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Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.

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Two associated resistance mechanisms were found in a nalidixic acid-susceptible (4 ,ug/ml) but fluoroquinolone-resistant (8 to 16 ,ug/ml) strain of Escherichia coli Q2 selected under norfloxacin therapy. As compared with the susceptible E. coli Ql isolated before treatment, changes in outer membrane proteins and lipopolysaccharides in Q2 were associated with a 1.5-to 3-fold decrease in the uptake of fluoroquinolones but not nalidixic acid. A 50% inhibition of DNA synthesis in toluene-permeabilized cells of the resistant strain E. coli Q2 required up to 500-fold increased quantities of fluoroquinolones, whereas such inhibition was obtained in both E. coli Ql and Q2 with similar amounts of nalidixic acid. Selection from E. coli Ql on norfloxacin of one-step resistant mutants resembling E. coli Q2 was unsuccessful. From these results we infer that a decrease in outer membrane permeability, associated with a peculiar alteration of the DNA gyrase, was responsible for the unusual quinolone resistance phenotype of E. coli Q2.Quinolones are synthetic antibiotics which can be grouped into the older compounds nalidixic acid, pipemidic acid, piromidic acid, and flumequine and the newer, fluorinated quinolones such as norfloxacin pefloxacin, ofloxacin, and ciprofloxacin. The latter have comparatively enlarged spectra of activity as well as better intrinsic activities. Many studies have been carried out with quinolone-resistant gramnegative bacilli. Two main mechanisms of resistance which may occur either alone or combined have been found (15): (i) modification of the DNA gyrase in either subunit A (8, 10, 15, 20) or B (16, 21), which can give rise to moderate or high levels of resistance, and (ii) decreased uptake of quinolones, which correlates with a decrease in the quantity of porins (5, 10, 13-15). Another not totally elucidated mechanism involving the marA gene may be responsible for low levels of resistance (7).It was the purpose of this study to describe the mechanism(s) which could explain the particular profiles of resistance to fluroroquinolones and susceptibility to nalidixic acid observed in a clinical isolate of Escherichia coli. Hinton agar by using a Steers-type replicator device with ca. 104 CFU per spot and were read after 18 h of incubation at 37°C. MICs of novobiocin were determined as the quantities of antibiotic necessary to totally inhibit the growth of 100 CFU per plate after 18 h of incubation at 37°C (21). Selection of resistant mutants. Mutants resistant to norfloxacin were selected in vitro on plates containing a gradient of the antibiotic from 0 to 8 pug/ml.Extraction of OM proteins and LPS. Cell membranes were prepared from 200 ml of exponential-phase cultures at an optical density at 650 nm of 0.4 as previously described (19). Outer membrane (OM) proteins were obtained as follows: 100 jig of total-membrane protein was incubated for 45 min at 25°C in 70 RI of sodium phosphate buffer (50 mM, pH 7.0) containing 0.3% of N-laurylsarcosine N197 (ICN Biochemicals, Cleveland, Ohio) and centrifuged at ...

-Pestiviruses have been isolated from live sheep pox Tunisian vaccines. Vaccination with these vaccines caused outbreaks of Border Disease in Tunisia. In order to study more precisely the pathogenicity of these isolates, three groups of eight four month old lambs from a pestivirus-free flock were infected by the intratracheal route with a French strain (AV) and two Tunisian isolates (SN3G and Lot21). Clinical, hematological, immunological and virological parameters were evaluated. The three groups developed mild fever and leucopaenia by day 3 to 6 post infection (pi). The differences in the weight curves were not significant. Viruses were isolated from the peripheral blood buffy coat cells by day 4 to 9 pi. Antibodies were present on day 16 pi following infection by the French strain and on day 21 pi with the Tunisian isolates. The results demonstrated that SN3G and Lot21 are almost similar to the French strain used as the reference strain. In field conditions, they could induce economical losses in naive flocks, alone or in association with other pathogens. (33) 492943701; e-mail: p.russo@sophia.afssa.fr 8 agneaux de 4 mois issus d'un troupeau indemne de pestivirus ont été infectés par voie intratrachéale avec une souche française de référence (AV) et 2 isolats tunisiens (SN3G et Lot21). Les données cliniques, hématologiques, immunologiques et virologiques ont été suivies. Les 3 groupes ont développé une légère hyperthermie et une leucopénie entre le 3 e et le 6 e jour post inoculation (pi). Les courbes de poids ne montraient pas de différence significative. Des pestivirus ont été isolés à partir des cellules blanches du sang périphérique entre le 4 e et le 9 e jour pi. Des anticorps ont été mis en évi-dence 16 jours pi avec la souche française et 21 jours pi avec les isolats tunisiens. L'ensemble des ré-sultats a montré que les 2 isolats tunisiens ont un pouvoir pathogène presque similaire à celui de la souche française; ils pourraient provoquer, dans les conditions du terrain, des pertes économiques, seuls ou en association avec d'autres agents pathogènes.Pestivirus / ovin / vaccins anti-clavelée / pouvoir pathogène

