The sources of materials were Teaching Veterinary Clinical Complex (TVCC), College of Veterinary Science, Khanapara; State Veterinary Dispensary, Chenikuthi; Govt. of Assam and the other private clinics within the Guwahati city. Dogs with the history of fever, purulent oculo-nasal discharges, pneumonia, pustular dermatitis and neurological disorders were considered as suspected cases of CD for the present study. Samples were collected from a total of 60 clinically suspected cases. Three ml of blood was aseptically collected in EDTA vacutainer and stored at−20°C for further analysis by RT-PCR.

Tissue samples from different organs showing specific lesions were collected in 10% formal saline solution at the ratio of 1:10 for histopathological examination. After proper fixation, tissue samples were processed for routine histopathological examination as per standard procedure (H&E) (Culling, 1974).

The most frequently observed clinical signs were respiratory distress characterized by pneumonia, coughing, laboured breathing and ocular and nasal discharge. Initially, nasal and ocular discharge was watery in nature, which later becomes mucopurulent leading to attachment of both the eyelids. This reports correlate with the findings of Mortella et al. (2006) and Zhao et al. (2015). High rise of body temperature from 102.8-105°F, anorexia, lethargy, dehydration and weight loss were noticed in all the animals at the initial phase of the disease. Similar findings were reported by Elia et al. (2015). Biphasic fever was recorded as a prominent clinical signs in almost all the affected animals. The initial rise of temperature was seen at the viraemic stage when virus was circulating in the blood, following rise of temperature was reported due to the secondary bacterial infections as CDV results immunosupression by affecting lymphoid organs. In young animals, pustular dermatitis was noticed in the ventral abdominal wall, inner side of the thigh and ears. Both hyperkeratosis and parakeratosis of the skin around the eyes and nose were observed in some of the dogs more than two years of age. Foot pads were hard to touch and painful due to hyperkeratosis (Fig. 1) which supports the findings of Carvallo et al. (2012). In some animals, neurological disorders were evident, which includes salivation, partial to generalized seizure, paddling movements and tremor.

Few chronic cases were reported with posterior paralysis. Neurological signs appear as CDV may affect the grey and white matter of the central nervous system (Amude et al., 2007).

RT-PCR was used as a diagnostic tool for the detection of CDV in the present study. Amplification of NP gene was found in 41.67 percent (n=25) cases out of total 60 sample tested. The positive samples and control showed clear band at 600bp against absence of band in negative control (Fig. 2). Since the NP gene is required for replication of the virus, the mRNA of the NP gene is transcribed more extensively in the infected cells (Shin et al., 1995) and appears to be a good marker of infection (Alcalde et al., 2013; Yi and Cheng 2014).

Necropsy was performed in six animals found positive in RT-PCR. The brain lesions observed were haemorrhages, congestions and meningitis (Fig. 3). Similar findings were also reported by Machida et al. (1993). The virus has the affinity towards central nervous system (Jones and Hunts, 2006) that could be the cause of gross alteration in the CNS. Lungs of the affected animals showed congestion, focal emphysema, dark patchy pneumonic areas in all the lobes especially in the diaphragmatic lobes indicate bronchopneumonia (Fig. 4). The affected lungs were dark in colour and heavy. Squeezing of the cut section of the lungs revealed frothy exudates coming out particularly from the bronchi and bronchioles. The tracheal lumen also contains mucopurulent exudets and the mocosa was congested. These findings correlate with the reports of earlier workers (Lan et al., 2006; Kubo et al., 2007). From the previous literature it was revealed that lung was the target organ for CDV and initial replication of the virus take place in the macrophages of the lungs (Beineke et al., 2009). The stomach was found to be empty with some watery contents in all the 6 positive cases. Haemorrhages were observed across the mucosal folds in the fundic and pyloric junction of the stomach. The intestinal mucosa showed congestion and haemorrhages with thickening of the intestinal wall in some cases. Similar findings were recorded by Galan et al. (2014) and Zhao et al. (2015).

