Experimental Chlamydia gallinacea infection in chickens does not protect against a subsequent experimental Chlamydia psittaci infection

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.

Keywords: Chickens; Chlamydia; Cross immunity; Cross protection.

Figure 1

Experimental design. A shows the experimental setup with group size and type of inoculum (coloured bacteria) and B the timeline of the experiment with sampling moments. From day 35 to day 42 daily pharyngeal and cloacal swabs were collected in the C. psittaci inoculated group GP and -P. In group GG and -G only daily cloacal swabs were collected.

Figure 2

qPCR results of cloacal swabs (A and B) and ELISA results of serum samples (C). In A results of individual cloacal swabs of group G in the Chlamydiaceae qPCR per timepoint are depicted in a heatmap. The darker the colour, the lower the Ct value as shown in the colour scale at the right side. Ct values > 40 or no qPCR signal are shown as Ct 41. The results of the negative control group (-) are not shown. B shows the mean Ct value of the cloacal swabs in time per group (of the Chlamydiaceae qPCR). The error bar indicates the SD. On the Y-axis the cycle treshold (Ct) value is depicted. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41. In C the mean OD (450 nm) value of the serum samples per group per timepoint is shown. The error bar indicates the SD.

Figure 3

C. psittaci qPCR results of group GP and -P. In A individual results of the C. psittaci qPCR of pharyngeal swabs per timepoint are depicted in a heatmap. The darker the colour, the lower the Ct value as shown in the colour scale at the right side. Ct values > 40 or no qPCR signal are shown as Ct 41. B shows the mean Ct value of the pharyngeal swabs pet timepoint per group (of the C. psittaci qPCR). The error bar indicates the SD. On the Y-axis the cycle treshold (Ct) value is depicted. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41. In C individual results of the C. psittaci qPCR of the cloacal swabs per timepoint are depicted in a heatmap. The darker the colour, the lower the Ct value as shown in the colour scale at the right side. Ct values > 40 or no qPCR signal are shown as Ct 41. D shows the mean Ct value of cloacal swabs in time per group (of the C. psittaci qPCR). The error bar indicates the SD. On the Y-axis the cycle treshold (Ct) value is depicted. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41.

Figure 4

Results of tissues in group GP and -P. A: the C. psittaci qPCR results of tissue samples are shown. On the Y-axis the cycle treshold (Ct) value is depicted. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41. The bar indicates the median. B: macroscopic enlargement of the spleen in a C. psittaci infected animal. C and D: histological examination of the spleen (same magnification, bar = 200 μm). Note the pronounced hyperplasia of the red (white arrows) and white pulp (black arrows) in the C. psittaci infected animal (D) compared to the animal infected with C. gallinacea (C).

Figure 5

Chlamydiaceae qPCR results of group GG and -G. In A individual results of the Chlamydiacea qPCR of the cloacal swabs per timepoint are depicted in a heatmap. The darker the colour, the lower the Ct value as shown in the colour scale at the right side. Ct values > 40 or no qPCR signal are shown as Ct 41. B shows the mean Ct value of cloacal swabs in time per group (of the Chlamydiaceae qPCR). The error bar indicates the SD. On the Y-axis the cycle treshold (Ct) value is depicted. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41. In C the Chlamydiacea qPCR results of tissue samples are shown. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41. The bar indicates the median.

Figure 6

Chlamydiaceae qPCR results of group -G and -P. A shows the mean Ct value of swabs in time per group of the Chlamydiaceae qPCR. The error bar indicates the SD. On the Y-axis the cycle treshold (Ct) value is depicted. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41. In B the Chlamydiaceae qPCR results of tissue samples are shown of group -G and group -P. The Y-axis has been rotated and Ct values > 40 or no qPCR signal are shown as Ct 41. The bar indicates the median.