First fertilizing-value typology of digestates: A decision-making tool for regulation.

Defined as the residue from anaerobic digestion (AD), digestate refers to a set of materials with varied biochemical compositions. The objective of this study was to establish a digestate typology according to its fertilizing-value with data from literature and internal unpublished databases. To establish a relatively big database allowing the application of advanced statistics, usual fertilizing-value parameters were used: dry matter, volatile solids, C/N, C/Organic-N, total N (TN), total ammoniacal nitrogen (TAN), TAN/TN, total P and total K. Statistical analysis was performed on a dataset of 91 raw digestates, 34 solid fractions and 25 liquid fractions after separation. The resulting typology outlined that fertilizing-values are linked to AD feedstock and process. As case study regulations, no digestate (without any post-treatment) fulfilled French standards and the latest European Union regulation proposal on fertilizers. Options to reach regulations' product categories were discussed according to the typology. For the first time, a digestate typology was established based on fertilizing value, which can be a useful tool enhancing digestate management and policy making.

A respirometric method for characterising the organic composition and biodegradation kinetics and the temperature influence on the biodegradation kinetics, for a mixture of sludge and bulking agent to be co-composted.

A respirometric method was set up to study kinetics of biological reactions involved in the treatment of organic wastes-sludge mixed with pine barks--by composting. Oxygen consumption rates of this type of mixture were monitored during 10-20 days, using a 10 l respirometric cell kept at constant temperature and moisture. Oxygen consumption kinetics were modelled and organic matter composition was characterised as biomass, easily-biodegradable, slowly-biodegradable and non-biodegradable organic matter. The influence of temperature on kinetics was tested. Results show that this respirometric method is a useful tool for the characterisation of solid organic matter biodegradability and for the modelling of the biological kinetics of the composting process.

Influence of the airflow rate on heat and mass transfers during sewage sludge and bulking agent composting.

The aim of this study was to characterize the gas flow during the composting, at a pilot scale, of a mixture of sludge and bulking agent, in order to model heat and mass transfers involved in the process. Thus, a closed 300-litre cylindrical pilot was fed with a mixture of wastewater treatment sludge and pine bark. Aeration was supplied from the bottom via an air blower and gases were collected at the top. Three experiments were led with constant gas flow rates and one with varying aeration rate. Temperatures within the pilot reactor were monitored all along the trials and their evolutions were discussed in term of heat transfers and parameters influencing the heat balance. Concurrently, Retention Time Distribution curves were obtained by injecting a pulse of methane in the entering airflow and by analysing the methane concentration in the exhaust gas, every two or three days during composting. The gas flow, within the composting medium, was characterized by a dispersion model, which is a deviation of the plug flow model. The dispersive effect of the flow was correlated to the evolution of the experimental temperature, and a convective dispersion model was used to describe the heat and mass transfers through the gas flow. These equations will be, in future work, coupled with heat production and mass degradation terms in order to model the global mass and heat balances of this composting process. Finally, axial dispersion coefficients of gases were determined and correlated with the airflow rate.

[Use of nonradioactive probes to detect the gene for 3-aminoside-acetyltransferase type IV in strains of Escherichia coli and Salmonella spp]. / Utilisation de sondes non radioactives pour la détection du gène de la 3-aminoside-acétyltransférase type IV chez des souches de Escherichia coli et Salmonella spp.

Forty-three isolates of Escherichia coli, Salmonella dublin and S. thyphimurium of animal origin were studied by colony hybridization using an intragenic probe specific for the gene encoding 3-aminoglycoside acetyltransferase type IV, which confers resistance to apramycin and gentamicin. Non-radioactive biotinylated and sulphonated DNA probes were used. No false-positive results were found and the positive results obtained were in agreement with those previously revealed with radioactive probes.

Survey of antimicrobial resistance in bacterial isolates from diseased cattle in France.

Since 1982, a national veterinary network has been involved in the monitoring of resistance to antimicrobial agents in the main pathogenic bacteria isolated from diseased cattle in France. It is based on 40 regional veterinary diagnostic laboratories and managed by a central reference laboratory (CNEVA Lyon). Highly standardized methods are used in the diagnostic laboratories. This network collects up-to-date information on antimicrobial resistance in veterinary isolates and gathers strains for specific studies on fastidious bacteria and for the analysis of mechanisms of resistance to antibiotics. Such a permanent survey is essential to establish a rational veterinary antibiotic policy. It could be connected to other compatible systems developed in other fields such as human medicine, food, and environment, to evaluate the importance of resistance and R-factors spread for public health. The limits and perspectives of this surveillance system are discussed.

