Cowdria ruminantium is recognized by a monoclonal antibody directed against the major outer membrane protein of Chlamydia trachomatis.

The relationship between Cowdria ruminantium and Chlamydia trachomatis was studied by immunofluorescence. A monoclonal antibody directed against the major outer membrane protein of C. trachomatis recognized rickettsial colonies of C. ruminantium in infected goat brain. No specific fluorescence was observed in non-infected brain. Two commercial Chlamydia-specific monoclonal antibodies as well as polyvalent anti-Chlamydia rabbit serum recognized C. trachomatis, but did not recognize Cowdria. Moreover, polyvalent Cowdria antiserum failed to recognize C. trachomatis cultivated in HeLa cells. It is concluded that Cowdria and Chlamydia are to a certain extent related, confirming similarities in ultrastructure and developmental cycle.

Serodiagnosis of respiratory syncytial virus (RSV) infection in children as measured by detection of RSV-specific immunoglobulins G, M, and A with enzyme-linked immunosorbent assay.

The diagnostic value of an enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV)-specific immunoglobulin G (IgG), IgM, and IgA in sera from infants and children with proven RSV infection, from a control group, and from patients with symptoms of viral respiratory disease was analyzed. Compared to virus isolation and RSV antigen detection methods, the sensitivity of this assay was 87% and the specificity was 79%. For IgG alone, these were 45 and 92%, for IgM alone they were 48 and 92%, and for IgA alone they were 74 and 95%, respectively.

Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays.

The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.

Detection of cytomegalovirus DNA in short term cultures.

Detection of human cytomegalovirus (CMV) by in situ DNA hybridisation six days after incubation of human diploid fibroblasts (ISDH-6) was evaluated prospectively in 205 urine samples, obtained from 57 kidney transplant and 17 bone marrow transplant recipients. The results were compared to those of conventional virus isolation (CVI) and the detection of CMV early antigens after one day of cultivation (EA-1). Of 42 samples positive for CMV by at least one of these methods, 40 (95%) were detected with ISDH-6. Thirty-five (83%) and 34 (81%) positive samples were found with CVI and EA-1, respectively. These data indicate that ISDH-6 is a sensitive method for detection of CMV. It can be used as a rapid and sensitive alternative to CVI in combination with EA-1.

Prevalence of Chlamydia trachomatis infection in a population of asymptomatic women in a screening program for cervical cancer.

The prevalence of Chlamydia trachomatis infection in a population of women with no symptoms of sexually transmitted disease was investigated. These women, aged 35-55 years, participated in a screening program for cervical cancer. With the use of a direct immunofluorescence method, 109 out of 2,470 smears tested were positive for Chlamydia trachomatis, indicating an overall prevalence of 4.4%. No changes in prevalence were found when five-year cohorts of this group were analyzed, indicating that age-dependent changes or epidemiological factors do not result in a different (decreased) prevalence over the ages 35 to 55 years. The prevalence of Trichomonas vaginalis and fungi, as detected by cytological screening, was lower than that observed for Chlamydia trachomatis: 3.1 and 2.1%, respectively. Of the 109 smears positive for Chlamydia trachomatis, 90 showed cervical cells with reactive changes (out of 1,490 smears with PAP II), whereas no cytological changes were found in 15 cases (out of 884 smears with PAP I). Changes suggestive of mild or moderate dysplasia were found in only four cases (out of 93 smears with PAP III). The results indicate that Chlamydia trachomatis is associated with reactive changes of endocervical cells and raise serious questions about whether prevention of possible secondary effects such as infertility and pelvic inflammatory disease can be achieved by a combined screening program for cervical cancer and Chlamydia trachomatis.

Favourable effect of detergent on antigen detection and comparison of enzyme linked detection systems in an ELISA for Chlamydia trachomatis.

The influence of addition of detergents to the antigen on sensitivity of an ELISA for the detection of Chlamydia trachomatis was investigated. Of the detergents tested, only octyl-beta-d-glucopyranoside and sodiumdesoxycholate gave respectively a two- to fourfold and an eightfold increase in sensitivity. The effect was only present within a narrow range of detergent concentrations. The optimal detergent concentration was strongly dependent on the protein concentration in the antigen preparation. For optimal detection of the bound chlamydial antigen, enzyme and biotin labeled secondary antibodies were compared. The biotin labeled antibodies were combined with enzyme labeled streptavidin-biotin complex (SBC). Color development was obtained with both types of conjugates by using either o-phenylenediamine (OPD) or an enzyme amplification system. The best results were obtained with the SBC method and OPD.

