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.

Comparison of early and late feline immunodeficiency virus encephalopathies.

DESIGN: The study of the early and late stages of encephalopathy following infection by the feline immunodeficiency virus (FIV) was carried out with laboratory and naturally infected cats. INTERVENTIONS: Animals infected experimentally were injected with three different isolates of the virus, administered either intracerebrally or intravenously, and sacrificed at 7 days, 1 and 6 months (intracerebral injection), and 2, 6 and 12 months (intravenous injection) post-inoculation, respectively. CONCLUSIONS: General features of encephalopathy were found to be identical, regardless of the method of inoculation or the viral strain used. Moderate gliosis and glial nodules, sometimes associated with perivascular infiltrates and white matter pallor, were observed at 1 month (intracerebral injection) and 2 months (intravenous injection), and remained unchanged until 12 months post-inoculation. The fact that these initial stages are identical for intravenously and intracerebrally inoculated cats suggests that the virus enters the brain very quickly in intravenously infected animals. Encephalopathy in cats naturally infected with FIV only consisted of gliosis, glial nodules, white matter pallor, meningeal perivascular calcification and meningitis. These lesions were more frequent and more severe in the group coinfected with feline leukaemia virus and feline infectious peritonitis virus. Although multinucleated cells were rare, the strong similarities between HIV and simian immunodeficiency virus encephalopathies at comparable stages support the view that FIV infection may represent an interesting model for a physiopathological approach of HIV infection of the central nervous system.

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.

Immunoblotting study of the antigenic relationships among eight serogroups of Leptospira.

Seven strains of Leptospira interrogans belonging to seven different serogroups, and one strain of Leptospira biflexa were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with gradient gels and immunoblotting with hyperimmune rabbit sera raised against each strain. The molecular masses of the proteins were calculated with a polynomial regression model. The SDS-PAGE patterns of the L. interrogans strains were similar and characterized by 24 common bands. This profile was not found for L. biflexa. The immunoblots obtained either with the seven anti-L. interrogans sera or the anti-L. biflexa serum allowed a clear distinction between the two species. Taken as a whole, the L. interrogans strain patterns revealed by the seven anti-L. interrogans sera were similar, sharing eight common major bands. A serovar- or serogroup-specific antigenic zone, ranging from 21 to 26 kDa, was also identified.

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.

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.

New topics on leptospirosis.

In the United States, leptospirosis takes the second place in human diseases transmitted by animals. The clinical features of leptospirosis are very various in man and domestic animals by the nature of the serovar involved and the animal species infected. There are many epidemiological cycles of leptospirosis but environmental conditions are always important.

Characterization of Pasteurella from gingival scrapings of dogs and cats.

Gingival scrapings of 62 dogs and cats were examined for the presence of Pasteurella. Isolation was performed in a medium supplemented with thiostrepton. Twenty-eight and 37 strains were obtained from 21 dogs and 26 cats, respectively, and classified in recently described species or subspecies of the genus Pasteurella (P.): P. multocida subspecies multocida and septica, P. canis, P. dagmatis and P. stomatis. Twenty-one strains were classified as atypical P. stomatis and one strain obtained from a cat remained unclassified. All strains were susceptible to the antibiotics studied. P. multocida and P. stomatis (including atypical strains) represented 65 and 30% of feline isolates, and 14 and 68% of canine isolates, respectively. Assuming that P. multocida, P. canis and P. dagmatis are potentially pathogenic for humans, and that P. stomatis has a low pathogenicity or non-pathogenic, 77 and 28% of examined cats and dogs harboured one or several pathogenic strains. This difference could explain the fact that Pasteurella infections in man are lower in dog bites rather than cat bites.

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.

Effect of stage of oestrous cycle on tylosin disposition in genital tract secretions of cows.

The influence of physiological state (oestrous or luteal phase) on tylosin disposition in genital secretions was examined after intravenous administration of tylosin to cows. Six healthy, cyclic and non-lactating dairy cows with controlled oestrous cycles were given a single slow intravenous injection of tylosin, at a dose of 10 mg kg-1. Plasma and genital fluid were regularly sampled up to 48 hours after injection. Tylosin diffused from blood to the genital tract and accumulated in genital secretions whatever the stage of the oestrous cycle: the secretion to plasma ratio was 3.45 +/- 2.54 during oestrous and 4.75 +/- 3.24 during the luteal phase. The area under the concentration-time curve (AUC) and the mean residence time (MRT) were significantly higher in genital secretions than in plasma. The AUC and the MRT in genital secretions showed no significant differences during the oestrous cycle. The minimal inhibitory concentrations (MIC) of tylosin for 25 strains of Actinomyces pyogenes were between 0.032 (12 strains) and 0.063 micrograms ml-1 (13 strains). Simulations based on the mean values of pharmacokinetic parameters determined in genital secretions, gave tylosin concentrations higher than the MIC for at least 36 hours. It was concluded that after an intravenous administration of tylosin, effective concentrations were achieved in genital secretions of the cyclic cow whatever its hormonal status which supported its use for the treatment of genital tract infections.

[Reaction of the complement fixation test on microtitration plates: application to the serology of myxomatosis. Comparative study of the results with the reaction of indirect immunofluorescence]. / Réaction de fixation du complément en plaques de microtitration: application à la sérologie de la myxomatose. Etude comparative des résultats avec la réaction d'immunofluorescence indirecte.

The authors describe a complement fixation technique on microtitration plates, using an antigen prepared from myxomas induced in rabbits. Compared with indirect immunofluorescence this technique was less cumbersome, more economical, easier to read and (as a conventional procedure) applicable in all laboratories. Results obtained with 165 serum samples tested by both methods showed good correlation and a specificity at least equal to that of indirect immunofluorescence. Taking into account its lower sensitivity, the positive threshold value for complement fixation under the described experimental conditions was a dilution of 1:4 (H50).

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.

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.

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.

Occupational hazard of unnoticed leptospirosis in water ways maintenance staff.

A serological survey was performed with M.A.T. on the maintenance staff of water ways and public works and lock keepers to study the occupational hazards of leptospirosis. The target population was 58 professional people, 40 working on French rivers and 18 on a channel in the same western area of France. When we compared serological tests between these two groups, no significant difference appeared. But when we linked fish handling to close contact with water, we found a higher number of positive tests in the channel group than in the rivers group (P less than 0.01). We conclude that people working on the channel were susceptible to unnoticed Leptospira infection.

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.

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.

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.