A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella typhimurium DT104.

A new chloramphenicol resistance gene from Salmonella typhimurium DT104, designated floR, also conferring resistance to florfenicol, was characterized. Sequence analysis of the deduced FloR protein suggested that it belongs to the 12-TMS (transmembrane segments) multidrug efflux pumps family. The floR gene, and the downstream sequenced tetR and tetA tetracycline resistance genes, were surrounded by two class 1 integrons. The first one contained the resistance gene aadA2 and a deleted sulI resistance gene. The second one contained the beta-lactamase gene pse1 and a complete sulI gene. Thus, the floR gene is included in a multiresistance locus of at least 12.5 kb. Its particular organization and chromosomal location could be involved in the antibioresistance pattern stability of the DT104 Salmonella typhimurium strains.

Monitoring of antibiotic resistance in bacteria of animal origin: epidemiological and microbiological methodologies.

The occurrence of antibiotic-resistant bacteria in food animals is a major public health threat. Information on the prevalence of resistance to specific drugs in both bacterial and animal species together with changes occurring over time, are necessary to understand the magnitude of the problem and to establish baselines for taking action. The aim of this paper is to define the minimum epidemiological and microbiological requirements for establishing a surveillance of antimicrobial resistance in bacteria of animal origin. Surveillance should involve different bacterial species, veterinary pathogens, zoonotic bacteria and commensal bacteria used as indicators. The collected data should be periodically updated and the reports distributed among practising veterinarians and regulatory authorities. These reports would be a useful tool for developing guidelines for the prudent use of antimicrobial agents in veterinary medicine and for action strategies.

The French antibiotic resistance monitoring programs.

Surveillance of antimicrobial resistance in bacteria from animal origin in France is organised by the French Agency for Food Safety (Agence Française de Sécurité Sanitaire des Aliments, AFSSA) through two types of networks. The first collects non-human zoonotic Salmonella strains in one centre (AFSSA, Paris) where they are tested for their antimicrobial susceptibility. The others, managed by AFSSA Lyon, deal with bovine pathogenic strains and are multicentric, that is they collecting antibiotic sensitivity and other data from the local public veterinary diagnostic laboratories. This requires standardisation of the methods used in each partner laboratory. Statistical analysis of any change in French resistance patterns can be monitored by these three networks either as a function of strain pathogenicity and/or of the ecological origin of the isolate. The system also encourages efficient collaboration between veterinarians and the laboratory. Such collaboration improves both the quality of routine antibiotic testing and understanding of the molecular mechanisms underlying resistance.

A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets.

This study, Vetoquinol S.A. epidemiosurveillance, was conducted from 1994 to 2001 in order to determine the susceptibility (by MIC determination) to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains from infected pets originated from six European countries. Isolates were collected from urinary infections (Escherichia coli), respiratory infections (Pasteurella multocida), dermatological infections (Staphylococcus intermedius, Pseudomonas aeruginosa) and otitis (S. intermedius, P. aeruginosa). The MIC distribution for each species was the same both before and after the launch of marbofloxacin in 1995. In E. coli, a resistant population was present before the use of marbofloxacin; this resistance was induced by co- or cross-resistance to other antibiotics used previously. Over this period, there was no significant evolution of MIC(90) for any bacterial species studied and no development of resistance was observed. Marbofloxacin was the most active antibiotic against P. multocida isolates and had the lowest MIC. No difference in MIC distribution was seen between the S. intermedius (unimodal distribution) isolated from dermatological infections and those from otitis. This was also true for P. aeruginosa. The use of marbofloxacin was not found to have induced a significant increase or spread of resistant bacteria.

Seven years survey of susceptibility to marbofloxacin of bovine pathogenic strains from eight European countries.

This study was conducted from 1994 to 2001 to determine the susceptibility of bovine pathogenic bacteria to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains originated in bovine diseases from eight European countries. They were isolated from gut infections (Escherichia coli, salmonellae), mastitis (E. coli, Staphylococcus aureus, Streptococcus uberis, S. agalactiae, S. dysgalactiae) and respiratory diseases (Mannheimia haemolytica, Pasteurella multocida, Haemophilus somnus). There was no change in the MIC distributions for each species after the launch of marbofloxacin in 1997. In E. coli, a resistant population was present before the use of marbofloxacin having been induced by co- or cross-resistance to other antibiotics used previously. Over this period the only a significant change seen was an increase in MIC(90) of E. coli from the gut (1.275 microg/ml in 1994/1995 to 5.098 microg/ml in 2001). All the salmonellae were susceptible to marbofloxacin with a MIC(90) = 0.073 microg/ml in 2001 without development of high level resistance. The use of marbofloxacin seems not to have favoured a significant increase and spreading of resistant bacteria.

