A qualitative risk assessment methodology for scientific expert panels.

Risk assessment can be either quantitative, i.e. providing a numeric estimate of the probability of risk and the magnitude of the consequences, or qualitative, using a descriptive approach. The French Agency for Food, Environmental and Occupational Health and Safety (ANSES), formerly the French Food Safety Agency (AFSSA), bases its assessments on the opinions of scientific panels, such as the ANSES Animal Health Scientific Panel (AH-SP). Owing to the lack of relevant data and the very short period of time usually allowed to assess animal health risks on particular topics, this panel has been using a qualitative risk method for evaluating animal health risks or crises for the past few years. Some experts have drawn attention to the limitations of this method, such as the need to extend the range of adjectives used for the lower probabilities and to develop a way to assess consequences. The aim of this paper is to describe the improved method now established by the AH-SP, taking into account the limitations of the first version. The authors describe a new set of levels for probabilities, as well as the items considered when addressing either animal or human health consequences.

Synergistic interactions between cefalexin and kanamycin in Mueller-Hinton broth medium and in milk.

AIMS: This study investigated the in vitro bactericidal activity of an intramammary drug product by comparing the kill kinetics of cefalexin and kanamycin, alone and in fixed ratio combination, against Streptococcus uberis, Staphylococcus aureus and Escherichia coli strains isolated from field cases of bovine mastitis. The effect of milk as a diluent on the rate of bacterial killing was also assessed. METHODS AND RESULTS: Antibacterial kill kinetics was determined against each bacterial strain in Mueller-Hinton broth (MHB) and in milk. In MHB, the fixed cefalexin : kanamycin combination (1.5 : 1 w/w) exhibited a clear synergistic bactericidal activity against the strains tested. The combination also showed an enhanced killing activity in milk, as compared to either agent alone. CONCLUSIONS: The data show the occurrence of synergistic interactions between cefalexin and kanamycin, resulting in a faster and enhanced bactericidal activity against major mastitis pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrated that the combination exhibited a larger and faster rate of kill of S. aureus, S. uberis and E. coli compared to either cefalexin or kanamycin alone, while using a lower total amount of antibiotic. Synergistic and additive effects were also observed when milk was used as a medium. The results support the use of this combination of narrow spectrum antibiotics to treat clinical mastitis via the intramammary route and provide data on its killing kinetics.

Antimicrobial resistance survey in a river receiving effluents from freshwater fish farms.

AIMS: The aims of this study were: (i) to determine the proportions of Aeromonas spp. resistant to florfenicol (FC), oxolinic acid (OA) and oxytetracycline (OTC) along a river receiving effluents from fish farms, and (ii) to assess the relevance of using this bacterial group as an indicator for studying the consequences of the use and release of these aquacultural antimicrobials in the freshwater environment, as compared with performing antimicrobial measurements in sediments. METHODS AND RESULTS: Sediment interstitial waters sampled along a river during two distinct climatic seasons were plated on an Aeromonas-selective medium supplemented or not with OA, OTC or FC. The October 2004 campaign showed an enrichment of OA- and OTC-resistant Aeromonas immediately downstream of the fish farms and a wastewater treatment plant. Two fish farms showed similar results in March 2005. In contrast, only 10 FC-resistant Aeromonas strains could be isolated, which revealed that minimum inhibitory concentrations of FC were greater than 64 microg ml(-1) and multiple antimicrobial resistances. Contamination of sediments by antimicrobials was detected but was not always co-localized with resistance peaks or known point sources of contamination. CONCLUSIONS: Aeromonas could be valuable indicators of OA, OTC and FC resistance in the freshwater environment. Fish farms contribute to the contamination of the river by antimicrobials and resistant bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the still very low proportion of FC-resistant Aeromonas, this study can be considered as a reference for further studies about this recently introduced veterinary antimicrobial agent.

Antimicrobial drug susceptibility of Staphylococcus intermedius clinical isolates from canine pyoderma.

A total of 50 Staphylococcus intermedius strains isolated in France from canine pyodermas in 2002 were investigated for their susceptibility to various antimicrobial drugs. Antimicrobial susceptibility was assessed using a 2-fold serial dilution method in Mueller-Hinton agar, and the minimal inhibitory concentrations (MICs) were determined. About 62% of the 50 strains tested were producers of beta-lactamase and categorized as penicillin-resistant. About 26% demonstrated resistance to sulphonamides, 46% to oxytetracycline, 30% to chloramphenicol, 28% to streptomycin, kanamycin, neomycin or erythromycin, 22% to clindamycin, 6% to doxycycline, 2% to gentamicin, enrofloxacin, marbofloxacin or pradofloxacin. Acquired resistance was not observed to a clavulanic acid-amoxicillin combination, oxacillin, cephalosporins (cephalexin, ceftiofur and cefquinome), trimethoprim, a sulphamethoxazole-trimethoprim combination and florfenicol. About 42% were simultaneously resistant to three or more antimicrobial classes (multiresistance). All isolates with acquired resistance to erythromycin were also resistant to streptomycin and neomycin/kanamycin. About 22% of isolates exhibited cross-resistance between erythromycin and clindamycin and all clindamycin-resistant isolates also exhibited resistance to erythromycin. Resistance to penicillin, oxytetracycline and chloramphenicol was also positively associated with resistance to erythromycin and streptomycin.

