Ultra Structural Changes Of The Skin Stratum Basal Layer And Basement

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Ultrastructural Changes of the Skin Stratum Basal Layer and Basement Membrane in Ovine Footrot Disease Al-Jashamy1* and S. Jasni2 1

Dept. of Microbiology and Parasitology, Faculty of Medicine, University Science Malaysia, Kubang Kerian, Kelantan. 2 Dept. of pathology and Microbiology,Faculty of Veterinary Medicine, University Putra Malaysia, Serdang, Selangor. * Corresponding email: [email protected]

ABSTRACT Ultrastructural changes in virulent footrot were mainly found in the stratum basal layer and intracellular space, where basal cells showed degenerative and necrotic changes. There were also gaps between the basal cell and the basement membrane. The ultrastructural changes of the basement membrane in the affected tissue were prominent. In the degenerative tissue of the virulent cases, both of the lamina lucida and lamina densa were discontinuous and the lucida was narrowed. In the regenerative tissue of virulent cases, giant basement membranes with many branches extending into the epidermis were present. The lucida and lamina densa were moderately thickened. The cytoplasm of basal cells had electron dense granules. Degeneration in the basal cell layer of the epidermis and the basement membrane in virulent form of footrot, which have not been reported previously, were observed in this study. KEYWORDS: Ultrastructural, Basement Membrane, Ovine Footrot

INTRODUCTION Footrot is a major disease affecting sheep. Dichelobacter nodosus has been implicated as a specific causative agent of virulent ovine footrot (Beveridge 1941). Dichelobacter nodosus is anaerobic Gram-negative rod shape microorganism with terminal enlargement (Skerman et al. 1981). Dichleobacter nodosus was isolated from cases of ovine footrot in Malaysia (Al-Jashamy et al. 2004). The ultrastructural histogenesis of the skin involves the early formation of immature desmosomes and early hemidesmosomes. The polyhedral cells of the stratum overlying the basal cell layer form mosaic and usually 5-10 layers thick. The keratinocytes are connected by desmosomes, with a dark appearance and contain numerous bundles of tonofilaments and hemidesmosomes along the basal border. Epidermal and dermal cells are separated by basement membrane (David et al. 1997; Ghadially 1997). There was no documentation on the ultrastructural changes in footrot lesions. The objective of the present study was to describe the ultrastructural changes of the skin stratum basal layer and basement membrane associated with virulent footrot. MATERIALS AND METHODS Five interdigital skin specimens measuring 1 mm3 from infected feet with active virulent ovine footrot and other five specimens from recovered (inactive) virulent cases after kept the infected animals in dry places were collected for this study. Specimens were examined under transmission electron microscopy (TEM). The specimens were fixed with 2.5 % glutaraldehyde for 24 hours at 4oC. The specimens were then washed three times in 0.1 M sodium cacodylate 36

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buffer at pH 7.3 for 10 minutes each, postfixed for 2 hours with 1% osmium tetroxide (OsO4). Fixed specimens were then rinsed and dehydrated in a graded acetone series. The specimens were embedded in resin. Semithin sections, 1 μm thick were cut by microtome (Ultracut E. Recher-jung, Austria). The sections were stained with toluidine blue and viewed prior to ultrathin sectioning in order to select suspected areas. Ultrathin sections of 60-90 nm cut from selected areas were then mounted onto 400-mesh-coper grids for ultrastructural studies. The sections were stained with uranly acetate for 10 minutes, washed with 50% filtered alcohol, counter stained with lead citrate for 10 minutes and washed with double distilled water. The sections were examined using a transmission electron microscope (Hitachi H7100, Japan). RESULTS Ultrastructural changes in virulent footrot were mainly found in the stratum basal layer and intracellular space. The affected basal cells showed degenerative, necrotic changes, and the cells were rounded, abnormal nuclei, vacuolation, fragmentation and dispersed cytoplasmic organelles, distended nuclear space and irregular cell membrane. There were also gaps between the basal cell and the basement membrane. Desmosomes and intermediate filaments were irregular and few in number (Figure 1). The ultrastructural changes of the basement membrane in the affected tissue were prominent.

Figure1: Transmission electron micrograph of skin of sheep infected with the D. nodosus virulent strain, showing stratum basal cells that are irregular in shape and have vacuolated nucleoli (thick arrow), fragmented and dispersed cytoplasmic organelles, distended nuclear space (arrow head), degeneration and irregular intracellular space (thin arrow). Lead citrate and uranyl acetate X 4,710.

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Figure3: Transmission electron micrograph of skin of sheep infected with the D. nodosus virulent strain (inactive case) showing: an increase in number of keratohyalin granules (G), desmosomes and melanosomes (M).Giant basement membrane with many branches extended through the epidermis (thick arrow) thickening of the lamina lucida and lamina densa (thin arrow), increased number of hemidesmosome (thick arrowhead). The reticular lamina is thickened and containing elastic fibers with regular distribution and orientations and very thin collagen fibrils (*). Lead citrate and uranyl acetate. X 79,380 and b, X 44,000.

