High and low virulence Staphylococcus aureus strains in a rabbit skin infection model

In the present study, an in vivo rabbit skin infection model was developed to reproduce the lesions caused by high and low virulence Staphylococcus aureus strains from rabbits. "O"-shaped dermal skin lesions were induced on the shaved flanks of anaesthetised rabbits using a tattoo pin and pincers. The induced lesions on the flanks of four groups of 10 rabbits were then inoculated by topical application of 0.1 ml of 10(8)cfu S. aureus bacteria. One group was inoculated with a typical high virulence (HV) S. aureus strain from rabbits, one group received an atypical HV strain and two groups were inoculated with low virulence (LV) strains. Five animals were kept as negative controls. The development, appearance and size of abscesses were scored daily for a period of 2 weeks. The infection model showed reproducible results for the different S. aureus inoculation groups. Inoculation of the skin with the typical HV strain resulted in significantly larger abscesses than those caused by the LV strains. The atypical HV strain caused abscesses of a size intermediate to that obtained with the HV and LV strains. In rabbits infected with LV strains, most of the lesions had healed by day 14 post-inoculation. The devised infection model is able to reliably reproduce the virulence properties of HV and LV S. aureus strains.     

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.     

Prevalence of genes encoding exfoliative toxins, leucotoxins and superantigens among high and low virulence rabbit Staphylococcus aureus strains

Staphylococcus aureus is an important cause of pododermatitis, subcutaneous abscesses and mastitis in rabbits. On rabbitry level, two types of S. aureus infections can be distinguished. In the first type, caused by low virulence strains, the infection affects only a small number of animals. The second type of infection is caused by high virulence strains and spreads throughout the rabbit flock. The pathogenic capacity of a particular S. aureus strain is attributed to a combination of invasive properties and extracellular factors such as toxin production. Therefore, 22 high virulence and 37 low virulence S. aureus isolates were compared regarding the prevalence of genes encoding exfoliative toxins, leucotoxins and superantigens. This study revealed a clearly significant difference between HV and LV rabbit S. aureus strains. All typical HV isolates were positive for the egc cluster, containing the enterotoxin(like) genes seg, sei, selm, seln, selo and selu, whereas these genes were not detected in any of the LV isolates. Further research is necessary to clarify the importance of the egc cluster in the pathogenesis of infections with high virulence S. aureus strains in rabbits.     

Genotypic and phenotypic screening of high and low virulence Staphylococcus aureus isolates from rabbits for biofilm formation and MSCRAMMs

At rabbit flock level, two types of Staphylococcus aureus infections can be distinguished. In the first type, caused by low virulence strains, the infection remains limited to a small number of animals. The second type of infection is caused by the high virulence strains, which spread throughout the rabbitry. The pathogenetic capacity of a particular S. aureus strain is attributed to a combination of extracellular factors and invasive properties such as adherence and biofilm formation. Twenty eight high virulence and 34 low virulence S. aureus isolates recovered from rabbits between 1998 and 2003 were used to study slime production on Congo red Agar (CRA) and prevalence of bap, icaA and icaD associated with biofilm formation. Furthermore these strains were screened for the presence of bbp, clfA, clfB, cna, ebpS, eno, fnbA, fnbB and fib encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The presence of icaA and icaD was not correlated with slime production on CRA. Bap was absent in all strains. All rabbit S. aureus strains harboured clfA and clfB. The prevalences of ebpS, eno, fnbA and fib did not reveal striking differences between high and low virulence strains. FnbB prevalence in high virulence isolates was lower than in low virulence isolates and cna was absent in high virulence strains. It was remarkable that only high virulence strains were positive for bbp. Further research is necessary to elucidate the significance of bbp in the pathogenesis of high virulence strains.     

Sequence analysis of a RAPD band differentiating high and low virulence Staphylococcus aureus strains from rabbits.