Detection of Chlamydia trachomatis in culture and urogenital smears by in situ DNA hybridization using a biotinylated DNA probe.

The detection of Chlamydia trachomatis by in situ DNA hybridization in urogenital smears was investigated using a commercially available biotinylated DNA probe. Intracellular staining of inclusion bodies was used as the criterion for positivity. Of 35 patients with a culture proven chlamydial infection 19 had smears in which C. trachomatis was detected by in situ DNA hybridization, indicating a sensitivity of 54%. Of 57 patients with a negative culture, two had positive smears by in situ DNA hybridization. To compare whether the criterion for positivity had influenced the sensitivity obtained with in situ DNA hybridization, 14 duplicate smears from culture positive patients were analysed with in situ DNA hybridization and immunofluorescence. Both methods detected intracellular inclusion bodies in seven of these smears, suggesting that the presence of infected cells mainly determines the sensitivity. The DNA probe did not cross-react with micro-organisms commonly found in the urogenital tract.

Immunological detection of cytomegalovirus early antigen on monolayers inoculated with urine specimens by centrifugation and cultured for 6 days as alternative to conventional virus isolation.

Detection methods for human cytomegalovirus were evaluated with 431 urine samples from 30 bone marrow and 88 kidney transplant recipients. Low-speed centrifugal inoculation was followed by early antigen (EA) detection by means of indirect immunofluorescence with a monoclonal antibody after 1 (EA-1) and 6 (EA-6) days of cultivation. The results were compared with those of conventional virus isolation (CVI). Of 68 positive samples, 49 (72%) were detected with EA-1, 58 (85%) were detected with EA-6, and 43 (63%) were detected with CVI. The combination of EA-1 and EA-6 showed positive results with 66 samples (97%), which is significantly better than with CVI (P less than 0.001). With the exception of one patient, all CVI-negative but EA-positive samples had either significant rises in immunoglobulin G (IgG) or IgA antibody titer or IgM antibodies present in the sera. These data indicate that the method with EA detection can replace CVI, provided that each sample is inoculated in duplicate. Sample 1 is examined after 1 day, and if it is negative, sample 2 is incubated for a further 5 days, followed by detection of cytomegalovirus.

Comparison of in situ DNA hybridization and immunological staining with conventional virus isolation for the detection of human cytomegalovirus infection in cell cultures.

The sensitivity of immunochemical staining and in situ DNA hybridization for the detection of human cytomegalovirus (HCMV) was compared with that of virus isolation. Human diploid fibroblasts were infected with serial, 10-fold dilutions of HCMV strain AD169 and examined at various intervals between 1 and 42 days after inoculation, using the three methods being compared. HCMV-DNA was detected by in situ hybridization using a biotin-labeled HCMV probe and CMV early antigen (EA) by immunochemistry using a specific monoclonal antibody. During the first 2 days after inoculation detection of EA appeared to be the most sensitive method. After the fifth day the sensitivity of the immunochemical and in situ hybridization methods was similar and equalled that of conventional virus isolation. However, 2-5 times more HCMV-DNA than HCMV-EA positive cells were detected. Our results indicate that both detection of HCMV-EA by immunological staining and HCMV-DNA by in situ hybridization are suitable methods for rapid and sensitive detection of HCMV infections.

Hamster model for herpes simplex virus infection of the central nervous system.

Adult Syrian hamsters were inoculated with a mouse brain passage of herpes simplex virus type 1 (HSV-1) along an intradermal and an oral route after local scarification. For both routes, clinical symptoms of central nervous system (CNS) involvement were seen in the period between five and 12 days post infection. Compared with the route via the buccal mucosa, CNS involvement by intradermal infection is easier to establish. With a minimum dose of 2 X 10(4) TCID50 virus via the intradermal route, an infection rate of 80% or more can be obtained reproducibly and detected simply by clinical observation without need of a survey of the humoral immune response. Scarification of the skin prior to inoculation was found to be essential for establishment of the CNS infection.

Role of granulocytes and monocytes in the prevention and therapy of experimental Staphylococcus epidermidis endocarditis in rabbits.

