In recent years, piggery has gained enormous popularity amongst Indian farmers because of its ability to convert low quality feed to pork, quick and fast returns, large litter size and fast growth rate. Recent livestock census report revealed the decline in pig population by 12.03% as compared to the previous census report, which might be due to various disease outbreaks affecting the economic viability and profitability of swine industry¹. There is an increasing trend in porcine bacterial diseases in several countries of the world among which Streptococcus suis has emerged as an emerging zoonotic pathogen to both pigs and human due to increasing intensification of farrowing management. Streptococcus suis (SS) is considered to be one of the most important bacterial pathogens, responsible for causing frequent outbreaks of meningitis, septicemia, endocarditis, arthritis, pneumonia, abortions and abscessation in pigs, and severe outbreaks of meningitis, septicemia, permanent hearing loss and endocarditis in human beings². After its first report in the early 1960s, it emerged as the most common cause of bacterial meningitis in pigs in the UK and nearly 30 other countries, causing over 300-million-dollar loss in the United States alone³.

The prevalence of S. suis infection in pigs and humans, is now recognized worldwide, with cases reported in several pig producing countries^4-6. The growing evidence suggests that the slaughtered pigs are the major reservoir of this pathogen⁴. In East and Southeast Asia, S. suis zoonosis is considered endemic due in part to the high density of pigs, relatively high number of backyard production farms, unhygienic slaughtering practices, wet markets, and consumption of undercooked pork products⁴. Since, similar types of practices are followed in India and a few sporadic reports of this zoonotic pathogen on local areas are in print^7,8, the authors hypothesize that the prevalence rate could be much higher and the prevalence is underreported. Therefore, the study intended to explore the magnitude of S. suis in Indian pigs, of distant locations with high density of pig population in Uttar Pradesh and North-eastern state.

A total of 664 tissue samples including tonsils (187), lungs (205), lymph nodes (56), spleen (164) and brain (52), were collected during August, 2017 to May 2018 at necropsy from the slaughterhouses at Lucknow, Bareilly, Delhi, Guwahati, and Chandigarh (Table 1). The representative tissues of palatine tonsils, lungs, lymph nodes, spleen and brain of slaughtered pigs were collected in 10% neutral buffered formalin (NBF) for histopathology and immunohistochemistry, and tissues were also stored in sterile cryovials at-20°C for the molecular diagnosis.

Out of 664 morbid tissue samples showing gross lesions, 41 samples (6.14%) were positive for S. suis DNA. The percentage frequency occurrence of positivity was more in the samples of Bareilly region (24.0%) while the lowest in Lucknow (5.45%) in the analyzed tissue samples. Out of different organs tested, lungs (58.5%) showed significantly higher detection for S. suis followed by palatine tonsils (26.8%). However, brains (2.4%) showed least number of positivity. The prevalence of S. suis infection in morbid tissue samples is presented in Table 1. Age wise significantly higher prevalence (P<0.05) was recorded in growers followed by finishers.

Out of 205 lungs showing pneumonic lesions, 24 cases were detected positive for S. suis. Among these cases (24), 15 lungs had lesions of bronchopneumonia, 5 cases had interstitial pneumonia, and 3 cases had enzootic pneumonic like lesions based on histopathological investigation. The enzootic pneumonic cases were further confirmed by using M. hyopneumoniae specific primer targeting 16srRNA gene amplifying 649bp. Out of 15 bronchopneumonic cases, the majority of the lungs showed the acute bronchopneumonia (6) followed by haemorrhagic pneumonia (4), sub-acute bronchopneumonia (2), sero-fibrinous bronchopneumonia (2) and necrotizing pneumonia (1). The bronchopneumonia lungs appeared congested, enlarged and consolidated cranio-ventral lobe (Fig 1a). Histopathologically, the small airways and air spaces were severely infiltrated with neutrophilic exudates (Fig 1b). In interstitial pneumonia, the affected lungs were pale in appearance, non-collapsible, meaty consistency interspersed with emphysema in various lobes of the lungs (Fig 1c). Histopathologically, lymphocytes, and mononuclear cells and macrophages were seen in large numbers in the alveolar septa (Fig 1d) and small amount of serofibrinous exudates in air spaces. The enzootic pneumonic cases with presence of S. suis showed the consolidation of apical lobe of both lungs with sharp demarcated area of consolidation (Fig 1e). Histologically, the hyperplastic bronchiolar epithelial cells, BALT hyperplasia, lymphoid aggregates, and massive perivascular and peribronchiolar lymphoid hyperplasia in the interstitium of the lung were noticed (Fig 1f). The immunostaining of PCR positive tissues of lungs revealed strong cytoplasmic immunoreactivity in the bronchiolar lining epithelial cell, and in the exudates of air spaces (Fig 3a). The pathology along with the presence of bacterial antigens in lungs confirms the association of S. suis with pneumonia.

