Genetic variability of PRRS virus in Austria: consequences for molecular diagnostics and viral quantification

Porcine reproductive and respiratory syndrome virus is a virus with a relatively high level of sequence variation, especially between the European and the American-type strains. This high degree of sequence variations will influence the accuracy of results obtained by molecular diagnostic methods, especially if mutations are located in the primer or probe binding regions of PCR or real-time PCR based assays. In order to provide the basis for the development of an accurate, region-tailored assay we have analysed samples obtained from several Austrian pig herds using a series of RT-PCR assays. Subsequent sequencing of the PCR products from ORF5 and phylogenetic analysis revealed the occurrence of both the European and the American-type of porcine reproductive and respiratory syndrome virus in Austria. Since vaccination with the American-type vaccine is not authorized in Austria the import of vaccinated animals is the most likely explanation for the occurrence of these strains. The study highlights the importance of a detailed study on the sequence variations occurring in the region of interest before the development of a reliable PCR-based assay.     

Proposals of the working group "Antibiotic resistance" for the configuration of microtitre plates to be used in routine antimicrobial susceptibility testing of bacterial pathogens from infections of large food-producing animals and mastitis cases

Two layouts for microtitre plates, which should serve for in-vitro susceptibility testing in routine diagnostics, have been set up by the working group "Antibiotic resistance" of the German Society for Veterinary Medicine. One of these layouts was designed for the testing of bacteria from cases of mastitis and the other for bacteria from infections in large food-producing animals. The choice of the antimicrobial agents and their concentrations to be included in these layouts were based on (1) the bacteria frequently associated with the respective diseases/animals, (2) the antimicrobial agents licensed for therapeutic use in these diseases/animals, (3) the currently available breakpoints, and (4) cross-resistances between the antimicrobial agerts so far known to occur in the respective bacteria.     

Expression and functional characterization of killer whale (Orcinus orca) interleukin-6 (IL-6) and development of a competitive immunoassay

Interleukin-6 (IL-6) is a cytokine that can reach detectable systemic levels and is a major inducer of the acute phase response. As such, clinical assays to identify this cytokine in mammalian sera are of diagnostic value. A 558 base-pair (bp) fragment of killer whale IL-6 was cloned and expressed as a 21 kDa protein in Escherichia coli. Biological activity of the recombinant killer whale IL-6 (rkwIL-6) was demonstrated using the IL-6-dependent B9 mouse hybridoma cell line; acute phase sera from a killer whale and supernatants from lipopolysaccharide (LPS)-stimulated killer whale peripheral blood mononuclear cells (PBMCs) also supported the proliferation of the B9 hybridoma. Rat anti-mouse IL-6 receptor antibody effectively blocked biological activity of all three sources of IL-6. Polyclonal antisera, specific for the recombinant protein, were obtained by successive immunization of a rabbit with rkwIL-6. The polyclonal antibody was capable of neutralizing the biological activity of both recombinant and native kwIL-6. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal rabbit anti-rkwIL-6 and the recombinant protein; sensitivity of the assay was in the range of 1 ng/ml. The ELISA was subsequently used to identify the presence of native IL-6 in acute phase sera of two species of delphinidae, a killer whale and a bottlenose dolphin. The application of quantitative cytokine assays as diagnostic tools for monitoring cetacean health are becoming feasible as many animals are now being trained for fluke presentation, making blood collection a routine procedure.     

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.     