Rabbits with endocarditis caused by Staphylococcus epidermidis were studied to determine the parts played by granulocytes and monocytes in the prevention or outcome of therapy with cloxacillin. Both monocytes and granulocytes influenced prophylaxis with cloxacillin. The amount of cloxacillin needed to prevent infection in 50% of the rabbits was significantly less in control rabbits than in those selectively depleted of monocytes, as it was also in rabbits selectively depleted of monocytes compared with those that had both granulocytopenia and monocytopenia. Granulocytes strongly potentiated the effect of cloxacillin during prophylaxis, whereas the contribution of monocytes was merely additive. Monocytes also contributed to the effect of therapy with cloxacillin, partially via a cloxacillin-independent mechanism and partially by potentiation of the effect of cloxacillin. Granulocytes did not appear to affect cloxacillin therapy. Results of this study suggest that currently used regimens for prophylaxis and treatment of S. epidermidis endocarditis may need to be adjusted for neutropenic patients.

Role of granulocytes in the induction of an experimental endocarditis with a dextran-producing Streptococcus sanguis and its dextran-negative mutant.

The role of granulocytes in the induction of endocarditis with a dextran-producing Streptococcus sanguis and a dextran-negative mutant of this strain was studied. The number of colony-forming units of Streptococcus sanguis needed to colonize the vegetations in 50% of the rabbits (ID50) was significantly lower for the parent strain than for the dextran-negative mutant. However, in granulocytopenic rabbits the ID50s of both strains did not differ measurably. Dextran-negative streptococci were more readily cleared from the circulation than dextran-positive, but in this respect no difference was found between control and granulocytopenic rabbits, which indicates that clearance cannot account for the difference in ID50 between the two strains in the control group. At serum concentrations of 5% and lower, in-vitro granulocytes phagocytosed the dextran-negative streptococci more rapidly than the dextran-positive. The intracellular killing of the streptococci was no influenced by dextran production. This study suggests that an impaired phagocytic removal of attached bacteria from the vegetational surface can be a factor promoting the induction of endocarditis by dextran-producing streptococci.

Role of granulocytes in the prevention and therapy of experimental Streptococcus sanguis endocarditis in rabbits.

The contributions of granulocytes to the prevention and therapy of Streptococcus sanguis endocarditis with procaine benzylpenicillin (PBP) was investigated in rabbits. Depletion of granulocytes by treatment with mechlorethamine appeared to have no significant effect on either the prophylactic or therapeutic activities of PBP. Administration of 3,000 IU of PBP before inoculation with S. sanguis retarded the course of the endocarditis for only 24 h whether granulocytes were normal or depressed in numbers. Prophylaxis with either 15,000 or 30,000 IU of PBP was equally effective in non-granulocytopenic and granulocytopenic rabbits. Treatment of established infections with PBP at doses of 3,000 to 300,000 IU of PBP at 12-h intervals for 48 h was equally effective in rabbits with normal and depressed numbers of granulocytes. The effect of 3,000 IU of PBP was equivalent, however, to that of granulocytes alone, as shown by the fact that the numbers of CFU per gram of vegetation in the granulocytopenic rabbits treated with this dose of PBP and in the non-PBP-treated control rabbits were not significantly different.

Role of granulocytes and monocytes in experimental Escherichia coli endocarditis.

The role of granulocytes and monocytes during the induction and course of Escherichia coli endocarditis was investigated in rabbits by selectively depleting monocytes from the circulation with the drug VP16-213 and granulocytes and monocytes with nitrogen mustard. For induction, the number of E. coli needed to infect the vegetations in 50% of the rabbits was significantly lower in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia or in control rabbits, whereas the number of E. coli needed to infect 50% of the rabbits did not differ between the latter two. During the course of the endocarditis in endocardial vegetations, the numbers of CFU per gram of vegetation were significantly higher in the rabbits with combined granulocytopenia and monocytopenia than in the monocytopenic and control rabbits but did not differ between the latter two. The numbers of granulocytes in the circulation and the numbers of CFU per gram of vegetation showed a significant negative correlation that was not measurably influenced by the duration of the disease but was dependent on the number of E. coli injected for the induction of endocarditis. Granulocytes were found to be most effective at the lowest numbers of bacteria injected. In the circulation, too, the numbers of CFU per milliliter were significantly higher in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia and control rabbits, and there was a significant negative correlation between the numbers of granulocytes and CFU per milliliter of blood. From these findings we conclude that granulocytes play a protective role during the induction and course of E. coli endocarditis in rabbits, whereas no role is demonstrable for monocytes.

Role of granulocytes and monocytes in experimental Staphylococcus epidermidis endocarditis.

