Histopathological Findings of Foot-Rot Disease Which Causes Deaths in a Sheep Flock

Acta Scientiae Veterinariae, 2017. 45(Suppl 1): 243. CASE REPORT Pub. 243 ISSN 1679-9216 Histopathological Findings of Foot-Rot Disease Which Causes Deaths in a Sheep Flock Turan Yaman, Ahmet Uyar, Omer Faruk Keles & Zabit Yener ABSTRACT Background: Foot-rot is a contagious bacterial infection of the feet of sheep that causes lameness. This infection leads to major economic losses in wool, meat, and dairy industries throughout the world. Pathogenesis of foot-rot can be described as the damage of the interdigital skin, invasion of fecal bacterium Fusobacterium necrophorum, and finally the involvement of Dichelobacter nodosus in the infection. In the worst cases, the disease becomes widespread, and systemic bacterial infections may occur. The present study aims to describe macroscopic and histopathological findings of foot-rot lesions formed in the foot, heart, liver, and lungs to draw attention to these aspects of the disease. Case: The material of the study consisted of a 3-year-old sheep. The investigation was composed of the disease history; post-mortem examination; and histopathological analysis of lung, liver, heart, and foot lesions. At the anamnesis, it was reported that the diseased animal had lameness and weakness with other clinical symptoms, and approximately 10 infected sheep had died within a 1-week period. At necropsy, malodorous ulcerative lesions between the nails; focal-disseminated foci in the liver, lung, and heart; yellowish matte thickening in the heart valves; and hydropericardium were detected. After necropsy, tissue samples taken from the skin, lungs, spleen, kidneys, and liver were fixed in a 10% buffered formalin solution, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Giemsa staining was done to detect the agents in the tissues. The slides were examined and photographed using a light microscope. Histopathological examination revealed that ulcerations between the nails were lesions shaped as the result of the foot rot disease. The focal-disseminated lesions in the internal organs were the foci of metastatic-embolic inflammation originating from the lesions between the nails. Rod-shaped bacteria were detected in foot, liver, and lung tissues by Giemsa staining. Discussion: Foot-rot has important economic and welfare impacts. A number of researches on the characterization of agents, regional incidence, pathogenesis, and macroscopic characterization and classification of lesions in the nail have been carried out on footrot disease. In the present case, the disease became systemic and numerous sheep died. Systemic infections are characterized embolic-metastatic inflammations formed in major vital organs such as the liver, lungs, brain, heart, and kidneys as the result of the vasculitis formed in the veins in the inflamed region, where the agents are spread by the bloodstream. Histopathologically, granulomatous foci were seen in the liver and lungs. In the middle of these foci were collapsed leukocytes and agents, and the surrounding area was composed of Langhans, foreign body giant cells, and mononuclear cells. Treatment with systemic infections is quite difficult. The disease can be treated, but the cost of treatment and care is very high. For this reason, foot-rot disease is among the most expensive sheep diseases requiring costly treatment. Because we believe foot-rot disease not only causes lameness but also leads to high mortality rates in sheep flocks, we presented the pathological findings to draw attention to these aspects of the disease. As far as we can investigate, no case reports have presented both macroscopic and microscopic findings of the disease that have formed in nails and internal organs. Keywords: foot-rot, liver, lung, histopathology, sheep. Received: 20 July 2017 Accepted: 25 October 2017 Published: 28 November 2017 Department of Pathology, Faculty of Veterinary Medicine, Yuzuncu Yil University, Van, Turkey. CORRESPONDENCE: T. Yaman [turanyaman@yyu. edu.tr - Tel.: +90 04322251128]. Department of Pathology Faculty of Veterinary Medicine, Yüzüncü Yil University. 65090 Van, Turkey. 1

