Dogs are one of the best-known examples of domestication¹. India has approximately 25 million dogs with an estimated dog: human ratio of 1:36. The dogs fall into 4 broad categories: pets (restricted and supervised); family dogs (partially restricted and wholly dependent); community dogs (unrestricted and partially dependent), and feral dogs (unrestricted and independent). Most dogs in India perhaps 80% would fall into the last 3 categories². The domestic dog Canis familiaris reportedly bears over 450 diseases approximately 360 of which are analogous to human diseases³. As a spontaneous model for many heritable human diseases, the dog provides an excellent system for the identification and study of disease loci⁴ particularly cancer loci⁵. Diseases of the respiratory system (respiratory apparatus) are one of the leading causes of morbidity and mortality in animals and a major source of economic losses. In the past few years, animal shelters have been recognized as a major risk factor for respiratory diseases in dogs and cats, a comparable situation to what is reported in human beings with nosocomial infections^6,7. The most common pathological lesions observed in the lungs were pneumonia, anthracosis, emphysema, congestion, and haemorrhages⁸. Hence, the present study was undertaken to find out the pulmonary pathological lesions in dogs in and around Chennai city, Tamilnadu, India.

The present study was conducted during the period from September, 2021 to January, 2022. The lung tissue samples were regularly collected from the dogs, who were brought for necropsy at the Department of Veterinary Pathology, Madras Veterinary College, TANUVAS, Chennai, Tamil Nadu, India. The gross pathological lesions of lungs were observed and recorded. The representative tissues were preserved in 10% neutral buffered formalin (NBF) for further histopathological analysis. The fixed tissue samples were cut at 4-5 mm and washed overnight in running tap water. Then the tissues were dehydrated in 3 changes of acetone for 30, 30 and 45 minutes followed by clearing in 3 changes of benzene for 30, 30 and 45 mins. Paraffin embedding was done with melted paraffin, which was maintained at 58-60°C and blocks were made using ‘L-blocks’. Sections were cut at a 4-5 µ thickness from each specimen using semi-automatic rotary cutting machine (MMRS 3500, Histoline Lab) and were stained with hematoxylin and eosin (H & E) stain⁹.

Different special staining like Masson’s Trichrome for collagen fibers, Periodic acid-Schiff (PAS) for the presence of carbohydrate, mucin, or mucopolysaccharides, Gram’s stain for the presence of Gram-positive and Gram-negative, and Perl’s Prussian blue for hemosiderin were performed as per the standard protocols¹⁰.

Sections of 3-4 µm thickness were mounted on 3-Aminopropyl Triethoxy-Silane coated slides. Incubated at 60°C for one hour. Deparaffinised in xylene for 3 min, in xylene and alcohol (1:1) for 3 min and then washed in graded alcohols. Washed in running tap water for 10 min. The slides were placed in Coplin jar in 1M Tris EDTA (pH 9) for antigen retrieval for 10 min, 120°C for NF-kB (Nuclear factor kappa B) (Pressure cooking – 7 min). Cooled at room temperature for 20 min. Rinsed with PBS (pH 7.4) twice for 5 min. Incubated with peroxide block (3% hydrogen peroxide in water) for 10 min. Rinsed with PBS twice for 5 min. Applied with Power Block for 10 min. Incubated with primary antibody NF-kB (Bought from Path-Insitu Company, Hyderabad) for 60 min at room temperature. Washed in PBS twice for 5 min. Treated with Super Enhancer^TM for 25 min. Washed in PBS twice for 5 min. Incubated with HRP polymer based secondary antibody for 30 min at room temperature. Washed in PBS twice for 5 min. Incubated with DAB + substrate buffer for 10 min. Rinsed in PBS twice for 5 min. Lightly counterstained with haematoxylin for 30 sec. Washed in running water for 5 min. Dried the slides at 37°C for 1h. Cleared with xylene and mounted with DPX. Examined under Olympus BX-51 microscope attached with image analyzer system (Image Proplus 5.1)

A total of 51 lung samples were collected during the study period. History regarding sex, age and breeds of different dogs that came for necropsy are presented in Table 1 and 2. The rate of death incidence was highest in the age group between 1-4 years (29.41%) and followed by the age group of 0-1 years (21.57%). Labrador breed showed more death incidence as compared to other breeds and the non-descriptive breed had the second most death rate.

All the lung samples collected during the study period showed different gross lesions, which are presented in Table 3. Circulatory disturbances showed more incidence percentage (35.30%); whereas, atelectasis, abscess and nodular growth each showed the lowest incidence percentage (5.88%). The incidence of different histopathological lesions was presented in Table 4.

In some cases, multiple, dark red, circular patchy areas were noticed and upon cut section, blood oozed out. Microscopically, there was the engorgement of blood vessels and the interstitial haemorrhage was also observed. Lungs showed diffuse edema and cut section revealed frothy exudates (Fig. 1). The alveolar spaces were filled with eosinophilic fluid (Fig. 2). There was a positive reaction of NF-kB markers in the macrophages with cytoplasmic expression (Fig. 3). Histopathological studies also revealed the occurrence of hemosiderosis in lungs. Brownish-yellow granules were seen within macrophages in H&E stain, which was further confirmed by Perl’s Prussian blue staining, where these granules are blue in colour (Fig. 4), which were confirmative of hemosiderosis. In anthracosis, grossly, lungs were darker in colour, oedematous, wet, heavy and the carbon particles appeared as very fine black or bluish-black pigment (Fig. 5). Microscopic examination revealed an accumulation of black particles in the alveolar wall, interlobular septa, and peribronchial tissue with the presence of haemorrhages (Fig. 6).