The role of granulocytes and monocytes during the induction and course of Staphylococcus epidermidis endocarditis was investigated by the selective depletion of monocytes with the drug VP16-213 and of both granulocytes and monocytes with nitrogen mustard. The induction of endocarditis was influenced only by the depletion of monocytes: the 50% infective dose differed significantly, being 3.4 X 10(5) CFU in control rabbits and 3.4 X 10(4) CFU in the monocyte-depleted rabbits, whereas no significant differences were found between the latter and those depleted of both granulocytes and monocytes. Also, control rabbits injected with 10(6) or 10(7) CFU had a significantly higher incidence of sterile vegetations than did rabbits selectively depleted of granulocytes or monocytes. Compared with baseline values, mean monocyte numbers at the time of bacterial inoculation were significantly increased in control rabbits whose vegetations remained sterile, whereas this effect was not seen in rabbits whose vegetations became infected. The course of the endocarditis appeared to be significantly influenced by both granulocytes and monocytes. Comparison showed that a decrease of the same numbers of these cells per microliter of blood was accompanied for the monocytes by an approximately fourfold higher increase of the number of staphylococci in the vegetations. The correlation between the number of granulocytes and of monocytes on the one hand and the number of staphylococci in the vegetations on the other was not substantially influenced by the duration of the disease or the number of staphylococci injected to induce the endocarditis. The number injected proved to be significantly correlated with the number of staphylococci in the vegetations. In rabbits with numbers of CFU per gram of vegetation exceeding 10(7), blood cultures were usually positive. This finding applied rarely to control rabbits, but generally to drug-treated rabbits. In the latter animals a significant correlation between the number of staphylococci in the vegetations and in the circulation was found. We conclude that only monocytes have a measurable effect on the induction of Staphylococcus epidermidis endocarditis but during its course both granulocytes and monocytes keep the endocardial infection in check.

Role of granulocytes in experimental Streptococcus sanguis endocarditis.

We investigated the role of granulocytes during the induction and course of experimental Streptococcus sanguis endocarditis in rabbits by depleting blood granulocytes with nitrogen mustard. The induction of the endocarditis was not influenced by granulocytopenia: the 50% infectious dose was 5.4 X 10(4) colony-forming units in normal and granulocytopenic rabbits. However, granulocytopenia influenced the curse of the endocarditis, as shown by a significant increase in the number of colony-forming units per gram of vegetation (P less than 0.02) from 24 to 72 h after the injection of 10(5) colony-forming units of S. sanguis. This rise did not occur in the control rabbits. Furthermore, bacteremia was significantly higher in the granulocytopenic rabbits (P less than 0.05) during the first 48 h compared with the control rabbits. This was not because of altered clearance of the streptococcus inoculum or seeding of streptococci from extracardiac bacterial foci. We concluded that granulocytes have no measurable effect on the induction of S. sanguis endocarditis, but during the course of the endocarditis, granulocytes keep the endocardial infection in check.

The role of bacterial adherence in the pathogenesis of infective endocarditis.

Bacterial adherence as a result of specific surface properties may be a contributory factor in the pathogenesis of bacterial endocarditis giving certain types of bacteria a selective advantage to cause this disease. Adherence could interact with other pathogenetic mechanisms, and this interaction could promote or hamper the development of endocarditis. Dextran production by streptococci, the activation of the clotting system by monocyte tissue thromboplastin, and phagocytic removal of bacteria from the vegetational surface by granulocytes and monocytes are examples of interacting mechanisms that could contribute to the pathogenesis of bacterial endocarditis.

Immunological studies on pancreatic phospholipase A2. Antigenic characterization of the NH2-terminal region.

Rabbit antisera elicited against pure pig, horse, ox, and sheep pancreatic phospholipase A2 revealed considerable immunological differences when tested by double immunodiffusion and microcomplement fixation assays. Snake venom phospholipases did not show any detectable cross-reactions with the pancreatic enzymes. Microcomplement fixation also clearly demonstrated conformational differences between porcine phospholipase A2 and its zymogen. NH2 terminally modified analogs of porcine phospholipase A2 could be clearly distinguished using the same assay. Moreover, strong evidence was obtained that Ala1-Arg6 is a part of an antigenic determinant. Radioimmune assay, using monovalent phospholipase-specific Fab fragments revealed a maximum number of three antigenic sites of phospholipase that can simultaneously be occupied by antibody. The Fab fragments were separated into three fractions, using three immunoadsorbent columns in series. These Fab fractions showed different inhibitory properties toward micellar binding of phospholipase A2. They also exhibited different protective effects against active center modification.