T. Yaman, A. Uyar, O.F. Keles & Z. Yener. 2017. Histopathological Findings of Foot-Rot Disease Which Causes Deaths in a Sheep Flock. Acta Scientiae Veterinariae. 45(Suppl 1): 243. INTRODUCTION ined and photographed using a light microscope (E400)1 equipped with a DS-Ri2 video camera (DS-U3)1. At necropsy, a necrotic-ulcerative dermatitis area was observed on the interdigital skin. The nails were undergoing deformation (Figure 1). Upon examination of the internal organs, sub-pleural, yellowish white, focal-disseminated, pinhead-sized foci were found in the lungs (Figure 3A). Yellowish white, focaldisseminated, pinhead-sized foci were also found in the liver (Figure 4A). In the heart, examination detected common focal centers of similar color at the myocar- Foot-rot is a common and contagious bacterial disease of sheep that causes lameness [19]. This disease leads to major economic losses in wool, meat, and dairy industries and reduces sheep welfare [15,20]. Foot-rot lesions show a clinical course that always starts at the interdigital skin, causing extensive necrosis and ulceration [6,9]. Humid and rainy climates, malnutrition and poor foot care and trauma are effective as predisposing factors [8]. Eventually, it results with the separation of the hoof horn from the sensitive tissue of the claw, with a gray scum in the formed cavity and a characteristic smell [12]. The disease occurs with the synergistic action of the Dichelobacter nodosus (D. nodosus) and Fusobacterium necrophorum (F. necrophorum) bacteria [11,20]. D. nodosus is a rod-shaped, Gram-negative, anaerobic bacterium that has proteases and keratinases that cause the disruption of nails [5,16]. This factor is the primary causal agent of footrot disease [6,19]. F. necrophorum is considered to be essential for the induction and progression of the disease [12]. It initially causes a mild and superficial localized dermatitis (interdigital dermatitis) and produces a number of toxins that cause necrosis of the superficial layer of the interdigital skin [12,18]. In severe cases, infection progresses to the foot joints and also inflames them. Thus, the disease becomes widespread, and systemic bacterial infections may occur [3]. This case report describes macroscopic and histopathological findings of foot-rot lesions formed between the nails and in the foot, heart, liver, and lungs to draw attention to the importance of foot-rot disease, which causes death in a sheep flock. Figure 1. Gross appearance of the necrotic-ulcerative (star) dermatitis on the interdigital area. CASE A 3-year-old sheep was brought dead to the Faculty of Veterinary Medicine of Yüzüncü Yil University. According to the sheep owner s statement, the disease had started approximately 1 week earlier and spread in the flock. After the owner had observed lameness and weakness in the flock, 10 of 85 sheep had died. It was also reported that no results were obtained from the treatment. After necropsy, tissue samples taken from the skin, lungs, spleen, kidneys, and liver were fixed in a 10% buffered formalin solution, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Giemsa staining was done to detect the agents in the tissues. The slides were exam- Figure 2. Histopathological appearance of necrotic-ulcerative dermatitis: Normal epidermis (arrow), ulceration area and broad coagulation necrosis (*) and a line consist of leukocytes and bacterial proliferation around the necrosis (arrowhead). 2

T. Yaman, A. Uyar, O.F. Keles & Z. Yener. 2017. Histopathological Findings of Foot-Rot Disease Which Causes Deaths in a Sheep Flock. Acta Scientiae Veterinariae. 45(Suppl 1): 243. blue necrotic mass formed by leukocytes and possibly by agents. Around the mass was an inflammatory reaction formed by macrophages and the giant cells of the foreign body and Langhans. In the heart, examination identified degenerative-necrotic changes in myocardial cells, as well as a metastatic valvular endocarditis table characterized by leukocyte infiltration diffused in the interstitium (Figure 5B). Rod-shaped bacteria were detected in foot, liver, and lung tissues by Giemsa staining. Figure 3. Embolic-metastatic granulomatous pneumonia: A- Sub-pleural, focal-disseminated pinhead-sized foci in the lungs (arrows). B- A necrotic mass formed by leukocytes and possibly by agents. Around the mass was an inflammatory reaction formed by macrophages and the giant cells of the foreign body (arrows) and Langhans (arrowhead). DISCUSSION In this manuscript, we reported pathological findings of foot-rot that led to death in a sheep flock. Foot-rot is often a difficult-to-treat disease that occurs in the feet of Equidae and ruminants, with necrotic lesions [15]. Sheep with foot-rot cannot walk, they lose weight, and their milk yields decrease. If treatment and care are delayed, the foot-rot will become wormed, and the animals that fall into cachexia will die. In severe cases, deaths are related to the spreading of the disease [3]. Systemic infections are characterized embolicmetastatic inflammations formed in major vital organs such as the liver, lungs, brain, heart, and kidneys as the result of the vasculitis