Role of the coypu (Myocastor coypus) in the epidemiology of leptospirosis in domestic animals and humans in France.

The coypu (Myocastor coypus), a rodent whose natural habitat is stagnant freshwater, has become a widespread pest in France within the last decade. This study investigated the prevalence of seropositivity and the renal carriage of leptospires in coypus in order to evaluate their role in terms of the risk of infection by Leptospira interrogans in domestic animals and humans. The study involved the application of serological and bacteriological methods to identify leptospires infection and/or carriage in 738 coypus trapped from 1996 to 1999 in six areas of France. Seroprevalence in samples ranged from 16.5 to 66%, and three field strains were isolated (two L. interrogans Icterohaemorrhagiae and one L. interrogans Sejroe). This first report on the isolation of leptospires from coypus in France emphasises the role of this animal in the epidemiology of leptospirosis.

Antimicrobial activity of enrofloxacin against Staphylococcus intermedius strains isolated from canine pyodermas.

This study examined and compared the minimal inhibition concentrations (MICs) of enrofloxacin against 393 Staphylococcus intermedius strains isolated in France from canine pyodermas during three different years, 1995 (174 isolates), 1997 (101 isolates) and 1999 (118 isolates). The MICs of enrofloxacin against these strains ranged from 0.063 to 64 mg L-1, with MIC50 and MIC90 equal to 0.125 and 0.25 mg L-1, respectively. Two resistant strains were found, but only among isolates collected in 1999. The data show that resistance to enrofloxacin among S. intermedius strains is still rare in dogs, but the selection in vitro of variants in which the MICs were increased 4-16-fold after 10 serial passages in subinhibitory concentrations of enrofloxacin suggests that inappropriate use might favour the development of resistant strains in vivo.

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family.

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.

Evidence of cross-protection within Leptospira interrogans in an experimental model.

Killed whole-cell preparations were used as bacterins against leptospirosis. As this type of protection is considered to be serogroup-specific, several serogroups were added to the usual vaccines, and the most pathogenic serovar was chosen for each group. Different leptospire extracts were evaluated for their protective capacity against acute lethal leptospirosis in gerbils (Meriones unguiculatus). Total extracts induced complete protection against homologous challenges and partial protection against heterologous challenges. LPS fractions protected against homologous but not heterologous challenges, whereas protein extract induced significant protection against both types of challenge. Thus, cross-protection within L. interrogans was related to the protein extract.

Post-antibiotic effect of cephalexin against isolates of Staphylococcus intermedius obtained from cases of canine pyoderma.

The post-antibiotic effect in vitro (PAE) of cephalexin was determined according to a broth dilution method against 5 isolates of Staphylococcus intermedius obtained from cases of canine pyoderma. Two durations of exposure and 3 concentrations were tested. The PAE increased when time of exposure or concentration increased. The mean PAE ranged from 0.7 to 3.3 h. The PAE of cephalexin against Staph-ylococcus intermedius may be clinically relevant when selecting a dosage regimen to treat pyoderma in dogs.

Virus load and neuropathology in the FIV model.

The FIV (feline immunodeficiency virus) induces in cats brain changes presenting similarities with those observed in human immunodeficiency virus infection. This FIV model was used to study the relationship between viral load in brain, in lymphoid organs and central nervous system (CNS) changes during the early and late stages of infection. Early brain changes were analyzed in animals experimentally infected with two different FIV isolates and sacrificed at 7 and 15 days, 1, 2, 6, and 12 months post inoculation (p.i.). Late CNS abnormalities were analyzed in naturally FIV-infected cats referred to the Veterinary School of Nantes. For each animal, one cerebral hemisphere was fixed and examined using routine techniques. The characterization of FIV replicating cells by in situ hybridization was performed on the other half frozen hemisphere on sections performed in the anterior and the median regions of the brain. During the early stages of infection, moderate gliosis with glial nodules and sometimes white matter pallor and meningitis were associated with few infected cells scattered in the brain. Infection was an early event as infected cells could be detected in brain at 7 p.i. For each cat, these findings were found identical in the two analyzed areas. During the late stages, brain lesions and the number of virus replicating cells increased especially in animals with perivascular infiltrates. The multinucleated giant cells encephalitis was never observed and the number of FIV replicating cells scattered in the whole brain was always low. This discrepancy between the number of replicating cells and the brain lesions, corroborates the hypotheses suggesting that brain injuries may be mediated via diffusive factors and amplification processes through cytokine cascades and cell activations.