RAPD typing revealed the presence of a nucleotide band in typical high virulence rabbit Staphylococcus aureus strains which was absent in low virulence strains and in an atypical high virulence strain. The nucleotide sequence of this band was determined. Primers within this sequence were developed and PCR products of eight typical high virulence, one atypical high virulence and nine low virulence rabbit S. aureus strains were sequenced. All low virulence strains and the atypical high virulence strain revealed a constant difference with the typical high virulence strains for nucleotide 377 of the 1055bp sequence. The eight typical high virulence strains possessed a guanine base on this site, while the other strains tested showed an adenine base. These findings support the hypothesis on the clonal origin of typical high virulence rabbit S. aureus strains. After comparison with databases, two open reading frames (ORF) were identified within the sequence, which appeared to encode two structural ribosomal proteins. The single nucleotide mutation does not affect the amino acid sequence of the protein it encodes for.     

International dissemination of a high virulence rabbit Staphylococcus aureus clone

High virulence rabbit Staphylococcus aureus strains cause chronic and spreading problems of mastitis, pododermatitis and subcutaneous abscesses on rabbit flock level, whereas infections with low virulence strains are limited to individual rabbits. In the present report, 13 high virulence rabbit S. aureus strains, selected out of a large collection of strains isolated in five European countries between 1983 and 2004, were genotyped using pulsed-field gel electrophoresis, spa typing, multilocus sequence typing (MLST) and accessory gene regulator (agr) group typing. Two low virulence rabbit S. aureus strains were also included in the study. The results indicate the clonal origin of high virulence rabbit S. aureus strains present in Europe. Furthermore, the results of MLST and spa typing form a basis for international epidemiology of rabbit S. aureus strains, as these DNA sequence-based typing techniques can easily be used for intercentre comparisons.     

Colonisation of rabbits with Staphylococcus aureus after experimental infection with high and low virulence strains

Four groups of 12 rabbits each were inoculated in the nose with strains with suspected differences in virulence. In the two groups infected with strains from severe outbreaks, belonging to a rabbit-pathogenic biotype-phage type combination, 6-12 rabbits were found positive at the successive bacteriological samplings over a period of 28 days. In the two other groups, infected with strains obtained from rabbitries without a history of staphylococcosis, the number of Staphylococcus aureus positive animals quickly became negative but increased again after 1 week to 1-5 positive animals until the end of the experiment. Two rabbits in each group inoculated with a high virulence strain developed purulent skin lesions, while in the groups inoculated with low virulence strains, all animals remained clinically healthy. Results indicate that colonisation capacity is an important virulence determinant in rabbit staphylococcosis.     

Interaction of sub-epithelial connective tissue components with Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis were examined for their ability to interact with 125I-labelled fibronectin, fibrinogen and type II collagen. Their relative surface hydrophobicity and production of extracellular capsule were also investigated. Almost all S. aureus strains bound fibronectin (mean value 23%), fibrinogen (mean value 12%) and type II collagen (mean value 16%). CNS bound fibronectin (mean value 6%) and type II collagen (mean value 7%), but not fibrinogen (mean value 2%). The specificity of binding of these proteins to S. aureus strain F1440 and to coagulase-negative Staphylococcus chromogenes strain BO52 was studied by adding an excess of unlabelled proteins. Fibronectin and collagen binding were observed to be specific, varying between 50 and 75%, whereas the specificity of fibrinogen binding to S. aureus strain F1440 was lower (26%). Most of the S. aureus strains (63%) showed very high surface hydrophobicity (autoaggregation) or lower hydrophobicity (29% of the strains) and the rest were hydrophilic. None of the CNS strains autoaggregated, 44% were classified as hydrophilic strains. Hydrophilic strains (except the reference strains) did not show extracellular capsule production. However, the encapsulated (reference) strains showed low binding to these proteins as compared to their unencapsulated variants. Pre-treatment of S. aureus strain F1440 and S. chromogenes strain BO52 with trypsin decreased their fibronectin binding capacity and surface hydrophobicity, whereas pre-treatment with bovine milk (except on collagen binding to strain F1440) did not significantly affect binding to these proteins. These data indicate that S. aureus and CNS isolated from bovine udder infection have the ability to bind to tissue matrix and plasma proteins which may be exposed in the traumatized or toxin-damaged udder epithelial lesions.     

