A play in four acts: Staphylococcus aureus abscess formation

Staphylococcus aureus is an important human pathogen that causes skin and soft tissue abscesses. Abscess formation is not unique to staphylococcal infection and purulent discharge has been widely considered a physiological feature of healing and tissue repair. Here we present a different view, whereby S. aureus deploys specific virulence factors to promote abscess lesions that are distinctive for this pathogen. In support of this model, only live S. aureus is able to form abscesses, requiring genes that act at one or more of four discrete stages during the development of these infectious lesions. Protein A and coagulases are distinctive virulence attributes for S. aureus, and humoral immune responses specific for these polypeptides provide protection against abscess formation in animal models of staphylococcal disease.     

Effect of different housing systems (single and group penning) on the health and welfare of commercial female rabbits

In recent decades, concern about rabbit welfare and sustainability has increased. The housing system is a very important factor for animal welfare. However, information about how different available housing types for female rabbits affect their health status is scarce, but this is an important factor for their welfare. Hence, the objective of this study was to evaluate the health status of female rabbits in five common housing systems: three different single-housing systems with distinct available surfaces and heights; a single-housing system with a platform; a collective system. Female rabbits in the collective and platform cages had greater cortisol concentrations in hair than those in the single-housing system with no platform. Haptoglobin concentrations and kit mortality rates during lactation were greater for the collective-cage female rabbits. The collective group had more culled females and more lesions than in the other groups. The main reasons for culling in all the groups were reproduction problems and presence of abscesses, and the collective group of females was the most affected. In conclusion, it appears that keeping females together in collective systems negatively affects their health status and welfare, while single-housing systems imply lower kit mortality rates during lactation and cortisol concentrations, and fewer lesions in female rabbits.     

Identification of novel staphylococcal virulence genes by in vivo expression technology

We have applied in vivo expression technology (IVET) to the study of staphylococcal virulence. Using a promoter trap that relies on genetic recombination as a reporter of gene expression, we identified 45 staphylococcal genes that are induced during infection in a murine renal abscess model. Of these, only six were known previously; 11 others have homology to known non-staphylococcal genes. The known staphylococcal genes include agrA , part of a key locus regulating numerous virulence products, and a glycerol ester hydrolase, which may enhance staphylococcal survival in abscesses. We constructed 11 strains containing mutations in previously unknown ivi genes. Of these strains, seven were significantly attenuated in virulence compared with the wild-type parent. The mutagenized ivi genes may encode novel staphylococcal virulence factors.     

Pathogenicity of Nocardia caviae,N. asteroides and N. brasiliensis

The pathogenicity ofNocardia caviae, N. asteroides andN. brasiliensis has been tested for white mice, guinea pigs and rabbits and chorio-allantoic membrane of the developing chick embryo. Altogether, 14 strains belonging to the 3Nocardia species originating from soil, human and animal sources in India or abroad were tested. All of them proved pathogenic though the degree of virulence varied from strain to strain. Incorporation of hog gastric mucin in the inoculum enhanced the virulence of all the 3Nocardia species for white mice.N. caviae strains were uniformly more virulent than those ofN. asteroides andN. brasiliensis.In the white mice inoculated intraperitoneally, a greater dissemination of the disease was apparent withN. caviae than withN. asteroides. Of the 6 strains ofN. caviae tested, 5 disseminated to the lung, 3 to the heart and 2 to the brain. InN. asteroides dissemination of the disease to the brain was observed with 2 of its 3 strains.N. brasiliensis showed no dissemination.N. caviae was found to be equally virulent for white mice, guinea pigs and rabbits. On the other hand,N. asteroides andN. brasiliensis were more virulent for white mice than for guinea pigs and rabbits. The lesions caused byN. caviae in mice, guinea pigs and rabbits persisted up to 4 weeks. In strong contrast to this the lesions due toN. asteroides andN. brasiliensis found in the guinea pigs and rabbits showed a strong tendency towards spontaneous clearance.Histologically, the lesions caused byN. caviae, N. asteroides andN. brasiliensis in mice, guinea pigs and rabbits were in the form of abscesses which showed an acute or chronic reaction. In the case ofN. caviae these abscesses showed both granules and freely dispersed cocco-bacillary bodies or filaments. As forN. asteroides it occurred in the form of cocco-bacillary bodies or filaments whereasN. brasiliensis consistently produced granules in the lesions.The lesions caused by the 3Nocardia species on the chorio-allantoic membrane of the developing chick embryo were in the form of abscesses which contained cocco-bacillary bodies and branching filaments but no granules.This forms a part of the thesis submitted by P.V.K. for Ph. D. degree, of the University of Delhi.     

