Equine granulocytic anaplasmosis (EGA) is a seasonal rickettsial disease of horses transmitted by Ixodes spp. ticks. The etiological agent is Anaplasma phagocytophilum, a coccobacillary gram-negative organism with a tropism for granulocytes. Clinical manifestations include fever, partial anorexia, depression, distal limb edema, petechiation, icterus, ataxia, and reluctance to move. Hematologic changes observed are thrombocytopenia, decreased packed-cell volume and marked leukopenia involving first lymphocytes and then granulocytes. Diagnosis is based on awareness of geographic area for infection, typical clinical signs, abnormal laboratory findings, and visualization of characteristic morulae in the cytoplasm of neutrophils and eosinophils in a peripheral blood smear. Treatment consists of the administration of tetracycline. The disease is being diagnosed with increasing frequency in the United States, Canada, Brazil and northern Europe.

Extracellular adherence protein (Eap) from Staphylococcus aureus has been reported to have strong anti-inflammatory properties, which make Eap a potential anti-inflammatory agent. However, Eap has also been demonstrated to trigger T-cell activation and to share structural homology with superantigens. In this study, we focused on whether Eap fulfilled the definition criteria for a superantigen. We demonstrate that T-cell activation by Eap is dependent on both major histocompatibility complex class II and intercellular adhesion molecule type 1, that cellular processing is required for Eap to elicit T-cell proliferation, and that the kinetics of proliferation resemble the profile of a conventional antigen and not that of a superantigen.

A mouse model resembling Streptococcus equi subspecies equi infection in the horse, strangles, was used to assess the protective effect of vaccination with selected recombinant proteins from S. equi subsp. equi. After challenge the infection was monitored by weight loss and by nasal colonisation with S. equi subsp. equi. Vaccination with a collagen-binding protein (CNE) and a collagen-like protein (SclC) resulted in protective antibodies, whereas a novel fibronectin-binding protein (FNEB) did not. Co-administration of CNE with EAG, a poorly immunogenic alpha2-macroglobulin-, albumin- and immunoglobulin G-binding protein, resulted in a significant synergistic effect and enhanced the protective immune response against EAG.

Antibodies against the fibrinogen binding protein (Fbe) of Staphylococcus epidermidis significantly increased macrophage phagocytosis. Antibodies against autolysin E were opsonic but to a lesser extent. Antibodies against a novel, putatively surface-located antigen were unable to enhance phagocytosis. The severity of systemic infection of mice with S. epidermidis was reduced if the bacteria were preopsonized with anti-Fbe prior to administration. Fbe is thus a strong candidate for protein vaccination against S. epidermidis infection, and antibodies against Fbe can be used to prevent or treat infections caused by S. epidermidis.

Since the 1930s, carbon dioxide (CO2) has been combined with cold storage for the preservation of food. However, its use for the prevention of surgical wound infection was long considered to be impractical. Now CO2 is widely used during laparoscopic procedures, and a method has been developed to create a CO2 atmosphere in an open wound. The aim of this study was to investigate the effect of CO2 on the growth of Staphylococcus aureus at body temperature.

Staphylococcus aureus bacteraemia (SAB) originating from local infections can lead to severe secondary infections such as endocarditis. The protective effect of antibodies against secondary infections was studied in a rat model, where a local joint infection leads to bacteraemia and endocarditis on damaged aortic valves. In this study, immunizations with a truncated D2-domain of the S. aureus fibronectin-binding protein displayed on a cow-pea mosaic virus (CPMV-D) carrier induced protection against endocarditis (P < 0.05). Opsonization of S. aureus with antibodies raised against CPMV-D stimulated both neutrophil activity and macrophage phagocytosis in vitro. Furthermore, intravenous administration of these antibodies protected mice from weight loss due to SAB.

ABSTRACT We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens, and cutoff levels (upper 95th percentile) against these antigens were determined. No correlation was seen between serum antibody levels against Clf and Efb. In acute-phase samples 27% of patients showed positive antibody levels against Clf and 10% showed positive levels against Efb, while in convalescent-phase samples 63% (26 of 41) showed a positive serology against Clf and 49% (20 of 41) showed a positive serology against Efb. Antibody levels against Efb were significantly lower in the acute-phase sera than in sera from healthy individuals ( P = 0.002). An antibody response against Clf was most frequent in patients suffering from osteitis plus septic arthritis and from endocarditis (80% positive). The antibody response against Efb appeared to develop later in the course of disease. A possible biological effect of measured antibodies was demonstrated with the help of an inhibition ELISA, in which both high-titer and low-titer sera inhibited the binding of bacteria to fibrinogen. In conclusion, we have demonstrated in vivo production of S. aureus fibrinogen binding proteins during deep S. aureus infections and a possible diagnostic and prophylactic role of the corresponding serum antibodies in such infections.