Detection of novel chlamydiae in cats with ocular disease

OBJECTIVE: To detect and characterize the full range of chlamydial infections in cats with ocular disease by use of polymerase chain reaction (PCR) assays, cytologic examination, immunohistochemical analysis, and evaluation of clinical information including status for feline herpesvirus-1 (FeHV-1). SAMPLE POPULATION: DNA extracted from 226 conjunctival samples obtained from cats with clinically diagnosed keratitis or conjunctivitis and 30 conjunctival samples from healthy cats. PROCEDURE: PCR assays for the 16S rRNA gene specific for the order Chlamydiales and a new Chlamydophila felis (formerly Chlamydia psittaci) species-specific 23S rRNA gene were performed. Seventy-four conjunctival samples were prepared with Romanowsky-type stain, grouped on the basis of inflammatory pattern, and screened for chlamydial inclusions by use of immunohistochemical analysis. Clinical information and FeHV-1 status were recorded. RESULTS: 26 (12%) specimens had positive results for the only known feline chlamydial pathogen, C felis. Surprisingly, an additional 88 (39%) were positive for non-C felis chlamydial DNA. Identification of non-C felis chlamydial DNA by direct sequencing revealed 16S rRNA gene sequences that were 99% homologous to the sequence for Neochlamydia hartmannellae, an amebic endosymbiont. Chlamydial prevalence was significantly higher in cats with ocular disease. CONCLUSIONS AND CLINICAL RELEVANCE: Application of a broad-range detection method resulted in identification of a new agent associated with ocular disease in cats. Finding chlamydia-like agents such as N hartmannellae in coinfections with their obligate amebic host, Hartmannella vermiformis, raises questions about the potential role of these microorganisms in causation or exacerbation of ocular disease in cats.     

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.     

Mineralization of [14C] glyphosate and its plant-associated residues in arable soils originating from different farming systems

The biomineralization of [¹⁴C]glyphosate, both in the free state and as ¹⁴C-residues associated with soybean cell-wall material, was studied in soil samples from four different agricultural farming systems. After 26 days, [¹⁴C]carbon dioxide production from free glyphosate accounted for 34–51% of the applied radiocarbon, and 45–55% was recovered from plant-associated residues. For three soils, the cumulative [¹⁴C]carbon dioxide production from free glyphosate was positively correlated with soil microbial biomass, determined by substrate-induced heat output measurement and by total adenylate content. The fourth soil, originating from a former hop plantation, and containing high concentrations of copper from long-term fungicide applications, did not fit this correlation but showed a significantly higher [¹⁴C]carbon dioxide production per unit of microbial biomass. Although the cumulative [¹⁴C]carbon dioxide production from plant-associated ¹⁴C-residues after 26 days was as high as from the free compound, it was not correlated with the soil microbial biomass. This indicates that the biodegradation of plant-associated herbicide residues, in contrast to that of the free compound, involves different degradation processes. These encompass either additional steps to degrade the plant matrix, presumably performed by different soil organisms, or fewer degradation steps since the plant-associated herbicide residues are likely to consist mainly of easily degradable metabolites. Moreover, the bioavailability of plant-associated pesticide residues seems to be dominated by the type and strength of their fixation in the plant matrix. ©1997 SCI     

Water disinfection by ozonation has advantages over UV irradiation in a brackish water recirculation aquaculture system for Pacific white shrimp (Litopenaeus vannamei)

By keeping tropical shrimp, like Litopenaeus vannamei, in recirculating aquaculture systems (RAS), valuable food for human consumption can be produced sustainable. L. vannamei tolerates low salinities, and therefore, the systems can operate under brackish water conditions. The stabilization of the microbial community in RAS might be difficult under high organic loads, and therefore, water treatment measures like UV irradiation or ozone application are commonly used for bacterial reduction. To investigate the impact of these measures, the effects of UV irradiation and ozone application were studied in small-scale brackish water RAS with a salinity of 15‰ stocked with L. vannamei. UV reactors with 7 and 9 W were used, and by ozonizers with a power of 5–50 mg/hr, the redox potential in the water was adjusted to 350 mV. Ozone had a stabilizing effect on the microbial composition in the water and on biofilms of tank surfaces and shrimp carapaces, prevented an increase of nitrite and accelerated the degradation of nitrate in the water. UV irradiation led to changes in the microbial composition and was less effective in optimizing the chemical water quality. Thus, the use of ozone could be recommended for water treatment in brackish water RAS for shrimp.