In Vivo and In Vitro Action of Norethindrone on Staphylococci

Norethindrone has been examined in vitro for antibacterial activity against 10 microorganisms. Turbidimetric techniques were used to assay the antibacterial activity of norethindrone. The organisms tested included Staphylococcus aureus, S. epidermidis, Micrococcus conglomeratus, Listeria monocytogenes, Streptococcus faecalis, Salmonella typhosa, Shigella flexnerii, Klebsiella pneumoniae, Escherichia coli, and Proteus vulgaris. Bacteriostatic action was shown only against the gram-positive microorganisms when they were grown anaerobically in Tryptic Soy Broth containing 10 to 50 mug of norethindrone per ml. The bacteriostatic action of norethindrone was exerted primarily during the first 8 hr of incubation and it was reduced by the presence of oxygen. Mestranol at a concentration of 1 to 10 mug/ml failed to exert any significant action on S. aureus. However, incorporation of 5 mug of mestranol per ml in the culture medium enhanced the bacteriostatic action of norethindrone on staphylococci. Enhancement of the bacteriostatic action of norethindrone could not be obtained by the addition of a concentration of 5 mug/ml of testosterone, 17alpha-estradiol, and 17beta-estradiol. Progesterone and 4-pregnen-20beta-ol-3-one under similar conditions showed an additive bacteriostatic effect when they were incorporated into the culture medium containing norethindrone. In vivo studies indicated that female, adult New Zealand rabbits, injected subcutaneously with two injections of 10 to 20 mug of norethindrone, 24 hr apart, and challenged intradermally with S. aureus 4 hr after the second injection, had fewer lesions with smaller areas of swelling and erythema as compared to control, nontreated rabbits. The protective effect of norethindrone on the development of staphylococcal lesion seemed related to hormone concentration. Thus, it was demonstrated with doses of 20, 15, and 10 mug, but not with doses of 1 and 5 mug. When the lesions were excised 48 to 92 hr after infection and when viable cell counts were made, rabbits treated with norethindrone showed significantly lower staphylococcal counts than the control rabbits. During the 1st day after infection with S. aureus, leukocytic counts of the norethindrone-treated rabbits remained normal, whereas control animals showed elevated leukocytic counts.     

Skeletal and dental pathology of free-ranging mountain gorillas

The mountain gorillas of the central Virungas have been the subject of field study for the last 30 years; however, our understanding of morbidity and mortality in these apes is limited. This paper describes pathological conditions of the skeleton and dentition of these animals and evaluates lesions in relation to behavioral and environmental data. The skeletal remains of 31 mountain gorillas from the Karisoke Research Center were examined for enamel wear, carious lesions, abscesses, periodontal disease, antemortem tooth loss, trauma, inflammation, arthritis, neoplasia, and developmental anomalies. Two infants, three juveniles, 13 adult males, and 13 adult females form the sample. Enamel wear in the permanent posterior dentition is moderate. Six periapical abscesses were seen; three are associated with antemortem tooth breakage. No carious lesions were observed. Pronounced calculus buildup and alveolar resorption are the most notable pathological conditions of the dentition and affect all adult animals. The primary affliction of the skeleton is arthritis, which affects 14 animals. Vertebral degenerative disease predominates, but there is also temporomandibular joint involvement. Fractures occur at seven locations in the postcranium. In addition, there are five cranial injuries, including a fractured sagittal crest, and a penetrating wound to the vault, which is believed to result from a bite. Also thought to result from a bite is a case of cranial osteomyelitis. The only other inflammatory responses are two cases of idiopathic periostitis and one idiopathic lytic lesion. Button osteomas affect two animals and are the only neoplastic conditions observed. Two animals are afflicted by developmental abnormalities: one animal by idiopathic vertebral fusion and the other by spinal scoliosis.     

