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Abstract The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

Abstract Chlamydia psittaci was considered the predominant chlamydial species in poultry until Chlamydia gallinacea was discovered in 2009. C. psittaci is a zoonotic obligate intracellular bacterium reported in more than 465 bird species including poultry. In poultry, infections can result in asymptomatic disease, but also in more severe systemic illness. The zoonotic potential of C. gallinacea has yet to be proven. Infections in poultry appear to be asymptomatic and in recent prevalence studies C. gallinacea was the main chlamydial species found in chickens. The high prevalence of C. gallinacea resulted in the question if an infection with C. gallinacea might protect against an infection with C. psittaci. To investigate possible cross protection, chickens were inoculated with C. gallinacea NL_G47 and subsequently inoculated with either a different strain of C. gallinacea (NL_F725) or C. psittaci. Chickens that had not been pre-inoculated with C. gallinacea NL_G47 were used as a C. gallinacea or C. psittaci infection control. In the groups that were inoculated with C. psittaci, no difference in pharyngeal or cloacal shedding, or in tissue dissemination was observed between the control group and the pre-inoculated group. In the groups inoculated with C. gallinacea NL_F725, shedding in cloacal swabs and tissues dissemination was lower in the group pre-inoculated with C. gallinacea NL_G47. These results indicate previous exposure to C. gallinacea does not protect against an infection with C. psittaci, but might protect against a new infection of C. gallinacea.