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Staphylococcus aureus is a leading cause of intramammary infections (IMI) and bovine mastitis is an important disease for the dairy industry. As this bacterium probably expresses specific genes for establishment of IMI, we studied the transcriptional profile of four S. aureus strains recovered from experimentally infected cows. Microbial RNA was extracted from bacteria isolated from milk, reverse-transcribed and labeled for hybridization to sub-genomic microarrays to detect candidate genes for further investigations. Several S. aureus genes were expressed during IMI; some were detected in samples from more than one strain, more than one cow and at more than one time point during infection. A selection of four genes showing strong expression and with putative functions in pathogenesis was further studied by qPCR. By comparing the expression in different media in vitro, we found that gene SACOL2171 was induced by iron restriction whereas the expression of the transcriptional regulator SACOL2325 and the ABC transporter SACOL0718-720 (vraFG) were induced by milk. In addition, the putative exotoxin SACOL0442 seemed to require the intramammary environment for expression. Gene-disrupted mutants for SACOL0720 and SACOL0442 showed no growth defect in vitro but were attenuated during bovine IMI, causing infections with significant reductions in bacterial and somatic cell counts. The milk from the mammary quarters infected with these mutants also showed better appearance and composition than milk from quarters infected with the wild type. In conclusion, we have identified genes that are most likely important for S. aureus IMI. These represent novel candidates to include in a vaccine., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fatal exudative dermatitis (FED) is a recently described condition affecting red squirrels (Sciurus vulgaris) on the Isle of Wight and Jersey (Simpson et al., 2010a). Staphylococcus aureus strains isolated from skin lesions in cases of FED were characterised by molecular and phenotypic approaches. The strains were found to belong to a single MLST clonal complex (CC49) representing either ST49 or a novel single locus variant thereof (ST1957), were closely related by other molecular typing approaches, and all possessed the leukotoxin M encoding gene (lukM). In contrast S. aureus was either not isolated from none-FED cases or belonged to distinct and diverse molecular types that, with one exception, did not encode lukM. All isolates from FED cases were susceptible to all antimicrobials tested, including penicillin, and all proved negative for mecA and mecC as well as 14 other staphylococcal toxin genes. As all squirrels affected by FED were infected with S. aureus of the same lineage and encoded the lukM gene, it is possible that strains of this lineage may be involved in the pathogenesis of the dermatitis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Pneumonic bighorn sheep (BHS) have been found to be culture- and/or sero-positive for Bibersteinia trehalosi, respiratory syncytial virus (RSV), and parainfluenza-3 virus (PI-3). The objective of this study was to determine whether these pathogens can cause fatal pneumonia in BHS. In the first study, two groups of four BHS each were intra-tracheally administered with leukotoxin-positive (Group I) or leukotoxin-negative (Group II) B. trehalosi. All four animals in Group I developed severe pneumonia, and two of them died within 3 days. The other two animals showed severe pneumonic lesions on euthanasia and necropsy. Animals in Group II neither died nor showed gross pneumonic lesions on necropsy, suggesting that leukotoxin-positive, but not leukotoxin-negative, B. trehalosi can cause fatal pneumonia in BHS. In the second study, two other groups of four BHS (Groups III and IV) were intra-nasally administered with a mixture of RSV and PI-3. Four days later, RSV/PI-3-inoculated Group IV and another group of four BHS (Group V, positive control) were intra-nasally administered with Mannheimia haemolytica, the pathogen that consistently causes fatal pneumonia in BHS. All four animals in group III developed pneumonia, but did not die during the study period. However all four animals in Group IV, and three animals in Group V developed severe pneumonia and died within two days of M. haemolytica inoculation. The fourth animal in Group V showed severe pneumonic lesions on euthanasia and necropsy. These findings suggest that RSV/PI-3 can cause non-fatal pneumonia, but are not necessary predisposing agents for M. haemolytica-caused pneumonia of BHS., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow's milk and bulk milk from dairy farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS-lukF and lukE-lukD genes were detected in 100 (62.5%) of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37 lukM-lukF-PV(P83) genes--positive isolates. In addition, we isolated a novel lukM-lukF-PV(P83)-carrying (designated phiLukM), and revealed that the lukM-lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage genomes. These results suggest horizontal transmission of lukM-lukF-PV(P83) genes by temperate bacteriophages in S. aureus of bovine origin.