Recurrent staphylococcal cellulitis and subcutaneous abscesses in a child with autoantibodies against IL-6

We investigated an otherwise healthy patient presenting two episodes of staphylococcal cellulitis and abscesses, accompanied by high fever and biological signs of inflammation but, paradoxically, with no detectable increase in serum levels of C-reactive protein (CRP), an IL-6-responsive protein synthesized in the liver. Following in vitro activation of whole blood cells from the patient with multiple cytokines, TLR agonists, heat-killed bacteria, and mitogens, we observed a profound and specific impairment of IL-6 secretion. However, the patient's PBMCs, activated in the same conditions but in the absence of the patient's plasma, secreted IL-6 normally. The patient's serum contained high titers of IgG1 autoantibodies against IL-6, which specifically neutralized IL-6 production by control PBMCs as well as IL-6 responses in the human hepatocellular carcinoma cell line Hep3B. These anti-IL-6 autoantibodies were detected over a period of 4 years, in the absence of any other autoantibodies. Our results indicate that these Abs probably prevented an increase in CRP concentration during infection and that impaired IL-6-mediated immunity may have contributed to staphylococcal disease. Patients with severe bacterial infections and low serum CRP concentrations should be tested for anti-IL-6 autoantibodies, especially in the presence of other clinical and biological signs of inflammation.     

Repression of the Staphylococcus aureus Accessory Gene Regulator in Serum and In Vivo

Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator (agr locus) as well as multiple virulence-associated genes in Staphylococcus aureus. Gene expression was examined during growth of S. aureus in vitro in standard laboratory medium and rabbit serum and in vivo in subcutaneous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxin B. Expression of RNAIII, the effector molecule of the agr locus, was dramatically repressed in serum and in vivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome (TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profiles in the various experimental conditions demonstrated no positive correlation between the expression of agr and any staphylococcal virulence factors examined. Disruption of the agr locus had only a minimal effect on the expression in vivo of the virulence factors examined. An effect of immunization on the expression of agr and virulence factors was also observed. These results suggest that agr activation is not necessary for development of staphylococcal TSS and that regulatory circuits responding to the in vivo environment override agr activity.     

Pathogenicity of Pasteurella multocida A:3 in Flemish giant and New Zealand white rabbits.

Pasteurella multocida A:3 was isolated during an outbreak of pasteurellosis in Flemish Giant (FG) rabbits. Since New Zealand White (NZW) rabbits housed in the same room were not as severely affected as FG rabbits, experimental inoculation was undertaken to determine if FG rabbits were more susceptible than NZW rabbits to pasteurellosis induced by this isolate. Rabbits of each breed were inoculated with P. multocida A:3 and observed for 3 weeks. Four of 5 FG rabbits developed severe clinical disease on days 6, 9, 12 and 14 after inoculation; whereas, the one affected NZW rabbit became ill 14 days after inoculation. All rabbits with clinical disease developed fibrinosuppurative pleuritis, pyothorax and pneumonia which was more severe in FG than NZW rabbits. At necropsy, P. multocida A:3 was isolated from multiple sites of the diseased rabbits. No significant difference (P = 0.099) in the prevalence of lesions between the two breeds was found; however, the score of pneumonia and pleuritis was 3 times greater in FG rabbits than NZW rabbits.     

