Anne Van Nerom

Induction of the respiratory burst in turtle peritoneal macrophages by Salmonella muenchen.

Peritoneal macrophages were collected from juvenile turtles 72h after intraperitoneal inoculation with a 3% Sephadex suspension. The macrophages were assayed for their chemiluminescent (CL) properties, reflecting their respiratory burst activity, after stimulation with Zymosan A, phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187. Except for fMLP, all triggering agents induced a marked CL response. Luminol was used as the chemiluminescent probe. When comparing CL responses in temperatures ranging from 15 to 35 degrees C, lower assay temperatures induced lower and slower CL responses. Stimulation with viable Salmonella muenchen resulted in a distinct response. Bacteria, inactivated by means of heat or acetone, induced a faster and stronger oxidative burst. Opsonization of either viable or heat-inactivated S. muenchen with non-inactivated anti-S. muenchen serum, prepared in turtles, induced faster and higher CL responses. On the other hand, opsonization of acetone-inactivated S. muenchen caused CL responses to be slower and weaker. S. muenchen, opsonized with heat-inactivated turtle anti S. muenchen serum, induced higher responses than non-opsonized bacteria, but slower and weaker responses than bacteria opsonized with native turtle antiserum. No response was recorded after stimulation with LPS and the supernatant of heat-inactivated bacteria.

Oxygenation activity of chicken blood phagocytes as measured by luminol- and lucigenin-dependent chemiluminescence

Luminol- and lucigenin-dependent chemiluminescence (CL) was used to compare activation of the respiratory burst of chicken peripheral blood monocytes and heterophils after stimulation with various agents. Monocytes and heterophils were obtained from the blood of three specific-pathogen-free chickens at 14 months of age and purified by a two-step discontinuous Percoll gradient. All cells responded to phorbol 12-myristate 13-acetate (PMA), zymosan A and calcium ionophore A23187 producing CL. The time course of luminol- and lucigenin-dependent CL was similar for both monocytes and heterophils after stimulation with PMA or zymosan A. Heterophils at lower cell number than monocytes responded with similar or higher peak maximum (PM) values. At the concentrations of stimuli used, the order of mean PM values was: zymosan A > PMA > A23187. Addition of 4 x 10(-6) M N-formyl-1-L-methionyl-L-leucyl-L-phenylalanine (fMLP) showed weak but significant CL activity at 1 x 10(6) monocytes per tube with luminol and at 5 x 10(6) monocytes per tube with lucigenin. No significant response to fMLP was observed with heterophils. The results indicate that the respiratory burst of chicken monocytes and heterophils can be measured by CL.

Lucigenin‐ and luminol‐enhanced chemiluminescence in turkey monocytes

Monocytes from 10 week-old specific pathogen-free turkeys were isolated from peripheral blood by density centrifugation and assayed for their oxidative activity by means of a luminometer. Chemiluminescence (CL) properties after stimulation with different soluble and particulate stimuli were compared in lucigenin- and luminol-enhanced assays. A distinct response could be measured with 12-phorbol 13-myristate acetate (PMA) and Zymosan A, but only a weak signal was obtained with calcium ionophore A23187. No oxidative activity could be induced with N-formyl-methionyl-phenylalanine. Peak maxima for both lucigenin- and luminol-enhanced CL were ranked: PMA > Zymosan A > calcium ionophore. The velocity of the lucigenin- and luminol-enhanced responses induced by calcium ionophore were of similar magnitude, but the lucigenin-enhanced responses of Zymosan A and PMA-stimulated monocytes were respectively about 5 and 10 times higher than those obtained in luminol-enhanced assays. No peroxidase activity could be detected in the purified turkey monocytes. As luminol-enhanced CL primarily results from the peroxidase activity, this lack of myeloperoxidase may explain the observed lower responses to the different stimuli, in the presence of a luminol. In contrast, lucigenin-enhanced CL is not related to peroxidase activity, but is a selective probe of oxidase activity. Irrespective of the myeloperoxidase deficiency, different soluble and particulate stimuli induced a significant and reproducible CL response in turkey monocytes, in the presence of both chemiluminigenic probes, lucigenin and luminol. The possibility of measuring the phagocyte oxygenation activity of turkey monocytes represents a useful tool for the study of monocyte mediated host defence in the turkey.

Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide.

The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro. Purified monocytes were inoculated with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M. hyorhinis, inoculation with C. psittaci did not induce NO production in these phagocytes. The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy. In contrast, upon co-inoculation of the monocytes with C. psittaci and M. hyorhinis, a reduced replication rate of C. psittaci was observed. N(G)-monomethyl-L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M. hyorhinis co-inoculated turkey monocytes. These results imply two considerations. First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection. Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo. Moreover, it was shown that avian M. gallisepticum strains were also able to induce NO in turkey monocytes. Considering the high prevalence of both C. psittaci and Mycoplasma sp. in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys.