Binding of mouse mannan-binding lectins to different bacterial pathogens of mice

Humans have one mannan-binding lectin (MBL) in circulation but rodents, pigs, rabbits and rhesus monkeys have two, MBL-A and MBL-C. Plasma forms of these proteins have similar mannan-binding activity in vitro, but might differ in their ability to bind other microbial targets. In these studies, we compared carbohydrate-dependent binding of mouse plasma MBL-A and MBL-C to mannan-sepharose beads and to intact bacteria isolated as pathogens from mice. After incubation of mouse plasma with intact bacteria, MBL-A and MBL-C were eluted with N-acetylglucosamine (GlcNAc) and identified in nonreducing SDS-PAGE using Western blot analysis and MBL-A or MBL-C specific monoclonal antibodies. GlcNAc eluates of plasma incubated with mannan-sepharose beads, Klebsiella oxytoca and Staphylococcus aureus contained similar bands (mainly approximately 50kDa) that were immunoreactive with MBL-C antibody. Furthermore, a smaller form of MBL-C (approximately 45kDa) was detected bound to Pseudomonas aeruginosa. By comparison, immunoreactive MBL-A (a ladder of approximately 175kDa and larger bands) was identified in these GlcNAc eluates from mannan-sepharose beads, S. aureus and K. oxytoca but not P. aeruginosa. These studies demonstrate that mouse MBL-A and MBL-C in plasma are not equivalent in their ability to recognize bacteria that are pathogens for mice.     

4株鸭源肠球菌的鉴定和致病性

对临床分离的4株鸭源肠球菌郑1株、郑2株、郑3株、北京株和1株粪肠球菌参考菌株进行了系统鉴定,并用SDS-PAGE和Western-blot技术对各菌株细胞壁蛋白图谱进行比较分析。结果5个菌株的形态、染色、生理生化特性均与粪肠球菌特性一致;它们均对青霉素、万古霉素和庆大霉素敏感而对四环素耐药;5个菌株人工感染雏鸭及小白鼠均有致病性,但各菌株间致病力存在差异,北京株最强,参考株最弱,其余3株介于北京株和参考株之间;各菌株的细胞壁蛋白经SDS-PAGE在相对分子质量33 370~131 690之间均显示数十条蛋白带,其中郑2株和北京株在相对分子质量66 840处均有1条染色较深的蛋白带,而用Western-blot分析显示抗北京株胞壁蛋白抗体只能检测到北京株相对分子质量为66 840的抗原蛋白。以上结果表明,这5个被检菌株为致病性粪肠球菌,且致病性以北京株最强。     

Virulence of wild and mutant strains of Salmonella typhimurium in ligated intestinal segments of calves, pigs, and rabbits

A ligated intestine model in calves, pigs, and rabbits was tested for its value as an indicator of virulence of potential vaccine strains of Salmonella typhimurium. A wild virulent strain (3860C), a laboratory strain LT2, and mutants of these 2 strains were evaluated. Inoculation of calf intestinal segments with strain 3860C revealed that fluid responses were greatest in the proximal portion of the small intestine and that doses greater than 10(7) organisms were required to produce fluid responses and mucosal damage. Immunoperoxidase-stained sections of intestine revealed that a large dose of Salmonella organisms was required before mucosal invasion could be detected. Aromatic (aroA), galactose epimerase (galE), and diaminopimelic acid (dap) mutants of strain 3860C all resulted in much less fluid response, mucosal invasion, and mucosal damage compared with those by the parent organism. Strain LT2 induced such weak responses that it was not possible to evaluate reductions in virulence of its mutants. In 6-week-old pigs, there was no fluid response to any strains; however, in 1-week-old pigs, there was fluid response to the wild strain and some of its mutants. In adult rabbits, fluid responses were not observed, except when the wild strain was inoculated in the proximal portion of the small intestine. The calf and 1-week-old pig models appeared to be best suited for assessment of virulence of mutant strains of S typhimurium.     