First description of sarcoptic mange in wild European rabbit (Oryctolagus cuniculus)

The European wild rabbit (Oryctolagus cuniculus) is a keystone species in the Iberian Mediterranean ecosystems being the staple prey of protected and endangered predators. Wild rabbits were once widespread, but the introduction of two viral diseases, myxomatosis in the 1950s and the rabbit hemorrhagic disease in 1989, resulted into a dramatic decline of its populations. Sarcoptic mange is a highly contagious parasitic infection caused by Sarcoptes scabiei. The first cases of sarcoptic mange in a wild rabbit population are recorded from a hunting area in Majorca (Balearic Islands, Spain). Five of 32 inspected rabbits (14.7%) were affected, with similar prevalences in summer and autumn. Sarcoptes scabiei were obtained from the edge of the lesions of two of the rabbits. The most frequently observed lesions were small areas of alopecia and crusts in the limbs. Affected limbs presented also a marked hypertrichosis and an apparent abnormal growth of the nails. One rabbit also presented lesions around mouth and nostrils. Parasitized rabbits were in significant lower body condition than healthy ones. According to previous reports of mange epizooties in other naïve wild species that caused marked short-term effects in their populations, this disease may pose a risk for the conservation of wild rabbit and their predators. Whether mange is endemic in rabbits from Majorca or it has been introduced is unknown. Game managers are encouraged to be aware of introducing sarcoptic mange during rabbit translocations.

     

Presence of phospholipid-neutral lipid complex structures in atherosclerotic lesions as detected by a novel monoclonal antibody.

A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic lesions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae is described. When (123)I-labeled ASH1a/256C antibody is injected intravenously into WHHL rabbits, it associates specifically with fatty streaks on the aorta. The antigen recognized by the antibody is lipid, based on extraction with chloroform and methanol from WHHL rabbit tissues. The antigen, purified by high performance liquid chromatography, was shown to be phosphatidylcholine (PC), which contains unsaturated fatty acyl groups based on analyses utilizing (1)H and (13)C nuclear magnetic resonance, Fourier transfer-infrared spectrum, and mass spectrometry. The antibody did not react with other classes of phospholipids or neutral lipids when tested using an enzyme-linked immunosorbent assay. When PC was mixed with either cholesterol, cholesteryl ester, or triacylglycerol, however, the reactivity of the antibody to PC increased up to 8-fold. Homogenates of aorta tissue obtained from normal and WHHL rabbits were fractionated using sucrose density gradient ultracentrifugation in which neutral lipid droplets, cellular membranes, and proteins are separated. The phospholipid content in cellular membrane fractions from WHHL rabbits was twice as high as that of normal rabbits, and there was an enormous difference in the antigenic activity in these fractions. The content of cholesterol in the cellular membrane fraction of WHHL rabbits was approximately 50 times higher than that of normal rabbits. Addition of neutral lipids to the cellular membrane fraction of normal rabbit markedly increased the antigenic activity. Atheromatous lesions in thickened WHHL rabbit aortic intima that were rich in lipid droplets were stained positively with ASH1a/256C immunohistochemically. These results strongly suggest that PC-neutral lipid complex domains are formed in atherosclerotic lesions.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Abstract Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli β-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli beta-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

