Evaluation of the efficacy of systemic danofloxacin in the treatment of induced acute Escherichia coli bovine mastitis.

The objective was to evaluate the efficacy of a single dose of danofloxacin (6 mg/kg bodyweight) given by the intravenous route for the treatment of acute bovine mastitis induced by intra-cisternal infusion of an Escherichia coli strain (26 cfu into one rear quarter of each cow). Twenty-three Prim'Holstein lactating cows were inoculated. To be challenged, the mammary glands had to be productive, free of pathogenic bacteria, and with somatic cell counts (SCC) of <200,000 cells/ml. The cows were treated on an individual basis when predetermined criteria involving both systemic and local clinical signs were satisfied. Allocation to treatment, danofloxacin or negative saline control, was performed according to a randomized treatment allocation plan. Monitoring during a 21-d period after inoculation included individual clinical examination, bacteriological examination and determination of SCC. Esch. coli was isolated from the milk of all inoculated quarters at the first milking post-inoculation and, together with reference to the clinical scores; the challenge was considered to be successful in 20 of the 23 cows. On study day 7 bacteriological cure rates with danofloxacin and saline control were 89% (8/9) and 44% (4/9) respectively. On days 14 and 21 all milk samples that could be collected were negative for Esch. coli in both groups of animals. Beneficial statistically significant differences were found at the end of the observation period (days 19-21 post treatment) between cows treated with danofloxacin and saline for SCC (P=0.0091) and earlier in the study for milk production (P=0.0003) and udder inflammation (P=0.004). Obvious beneficial trends were recorded in the danofloxacin group for rectal temperature, milk quality, general behaviour and appetite. Danofloxacin-treated cows showed statistically significant lower local clinical scores and a more rapid return to pre-inoculation values. It was concluded that systemically administered danofloxacin is effective in terms of bacteriological results, milk production and both systemic and local signs when used in the treatment of induced acute Esch. coli mastitis. Danofloxacin hastens recovery and return to productivity compared with potential self cure.

S. aureus IgG-binding proteins SpA and Sbi: host specificity and mechanisms of immune complex formation.

The evasion of the host immune response is central to the pathogenicity of Staphylococcus aureus, and is facilitated by the ability of the cell wall-associated protein A (SpA) to bind immunoglobulin G Fc fragments, thereby impeding phacocytosis and classical pathway complement fixation. SpA also acts as a B-cell superantigen through interactions with the heavy-chain variable part of Fab fragments, and sequesters immunoglobulins by forming large insoluble immune complexes with human IgG. Here we show that the formation of insoluble immune complexes is mediated by the binding of (VH3+) Fab fragments in addition to Fc, and that SpA forms soluble complexes with IgG Fc fragments. We compared these results with those for Sbi, a second staphylococcal immunoglobulin-binding protein, and note that this protein requires only the Fc fragment for precipitation with human IgG. Homology models of immunoglobulin-binding domains of SpA and Sbi in complex with Fc reveal the molecular basis of the Fab-independent formation of insoluble complexes by Sbi. Finally, we compared the sequences of the spa and sbi genes from human strains to those infecting a range of animal hosts to determine whether Sbi and SpA have acquired specificity for host IgG. We note remarkable sequence conservation within the IgG-binding domains of these genes, consistent with a lack of host specificity. The Fab-independent binding of IgG by Sbi could have significant clinical implications. The use of SpA in immunoadsorption therapy can cause severe side-effects, thought to be mediated by Fc gamma R recognition and complement fixation. The formation of insoluble immune complexes with Sbi occurs only via Fc binding and free Fc regions are unlikely to be available for Fc gamma R recognition and complement fixation.

Evaluation of tandem repeats for MLVA typing of Streptococcus uberis isolated from bovine mastitis.

BACKGROUND: Streptococcus uberis is a common cause of bovine mastitis and recommended control measures, based on improved milking practice, teat dipping and antibiotic treatment at drying-off, are poorly efficient against this environmental pathogen. A simple and efficient typing method would be helpful in identifying S.uberis sources, virulent strains and cow to cow transmission. The potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats) for S. uberis mastitis isolates genotyping was investigated. RESULTS: The genomic sequence of Streptococcus uberis (strain 0104J) was analyzed for potential variable number tandem repeats (VNTRs). Twenty-five tandem repeats were identified and amplified by PCR with DNA samples from 24 S. uberis strains. A set of seven TRs were found to be polymorphic and used for MLVA typing of 88 S. uberis isolates. A total of 82 MLVA types were obtained with 22 types among 26 strains isolated from the milk of mastitic cows belonging to our experimental herd, and 61 types for 62 epidemiologically unrelated strains, i.e. collected in different herds and areas. CONCLUSION: The MLVA method can be applied to S. uberis genotyping and constitutes an interesting complement to existing typing methods. This method, which is easy to perform, low cost and can be used in routine, could facilitate investigations of the epidemiology of S. uberis mastitis in dairy cows.

Differentiation of bovine Staphylococcus aureus isolates by use of polymorphic tandem repeat typing.

