Figure 1.
Histopathology of the organs from the immunized animals, the control animals, and the naïve control animal.
Tissue sections were stained with hematoxylin and eosin for pathological examination after infection with Y. pestis. (a) Tissue sections from the naïve control animal that was neither immunized with plague vaccines or aluminum hydroxide adjuvant nor infected with Y. pestis. (b) Tissue sections from the control animal that only received aluminum hydroxide adjuvant before it was infected by virulent Y. pestis strain 141. (c) Tissue sections from the animals immunized with SV1 and then infected with virulent Y. pestis strain 141. (d) Tissue sections from the animals immunized with SV2 and then infected with virulent Y. pestis strain 141. (e) Tissue sections from the animals immunized with EV76 and then infected with virulent Y. pestis strain 141.
Figure 2.
Giemsa staining was done to visualize the bacteria in tissues.
The bacteria were clearly visible within the tissues from the lungs, livers, kidneys, lymph nodes, brains, spleens, and blood from heart of the control animals that were only immunized with aluminum hydroxide adjuvant before they were infected with virulent Y. pestis strain 141. The lungs showed several large clumps of bacteria in the alveolar septum (a, arrows). Bacteria were also found inside blood vessels in the lungs (b, arrows) and blood from heart (d, arrows). Several large clumps of bacteria were shown in the hepatic sinus (c, arrows). Medullary cords of the lymph nodes were damaged and occupied by a large number of bacteria (e, arrows). The spleen show many large clumps of bacteria (f, arrows). Smaller clumps of bacteria are shown within the glomcrulus (g, arrows) and blood vessels in brain tissue (h, arrows).
Figure 3.
Transmission electron micrographs of the organs of the control animals.
Transmission electron micrographs of the lymph nodes, spleen, liver, and lungs of the control animals that were only immunized with aluminum hydroxide adjuvant and then infected with virulent Y. pestis strain 141. The tissues were fixed in 3.1% glutaraldehyde solution and then postfixed in 1% osmium tetroxide. Afterwards, the tissue samples were dehydrated with a serial alcohol gradient, and then embedded in Epon812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a Philips Tecnai 10 transmission electron microscope. Ultrastructural examination shows that lymph node tissue structures were destroyed and a large number of bacteria are found in the interstitium (a). The Splenic pulp shows a large number of bacteria and its structure was destroyed (b). A large number of bacteria are found around Kupffer's cells (c). Three Y. pestis were observed in blood vessel of lungs (d).
Figure 4.
The F1 antigen of Y. pestis was identified by Immunohistochemitry staining.
The F1 antigen of Y. pestis was identified by Immunohistochemitry staining in formalin-fixed paraffin-embedded tissues from the control animals that were only immunized with aluminum hydroxide adjuvant and then infected with virulent Y. pestis strain 141. The sections were incubated for 12 h with the purified rabbit anti-F1 antigen of Y. pestis polyclonal antibody at 4°C, and incubated for 10–20 min with the polyperoxidase-anti-rabbit IgG at 37°C. The slides were stained with 3, 3′-diaminobenzidine tetrahydrochloride (DAB). Finally, the sections were rinsed, counterstained, dehydrated, cleaned, mounted and examined by light microscopy. Bacteria were visualized as those expressing F1 antigen (brown and yellow stain). Numerous bacteria were observed in the tissues of the lymph node (a), spleen (b), liver (c), kidneys (d) or lungs (e). No bacterium was found in the control spleen (f).
Figure 5.
Ultrastructure of the glomerular basement membranes of the immunized animals after the Y. pestis challenge.
The control animals infected with Y. pestis were observed under a transmission electron microscope. Transmission electron micrograph of the glomerular basement membrane (GBM) of the control animal that that were only immunized with aluminum hydroxide adjuvant and then infected with virulent Y. pestis strain 141 (a), the animal immunized with EV76 (b), the animal immunized with subunit vaccine SV1 (c), and the animal immunized with subunit vaccine SV2 (d).