Correlation of antigenic expression with progress in antibiotic therapy of acute human brucellosis.

Human brucellosis is a zoonotic disease which is endemic in Saudi Arabia. The aim of this study was to investigate the humoral immune responses and identify the target antigens that persist at different stages in human brucellosis during antibiotic therapy. To do this, an acute case of accidental nosocomial infection was studied experimentally. Blood was collected from the patient at the time of diagnosis, and at weekly intervals during therapy until remission. IgG and IgM immunoblotting was used to characterize specific antigenic determinants, and ELISA antibody titration was performed to quantify the circulating antibodies. Results indicated that protein bands of 12-13.5 kDa bound IgG in the patient's sera but did not bind IgM on immunoblots and are probably not specific for, or important in, early stage infections. However, an 18 kDa band persisted during infection through remission. The pivotal and most important findings were that the number of protein bands seen on immunoblots, the magnitude of ELISA antibody titres and the concomitant changes in the intensity of the polypeptide bands of 42-43 kDa were positively correlated with the stage of infection. High numbers of anti-IgG and -IgM immunoblot bands coupled with high ELISA antibody titres and a concomitant increase in intensity of the 42-43 kDa bands were positively correlated with acute and severe infection. Conversely, a reduction in the number of polypeptide bands as well as a decrease in the intensity, until the complete disappearance of the 42-43 kDa bands, coupled with low (baseline) ELISA antibody titration values indicated successful treatment and remission. The routine use of the methods described here to ascertain the stage of the disease, assess the progress of antimicrobial therapy and monitor cases of relapse in human brucellosis is suggested.

Isolation and characterization of Mycoplasma arginini from camels (Camelus dromedarius) with pneumonia.

Post-mortem examinations of 100 camels with pneumonic lesions were made at a local abattoir for Mycoplasma species. Sixteen isolates with indistinguishable biochemical and immunological characters were identified. The biochemical profile of these isolates showed that they were sensitive to digitonin, negative for urease production, glucose fermentation, and phosphatase activity but were positive for arginine hydrolysis. The identity of these isolates was further confirmed by disk growth inhibition test using a panel of specific antisera against selected reference Mycoplasma spp. Based on the biochemical profile and growth inhibition results, the camel isolates were identified as M. arginini. The pathological findings associated with M. arginini isolation consisted mostly of chronic interstitial pneumonia. The isolation rate of M. arginini from these specimens was 8.8%. These results suggest that the role of M. arginini in pneumonia in camels should be explored in greater detail.

The parotid, mandibular and lateral retropharyngeal lymph nodes of the camel (Camelus dromedarius).

The parotid, mandibular and lateral retropharyngeal lymph nodes of the dromedary camel were examined using both light and electron microscopy. All three lymph nodes were lobulated. They did not show the characteristic medulla, cortex and paracortex of typical lymph nodes. Instead, they contained lymphatic nodules, dense anodular lymphoid tissue and diffuse lymphoid tissue dispersed throughout the lymph node. Networks of sinuses were present in the diffuse lymphoid tissue. The diffuse lymphoid tissue in the periphery of all lymph nodes examined was characterized by numerous erythrocytes within and around its network of sinuses. The nodal sinuses were contiguous with the septal vessels, which are considered the possible source of erythrocytes seen in this study. The lymph nodes that were seen in this study resembled the haemolymph nodes of other mammals with regard to their content of erythrocytes but were unique in being located in sites that were typical of ordinary lymph nodes. Morphometric analysis has shown that the percentage volume densities of the stroma and the various parenchymal components were similar in the three lymph nodes.

Preparation and characterization of a biodegradable microparticle antigen/cytokine delivery system.

Biodegradable microparticles (MPs) were prepared to contain dinitrophenylated bovine serum albumin (DNP-BSA) and the cytokines interleukin (IL)-5 and IL-6. The conformational integrity of these MPs was examined by scanning electron microscopy. DNP-BSA was evaluated, postentrapment, by a bicinchoninic acid assay, SDS-PAGE, isoelectric focusing, Western blotting, ELISA and spectrophotometric analysis. The bioactivity of the ILs postentrapment was measured using bioassays. Ocular-topical (OT) and intraperitoneal (IP) administration of loaded MPs induced serum IgG, tear IgA and vaginal wash (VW) IgA responses which persisted up to 45 days post secondary immunization (P2o). OT and IP delivery elicited serum IgG and tear IgA responses up to 140 days post tertiary immunization (P3o). VW IgA responses persisted up to 45 days and 140 days P3o OT and IP delivery, respectively. Overall, the inclusion of cytokines in antigen containing MPs enhanced tear IgA antibody levels following OT delivery P2o, while elevated VW IgA responses occurred following IP delivery P2o and P3o. These data demonstrate that antigen/cytokine loaded MPs can potentiate long-term mucosal antibody responses at both target and distal effector sites as well as elicit circulating antibodies.