Staphylococcus aureus is a common cause of bovine mastitis. A simple and efficient typing method would be helpful in understanding S. aureus sources and spread. Ninety-six S. aureus strains, isolated between 1961 and 2003 from the milk of 90 dairy cows belonging to 75 French herds, were subjected to multiple-locus variable-number tandem repeats analysis (MLVA) by PCR. The conjunction of clfA, clfB, SAV1078 and fnb gene tandem repeats (TRs) enabled the definition of 61 types. When coa, spa, sdrC, sdrD and sspA TRs were used individually as additional markers, 63, 68, 67, 65 and 67 types were defined, respectively, versus 77 types when they were all included in the method. These additional TRs did not improve the differentiation of isolates collected in the same farm. The MLVA procedure using the tandem repeats embedded in clfA, clfB, SAV1078 and fnb loci as a basic combination at the herd level or associated with other TRs such as spa, sdrC, sdrD, sspA and coa can be a valuable tool for bovine S. aureus epidemiological studies.

Interdependence of udder quarters for new intramammary infection during the dry period in cows submitted to selective antibiotic therapy.

The aim was to investigate the interdependence of udder quarters within cow towards the incidence of intramammary infections during the dry period in herds under selective dry-cow antibiotic therapy. A total of 368 cows among 28 herds were included in a survey. Quarter milk samples collected at the last milking before drying-off and on day 3 after calving were submitted to microbiological procedures. An expected distribution of cows according to their number of newly infected quarters was calculated based on a binomial probability distribution from the overall quarter incidence (or from the quarter incidence in each herd) and compared with the observed distribution. Incidence of newly infected quarters ranged from 0.0 to 39.3%, depending on the herd (median: 17.7%). Interdependence of quarters towards new infection during the dry period was observed whatever the pathogen type, for both treated and untreated cows. Calculation of an expected distribution of cows according to their number of newly infected quarters using the quarter incidence in each herd (instead of the overall quarter incidence) reduced the distance to the observed distribution, but interdependence was still observed. Our results support the application of selective antibiotic therapy at the cow level rather than at the quarter level.

Transcriptional profiles of regulatory and virulence factors of Staphylococcus aureus of bovine origin: oxygen impact and strain-to-strain variations.

Staphylococcus aureus is responsible for a large panel of infections in humans and animals. In cows, S. aureus provokes chronic intramammary infections. Little information is available about the regulation of virulence factors in bovine isolates. Moreover, oxygenation, which is low in an inflamed mammary gland, could play an important role during the infectious process. We investigated the impact of oxygen on regulatory and virulence factors transcription for three S. aureus bovine isolates cultivated in CYPG medium into a fermentor under moderate oxygenation or low oxygenation. A selective panel of regulatory factors and virulence factors was studied through their mRNA profiles by real-time PCR according to growth phases and oxygenation. RNAIII, rot and sarR genes, for the regulatory factors, and asp23 and cflA genes, for the virulence factors, were strongly expressed, whatever the oxygenation and the strains. Under low oxygenation, whatever the strain, an enhanced expression of srr, clfA and spa genes was detected. Some regulators such as sae, sarA and sigB were differentially transcribed according to the strain and the oxygenation condition. This study sustains the complexity of S. aureus genes global regulation and suggests the coexistence of different pathways that can be activated depending on the strain and the oxygen availability.

Leucotoxic activities of Staphylococcus aureus strains isolated from cows, ewes, and goats with mastitis: importance of LukM/LukF'-PV leukotoxin.

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF'-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF'-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF'-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF'-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinity-purified antibodies to LukM or to LukF'-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF'-PV positive or negative isolates. These results establish that LukM/LukF'-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF'-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.

Milk complement and the opsonophagocytosis and killing of Staphylococcus aureus mastitis isolates by bovine neutrophils.

Phagocytosis of bacteria by bovine polymorphonuclear neutrophils (PMN) has long been regarded as essential for host defense against mastitis infection. Complement-mediated opsonisation by complement component 3 (C3) binding is an important component of the innate immune system. We investigated the role of milk complement as an opsonin and its involvement in the phagocytosis and killing of Staphylococcus aureus isolates from cases of bovine mastitis by bovine blood PMN. We show that deposition of milk C3 component occurred on six different isolates of S. aureus and that the alternative pathway was the sole complement pathway operating in milk of uninflamed mammary gland. This deposition was shown to occur at the same location as the capsule, but not on capsular antigen. Milk complement enhanced the chemiluminescence response of PMN induced by S. aureus. Nevertheless, the association of S. aureus to cells and the overall killing of bacteria by bovine PMN were not affected by the presence of milk complement. Therefore, as all milk samples contained antibodies to capsular polysaccharide type 5 and to other surface antigens, it is likely that milk antibodies were responsible for these two phagocytic events. Results of this study suggest that the deposition of milk complement components on the surface of S. aureus does not contribute to the defence of the mammary gland against S. aureus.

Analysis of the genetic variability of genes encoding the RNA III-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis.

The expression of Staphylococcus aureus virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the agr locus. RNA III is activated by at least two two-component systems, one encoded by the agr locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of S. aureus bovine mastitis isolates. Twelve agr restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose agr sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those agr variants allowed the linking of all types in an evolution scheme and their grouping in one of the four agr interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1. agr group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an agr group-specific multiplex PCR, we confirmed the above classification of strains in the agr interference groups. Four allelic variants of trap were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the trap 1 type (71.8%). Whereas no relationships between agr group and trap types were found, strains of similar agr restriction type were also of similar trap type (with the exception of strains belonging to the agr R IV-A5 and R VI-A8 types). Our analysis indicates that S. aureus isolated from cows has predominantly a clonal structure and that the highly prevalent agr R III-A1, trap 1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.