Figure 1.
Mortality of rainbow trout during experimental bath infection with Y. ruckeri.
The fish (n = 112) were exposed to Y. ruckeri O1 (1×108 CFU/ml) in one aquarium for 1 hour and then moved to an aquarium with clean water for 21 days of observation. There was significantly cumulative mortality in the infected group compared with the un-infected group by use of Kaplan-Meier survival analysis (p<0.01).
Figure 2.
Detection of Y. ruckeri in blood of infected trout.
Blood was sampled from uninfected control and Y. ruckeri infected fish during the challenge experiments (n = 4). Blood samples were taken from 1 hpi until 3 days post infection during the initial challenge (solid line). Since all infected trout had Y. ruckeri in the blood already at 1 hpi, an additional experiment was conducted and blood samples were taken between 1 mpiand 1 hpi (dashed line). Y. ruckeri were re-isolated from all blood samples obtained from all infected fish at all-time points and are given as medians with ranges in the figure. Kruskal-Wallis test with Dunn's multiple comparison post-test was used to test for difference in the quantity of Y. ruckeri in the blood in infected relative to uninfected rainbow trout. The infected group had significantly increased numbers of Y. ruckeri in the blood 40 mpi and 1 hpi p = 0.0279 and p = 0.0076, respectively. * = p<0.05.
Figure 3.
Anatomy of the gills of rainbow trout based on detection of autofluorescence.
3D organization and images of vertical sections of the gills from a healthy non-infected rainbow trout. The image are taken from the following angles: (A) proximally, (B) distally, (C) overlaid images of autofluorescent showing the anatomy and TXR red channel (negative control showing no binding of Y. ruckeri specific antibodies). (D-F) 2D sections generated from a 3D model of C along (D) coronal axis, (E) sagital axis, (F) transveral axis. Esophagus, Es; hypobranchial, Hb; ceratobrancial, Cb.
Figure 4.
Detection of Y. ruckeri in the gills of infected rainbow trout by OPT.
(A, C and F) Red spots showing specifically stained Y. ruckeri fluorescent and green is autofluorescent showing the gill morphology. B, D and G show the overview of the Y. ruckeri positive signals detected in the sample (grey outline).E, H and I show the Y. ruckeri specific staining in vertical sliced sections. A and B are the gills from an uninfected rainbow trout (negative control) sampled pre infection, showing no Y. ruckeri specific staining (red). (C−E) Gills sampled at 1 mpi demonstating that Y. ruckeri is attached to the gill lamellae and it is detected inside the lamellae (E). (F−I) Show the progression of Y. ruckeri infection over time, detected in the gills and esophagus at 7 dpi.
Figure 5.
Detection of Y. ruckeri in skin and kidney.
(A) 3D spatial organization of skin and underlying muscles surrounding the lateral line canal (sampled 10 mpi). (a) Shows the 3D structure based on autofluorescence (green) and only Y. ruckeri present on the outer surfaces of the 3D sample is displayed. Picture (b) shows (a) as a flattened 2D image, resulting in an overlaid figure enabling the display of every Y. ruckeri positive signal throughout the entire 3D model in a single 2D section. (c) The vertical section of the skin including Y. ruckeri positive signal from the deeper part of the connective tissue (arrowhead). Lateral line canal (Lc); scale, Sc; skeletal muscle, Mu; dorsal rib bone, Dr. (B) The 3D spatial organization of whole kidney sampled at 3 dpi. In order to scan the whole kidney, it was cut into three pieces. (a) The overlay of autofluorescence and Y. ruckeri specific staining generate an image which shows that Y. ruckeri positive signals could be detected throughout the kidney. Green (a) and gray (b) colors display the anatomy of the whole kidney in 3D and flattened 2D, respectively.
Figure 6.
Immunohistochemistry of gill lamellae from un-infected and Y. ruckeri infected rainbow trout.
The sections are from (A) uninfected control, (B) 1 mpi, (C) 10 mpi, (D) 6 hpi, (E) 48 hpi and (F) 7 dpi and were stained with rabbit anti-Y. ruckeri polyclonal antibody and HRP conjugated goat anti-rabbit IgG antibody. The nuclei were counterstained with hematoxylin. Y. ruckeri was detected inside the gill lamellae already at 1 mpi and the blood capillaries in the gill lamellae at 7 dpi, indicating systemic infection.
Figure 7.
Immunohistochemistry of internal organs from Y. ruckeri infected rainbow trout.
The sections are from (A) liver, (B) spleen, (C) heart, (D) brain (optic tectum) and (E) intestine sampled 7 dpi, and (F) trunk kidney sampled 3 dpi, and stained as described in Fig. 6.
Figure 8.
Double fluorescent immunohistochemistry of the gill lamellae from un-infected and Y. ruckeri infected rainbow trout.
The Y. ruckeri+ and NKA+ cells are visualized in red and green, respectively. Sections of gill lamellae from (A) un-infected control, (B) 10 mpi, (C) 6 hpi and (D) 7 dpi are stained, as described in materials and methods. The arrowheads indicate the Y. ruckeri+ cells. No Y. ruckeri (red) were detected in the chloride cells (green).
Figure 9.
Summarizing the progression of the Y. ruckeri infection in blood and organs.
Arrows indicate the presence of Y. ruckeri in the organ at the time point, where bacterial cells were observed by OPT, and/or IHC. The solid line indicates the change of bacteria counts in blood obtained from this study.