Toxic properties of the cell wall of gram-positive bacteria

The biological activity of Odontomyces viscosus, which has been reported to cause periodontal disease in hamsters, was examined. The microorganism was cultured anaerobically in Brain Heart Infusion broth, and the cells were harvested. The washed cells were injected intradermally into the abdomen of rabbits. After 72 hr, a well-defined, firm, raised nodule (about 1.0 by 1.5 cm) with an erythematous border was seen at the injection site. Suspensions of cell wall and cytoplasmic material were injected intradermally, and the lesions appeared only at the site of cell wall injection. The cell walls, which were then treated with trypsin, pepsin, and ribonuclease, again produced the characteristic lesion. These nodular dermal lesions persisted for a minimal time of 10 days. The enzymatically treated cell walls were then hydrolyzed with 1 n HCl, and such hydrolysis up to 1 hr failed to alter the toxic activity of the cell walls. Similar dermal nodular lesions were obtained by injection of enzymatically treated cell walls of strains of Staphylococcus aureus, Streptococcus groups B, C, E, F, K, Lactobacillus casei, and Actinomyces israelii. Treatment with hot and cold trichloroacetic acid solutions and proteolytic enzymes, or with formamide, yielded insoluble fractions which produced the characteristic nodular lesions. The size of the lesion resulting from injection of these fractions was proportional to the amount of the injected material. The active fraction, which does not appear susceptible to hydrolysis by lysozyme, is thought to be cell wall mucopeptide. Histological studies showed skin abscesses due to the toxic reaction; however, in addition to the acute inflammatory reaction, there was local eosinophilia.     

Rabbit pasteurellosis: respiratory and renal pathology of control and immunized rabbits after challenge with Pasteurella multocida

Gross and microscopic lesions of pasteurellosis were studied in control and immunized pasteurella-free rabbits after challenge with virulent Pasteurella multocida. Pathologic responses were compared in rabbits immunized intravenously or mucosally with P. multocida or with J5, a cross protective core LPS mutant of E. coli. All rabbits were challenged conjunctivally with approximately 2xLD50 of P. multocida. Rabbits were necropsied and examined for histopathology of the respiratory tract and kidneys. Lung lesions varied in severity depending on the duration of the disease, the route of vaccination, and the vaccine used. The most severe lung lesions occurred in rabbits vaccinated intravenously with P. multocida and challenged with the same strain. Some of these rabbits had purulent bronchopneumonia and pleuropneumonia. Lung lesions were absent or less severe in rabbits vaccinated by a mucosal (aerosol, conjunctival) route and in unvaccinated controls. In these animals there was no bronchopneumonia or pleuropneumonia, and bronchiolitis, if present, was less severe. Kidney lesions were found only in rabbits vaccinated intravenously. There was an interstitial nephritis, some collagen deposition, mononuclear cell infiltration, and a loss of tubular architecture in the cortex. Some glomeruli were affected. These results indicate that intravenous immunization contributes to the formation of lesions whereas mucosal immunization prevented lesion formation to some degree.     

Temporal and quantitative analysis of expression of metalloproteinases (MMPs) and their endogenous inhibitors in atherosclerotic lesions

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of vascular diseases, such as atherosclerosis, plaque rupture and aneurysms. Although several MMPs have been demonstrated in the lesions of atherosclerosis, their expression profiles during the initiation and progression of lesions have not been fully determined. We hypothesized that the expression of various MMPs, along with their endogenous inhibitors, may be differentially regulated dependent upon the lesion progression. Therefore, we made a temporal and quantitative analysis of the mRNA and protein expression of MMPs and tissue inhibitors of metalloproteinases expressed in the different stages of atherosclerotic lesions of rabbits and humans. We found that MMP-1, MMP-12 and MMP-13 expression was nearly absent in the normal arterial wall, but was remarkably increased with lesion progression. Furthermore, the expression of these MMPs in the lesions was closely associated with intimal macrophages and monocyte chemoattractant protein-1 expression, suggesting that the intimal macrophages are the major source of production of these MMPs. MMP-3 and MT1-MMP were also significantly upregulated in the early-stage lesions and fatty streaks compared to the normal aortas of rabbits. Our results indicate that MMP-1, -12, and -13 derived from intimal macrophages may play a pivotal role in both lesion initiation and progression, and therefore are potential therapeutic targets for the treatment of plaque rupture and aneurysm formation.     