Multiplex PCR assay for the detection of high virulence rabbit Staphylococcus aureus strains

High virulence rabbit Staphylococcus aureus strains, which are clonal in origin, are responsible for the spread of chronic staphylococcosis at the rabbit flock level. The aim of the present study was to develop a multiplex PCR assay that can be used for the identification of these high virulence strains. Two targets of the assay were the bbp and the selm genes, which have recently been shown to occur specifically in high virulence isolates. A third target was a sequence designated "flank", which was derived from a previously generated high virulence specific RAPD pattern. Furthermore, the femA gene, which is specific for S. aureus, was incorporated in order to avoid false negative results due to insufficient DNA preparation. The multiplex PCR was successful at differentiating the 26 typical high virulence and 50 low virulence rabbit S. aureus strains incorporated in the present study. Therefore it is useful for the initial screening of newly acquired breeding stock, in order to prevent the intake of high virulence strains in rabbitries.     

Colonization of rabbits with Staphylococcus aureus in flocks with and without chronic staphylococcosis.

Rabbits of 19 rabbitries were examined for the presence of Staphylococcus aureus in nine different body sites. Seven rabbitries experienced epidemically spreading signs of staphylococcosis while the other 12 rabbitries did not. S. aureus was isolated in all seven flocks that suffered from chronic problems of staphylococcosis and in 11 of the 12 clinically healthy flocks. The mean percentage of infected animals in these two groups was 90 and 43.3%, respectively. S. aureus was isolated from all body sites examined, but the ear and the perineum were often more intensely colonized. The number of animals colonized with S. aureus and the mean number of positive body sites in S. aureus positive rabbits were significantly higher in rabbitries with chronic staphylococcosis. This indicates that colonization capacity of S. aureus plays a role in epidemically spreading disease in rabbits. S. aureus isolates belonged to five different biotypes and 23 different phage types. Several different types simultaneously circulated in contaminated rabbitries and even simultaneously infected individual rabbits. Strains that belonged to the biotype-phage type combination mixed CV-C, 3A/3C/55/71 only occurred in rabbitries chronically dealing with signs of staphylococcosis. This may indicate a relationship between phenotypic strain properties and virulence of S. aureus.     

A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.     

Differentiation between high and low virulence Staphylococcus aureus strains from rabbits by randomly amplified polymorphic DNA (RAPD) analysis

Randomly Amplified Polymorphic DNA (RAPD) typing was performed on 53 rabbit Staphylococcus aureus strains. Twenty-three strains isolated in 13 different rabbitries with chronic problems of staphylococcosis, showed the same RAPD banding pattern. Twenty of these strains belonged to the 'mixed CV-C' biotype and to the phage-type 3A/3C/55/71, previously described to be highly virulent in rabbits, and three strains belonged to other biotypes or phage-types. None of the strains isolated from rabbitries without chronic problems of staphylococcosis showed this specific RAPD pattern. RAPD analysis can be used as a rapid and reliable test method to differentiate between the characteristic genotype corresponding to high virulence and other S. aureus strains from rabbits. This is useful for the diagnosis and prevention of the introduction of these highly virulent strains in industrial rabbitries.     

Dissemination of the superantigen encoding genes seeL, seeM, szeL and szeM in Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Bacterial superantigens are one of the major virulence factors produced by Streptococcus pyogenes and Staphylococcus aureus. The two novel superantigen encoding genes seeM and seeL were described for S. equi subsp. equi which is known as the causative agent of strangles in equids. In the present study previously characterized S. equi subsp. equi strains and strains of various other animal pathogenic streptococcal species and subspecies were investigated for the presence of the superantigen encoding genes seeM and seeL by polymerase chain reaction. According to these studies seeL and seeM appeared to be a constant characteristic of all investigated S. equi subsp. equi strains. Surprisingly, one S. equi subsp. zooepidemicus strain (S.z. 122) was also positive for both genes. The species identity of this S. equi subsp. zooepidemicus strain could additionally be confirmed by sequencing the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. The superantigen encoding genes could not be found among additionally investigated S. equi subsp. zooepidemicus strains or among strains of seven other streptococcal species. The seeL and seeM genes of the S. equi subsp. equi strain S.e. CF32 and the genes szeL and szeM of the S. equi subsp. zooepidemicus strain S.z. 122 were cloned and sequenced. A sequence comparison revealed a high degree of sequence homology between seeL, szeL, speL and seeM, szeM and speM, respectively. The superantigenic toxins L and M seemed to be widely distributed virulence factors of S. equi subsp. equi, rare among S. equi subsp. zooepidemicus but did not occur among a number of other animal pathogenic streptococcal species.     