Early stages of rabbit haemorrhagic disease virus infection monitored by polymerase chain reaction.

In order to define more accurately the initial events that take place during rabbit haemorrhagic disease virus (RHDV) infection, different organs of experimentally infected rabbits were analysed for the presence of the virus and correlated with histopathological observations. A total of 24 rabbits were intranasally inoculated with a viral suspension, and tissue samples were taken from the liver, spleen, kidney, lung, thymus, lymph node and tonsil at different intervals post-inoculation (2, 4, 6, 12, 18, 24, 30, 36, 48, 50, 51, 70 and 72 h). Histopathological observations revealed the presence of the first significant lesions at 30 h post-inoculation (p.i.) in the liver. Using an ELISA and a haemagglutination test (HAT), the virus was detected in the liver at 36 h p.i. The reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the RHDV RNA was present as early as 18 h p.i. in the liver and spleen, whereas thymus, kidney, tonsil and lymph node were found to be positive after more than 36 h p.i. The lungs presented a variable positivity between 0 and 36 h p.i., but remained positive after this time.

Comparative studies of the efficacy of a recombinant feline leukaemia virus vaccine.

The efficacy of three feline leukaemia virus (FeLV) vaccines was compared. Kittens were immunised with either a recombinant subunit vaccine, Leucogen, or one of two inactivated virus vaccines, Leukocell 2 or Leucat. On subsequent challenge by intraperitoneal inoculation of FeLV of subgroup A (FeLV-A), only Leucogen gave significant protection. In a second experiment, kittens vaccinated with Leucogen were protected against oronasal challenge with a phenotypic mixture of FeLV of subgroups A, B and C. These results indicate that a recombinant vaccine, containing only the protein moiety of the surface glycoprotein of FeLV-A, can provide better protection than the inactivated virus vaccines tested against challenge with virus of the same subgroup, and can also protect against challenge by all three subgroups of FeLV.

Detection of antibodies to rabbit haemorrhagic disease virus: an immunoblotting method using virus-coated human erythrocyte membranes.

The virus of rabbit haemorrhagic disease (RHDV) was purified from infected rabbit liver homogenate by using its property to bind to human red blood cells. Lysates from virus coated cells contained a 60 kDa protein identified as the major viral protein. Immunoblots prepared with that preparation were proved to be useful for immunochemical analysis since the 60 kDa component was intensively stained by subsequent incubation with rabbit sera from infected rabbits and with a secondary labelled antibody. The sera from 114 rabbits were analysed with this test and the data were compared with those obtained by using the haemagglutination inhibition test (HIT). Among the 114 field sera tested by Western blot, 86 contained antibodies to the 60 kDa RHDV antigen whereas only 76 showed positive reaction by HIT. The sensitivity and the specificity of the Western blot were 0.85 and 0.45, respectively, with a concordance between the two techniques of 0.72. Additionally, the European brown hare syndrome virus antibodies reacted with the 60 kDa RHDV protein on immunoblots.

Characterization of group EF-4 bacteria from the oral cavity of dogs.

Samples from gingival scrapings of dogs were examined for the presence of CDC Groups EF-4 bacteria. Isolation procedures were performed in 5% sheep blood agar supplemented with thiostrepton and trimethoprim (10 mg/l). Fifty nine EF-4 strains were isolated from 92% of 49 dogs. Among the Group EF-4 bacteria, the majority of isolates belonged to the arginine-negative (biovar "b") Group EF-4 (42 strains recovered in 82% of dogs). Seventeen arginine-positive strains (biovar "a") were recovered only from 35% of dogs. The strains were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The analysis of electrophoretic protein pattern of these bacteria supported the results of conventional testing, confirmed the distinction between the biovars "a" and "b" of Group EF-4 and supported the division of biovar EF-4b into two subgroups of either producing or non-producing acid from gluconate.

Partial characterization of the human erythrocyte receptor for rabbit haemorrhagic disease virus.

An important, well known property of the rabbit haemorrhagic disease virus is its ability to agglutinate human red blood cells. Accordingly, red cells from human adult donors were agglutinated despite their blood group ABO status, and treatments with proteases or glycosidases did not prevent agglutination. However, we discovered that the cells from human umbilical cords or foetuses were not agglutinated. In order to identify the viral receptor on human erythrocytes, glycolipids and glycoproteins from adult red cells were separated and tested for their potency in inhibiting agglutination. The bulk of the biological activity was associated with the highly glycosylated glycolipids (polyglycosylceramides), whereas a lower but significant activity was also associated with neutral glycolipids. No activity was found in the lipid-free sialoglycoprotein fractions. All these data strongly suggest that the RHDV receptor on human red cells corresponds to a development antigen which is not expressed on foetal cells and is mainly carried by glycolipids. Faint activity was also found in membranes from sheep red cells, suggesting that a similar glycolipid component is carried by these animal cells.