Bacterial resistance monitoring in animals: the French national experiences of surveillance schemes.

In France, bacterial resistance monitoring in animals is based on a national network of local veterinary laboratories organised by CNEVA following two methodologies. As part of the epidemiological surveillance of Salmonella, LCHA in Paris has been receiving since 1969 most of the strains isolated from animals (sick and healthy carriers) but also from food, feed and environment. For many years sensitivity tests have been carried out on strains collected: animal ones, mainly from cattle and poultry, show multiple antibiotic resistance more frequently than those of food or environment. Taking into account of the importance of antibiotic resistance in E. coli and Salmonella isolated from sick calves, LPB in Lyon has set up since 1982 a national system for collecting and analysing antimicrobial sensitivity patterns of main bovine pathogens determined routinely by local veterinary laboratories. In order to ensure comparability and coherency of data from different laboratories it was necessary to introduce standard techniques and reference reagents. The major bacterial species being tested concern enteric, respiratory and mammary flora. This network is useful to detect new resistances. The resistant strains collected are useful to carry out studies on the mechanism of resistance and the efficacy of new molecules. Results constitute the basis of a real predictive epidemiology necessary to lay down a policy for rational use of antimicrobials in animal breeding and veterinary medicine.

Application of dot immunobinding on membrane filtration (MF dot) to the study of relationships within "M. mycoides cluster" and within "glucose and arginine-negative cluster" of ruminant mycoplasmas.

A total of 189 isolates from cattle, sheep and goats, allocated to two groups on biochemical grounds, have been examined by a dot immunobinding membrane-filtration (MF dot) method. Seventy glucose fermenting isolates, showing relationships with the "Mycoplasma mycoides cluster", have been compared by MF dot against polyclonal hyperimmunesera prepared against the following reference strains: M. mycoides subsp. mycoides, small colony type (SC), strain PG1 and large colony type (LC) strain Y Goat; M. capricolum strain California Kid (CK); M. species bovine serogroup 7 strain PG50, and, ovine/caprine serogroup 11 strain 2-D. The isolates fell into 5 main groups: (a) 14 serologically homogeneous isolates similar to subsp. mycoides SC PG1 (b) 4 homogeneous isolates similar to PG50 (c) 14 isolates all serologically similar to Y Goat, but heterogeneously reactive with subsp. capri PG3 and M. capricolum CK antisera (d) 7 isolates serologically similar to subsp. capri PG3, but heterogeneously reactive with subsp. mycoides SC PG1, M. capricolum CK and 2-D antisera (e) 28 isolates strongly reactive with both M. capricolum CK and serogroup 7 PG50 antisera. 119 isolates that were all glucose and arginine negative were also compared by the MF dot method with the reference strains. Most of these could be classified definitely as M. bovis (78 isolates), M. agalactiae (21 isolates) and serogroup 11 (5 isolates). 13 isolates gave a strong reaction with both M. bovigenitalium and serogroup p11 antisera. 2 isolates showed an unclassifiable pattern. The results confirm that the glucose and arginine-negative cluster strains that reacted with 2-D antiserum, also share serological relationships with the "M. mycoides cluster", albeit with a very heterogeneous pattern.

Resistance to trimethoprim and 2,4-diamino-6,7-diisopropyl-pteridine (0/129) in Pasteurella haemolytica.

Thirteen strains of Pasteurella haemolytica resistant to moderate levels of trimethoprim (MICs from 8 to 64 micrograms/ml) and 0/129 (MICs from 16 to 64 micrograms/ml) were isolated from bovine specimens. Two strains, CNP330 and CNP334, were studied and found to harbour various plasmids but all attempts to cure trimethoprim resistance were unsuccessful. Resistance characters were not transferable to Escherichia coli or to Pasteurella multocida by conjugation and to E. coli by transformation. The resistance gene(s) was therefore tentatively assigned to a chromosomal location and cloned into E. coli where it conferred trimethoprim resistance. Trans-complementation analysis of a dihydrofolate reductase-deficient mutant of E. coli showed that trimethoprim resistance was secondary to synthesis of a dihydrofolate reductase. DNA/DNA hybridization of the hybrid plasmid and of strains CNP330 and CNP334 with probes specific for dihydrofolate reductase types I to V were negative, indicating that cross-resistance to trimethoprim and 0/129 in P. haemolytica was due to the acquisition by P. haemolytica of a new resistance determinant.

