Introduction

Sheep pox is an acute, contagious and often fatal disease of sheep, caused by a member of the Capripoxvirus group and is characterized by generalized pox lesions throughout the skin and mucous membranes, a persistent fever, lymphadenitis, and often a focal viral pneumonia with lesions distributed uniformly throughout the lungs, Bakos and Brag (1957). Under field conditions, the incubation period of Sheep pox is between 4 to 8 days. The skin lesion first appears as an erythematous area (macula). This lesion progresses to slightly blanched, raised lesion that presents erythema with edema in the central part (papule). In most of the cases, pneumonia may occur with labored breathing and a respiratory rate approaching 90 per minute. Pox lesions in the lungs may be severe and extensive; the lesions are focal and uniformly distributed throughout the lungs as the result of hematogenous infection, Davies (1976). Early lesions are congested areas; these then progress to discrete areas of congestion and edema and finally to white nodules. Lambs under one month of age may suffer a very severe generalized form of Sheep pox. The signs are exaggerated, and there is an increased mortality in young animals.

Various lesions due to sheep pox have been extensively studied by many researchers but nature and type of lesions produced by this virus in the lungs have been given very less attention so far. Hence present study was designed with the objective of determining pathology and histochemistry of sheep pox virus induced lesions in lungs of sheep.

Material and Methods

The present postmortem study was conducted on sheep that had died spontaneously during a sheep pox outbreak under field conditions in Kashmir valley. Following postmortem examination, lungs were first examined in situ and any lesions observed were noted. Then whole lungs from all the animals were collected and thoroughly screened by visual examination, palpation and dissection. Representative tissue pieces from grossly affected lungs were collected in 10% neutral buffered formalin for histopathological and histochemical examination.

Histopathological Examination

For histopathological examination tissues were processed by routine paraffin embedding technique. Briefly, the fixed tissue samples were cut into pieces of 2-3mm thickness and washed thoroughly with water for several hours before putting in ascending grades of alcohol for dehydration, followed by clearance in benzene and embedded in paraffin. Sections of 4-5 micron thickness were cut and stained with Harri’s Haematoxyllin and eosin method, Luna (1968). Duplicate sections were stained by Page-Green method for demonstration of viral inclusions Luna (1968).

Histochemical study

Histochemical study was carried on paraffin sections. Duplicate sections were selected on the basis of histopathological examination and were stained for elastin by Hart’s method, Luna (1968); neutral and acid mucopolysaccharide by Combined Alcian Blue PAS technique, Bancroft and gamble (2002); and mast cells by Toludine blue Stain, Humason (1979).

Results and Discussion

Pathology

Grossly, the lungs presented multiple variable sized nodular areas particularly in diaphragmatic lobes, with slightly grayish-white to yellowish red circular hemorrhagic areas (Fig 1). Cut sections of larger nodules were grey, firm and surrounded by edematous connective tissue (Fig 2).Histopathologically, the nodular lesions were characterized by interstitial pneumonia. There was proliferation and desquamation of the alveolar and bronchiolar epithelial cells along with perivascular lymphoid aggregation and peribronchiolar lymphoid hyperplasia (Fig 3.). Proliferative bronchiolitis and alveolitis were also observed giving the parenchyma appearance of a gland-like structure (Fig 4). Severe congestion of blood vessels and oedema was also noticed. Interalveolar septae were very much thickened and infiltrated with lymphocytes, neutrophils and macrophages (Fig 5). Occasionally syncytial cell formation as a result of fusion of macrophages resulting in the formation of the multinucleated cells was also observed. Eosinophilic intra-cytoplasmic inclusions were noticed in the pulmonary macrophages and bronchial epithelial cells (Fig 6). Alveoli adjacent to the lesion revealed epithelialisation. In the present study, the characteristic gross and histopathological lesions observed were in accordance with earlier reports, Afshar et al (1986); Hailat et al (1994); Ozmen et al (2009); Betyut (2010). Further the  proliferative nature of the pock lesions in the lungs has been attributed to the expression of pulmonary surfactant proteins, thyroid transcription factor-1 and proliferating cell nuclear antigen in the cytoplasm of type II pneumocytes and other epithelial cells as well as due to infiltration of CD3(+) T and CD79alphacy(+) B lymphocytes, Beytut (2010).

Histochemistry

Elastic fibres were thickened and were concentrically arranged around the alveoli. Disruption and disorientation of elastic fibres was also prominent in the the affected areas (Fig 7). Similar observations have been reported by earlier workers, Kuhn (1978); Langston and Fagan (1978) and Kuhn and Starcher (1980). It has been suggested that following acute destruction, the elastin content of lung regenerates; however, the newly synthesized elastin is in the form of highly disorganized fibers. The hyperplasic bronchial epithelium and glandular cells were positive for acid mucopolysaccharides where as alveolar wall was positive for neutral mucopolysaccharides (Fig 8). This may be attributed to their probable role in the inflammation Darzi et al (2003); Shah (2008). Also, increased amounts of mucopolysaccharides in and around the lesions may be attributed to prolonged irritative action of different insults, which are believed to determine hypersecretion of these substances, Lupu et al (1959).

No mast cell reaction was evident in and around the lesions. Mast cells have been found to play an important role in lung inflammation, producing compounds and cytokines that induce acute phase response and induce neutrophil infiltration besides acting as antigen presenting cells Abraham et al (1996); Abraham et al (1997). It has been suggested that mast cells disappear due to degranulation during an acute inflammatory response, as observed in pock lesions in the lungs depending on intensity of lesion and time of exposure, Cheville (1994).

The results of this study showed that sheep pox virus induces marked proliferation of type II pneumocytes, bronchiolar epithelial cells and also lung lesions in diseased sheep were mainly proliferative, which is characteristic of interstitial pneumonia.

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