Fatal disease mimicking leptospirosis in a dog, caused by Aeromonas hydrophila.

A dog was treated for leptospirosis on clinical and epidemiological arguments. The amoxicillin treatment was not successful. Pure culture of Aeromonas hydrophila was then obtained from liver and kidney, indicating that the septicemia was due to this bacteria commonly found in waters.

Early stages of feline immunodeficiency virus infection in lymph nodes and spleen.

Analysis of the early stages of infection within the lymphoid organs is crucial for the understanding of the physiopathology of HIV infection. Such analysis can only be performed using animal models. Cats were infected with two strains of FIV and killed at regular intervals for a classic pathologic study along with a quantification of the viral load by in situ hybridization in the spleen and the lymph nodes. The pathological study showed a persistent follicular reaction, which peaked 15 days postinoculation (p.i.). The in situ hybridization study showed two types of labeling. The first was spot labeling corresponding to cells actively replicating the virus. The second consisted of a more diffuse labeling linked to the follicular dendritic cells (FDCs) demonstrating by colocalization of virus detected by in situ hybridization associated with the FDCs, specifically labeled by immunohistochemistry. The number of productive cells is few and identical for the two viruses tested. Despite a slight peak at 15 days p.i., the number of infected cells persists while slightly decreasing over time. The FDC virus load appears jointly with the appearance of antibody and remains permanent until the end of the study at 3 years p.i. These results show that in the FIV model, there is a chronic permanent infection in the lymphoid organs. Furthermore, as compared with the SIV-macaque model, there is a correlation between the low number of infected cells detected in these organs in the early phase and the extended length of the asymptomatic period, which contrasts with the high level of the FDC virus load lasting during the same period.

Recognition of Leptospira interrogans antigens by vaccinated or infected dogs.

Antigenic recognition of leptospiral antigens by vaccinated or infected dogs was studied by microagglutination test (MAT) and by western blots. In western blots, serovar specific antigens detected by MAT migrated in the 18-31 kDa zone. The 25-31 zone seemed to be linked to antigens indicating virulence of the strain. These antigens are LPS. The first antibodies made after infection are produced against LPS migrating in the 14 kDa zone. Many protein antigens are common in leptospires belonging to different serogroups. Virulent strains exhibited specific antigens in the 45 and 32-34 kDa zones.

Serological analysis by A60 antigen ELISA and BCG immunoblotting in domestic carnivores experimentally vaccinated with mycobacterium bovis BCG.

The serological response to Mycobacterium bovis BCG vaccination was studied in 3 cats, 3 dogs and 3 puppies. The animals received two doses of 0.1 mg of BCG and were studied over a period of 8 months simultaneously by ELISA with antigen A60 (Anda Biologicals, Strasbourg, France) and immunoblotting with BCG. The two methods detected an increase of antibodies at 3 or 5 weeks. In the cats, specific antibody titer remained high and stable for more than one year, in the dogs they diminished quickly within 23 weeks. Carnivores elicit a serological response against specific protein antigens in the bands corresponding to 18 and 25kDa, 30 and 45kDa, and especially 35 and 42kDa bands, and to minor bands at higher molecular weight. Non-specific bands at 50-55 kDa and 70 kDa, assigned to heat shock proteins, were enhanced by BCG vaccination. Cat immunosera recognized on A60 immunoblots the specific homologous bands at 20-25 kDa and 32 kDa, but also two other dominant bands: one was partially specific (65 kDa) and the other was absent from the M. bovis and M. paratuberculosis profiles. The suitability of A60 ELISA for detection of mycobacterial infection in carnivores may be highlighted by immunoblot analysis.

Early events in lymph nodes during infection with SIV and FIV.

To elucidate the initial pathogenic events in lymphoid organs, the major reservoir of virus in HIV infection, follow-ups of viral load, pathological changes and target cells were performed in the rhesus macaque SIVmac251 model and in the cat FIV model. Lymph nodes (LN) obtained from animals sacrificed at early time points following experimental inoculation were analysed by in situ hybridization for virus load and by combined immunohistochemistry and in situ hybridization for virus cellular tropism. In the SIV model, the LN presented a high viral load at 7 days post inoculation (p.i.); at this stage, macrophages and T4 lymphocytes were identified as the target cells of the virus. A shift in the pattern of viral infection was observed at 2 weeks p.i., with a concentration of viral RNA in follicular dendritic cells (FDC) in the germinal centres of the developing lymphoid follicles. This FDC-associated virus persisted at high levels for 2 months p.i. in the FIV model, the number of infected cells detected in LN was very low compared with that found in the SIV model, and a similar role played by FDC was found.