Analysis of the variability in expression of Mycoplasma gallisepticum surface antigens.

The in vitro expression of surface epitopes for different strains of Mycoplasma gallisepticum (MG) was studied with a panel of monoclonal antibodies (mAbs) using indirect colony immunostaining and Western blot (WB) analyses. Immunostaining of colonies with mAbs showed that five epitopes had different degrees of variable expression, while one epitope was permanently expressed in vitro. Colonies that failed to express the studied epitopes had the potential of phenotypically switching the expression of these epitopes in vitro. Variable and permanently expressed epitopes were associated with more than one protein and not all mAb-defined proteins were responsible for the immunostaining of intact MG colonies. The ability of MG to variably express their surface epitopes maybe the mechanism utilized by the microorganism to avoid the host immune response.

Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies.

Twelve monoclonal antibodies (Mabs) against Mycoplasma gallisepticum (Mg) strains F, R, S6(208) and PET2 were used for analysis of epitopes of 22 Mg strains. Six Mabs recognized surface epitopes in the majority of strains, but did not react with variant strains like K 503 and K 703. Two Mabs reacted with epitopes on about 56 kilodalton (kDa) proteins and showing consistent expression on Mg colonies. Three Mabs recognized three different variable surface epitopes associated with about 67 kDa proteins and one Mab variable epitope on about 33 and 80 kDa proteins. Two-dimensional immunoblotting showed considerable differences in the charge of proteins bearing variable surface epitopes in different Mg strains. Subcloning of four low passage Mg strains using Mabs for screening populations that derived from a single colony with defined surface epitopes showed that some colonies may switch surface epitopes associated with 67 and 80 kDa proteins. This switching was reversible and generated subpopulations of Mg expressing different combinations of surface epitopes. Phenotypic switching of epitopes probably occurs also in vivo and may be the mechanism enabling Mg to evade the host immune response.

Inhibition of lymphocyte adherence to rat lacrimal acinar epithelium by interleukin-4 and transforming growth factor-beta 1.

Mature circulating lymphocyte populations specifically bind to lacrimal gland acinar epithelium in vitro and this adherence is thought to contribute to the accumulation of lymphoid subsets within lacrimal tissue in vivo. The regulatory role of interleukin-4 (IL-4) and transforming growth factor-beta (TGF-beta) in this adherence process was examined using an in vitro binding assay. Pretreatment of thoracic duct lymphocytes (TDLs) with increasing concentrations of IL-4 or TGF-beta for 1 hr resulted in a dose-dependent inhibition of lymphocyte binding to lacrimal gland acinar epithelium. In contrast, the binding of TDLs to high endothelial venules of cervical lymph node was not inhibited by either cytokine. Further, IL-4 and TGF-beta pretreatment did not alter the expression of lymph node or Peyer's patch homing receptors as well as the LFA-1, VLA-4, or CD44 adhesion molecules on TDLs. These results suggest that the interaction of lymphocytes with lacrimal gland acinar epithelium may be regulated by a receptor-mediated mechanism that differs from those governing HEV recognition.

Evidence for a common epitope on the surface of Mycoplasma gallisepticum defined by monoclonal antibody.

An antigen containing a common epitope in most strains of Mycoplasma gallisepticum was purified by isoelectric focusing and used in the production of monoclonal antibodies (mAb). Of several mAb produced, only one mAb reacted with focused component and with all six strains of M. gallisepticum except strain 6/85. This mAb was designated MG3D6.A5, and it was subsequently purified with immobilized rProtein Atm. The MG3D6.A5 mAb recognized a common epitope on a molecule with relative molecular weight of 98 kilodaltons (kDa), termed p98. No binding was observed when the MG3D6.A5 mAb was reacted against antigens extracted from other mycoplasma species, indicating its species-specificity. Physicochemical studies revealed that p98 had an isoelectric point of 5.2, was stable to heat, and was resistant to periodate oxidation but sensitive to trypsin treatment, suggesting that p98 is a nonglycosylated protein. Furthermore, ultrastructural studies with colloidal gold revealed that M. gallisepticum cells were selectively stained with MG3D6.A5 mAb to p98. The latter was focally distributed on the surface of a mycoplasma cell membrane near the attachment organelle. These results suggest that p98 is a highly conserved protein in M. gallisepticum strains, is immunogenic, and is surface-accessible; its binding specificity to MG3D6.A5 mAb could be used to identify M. gallisepticum in multiple cultures.