Grossly, emphysematous lungs were non collapsible, enlarged in size, dry, and easily compressed by finger. There was presence of pale or white areas projecting above the surrounding tissues. Histopathologically, there were diffuse, distended, coalescing and broken alveoli i.e. formation of giant alveoli along with congestion (Fig. 7). Lungs showed focal collapsed, not fully and the collapsed part sank in the water. The dark reddish affected part was depressed from the level of surrounding healthy part and leathery in consistency (Fig. 8). Microscopically, the affected part revealed collapsed alveoli without air and the presence of congestion and haemorrhage (Fig. 9)

Pneumonia was the 3^rd most finding in the present study. Pneumonic lungs showed congestion and haemorrhages with areas of consolidation. In some cases, lungs showed focal pale red areas (Fig. 10). In interstitial pneumonia, mottling of lungs with thickened interalveolar septa was observed. Microscopical examination revealed thickening of interalveolar septa with mononuclear cells infiltration (Fig. 11) and varying degrees of serous or fibrinous exudates were observed in interstitial pneumonia cases. Congestion and haemorrhages with alveolar exudations were also observed.

In cases with bronchopneumonia, the anterior and ventral portion of lungs were severely affected as compared to other parts. Bronchopneumonia was indicated by the presence of congestion along with sloughed cells inside the bronchus and inflammatory reactions surrounding the bronchus (Fig. 12). In the majority of the cases, infiltrations were of mononuclear types and in some cases, infiltrations by neutrophils and eosinophils were observed. Broncho-interstitial pneumonia was more common compared to other types of pneumonia. Microscopic examination revealed infiltration of mononuclear cells in both interstitial and around the bronchus (Fig. 13). In some cases, the bronchiolar lumen contains sloughed cells, erythrocytes, mononuclear cells and eosinophilic exudates, and in others fibrino-cellular exudates with sloughed cells (Fig. 14). There was hyperplasia of bronchiolar cells and the presence of angiectasis. PAS stain revealed a magenta to the intense pink colour of hyperplastic bronchiolar cells (Fig. 15).

There was multiple, varying size of abscess cavity were present in lungs and upon cut section, the cavities were filled with thick purulent fluid. In some cases, there was the presence of multiple, variable sized yellowish nodular growths (Fig. 16). Microscopically, congestion, haemorrhages along with edema were seen. Multiple bacterial granulomas were observed indicated by the presence of infiltration of mononuclear cells along with necrotic areas (Fig. 17) and the granuloma was surrounded by fibrous tissues i.e. confirmed by Masson’s trichrome stain. The Gram-positive bacterial colonies were identified by Gram’s staining (Fig. 18).

The respiratory tract is frequently exposed to different infectious agents and environmental pollutants, which can reach the upper and lower respiratory tract aerogenously or hematogenously^11,12. Some of the respiratory pathogens act as the primary cause of infections and some others cause infection as secondary, opportunistic invaders after host defense mechanisms get disrupted by other factors like immunosuppression, environment, stress, toxins, and concurrent infection¹³. In the present study, the death incidence in the age group between 1-4 years and 0-1 years was more, which further showed that animals in this group could have compromised immunity or those associated with stress due to weaning or absence of maternal immunity¹⁴. Labrador breed showed more death incidence followed by non-descriptive breed, which could be due to more number of owners keeping of these breeds as a pet.

In the majority of cases, congestion, haemorrhages, and edema were recorded in lungs, which were earlier described by Bruchim et al.¹⁵ in dogs affected with fatal heat strokes, which could be these conditions and edema may be due to irritation by dust particles¹⁶, which are present more in urban areas. There was a positive reaction of NF-kB markers in the macrophages with cytoplasmic expression with pulmonary edema¹⁶. There were diffuse, distended, and ruptured alveoli with congestion in emphysematous lungs¹⁷. Bettini et al. (2010) found that anthracosis was common among dogs, who lived in urban areas due to environmental or air pollution, which was found to be similar in the present study.

Broncho-interstitial pneumonia was more common compared to other types of pneumonia. The gross and histopathological lesions in different types of pneumonia were also reported in the previous reports^8,19,20. In interstitial pneumonia, thickening of interalveolar septa and infiltration of inflammatory cells predominantly mononuclear types with varying degrees of serous or fibrinous exudates were observed²¹. But in bronchointerstitial pneumonia, there was infiltration of mononuclear cells, both in the interstitium and around the bronchus²². Chvala et al. (2007) reported fibrinous pneumonia in lungs of dogs affected with canine distemper, canine adenovirus type 2, and Mycoplasma cyanos. Similar lesions were recorded during the present study, which could be due to these conditions. Multiple bacterial granulomas were observed and the Gram-positive bacterial colonies were identified by Gram’s staining. Similar findings were in accordance with Sastry and Rao (2013).

The present research was conducted to study the incidence of different pathological lesions of canine lungs collected from the routine necropsy. The age group between 1-4 years showed more death rate followed by the age group of 0 to 1 year and the Labrador breed showed more death incidence as compared to other breeds and the non-descriptive breed had the second most death rate. Among pathological lesions, circulatory disturbances showed more incidence percentage; whereas, atelectasis, abscess, and nodular growth each showed the lowest incidence percentage. Bronchointerstitial pneumonia was more common compared to other types of pneumonia. Anthracosis was also common among dogs, who lived in Chennai city due to environmental or air pollution. Therefore, the results of present study will be useful for treating the animals and by knowing the incidence pattern of different pathological conditions in urban areas.

References