Leukotoxins are a group of exotoxins that produce their primary toxic effects against leukocytes, especially polymorphonuclear cells (PMNs). Leukotoxins include a variety of chemicals ranging from 9,10-epoxy 12-octadecenoate, a fatty acid derivative secreted by leukocytes themselves, to proteins such as RTX (repeats in toxin). This review focuses on leukotoxins of three species of gram-negative bacteria, Mannheimia (Pasteurella) haemolytica, Actinobacillus actinomycetemcomitans, and Fusobacterium necrophorum.

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays. Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described. The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41%; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2%; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.

Milk from mastitis-free goats from French herds was examined for the presence of coagulase-negative staphylococci (CNS), and 165 positive isolates were evaluated for their capacity to produce exoproteins. Most isolates were identified as Staphylococcus caprae (N = 91) or S. xylosus (N = 36), but members of at least nine other species were present. Overall, some 57% of isolates produced toxins with phenotypic properties of alpha-hemolysin, and 75% produced toxins resembling S. aureus beta and/or delta-hemolysins. Thermostable desoxyribonuclease (TNAse) was secreted by 29% of isolates and thermolabile DNAse by 66%. Slime was produced by 42% of our cultures and, although none of them showed activity to insoluble elastase, over 70% lysed the soluble substrate. No clinical consequences were observed to correlate with exoprotein production, which proved to be inconstant within individual CNS species.

The effect of cultural conditions on the production of leukotoxin by biotypes A and B of F. necrophorum was investigated. Biotypes A and B were grown in prereduced, anaerobically sterilized, brain-heart infusion (BHI) broth. The average leukotoxin titer of culture supernatant was 18 times higher from biotype A strains than from biotype B strains. Leukotoxin activity peaked during the late-log and early-stationary phases of growth, then declined precipitously in both biotypes. F. necrophorum biotype A was grown in different media (BHI, liver infusion, and Eugon broths), at various pH (6.6, 7.3, 7.7, and 8.2), incubation temperatures (30, 35, 39, and 43 degrees C), redox potentials (-352 to +375 mV), and iron concentrations (less than 0.2, 4.2, 42.1, and 361.4 microM). Anaerobic BHI broth with pH from 6.6 to 7.7 at 39 degrees C incubation temperature supported maximal F. necrophorum growth and leukotoxin production. The optimum redox potential for F. necrophorum growth was in the range of -230 to -280 mV. However, the presence of titanium III citrate or dithiothreitol (7.78 mM) in the medium decreased (P less than 0.05) the leukotoxicity of F. necrophorum. Low iron concentration (less than 0.2 microM) decreased (P less than 0.05) growth rate but not leukotoxin activity of F. necrophorum, whereas high iron concentration inhibited the leukotoxin activity.

Single strains of 5 different P. haemolytica serotypes (1, 2, 5, 6 and 9) and an untypable strain were compared in an attempt to detect differences which might be related to virulence. All but the untypable strain caused extensive lesions when injected into the lungs of healthy cattle. Each strain was found to be encapsulated and to be toxic in vitro for bovine leukocytes. Each strain also produced leukotoxin in vitro. The toxins varied, however, in total toxic activity and in the kinetics of leukotoxin production. Vaccination of cattle with each of the serotype strains elicited antibodies to organism somatic antigens and, to various degrees, the production of leukotoxin-neutralizing antibodies which showed no strain specificity in cross-neutralization studies. Although each of the serotype strains appeared to be a potential bovine pathogen, subtle differences were observed which may explain the importance of Serotype 1 strains in bovine pneumonic pasteurellosis.