Out of 187 palatine tonsils tested, 11 (17.11%) were positive for S. suis. The tonsils of naturally infected pigs showed marked vascular congestion (6.4%), severe haemorrhagic tonsillar flaps (2.1%), and multifocal necrotic ulcers in the epiglottis area (2.6%). The lymphoid follicles in majority of the cases were depleted and crypts epithelium revealed degenerative changes with the heavy infiltration of inflammatory exudates (neutrophils) and necrotic debris in the lumen (Fig 2). The spleen showed depletion of lymphocytes in the white pulp area, and congestion and hemorrhages in the red pulp (Fig 2(e, f)). The lymph nodes also showed the depletion of lymphoid follicles along with severe congestion, haemorrhages and focal areas of necrosis in vascular compartment (Fig 2(c, d)). Grossly, the brain affected with S. suis showed no appreciable gross lesions. However, on microscopic examination, it showed meningitis characterized by the mild infiltration of mononuclear cells in the leptomeningeal space (Fig 2g) along with the perivascular cuffing with mononuclear cells in the brain parenchyma (Fig 2h).

A total of 41 DNAs were found to be positive for S. Suis in morbid tissues yielding 566bp (Fig 4). The sequences of S. suis isolates from Japan and the United Kingdom showed lesser divergence with Indian isolates as compared to Chinese isolates. The estimates of evolutionary divergence and subsequent phylogenetic tree analysis constructed between Indian S. suis isolates and related sequences were presented in Fig 5. Phylogenetic analysis of the 4 sequenced PCR products revealed the close resemblance with one Indian isolate and one Chinese isolate while the other three isolates showed resemblance with the isolates from Japan, United Kingdom and China. Further, two Indian isolates (MN993282, MN993283) were observed to emerge in the similar way (Fig 5).

Streptococcus suis is a swine bacterial pathogen of emerging zoonotic significance. The paucity of data on prevalence of S. suis in Indian pigs despite causing severe disease in humans, including meningitis, septicemia, and endocarditis, prompted us to take up the study on patho-epidemiological aspect of S. suis involving slaughtered pigs. Altogether, 664 morbid tissue samples were collected from slaughtered pigs of 4 states. The 41 (6.14%) cases were positive for S. suis by PCR followed by sequencing methods. The similar rate of prevalence has also been reported in earlier reports^7,13. However, Devi et al.⁸ documented low prevalence of S. suis infections (1.41%) in pigs of Guwahati region. The degree of pathology in vital organs corroborated well with higher positivity in lungs (58.5%) followed by palatine tonsils (26.8%), spleen (7.3%), and lymph nodes (4.8%) indicating graded tissue tropism of S. suis. S. suis is recognized as a major respiratory pathogen of pigs and the same holds true in our study. S. suis has been the found as the major cause of bronchopneumonia in most of the earlier reports ^4,7. In some countries, it is regarded as the primary agent for pneumonia⁴. It has also been reported in greater frequency in cases of acute haemorrhagic pneumonia together with the presence of S. suis bacterial colonies in those lesions. The acuteness of pneumonia could be due to the toxins and superantigens liberated by this haemolytic bacteria compromising the lungs defenses. Our observations also fall in line with the findings of previous report⁷. The association of S. suis in interstitial pneumonic cases has also been highlighted by others^13,14,15. This might have happened due to prior injury by the viral pathogens like PRRS, PCV and Swine influenza virus, which might have caused immune suppression, thus favouring the growth of the opportunistic pathogen like S. suis for causing the pathologies. The association of S. suis in Enzootic pneumonic cases was documented in earlier report¹⁶. The higher positivity of S. suis DNA in lungs was corroborated with antigen distribution in tissues, and pathological changes. Next to lungs, higher numbers of cases were recorded in palatine tonsils. The upper respiratory tract of pigs, particularly the palatine tonsils is the preferred site for sampling to investigate the prevalence of S. suis infections in both live and slaughtered pigs^17,18. In palatine tonsils, the pathology and intensity of S. suis antigen distribution matched well suggesting the palatine tonsils as potential portal of entry of this pathogen to the systemic blood circulation¹⁹.The detection of S. suis in spleen and lymph nodes indicated the site for its replication in the lymphocytes. In spleen and lymph nodes, S. suis antigens were demonstrated in the immune cells (lymphocytes) which might be due to the subsequent lymphogenous route to the draining lymph nodes and through the hematogenous route to the spleen and lymph nodes. Further, S. suis has also been shown to be present in nearly half of the submaxillary lymph nodes of pigs ^17,20,21. In the present study, low positivity was recorded in the brain of slaughtered pigs as against natural cases although, S. suis is the primary agent for causing meningitis in post weaned piglets⁴. S. suis induced the classical lesions of meningitis characterized by meningeal vessels congestion, infiltration with mononuclear cells in the subarachnoid space, and the perivascular cuffing in the brain parenchyma were largely supported by the findings of previous worker²². It may be hypothesized that S. suis might have reached the brain via the blood after colonizing the nasopharynx and caused severe lesions in the brain. The amplified partial fragment of gdh gene sequencing data in our study matched with available gdh sequences at the NCBI database showing close homology with the sequences from Japan, United Kingdom and China, confirms the prevalence of S. suis pathogen in the Indian swine population. Moreover, further studies with considering larger fragments and the complete genome are needed to develop appropriate diagnostics and vaccines for the effective control of the disease.

The present investigation reports 6.14% of prevalence of S. suis in the tissue samples of slaughtered pigs belonging to 5 localities of 4 states/union territory of India. A panel of pathological, IHC, and PCR tests was conducted on morbid tissue samples to confirm the prevalence of S. suis in naturally infected slaughtered pigs. The study suggested that there is a need for the public awareness, personnel particularly engaged in pig sector. Hence, continuous surveillance and monitoring of this zoonotic pathogen in different geographical locations of the country is warranted to understand the disease dynamics for its effective control.

References