The kidneys were congested and haemorrhagic with presence of necrotic foci over the cortex. This finding is correlates with Jones and Hunts (2006). The urinary bladder was found distended and on opening blood tinged urine was came out. The mucosal surface of the bladder revealed petecheal to diffuse ecchymotic haemorrhages spread over the whole mucous membrane. Congestion with mild to moderate enlargement of the lymphoid organs including spleen was noticed. The lesions were marked in the mesenteric lymph nodes. This gross alteration may be due to the affinity of the CDV to the lymphoid organs (Kumagai et al., 2003).

Non-supurative encephalomyelitis was the most prominent microscopic change in the brain. Other changes in the brain were multifocal areas of demyelination in the cerebrum and cerebellum (Fig. 5). Oligodendrocytes were metabolically active cell in the central nervous system and play an important role for myelin renewal and when brain tissues were infected by CDV, degeneration of oligodendrocyte was often occurred because of metabolic impairment which may be the cause of demyelination (Yao-qian et al., 2013). Most of the neurons were degenerated and marked perivascular cuffing predominantly with lymphocytes and plasma cells. Degeneration of the Purkinje cells seen in few cases of this study was also reported by Lisiak et al. (1979). Several eosinophilic intranuclear and intracytoplasmic inclusion bodies were observed in the neuron and glial cells (Fig. 6) which are characteristic histopathological changes recorded by various workers (Lan et al., 2006; Kubo et al., 2007; Renteria-Solis et al., 2014). Alveolar septa showed marked thickening due to presence of oedematous exudates, haemorrhage and infiltrating cells indicative of interstitial pneumonia (Fig. 7). The alveolar lumen was occluded with serofibrinous exudates, polymorphonuclear cells and mononuclear cells with hyperplasia of the alveolar lining epithelium. Sqoumous metaplasis of bronchiolar epithelia were occasionally seen in the lungs. These findings are in accordance with Apple et al. (1994). Inclusion bodies, both intranuclear and intracytoplasmic type were also observed in the alveolar macrophages, epithelial cells of the bronchioles, kidney, stomach and urinary bladder (Fig. 8,9,10,11), which were considered as a characteristic histopathological lesions in CD cases (Lan et al., 2006; Zhao et al., 2015; Green and Vandevelde, 2011; Kubo et al., 2007). This is in agreement with the present findings where inclusion bodies could be demonstrated in all the carcasses. The inclusion bodies recorded in both the cells were found to be round with intense eosinophilic appearance. The stomach revealed congestion, haemorrhage, degeneration and desquamation of lining epithelial cells which is also recorded by Galan et al. (2014). The stomach showed few necrotic cells with eosinophilic intranuclear inclusion bodies in the epithelial cells of intestinal crypts and gastric glands (Kubo et al., 2007). The CD virus is pantropic and has special affinity to the epithelial cells of the body where virus replicates, might damage to the stomach mucosa (Jones and Hunts, 2006). Intestine revealed necrosis and detachment of the villus epithelium and infiltration of mononuclear cells in the lamina propia. Similar findings reported by Zhao et al. (2015). The epitheliotropic nature of the virus might be the reason of histopathological alterations found in the present study (Green and Vandevelde, 2011). Degeneration, necrosis and sloughing of transitional epithelium with congestion and haemorrhages in the submucosal layer were noticed. Mild infiltration with mononuclear infiltrating cells was observed in the sub mucosa and muscularis layer. These findings correlate with Zhao et al. (2015). Spleen and lymph nodes revealed congestion and haemorrhages with mild to moderate depletion of lymphocytes (Fig. 12) in the splenic white pulp and in the follicles as well as. The canine distemper virus has affinity towards the lymphoid organs and replicates in the lymphoid cells, so, it could be the cause of lymphoid depletion (Lan et al., 2006).