Probable transmission between animals of a plasmid encoding aminoglycoside 3-N-acetyltransferase IV and dihydrofolate reductase I.

Ten aminoglycoside-resistant Escherichia coli isolated from the faeces of healthy or diarrhoeic animals reared in the same herd were studied. These strains were resistant to high levels of apramycin and low levels of gentamicin. They were also resistant to streptomycin, tetracycline, trimethoprim and some to ampicillin, chloramphenicol, kanamycin or nalidixic acid. Two strains, isolated from a calf and a lamb, respectively, belonged to the same biotype. All the transconjugants resistant to gentamicin-apramycin were also resistant to streptomycin, tetracycline and trimethoprim. In all cases, these resistances were encoded by plasmids of 100 kb. Analysis of these plasmids by agarose gel electrophoresis after digestion by EcoRI or BamHI revealed their similarity. Hybridization with a 500-bp HpaI insert of plasmid pFE872 was observed with DNA from field strains and their transconjugants, demonstrating the presence on the 100-kb plasmids of the gene coding for a dihydrofolate reductase I. A single plasmid, designated pIP1831, could be observed in identical or different strains isolated from calves or lambs, suggesting the transmission of strains and plasmids between animals of different species.

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals.

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.

[Laboratory diagnosis of respiratory diseases of cattle]. / Diagnostic de laboratoire des affections respiratoires des bovins.

The authors describe the procedure of laboratory diagnosis for bovine respiratory diseases: direct diagnosis by isolation and for identification of bacteria or viruses and indirect diagnosis by serological methods. They specify the restraints and limits of this diagnosis and the significance results which are obtained and connected with knowledge of anamnestic information.

Manifestation and epidemiology of contagious bovine pleuropneumonia in Europe.

The authors describe the clinical profile and epidemiology of contagious bovine pleuropneumonia in Europe. This disease, once considered to have been eradicated several years ago, has now become endemic in southern countries of Europe. The status of contagious bovine pleuropneumonia in Portugal and Italy, and the evolution of the disease during the last ten years, are analysed in detail, in addition to the measures undertaken for control and eradication. The authors also refer to hosts and possible reservoirs of the infection.

Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies.

Seventeen monoclonal antibodies (MAbs) raised against Mycoplasma mycoides ssp. mycoides small colony type (Mmm SC), the causative agent of contagious bovine pleuropneumonia (CBPP), were partially characterised. Six MAbs recognising a main protein of 70 kDa and showing reciprocal competition were found to be specific for Mmm SC, while the other MAbs showed different patterns of reactivity with Mycoplasma spp. within the mycoides "cluster". Sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of MAbs enabled the detection of Mycoplasma of the mycoides cluster in pathology samples and the specific identification of Mmm SC from the initial culture passages, overcoming the necessity for difficult and time-consuming biochemical and growth inhibition assays. A competitive ELISA based on Mmm SC-specific MAbs was able to measure anti-CBPP antibodies in cattle sera, and detected only the set of antibodies directed at Mmm SC-specific epitopes, thus avoiding the false-positive reactions occasionally observed with the complement fixation test.

[Evolution of antimicrobial susceptibility in salmonellas from bovine origin in France.]. / Evolution de la sensibilité aux antibiotiques des salmonelles d'origine bovine en France.

Animals are a wide source of salmonellas for humans and their environment. Domestic ruminants play an important role because of their high susceptibility to salmonellas infection giving an acute disease with massive salmonella excretion and mortality if lack of treatment. As in human medicine use of antibiotics at therapeutic doses in animals can lead to the selection of resistant strains may be pathogenic for humans by direct contact or through environment and food. Taking into account the importance of salmonellosis on calves in rearing units, CNEVA Lyon has set up since 1982 a national network for antimicrobial resistance monitoring of main bacterial pathogens in bovine. This network allowed to detect a recent evolution of antimicrobial susceptibility in salmonella from dairy cows related with new problem of salmonellosis in cattle.