucerne and led to a reduced BNF. Under the dry and unfavourable conditions in both study years, the nitrogen release from the legume mulch was retarded and BNF in mulched lucerne was not reduced. Assuming low gaseous N losses by mulching (15–30 kg N ha⁻¹), the green manure system reached a positive N balance (+137 to +186 kg N ha⁻¹) for the subsequent crops because abundant residues remained on the field.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.