Protection against airsacculitis with sequential systemic and local immunization of chickens using killed Mycoplasma gallisepticum bacterin with iota carrageenan adjuvant.

The induction of protective immunity to Mycoplasma gallisepticum (MG) by bacterins containing 0.2% iota carrageenan (iCGN) as an adjuvant has been studied. Various combinations of intracoelomic (i.c.), intratracheal (i.t.), intranasal (i.n.), intravenous (i.v.), subcutaneous (s.c.) and oral immunization routes were evaluated. Vaccinated and non-vaccinated groups were compared with a group vaccinated s.c. with a commercial bacterin. Primary i.c. immunization with the bacterin was as effective as commercial bacterin and was more effective when followed by i.n. or i.t. immunization. Oral immunization was ineffective, in contrast to observations reported with mice. The i.c./i.n. and i.c./i.t. combinations were the most effective, produced the highest levels of anti-MG IgG and IgA in serum and tracheobronchial washes, and sometimes provided 100% protection against air sac lesions. Chickens vaccinated by the i.c./i.n. or i.c./i.t. routes had the fewest virulent organisms in their respiratory tract secretions. These results demonstrated that i.c. immunization followed by local immunization with the bacterin is most efficacious in protecting chickens against airsacculitis.

Sequential intracoelomic and intrabursal immunization of chickens with inactivated Mycoplasma gallisepticum bacterin and iota carrageenan adjuvant.

Chickens immunized by sequential intracoelomic (analogous to intraperitoneal route in mammals) and intrabursal (i.c./i.b.) routes with inactivated Mycoplasma gallisepticum (MG) bacterin mixed with 0.2% iota carrageenan (iCGN) as an adjuvant were resistant to airsacculitis induced by a subsequent aerosol challenge with virulent R strain MG. In contrast, immunization by the i.c./i.b. routes with inactivated bacterin without the adjuvant or with 0.2% iCGN did not confer significant protection. Chickens immunized by the i.c./i.b. routes with the adjuvanted bacterin had increased levels of circulating and local anti-MG IgG but not IgM or IgA. Tracheal populations of MG were reduced when compared with unimmunized controls.

An enzyme-linked immunosorbent assay for the detection of specific IgG antibody to Mycoplasma gallisepticum in sera and tracheobronchial washes.

A sensitive indirect ELISA is reported for the detection and quantitation of specific IgG to Mycoplasma gallisepticum (MG) in sera and tracheobronchial washes (TBW) of MG-infected chickens. The sensitivity of the assay was ensured by the use of mouse monoclonal antibody to chicken IgG bound to a prospective anti-MG containing sample that was complexed with MG antigen immobilized on a solid phase. The level of specific IgG antibody in a test sample was detected by using peroxidase-conjugated goat anti-mouse IgG. Serum samples with various levels of anti-MG IgG activity were used to construct a standard curve response at a single working dilution by using Logit-Log curve fitting. The assay was simple, reliable, and specific and was used to monitor the appearance of specific anti-MG IgG in chicken sera and TBW at various intervals after the onset of mycoplasma-induced respiratory disease. The IgG response reached a plateau at 2 and 4 weeks postinfection in TBW and sera, respectively; then the response waned but still was detectable at a significant level for up to 25 weeks postinfection.

Suppression of humoral immunity in chickens with carrageenans.

Carrageenans (CGN), sulphated polygalactans, have been reported to be cytotoxic for macrophages in vitro. On this basis, the effect of the 3 major CGN types on humoral immune responses in chickens was investigated. Carrageenan had no effect on body and lymphoid organ weights. Histologically, CGN produced a significant proliferation of reticuloendothelial cells in liver and spleen, but no changes were observed in lymphocyte populations of the bursa of Fabricius, thymus, or spleen. Intracoelomic pretreatment with high doses of CGN induced a marked suppression of primary responses to sheep red blood cells (SRBC) given by the same route. However, if SRBC were injected intravenously into chickens already treated intracoelomically with CGN, no evidence of suppression was demonstrated. Antibody responses to Brucella abortus (BA), a T-independent antigen, were not affected by intracoelomic treatment with CGN. Intravenous pretreatment with CGN did not alter antibody responses to SRBC and BA given by the same route.