ABSTRACT:Border disease virus (BDV) causes high mortality in Pyrenean chamois (Rupicapra pyrenaica) on the French and Spanish sides of the Pyrenees Mountains. We investigated the pathology induced by BDV in pregnant chamois via experimental infection. Three females were inoculated during the second third of pregnancy with a BDV-4 subgroup strain isolated from a wild Pyrenean chamois during an acute epizootic. A fourth pregnant chamois and one nonpregnant ewe were kept as negative controls. Animals were monitored to assess clinical signs, hematology, viremia, and serology. Postmortem examinations included necropsy, histopathology, and quantification of viral RNA in organs. Pregnancy was unsuccessful in all inoculated animals. One died 24 days postinoculation (dpi) without showing any precursory clinical signs. The second animal had profuse diarrhea from 13 dpi to its death at 51 dpi. The third aborted at 46 dpi and was euthanized at 51 dpi. All animals were viremic from 4 dpi until death. Neutralizing antibodies against BDV-4 were detected from 12 dpi. Necropsies showed generalized lymphadenomegaly, associated in one case with disseminated petechial hemorrhages in the digestive tract. Seventyeight of 79 organs from inoculated adults and their fetuses had detectable viral RNA. The main histologic lesions in adults were mild lymphohistiocytic encephalitis associated with moderate or moderately severe lymphoid depletion. Control animals remained negative for virus (in blood and organs), antibody, and lesions upon postmortem examination. BDV infection during pregnancy in Pyrenean chamois causes severe disease leading to abortion, then death. Despite 100% fetal death following inoculation, viral RNA was recovered from all organs of infected fetuses, suggesting that persistently infected offspring could be born. Our results may help explain the reported decrease in chamois populations in several areas and suggest that great care must be taken when interpreting infection status for wildlife.

-Contagious agalactia affects goats and sheep. In most infected sheep, the causal agent, Mycoplasma agalactiae, induces mastitis and/or agalactia, keratoconjunctivitis and arthritis. However, a few strains of M. agalactiae were isolated from tank milk from flocks without any clinical signs. The present study was undertaken to compare these apparently "asymptomatic" strains to classical virulent strains in order to assess the pathogenicity of four "asymptomatic" strains. Six groups of lactating ewes were inoculated by the intramammary route with 10 8 viable mycoplasmas of each strain. The clinical signs were regularly evaluated; the excretion of bacteria in milk and the serological response were measured. Ewes were necropsied 7 weeks after inoculation and the level of infection in retromammary lymph nodes was determined. Among the 4 apparently "asymptomatic" strains, 2 were fully virulent as were the strains isolated from diseased animals, and the other 2 induced somewhat less severe clinical symptoms. The other parameters, in particular the level of excretion in milk and the level of infection of regional lymph nodes following necropsy were similar for all strains. Mean antibody response was also comparable between the apparently "asymptomatic" and virulent strains, in spite of great individual variability. This observation shows that flocks without any clinical sign from which M. agalactiae is isolated in bulk milk, must be kept under strict control since mycoplasmas may induce severe outbreaks later with changing conditions of breeding.contagious agalactia / Mycoplasma agalactiae / virulence / experimental infection / sheep disease Résumé -Inoculation de Mycoplasma agalactiae par voie intramammaire chez des brebis en lactation : virulence comparée de 6 souches de terrain. L'agalactie contagieuse affecte les chèvres et les moutons. Chez la plupart des moutons infectés, l'agent causal, Mycoplasma agalactiae, induit des mammites et/ou de l'agalactie, des kératoconjonctivites et des arthrites. Cependant, quelques Vet. Res. 31 (2000) 329-337 329

Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.