Raising Antibodies to Staphylococcal Enterotoxins A and B

A method is described for raising specific antibodies to staphylococcal enterotoxins A (SEA) and B (SEB) by intravenous inoculation of rabbits with small doses of enterotoxin diluted in sterile physiological saline. A course of six injections over a period of two weeks was given on four occasions. After the third course, 3/5 rabbits given SEA had a titre ± 1/50 and 4/6 given SEB had a titre of 1/50. Titres were not appreciably enhanced by the fourth series of injections. Only minor non-specific reactions which would require no absorption were found at the end of the series. It is believed that the findings in this study would be reproducible and that the method is likely to be suitable for raising antisera to other enterotoxins. Reference is also made to preliminary experiments in which latex sensitized with SEA or SEB was inoculated intravenously into rabbits.     

Reinfusion of UV-irradiated blood in the treatment of infection. I. Experimental infection with staphylococci

During the last 10 years, reinfusion of UV-irradiated blood has been rediscovered again as a therapeutic method suitable in the treatment of a variety of diseases. The described series of model experiments on rabbits confirm its beneficial effects in the treatment of staphylococcal infection: the control animals reinfused blood not exposed to UV radiation died all within 48 hours after injected with a suspension of live Staphylococcus aureus culture; all rabbits reinfused UV-irradiated blood (2 ml per kg body weight) survived the whole period of observation (30 days); reinfusion of UV-irradiated blood in a volume reduced to 1 ml.kg-1 body weight prolonged the animals' life-span to 96 or 120 hours.     

Intravenous Shiga toxin 2 promotes enteritis and renal injury characterized by polymorphonuclear leukocyte infiltration and thrombosis in Dutch Belted rabbits

Enterohemorrhagic Escherichia coli (EHEC) infection causes hemolytic uremic syndrome, a leading cause of acute renal failure in children. Dutch Belted (DB) rabbits are susceptible to EHEC-induced disease. Using real-time quantitative RT-PCR we measured the renal mRNA expression of cytokines and fibrinolytic factors in DB rabbits challenged with intravenous Shiga toxin 2 (Stx2) (1200 ng/kg). Group 1 rabbits received an incremental dose during an 8-day period whereas Group 2 rabbits received a single dose. Group 1 rabbits developed mild disease. In contrast, Group 2 rabbits developed severe diarrhea, higher levels of circulating polymorphonuclear leukocytes, increased mean platelet volume, and increased fibrinogen levels. Group 2 rabbits developed polymorphonuclear leukocyte infiltration in the intestine and kidney as well as glomerular congestion, luminal constriction, and mesangial glomerulonephropathy. These renal lesions were associated with up-regulation of interleukin-8 (P<0.006), plasminogen activator inhibitor-1 (P<0.04), and tissue plasminogen activator (P<0.05). Circulating Stx2 promoted dose-dependent enteritis and renal injury characterized by inflammation and impaired fibrinolysis leading to thrombosis.     

Genomic Analysis of Companion Rabbit Staphylococcus aureus

In addition to being an important human pathogen, Staphylococcus aureus is able to cause a variety of infections in numerous other host species. While the S. aureus strains causing infection in several of these hosts have been well characterised, this is not the case for companion rabbits (Oryctolagus cuniculus), where little data are available on S. aureus strains from this host. To address this deficiency we have performed antimicrobial susceptibility testing and genome sequencing on a collection of S. aureus isolates from companion rabbits. The findings show a diverse S. aureus population is able to cause infection in this host, and while antimicrobial resistance was uncommon, the isolates possess a range of known and putative virulence factors consistent with a diverse clinical presentation in companion rabbits including severe abscesses. We additionally show that companion rabbit isolates carry polymorphisms within dltB as described as underlying host-adaption of S. aureus to farmed rabbits. The availability of S. aureus genome sequences from companion rabbits provides an important aid to understanding the pathogenesis of disease in this host and in the clinical management and surveillance of these infections.