Figure2: Transmission electron micrograph of skin of sheep infected with the D. nodosus virulent strain, showing that basement membranes in the degenerative tissue are narrowed in the lucida (thin arrow), attenuated and discontinuous in the lamina densa (thin arrowhead). There is lysis of the hemidesmsome (thick arrow), variable thickening of the reticular lamina with elastic fibers. Some elastic fibers have amorphous dense material (thick arrowhead). Lead citrate and uranyl acetate, X 35,467.

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In the degenerative tissue of the virulent cases in both of the lamina lucida and lamina densa were discontinuous and the lucida was narrowed. Lysis of the hemidesmosome, electron-dense anchoring fibrils, and microfibrillar bundles appeared at different orientations. The thickeness of the reticular lamina was not uniform and some elastic fibers had amorphous electron dense materials (Figure 2). In the regenerative tissue of virulent cases, giant basement membranes with many branches extending into the epidermis were present. The lucida and lamina densa were moderately thickened. Hemidesmosomes were attached to keratin filaments and anchoring fibrils and microfibrillar bundles had extended in various directions. The cytoplasm of basal cells had electron dense granules. Keratohyalin granules between the keratinocytes had an increased number of desmosomes and melanosomes. The reticular lamina was markedly thickened and contained elastic fibers and thin collagen fibrils. The elastic fibers had a regular distribution and orientation (Figure 3). DISCUSSION Although many studies have been conducted on the pathogenesis of footrot, there is no documentation on the ultrastructural alterations of the affected feet in sheep and other susceptible animals. The scanning electron microscopy of foot lesions caused by virulent strain of D. nodosus showed a severe digestion of the horny layer substance around the bacterial cells. The similar observations were seen with Moraxella bovis in bovine keratoconjunctivitis (Hirsh and Zee, 1999). The use of electron microscopy of footrot lesions in the present study has clearly revealed the presence of D. nodosus ultrastructural changes in the epidermis and dermis affected. Cellular regeneration was evident in cases of inactive virulent D. nodosus under non-suitable conditions in which D. nodosus produces less amount of proteolytic enzyme. An increase in the intercellular space and a decrease in the number of intercellular bridges or desmosomes between keratinocytes were obvious in the virulent footrot seen in this study. The keratinocytes had loose bundles and amorphous intermediate filaments, tonofibrils and intracellular tonofilaments. Intracellular spaces have glycosaminoglycans, while the intercellular cement and desmosomes contain similar keratin based elements. Previous reports have indicated that the intercellular cement substances contain a gel-like substance that provides cohesion between the keratinocytes (David et al. 1997). The intermediate filaments, tonofibrils and tonofilaments structures have prekeratin or prekeratin-like protein called cytokeratins (Franke et al. 1978; Franke et al. 1980). Desmosome and the plaque are also composed of cytokeratin (Schmelz et al. 1986). In the present study, virulent footrot caused the interface and dermoepidermal junction to undergo ultrastructural changes. The presence of regeneration and formation of a giant basement membrane in inactive virulent footrot suggests that the D. nodosus enzyme has a sccharolytic activity on the basement membrane and that the basement membrane has the ability to regenerate and rebuild when this enzymatic activity stops. The basement membrane consists of neural mucopolysaccharides. This suggests that the D. nodosus utilize these substrates for its survival. These ultrastructural changes of the interdigital skin infected with virulent footrot provide further understanding of footrot pathogenesis and potential activity of proteolytic enzymes of D. nodosus on the skin cells organelles. Dewhirst et al. (1990) reported that D. nodosus had saccharolytic ability. The marked reduction in the numbers of desmosomes and the loss of structural cohesion between adjacent epithelial cells in the affected foot may indicate that the wide intercellular gaps between the epithelial cells and the tissue result in edema because of high cellular permeability due to the proteolytic bacterial enzymes. The pathological picture is supported by the finding of Ghadially (1997) who indicated the intermediate junction and desmosomes were impermeable and thus provide a firm mechanical seal between cells. Therefore, the D. nodosus located in distended intercellular gaps of epidermis may have a chance to persist for a long duration in chronic foot 39