This chapter presents a model of hematogenous osteomyelitis in the rat, which does not use any foreign body or sclerosing agent. Female Wistar weighing approximately 185 g, are anesthetized, surgically manipulated at the tibia or mandible, and subsequently challenged with S. aureus. Two weeks postinoculation, rats are sacrificed and assessed. Between inoculation and sacrifice, animals may be monitored radiographically for signs of infection. Since radiographic evaluation requires anesthesia of the animals and is very time-consuming, using a direct digital method that cuts down on working time is recommended. Radiographic parameters of osteomyelitis include general loss of bone architecture, bone destruction, sequestrum formation, and presence of periosteal bone. Most dramatic changes can be observed after two weeks and include diffuse loss of bone from the medulla extending to the cortex. Also, elevation and disruption of the normally smooth periosteum and new bone deposition become apparent.

Hematogenous osteomyelitis was produced in the tibia or the mandible of rats by drilling a hole into the bone, injecting sodium morrhuate, and inoculating Staphylococcus aureus Phillips into the femoral vein. Animals were sacrificed after 2 weeks and examined. The infection was characterized grossly and radiographically by bone deformation, histopathologically by a characteristic suppurative reaction, and microbiologically by the recovery of S. aureus Phillips from the infected tissue. These findings indicate that the model mimics human osteomyelitis with respect to its inflammatory bone changes. In contrast to earlier rat models in which bacteria were injected directly into the bone, this new experimental model allows study of the initiating events of osteomyelitis such as bacterial attachment and might assist as a model for both prophylactic and therapeutic trials.

A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus. The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibrinogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus.

Mice were immunized with fusion proteins encompassing the fibronectin-binding domain of a staphylococcal fibronectin-binding protein (FnBP-A). A specific antibody response against the fibronectin-binding part of the fusion proteins was detected in the serum of all vaccinated animals. The protective potential of these vaccinations was evaluated in a mouse mastitis model, using Staphylococcus aureus, strain SA113, for challenge. The mice vaccinated with FnBP fusion proteins showed a decreased number of bacteria recovered from the mammary glands and significantly reduced cases of severe mastitis. Histopathological examination of tissue from challenged glands of vaccinated mice revealed either no pathological reactions or disseminated inflammatory reactions with focal necrosis whereas four of six examined tissues from challenged glands of non-vaccinated animals showed total necrosis. A combination of FnBP fusion protein with staphylococcal α-toxoid did not increase the efficacy of the vaccination and animals vaccinated with α-toxoid alone were as sensitive to challenge as those from the non-vaccinated control group. Thus vaccination of mice with recombinant FnBP resulted in significant protection against challenge with S. aureus.

Rats were immunized with a fusion protein (gal-FnBP) encompassing beta-galactosidase and the domains of fibronectin binding protein from Staphylococcus aureus responsible for binding to fibronectin. Antibodies against gal-FnBP were shown to block the binding of S. aureus to immobilized fibronectin in vitro. Endocarditis in immunized and non-immunized control rats was induced by catheterization via the right carotid artery, resulting in damaged aortic heart valves which became covered by fibrinogen and fibronectin. The catheterized rats were then infected intravenously with 1 x 10(5) cells of S. aureus. The number of bacteria associated with aortic valves was determined 1 1/2 days after the challenge infection and a significant difference in bacterial numbers between immunized and non-immunized groups was then observed (p < 0.05).

Maltose binding protein (MBP) fused to STb, a heatstable enterotoxin of Escherichia coli, was secreted into the periplasm. A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb. The gene fusion is under the control of the strong and inducible Ptac promoter. Three hours after induction with IPTG, cells were harvested. Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin. The fusion protein purified in this way was biologically active in ligated intestinal segments of rats. Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column. The toxin was eluted at approximately 38% acetonitrile. The 5000-Da toxin was shown to be pure by SDS-PAGE and immunoblotting. The recovered enterotoxin was active in the rat loop assay. Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb. The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.6. Typically, 8 liters of bacterial culture resulted in 2.2 mg of pure STb. This genetic construction provides a readily obtainable source of biologically active STb toxin.

A protein (gal-FnBP), constructed by fusion of the genes encoding beta-galactosidase of Escherichia coli and the binding domains of fibronectin-binding protein (FnBP) of Staphylococcus aureus was used. FnBP is a surface protein responsible for attachment of bacteria to extracellular matrix of various host tissues. Gal-FnBP is more stable and can be produced in larger quantities than native FnBP. The binding specificity of this fusion protein was established in a Western blot analysis. Treatment of gal-FnBP with formalin inactivated the binding capacity of the protein but immunogenicity was retained. Immunisation of mice with formalin-treated gal-FnBP resulted in high antibody titres against the fibronectin-binding part of this fusion protein. These antibodies were measured by their ability to block the specific binding of fibronectin to gal-FnBP in a blocking assay. Sera raised against formalin-treated gal-FnBP and non-treated gal-FnBP blocked this binding to 40 and 25 % respectively, thereby indicating the usefulness of gal-FnBP as a vaccine component.