Oxygen radicals and nitric oxide production by turkey respiratory macrophages

The influence of different induction protocols on the recovery of elicited turkey respiratory macrophages (RM), and on their oxygenation activity and nitric oxide (NO) production was examined. RM were induced in three week old specific pathogen free turkeys with Sephadex G-50, Thioglycollate broth, and an emulsion of incomplete Freunds adjuvant (IFA), supplemented either with Mycoplasma hyorhinis grown in Modified Channock broth (IFA-M. hyorhinis) or with Modified Channock broth (IFA-Broth). The RM were recovered by lavage of the lungs and air sacs and were purified by centrifugation through a Percoll suspension. Their oxygenation activity was evaluated in luminol-enhanced chemiluminescence assays, following stimulation with Zymosan A. The NO production was evaluated by incubating the RM with lipopolysaccharide (LPS) from Salmonella enteritidis for 24 or 48 hours. The number of recovered RM was slightly, but not significantly lower for Sephadex G-50 and IFA-Broth than for Thioglycollate broth and IFA-M. hyorhinis. RM elicited with Sephadex G-50 and IFA-Broth showed a significantly higher oxidative burst response to Zymosan A, compared to the Thioglycollate and IFA-M. hyorhinis elicited RM. Although all elicited RM showed a high NO production upon stimulation with LPS, no significant differences were seen in the NO production of the RM obtained following the different induction treatments. Our results point out that care should be taken when applying elicited RM for in vitro assays, as distinct levels of oxygenation activity were obtained using different induction protocols.

Mucosal and systemic humoral immune response of turkeys after infection and reinfection with a Chlamydia psittaci serovar D strain

The purpose of this study was to examine the effects of Chlamydia-specific antibodies in tears and tracheal washings (IgA and IgG) and sera (IgG) on chlamydial excretion during the course of an experimental infection and reinfection of turkeys with Chlamydia psittaci. Two groups of turkeys were experimentally infected with a serovar D strain of Chlamydia psittaci, either at the age of 7 days or at the age of 35 days. A third group was infected at the age of 7 days and reinfected with the same strain at 35 days of age. A control group consisted of sham infected turkeys. All turkeys were observed daily for clinical symptoms. At the age of 49 days, the turkeys were euthanized and examined for macroscopic lesions. Following primary infection and reinfection, turkeys were equally depressed and dyspneic. Necropsy findings revealed no significant differences in the lesions of the birds which received both the prime and challenge infection and the birds, which received only a single infection. Anti-chlamydial antibodies in sera, tears, and tracheal washings were determined by IgA and IgG immunoblot assays. A clear local and systemic antibody response towards a broad range of chlamydial antigens was already seen 10 to 14 days following the experimental infections at both 7 and 35 days of age. In spite of the presence of Chlamydia-specific antibodies in tears, tracheal washings, and sera, chlamydial excretion was observed in all infected and reinfected turkeys throughout the experiment. In most turkeys, this chlamydial excretion was detected in three or four tissues sampled at set times, i.e., the conjunctiva, nostrils, trachea, and cloaca.

Oxidative activity of turkey monocytes, following the inoculation with Chlamydia psittaci.

Chemiluminescence (CL) was used to investigate the competence of turkey monocytes to mount a respiratory burst response upon interaction with Chlamydia psittaci. The oxidative activity of purified turkey monocytes, following inoculation with the avian C. psittaci serovar D strain 92/1293, was studied using luminol- and lucigenin-enhanced CL. Purified turkey monocytes were inoculated with C. psittaci at multiplicity of infection (MOI) of approximately 100, 10 and 1. In the presence of luminol, no detectable CL or only a weak CL response was obtained, and if present it increased with increasing MOI. Either sham inoculated monocytes, or monocyte-free control assays supplemented with C. psittaci, gave no detectable luminol-enhanced CL responses. In the lucigenin-enhanced assays, monocytes inoculated with C. psittaci demonstrated an immediate CL peak, the height of which was proportional to the MOI used. Following inoculations at a MOI 1, a faint second peak was observed, when applying high concentrations of lucigenin. Sham inoculated monocytes gave no detectable lucigenin-enhanced CL responses. However, in the presence of lucigenin, the addition of C psittaci to monocyte-free controls also resulted in an immediate CL peak, though no second peak was detected. This immediate lucigenin-dependent CL peak induced by C. psittaci was similar to the one observed in the presence of monocytes, and was not inhibited by superoxide dismutase. We demonstrated that this avian C. psittaci strain induces only a very weak respiratory burst response in turkey monocytes. In contrast, C. psittaci itself elicited an intense non-superoxide mediated lucigenin-dependent CL, indicating that in chlamydial research the detection of superoxide, using lucigenin, should be confirmed with a specific superoxide inhibitor.