formed in the veins in the inflamed region, where the agents are spread by the bloodstream [3]. Treatment with systemic infections is quite difficult. The disease can be treated, but the cost of treatment and care is very high. For this reason, foot-rot disease is among the most expensive sheep diseases requiring costly treatment [3]. The severity of foot-rot may vary depending on the virulence factors of D. nodosus strains [1]. Benign strains of D. nodosus cause interdigital skin inflammation, which is impossible to distinguish clinically from interdigital dermatitis caused by F. necrophorum. In benign footrot infections, spontaneous regression occurs even in the most affected animals after normalization of the environmental conditions [12]. Malignant footrot is characterized by the separation of the corneal tissue and deeper parts of the hoof. In this latter case, virulent strains produce proteases that allow the corneal tissue of the nail to deteriorate [7]. The initiation of detachment is a consequence of protease enzyme activity prepared by D. nodosus organisms [10]. Macroscopic examination first reveals that the skin in the interdigital region and along the corona becomes red, swollen, and moistened with a sticky Figure 4. Embolic-metastatic granulomatous hepatitis: A- Multifocal, pinhead-sized foci in the liver (arrows). B- A necrotic mass formed by leukocytes and possibly by agents. Around the mass was an inflammatory reaction formed by macrophages and the giant cells of the foreign body (arrows). Figure 5. A- Yellowish matte thickening in the right atrioventricular (tricuspid valve) valves. B- Degenerative-necrotic changes in myocardial cells and extensive leukocyte infiltration in the interstitium. dium, especially yellowish matte thickening in the right atrioventricular (tricuspid valve) valves (Figure 5A). Hydropericardium was also present. Histopathological examination of the tissue specimens taken from the lesion showed an inflammatory line consisting of normal epidermis, an ulceration area, and broad coagulation necrosis, with bluish dense leukocytes and bacterial proliferation around the necrosis (Figure 2). The histopathological examination of the tissue specimens taken from the lungs and liver revealed embolic-metastatic granulomatous pneumonia (Figure 3B) and embolic-metastatic granulomatous hepatitis (Figure 4B) scenes. In these granulomatous lesions in both organs, examination revealed a pinkish 3

T. Yaman, A. Uyar, O.F. Keles & Z. Yener. 2017. Histopathological Findings of Foot-Rot Disease Which Causes Deaths in a Sheep Flock. Acta Scientiae Veterinariae. 45(Suppl 1): 243. exudate. The amount of the emerging exudate gradually increases and becomes malodorous. Then, this inflammation spreads to the heel. In addition, the infection continues to progress with necrosis of the sole and results in erosive-ulcerative dermatitis [17]. A bad smell, characteristic of anaerobic bacterial activity, accompanies the development of clinical findings with an accumulation of gray pasty scum between the sensitive dermis and epidermal horn [10]. In this case, similar macroscopic findings in the nails were detected. Also, focal-disseminated metastatic-embolic foci in the liver, lung, and heart; yellowish matte thickening in the heart valves; and hydropericardium were detected. Microscopically, at onset, there is a moderate leukocyte infiltration in the granulosum and spinosum layer of the epidermis of the nail. A mild inflammation, recognized by a leakage of fairly rich edema with the inflammatory cells, is seen on corium ungulae. As all these events progress, necrosis develops in the upper parts of the epithelial tissue. In progressive cases, ulceration and extensive coagulation necrosis occur [3]. In the present case, ulcerations and large coagulation necrosis areas were presented. Around these necrosis areas was a line of intense leukocyte and bacterial proliferation. As a result of systemic infection, granulomatous foci were seen in the liver and lungs. In the middle of these foci were collapsed leukocytes and agents, and the surrounding area was composed of Langhans, foreign body giant cells, and mononuclear cells. We think that these foci are the result of metastasis of Dichelobacter nodosus. Because the foci of Fusobacterium necrophorum are characterized by areas of coagulation necrosis. A number of researches on the characterization of agents [4], regional incidence [14], pathogenesis [13], and macroscopic characterization and classification of lesions in the nail [2] have been carried out on foot-rot disease. In the present case, the disease became systemic and numerous sheep died. Because we believe foot-rot disease not only causes lameness but also leads to high mortality rates in sheep flocks, we presented the pathological findings to draw attention to these aspects of the foot-rot disease. As far as we can investigate, no case reports have presented both macroscopic and microscopic findings of the disease that have formed in nails and internal organs. Also, this information provides an improved understanding of the course and pathogenesis of foot-rot. MANUFACTURER 1 Nikon Corporation. Tokyo, Japan. Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES 1 Abbott K.A. & Egerton J.R. 2003. Effect of climatic region on the clinical expression of footrot of lesser clinical severity (intermediate footrot) in sheep. Australian Veterinary Journal. 81(12): 756-762. 2 Aguiar G., Simoes S.V., Silva T.R., Assis A.C., Medeiros J., Garino Jr. F. & Riet-Correa F. 2011. Foot-rot and other foot diseases of goat and sheep in the semiarid region of northeastern Brazil. Pesquisa Veterinaria Brasileira. 31(10): 879-884. 3 Alibasoglu M. & Yesildere T. 1988. Veteriner Sistemik Patoloji. 2nd edn. Ankara: Konia, 118-119p. 4 Baran V., Yayla S., Kilic E., Ozaydın I., Aksoy O. & Ermutlu C.S. 2015. The effects of pasture characteristics and seasonal differences on sheep foot diseases: A field study on the Kars and Iğdır regions - Turkey. Kafkas Universitesi Veteriner Fakultesi Dergisi. 21(3): 377-382. 5 Bennett G., Hickford J., Sedcole R. & Zhou H. 2009. Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot. Anaerobe. 15(4): 173-176. 6 Beveridge W.I.B. & Gregory T.S. 1941. Foot-rot in sheep: A transmissible disease due to infection with Fusiformis nodosus (n. sp.). Bulletin no. 140. Melbourne: Council for Scientific and Industrial Research, 56p. 7 Billington S.J., Johnston J.L. & Rood J.I. 1996. Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus. FEMS Microbiology Letters. 145(2): 147-156. 8 Celebi O., Otlu S., Sahin M., Buyuk F., Ermutlu C.S., Saglam A.G., Celik E. & Akca D. 2016. The isolation of Dichelobacter nodosus and identification by PCR from ovine footrot in Kars district, Turkey. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 22(2): 221-224. 9 Deane H.M. & Jensen R. 1955. The pathology of contagious foot rot in sheep. American Journal of Veterinary Research. 16(59): 203. 4

T. Yaman, A. Uyar, O.F. Keles & Z. Yener. 2017. Histopathological Findings of Foot-Rot Disease Which Causes Deaths in a Sheep Flock. Acta Scientiae Veterinariae. 45(Suppl 1): 243. 10 Egerton J.R., Roberts D.S. & Parsonson I.M. 1969. The aetiology and pathogenesis of ovine foot-rot: I. A histological study of the bacterial invasion. Journal of Comparative Pathology. 79(2): 207IN7-216. 11 Foddai A., Green L.E., Mason S.A. & Kaler J. 2012. Evaluating observer agreement of scoring systems for foot integrity and footrot lesions in sheep. BMC Veterinary Research. 8(1): 65. 12 Green L.E. & George T.R.N. 2008. Assessment of current knowledge of footrot in sheep with particular reference to Dichelobacter nodosus and implications for elimination or control strategies for sheep in Great Britain. The Veterinary Journal. 175(2): 173-180. 13 Kennan R.M., Han X., Porter C.J. & Rood J.I. 2011. The pathogenesis of ovine footrot. Veterinary Microbiology. 153(1): 59-66. 14 Moore L.J., Wassink G.J., Green L.E. & Grogono-Thomas R. 2005. The detection and characterisation of Dichelobacter nodosus from cases of ovine footrot in England and Wales. Veterinary Microbiology. 108(1): 57-67. 15 Petrov K.K. & Dicks L.M. 2013. Footrot in clawed and hoofed animals: Symptoms, causes and treatments. Biotechnology & Biotechnological Equipment. 27(1): 3470-3477. 16 Raadsma H.W. & Egerton J.R. 2013. A review of footrot in sheep: aetiology, risk factors and control methods. Livestock Science. 156(1): 106-114. 17 Riet-Correa F. 1987. Enfermedades Del aparato locomotor. In: Marlan J.B., Campo A.D. & Mari J.J. (Eds). Enfermedades de los Lanares. 2nd edn. Hemisferio Sur, Montevideo, pp.219-238. 18 Wani S.A. & Samanta I. 2006. Current understanding of the aetiology and laboratory diagnosis of footrot. The Veterinary Journal. 171(3): 421-428. 19 Witcomb L.A., Green L.E., Kaler J., Ul-Hassan A., Calvo-Bado L.A., Medley G.F. & Wellington E.M. 2014. A longitudinal study of the role of Dichelobacter nodosus and Fusobacterium necrophorum load in initiation and severity of footrot in sheep. Preventive Veterinary Medicine. 115(1): 48-55. 20 Zhou H., Bennett G. & Hickford J.G. 2009. Variation in Fusobacterium necrophorum strains present on the hooves of footrot infected sheep, goats and cattle. Veterinary Microbiology. 135(3): 363-367. www.ufrgs.br/actavet CR243 5