Figure 1.
Experimental EHV 5 infection; gross pathology (horse E3, E4).
Nodules of fibrosis (A, between arrows, and at asterisk; horse E4) were evident beneath the pleura (cut section, B; horse E4). The nodules of fibrosis extended into the underlying alveolar parenchyma (B,C; horse E3,E4 respectively).
Figure 2.
Experimental EHV 5 infection; histopathology (horse E4, C1).
Nodules of fibrosis (horse E4 arrows, A, B) were present within the alveolar parenchyma of infected horses. The collagen (pink) disrupted the normal alveolar architecture; occasional foci of lymphocytic inflammation are evident. Higher magnification of the fibrosis with interspersed small blood vessels and mild lymphocytic infiltrates (C). Inset (C) shows normal alveolar architecture of control horse lung (C1). Magnification: A - 2x; B - 10x; C - 20x.
Figure 3.
Experimental EHV 5 infection; polarized light microscopy, picrosirius red histochemistry (horse C1, E1).
The collagen-specific dye, picrosirius red binds to collagen and fluoresces under polarized light. Within the lung parenchyma, collagen within control horse lung (C1) is restricted to branch points of alveoli and surrounding airways and blood vessels (A). Collagen is significantly increased in the alveolar parenchyma of the lungs of EHV 5 infected horses (horse E1, B). Magnification: A, B - 10x.
Figure 4.
Immunohistochemistry for α-smooth muscle actin for myofibroblasts (MFB) (horses C1, E4).
In control horse lung smooth muscle actin is present around blood vessels and within occasional alveolar interstitial MFB (A). Numerous MFB are present within the nodules of alveolar fibrosis in the lungs of EHV 5 infected horses. Similar distribution of MFB is present within the foci of alveolar fibrosis in horses with naturally-acquired EMPF (C). Inset (A) negative control, mouse serum IgG substitution for primary antibody. Magnification: A-C - 20x.
Figure 5.
Immunohistochemistry for EHV 5 (horse C2, E4).
Control horse (C2) has detectable EHV 5 antigen within scattered alveolar macrophages (arrowhead). EHV 5 infected horses (B) had detectable EHV 5 antigen within the nodules of fibrosis, including the honeycomb epithelial cells (arrow), interstitial fibroblasts, and macrophages. Similar distribution of EHV 5 was found in the lungs of naturally-acquired EMPF (C). Inset (A) negative control, rabbit serum IgG substituted for primary antibody. Magnification: A-C - 20x.
Figure 6.
Immunohistochemistry for cytokeratin (horse E4).
Cytokeratin expression within the cells of the honeycomb regions of lung (arrows) in the regions of nodular fibrosis in EHV 5 infected horses confirms their epithelial identity – the same cells expressing EHV 5 antigen in Figure 5B (arrow). Magnification 20x.
Figure 7.
Collagen concentration (µg/mg wet tissue) was determined for all horses using the Sircol assay (Biocolor Ltd, UK) in control (C1, C2) and EHV 5 inoculated (E1-E6). Collagen concentration was increased in all EHV 5 infected horses. Mean collagen was significantly increased (80 µg/mg) in EHV 5 inoculated horses compared to controls (26 µg/mg) (p < 0.5).
Figure 8.
Morphometry for interstitial lung collagen (horse C1, E1 shown).
Picrosirius red stained control horse (A) and EHV 5 infected (B); brightfield microscopy. Representative regions analyzed control horse lung (C, shown in ‘a’ above) and EHV 5 infected lung (D, E, shown in ‘b’ above); polarized light microscopy. EHV 5 infected horses had a significant increase in percent collagen within the alveolar parenchyma (F). Magnification: A, B - 2x; C-E - 20x.
Figure 9.
PCR detection of EHV 5 DNA (gH gene) from cell culture and equine tissues (Horse E4).
PCR amplicons from serial 10 fold dilutions of DNA extracted from 100 µl of stock virus A (A) or virus B (B) grown in RK-13 cells: lane 1 = 100 base pair DNA ladder; lanes 2 -10 = dilutions of viral DNA from 0 to 108. Example of EHV 5 detection in tissues collected postmortem (C). Lane 1 = 100 base pair ladder; lane 15 = EHV 5 positive control representing approximately 100 PCR detectable units of viral DNA; lane 16 = negative control. Tissue PCR results: lane 2 (nasal mucosa); lane 3 (right caudal lung lobe); lane 4 = mesenteric lymph node; lane 5 = spleen; lane 6 = liver; lane 7 = kidney; lane 8 = tracheobronchial lymph node; lane 9 = left caudal lung lobe; lane 10 = accessory lung lobe; lane 11 = submandibular lymph node; lane 12 = bone marrow; lane 13 = right cranial lung lobe. . Nucleic acid sequence analysis failed to verify that all of the amplicons were from EHV 5 inoculated into the horses (see Table 3).