Enhanced virulence of Staphylococcus aureus from bovine mastitis induced by growth in milk whey

The virulence towards mice of Staphylococcus aureus strains from bovine mastitis was enhanced upon growth in milk whey compared to homologous organisms grown in tryptic soy broth (TSB). In the mouse mastitis model, S. aureus grown in milk whey caused more severe lesions than homologous strains grown in TSB. Staphylococcus aureus strain F1440 grown in milk whey induced 75% mortality and local necrotic reaction in subcutaneously inoculated mice, whereas the homologous strain grown in TSB caused only 5% mortality and slight skin reaction. Extracellular capsule on milk whey-grown, S. aureus could not be demonstrated. However, diffuse type colony morphology could be correlated with an increased virulence of S. aureus towards mice.     

Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens

Capsule thickness of avian Pasteurella multocida type A strains was determined by transmission electron microscopy after labeling with polycationic ferritin and compared with their pathogenicity for chickens. The capsule thickness of P. multocida strains Pm-18 and X-73 was 81.4 and 50.1 nm on average, respectively. These strains were highly virulent for chicken, whereas the less virulent strains Pm-1 and Pm-3 had a thin and irregular capsule, 21.0 and 29.8 nm on average, respectively. However, the thickest capsule was observed in strain P-1059, 101.2 nm on average, and the strain revealed moderate virulence. The noncapsulated variant P-1059B, which was derived from strain P-1059, revealed low virulence. The six P. multocida strains were examined with regard to protein content on the capsule of organisms. Amounts of total proteins of crude capsular extract (CCE) from capsulated strains were approximately twice those of the noncapsulated strains. The amount of an antigenic 39 kDa protein in the CCE were found to correlate with the capsule thickness, since heavily capsulated strains exhibited the greatest amount, whereas noncapsulated strains including noncapsulated and low virulent variant P-1059B possessed little 39 kDa protein. The results demonstrated that the capsule thickness and the quantity of a 39 kDa capsular protein of avian P. multocida type A strains correlated with their pathogenicity for chickens.     

Protein variability among Mycoplasma hyopneumoniae isolates

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.     

Immunoglobulin G responses in 21 dogs with skin diseases to antigens from different isolates of Staphylococcus intermedius

The aim of this study was to characterise the immunoglobulin G (IgG) response in 21 dogs with or without pyoderma to antigens from six isolates of Staphylococcus intermedius. The staphylococcal proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred electrophoretically on to a membrane and subjected to immunoblotting with the dogs' serum. Gels containing separated proteins from the six isolates revealed 29 to 33 distinct bands with molecular weights ranging from 20 to 230 kDa. All the dogs' sera contained IgG that recognised 12 to 24 bands (mean 17), regardless of whether the dogs had pyoderma. The recognised proteins had molecular weights ranging from 20 to 198 kDa but the majority had molecular weights below 75 kDa. The most intense band in all six isolates had a molecular weight of 28 to 29 kDa. The antibody responses to the six isolates were essentially similar except that there were significantly more bands in the response to isolate 2 than to isolate 6, and occasional differences in the intensity of individual bands. All 21 dogs mounted an IgG response to multiple antigens in S intermedius, which differed only marginally between the six isolates. This lack of variation provides evidence that the host's response to different isolates of S intermedius is not a major factor in canine pyoderma.