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lesions. Furthermore, D. nodosus was found to be surrounded by a clear zone of degenerative elastin and collagen fibers as well as many degenerate and lysed bacteria close to active neutrophils and macrophages. Dichelobacter nodosus can enter the dermis from the degenerate basal cell and basement membrane and progress toward the dermis. The bacteria were later destroyed by phagocytes and the humoral bactericidal system as described by Roberts and Egerton (1969); Whittington et al. (1990). The occurrence and severity of footrot lesions in the sheep depends on rainfall, temperature and the virulence of D. nodosus strains. A combination of high incidence and persistence of the disease in most affected sheep was reflected in a sharp increase in the prevalence of footrot. The outbreaks are usually confined to regions that have a sufficiently high annual average rainfall and experience dampness, warm weather and a susceptible host together with a source of infection. Wet conditions cause softening of the soft and hard hoof allowing easier access to the invasive bacteria (Stewart and Claxton 1993; Locke and Coombes 1994). Warm conditions facilitate the growth of the bacteria (White,1991; Glynn, 1993). The ultrastructural study showed basement membrane degeneration and bacterial cell division and high affinity of D. nodosus to the elastin fibres in the dermis. Therefore, the virulent strains of D. nodosus also exhibited highly proteolytic activity in the dermis. These results suggested that sequels of the disease were very harmful to the foot and the regeneration of the new dermis and epidermis was difficult to rebuild. Thus lead to lameness. CONCLUSION Today we can use the electron microscope to reveal ultrastructural changes leading to footrot. Such findings should help in understanding the pathogenesis of footrot disease and the effect of the proteolytic enzymes of D. nodosus on the skin cells organelles, and basement membrane that may not be revealed completely by light microscopy. The ultrastructural study showed basement membrane has an ability to rebuild its structures. ACKNOWLEDGEMENTS This study was supported by the Malaysian Government’s IRPA No 51488. REFERENCES Al-Jashamy, K. Jasni, S. Al-Salihi K., and Sheikh-Omar A.R. (2004). Immunocytochemical labelling of Dichelobacter nodosus fimbriae using an immuogold technique. Annals of microscopy, Vol. 4: 45-47. Beveridge, W. I. B. (1941). Footrot in sheep a transmissible disease due to infection with Fusiformis nodosus studies on its epidemiology and control. Bulletin of Council for Scientific and Industrial Res. Aust. 140: 1-26. Claxton, P. D., Ribeiro, L. A. and Egerton, J. R. (1983). Classification of Bacteroides nodosus by agglutination tests. Aust. Vet. J. 60 (11), 331-334. David, E., Rosalie, E., Christine, J. and Bernett, J. J. (1997). Lever’s histopathology of the skin. Eighth edition, by Lippincott-Raven Publishers. pp. 5-50, 1020-1044. Dewhirst, F. E., Paster, B. J., Fontaine, S. L. and Rood, J. I. (1990). Transfer of Kingella indogenes (Snell and Lapage 1976) to the genus Suttonella gen. nov. as Suttonella indogenes comb. nov. transfer of Bacteroides nodosus (Beveridge 1941) to the genus Dichelobacter gen. nov. as Dichelobacter nodosus comb. nov.; and assignment of the Genera Cardiobacterium, Dichelobacter and Suttonella to Cardiobacteriaceae Fam. nov. in the gamma division of Proteobacteria on the basis of 16S rRNA esquence comparission. Int. Syst. Bact.40 (4), 426-433. 40

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Franke, W.W., Weber, K., Osborn, M., Schmid, E. and Freudenstein, C. (1978). Antibody to prekeratin. Decoration of tonofilaments-like arrays in various cells of epithelial character. Exp. Cell Res. 116, 4429. Franke, W.W., Schmid, E., Freudenstein, C., Appelhans, B., Osborn, M., Weber, K. and Keennan, T. W. (1980). Intermediate-size filaments of the prekeratin type myoepithelial cells. J. Cell Biol. 84, 633. Ghadially, F. N. (1997). Ultrastructural pathology of the cell and matrix. 4th ed, Vol. 2. ButterworthHeinmann. USA. pp. 822-936, 1181-1355. Glynn, T. (1993). Benign footrot-An epidemiological investigation into the occurrence of effects on production, response to treatment and influence of environment factors. Aust. Vet. J. 70. 7-12. Hirsa, D. C and Zee, Y. C. (1999). Veterinary Microbiology. 1st ed. Blackwell science, Inc, USA, pp 151-154. Lock R. H. and Coombes N. E. (1994). Prevalence of virulent footrot in sheep flocks in southern New South Wales. Aust. Vet. J. 71 (10) 348-349. Roberts, D. S. and Egerton, J. R. (1969). The aetiology and pathogenesis of ovine footrot II: the pathogenic association of Fusiformis nodosus and F. necrophorum. J. Comp. Pathol. 79, 217-227. Schmelz, M., Duden, R., Cowin, P. and Franke, W.W. (1986). A constitutive transmembrane glycoprotein of Mr165000 (desmoglein) in epidermal and ono-epidermal desmosomes. II. Immunolocalisation and microinjection studies. Eur. J. Cell Biol. 442, 1884. Skerman, T. M., Erasmuson, S. K. and Every, D. (1981). Differentiation of Bacteroides nodosus biotypes and colony in relation to their virulent and immunoprotective properties in sheep. Infect. Immun. 31, 788-95. Stewart, D. J. and Claxton, P. D. (1993). Ovine footrot clinical diagnosis and bacteriology. In Australian Standard Diagnosis Techniques for Animals Disease. CSIRO. pp, 1-26. White, A. (1991). Footrot in sheep. Vet. Ann. 31, 85-89. Whittington R. J., Marshall. D. J., Walker R. I. and Turner, M. J (1990). Serum antibody responses in sheep after natural infection with Bacteroides nodosus. Aust. Vet. J. 67 (3), 98-101.

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