Pathomolecular epidemiology, antimicrobial resistance, and virulence genes of Streptococcus dysgalactiae subsp. equisimilis isolates from slaughtered pigs in India.

AIMS: We aimed to investigate the prevalence, pathology, and characterization of Streptococcus dysgalactiae subsp. equisimilis (SDSE) in slaughtered pigs of India. METHODS AND RESULTS: We collected 1254 morbid tissues (lungs-627 and spleen-627) and 627 heart-blood from 627 slaughtered pigs. The bacterial isolation, antibiogram, virulence gene profiling, and mouse pathogenicity testing were performed for the detection and characterization of SDSE. A total of 177 isolates (heart-blood-160 and tissues-17) were recovered from 627 slaughtered pigs with higher isolation rate in heart-blood (25.51%). The prevalence of SDSE was 11% in morbid tissues by polymerase chain reaction. Majority of isolates showed higher detection of streptolysin O, followed by streptokinase and extracellular phospholipase A virulence genes with higher degree of resistance to azithromycin, clindamycin, erythromycin, and penicillin antibiotics. Mouse pathogenicity testing confirmed virulence based on histopathological lesions and re-isolation of SDSE. CONCLUSIONS: Our findings highlight the high prevalence of SDSE in slaughtered pigs. The presence of virulence genes and mouse pathogenicity testing confirm their pathogenic potential.

AFB1 induced free radicals cause encephalopathy in goat kids via intrinsic pathway of apoptosis: pathological and immunohistochemical confirmation of non-hepatic neuroaflatoxicosis.

Aflatoxins, particularly AFB1, are the most common feed contaminants worldwide, causing significant economic losses to the livestock sector. The current paper describes an outbreak of aflatoxicosis in a herd of 160 male young goat kids (3-4 months), of which 68 young kids succumbed over a period of 25 days after showing neurological signs of abnormal gait, progressive paralysis and head pressing. The haematobiochemical investigation showed reduced haemoglobin, leucocyte count, PCV level, increased levels of AST, ALT, glucose, BUN, creatinine and reduced level of total protein. Grossly, kids had pale mucous membranes, pale and swollen liver; right apical lobe consolidation, and petechiation of the synovial membrane of the hock joints. The microscopic changes were characterized by multifocal hemorrhages, status spongiosus/ vacuolation, vasculitis, focal to diffuse gliosis, satellitosis, and ischemic apoptotic neurons in different parts of the brain and spinal cord. These changes corresponded well with strong immunoreactivity for AFB1 in neurons, glia cells (oligodendrocytes, astrocytes, and ependymal cells) in various anatomical sites of the brain. The higher values of LPO and reduced levels of antioxidant enzymes (Catalase, SOD, GSH) with strong immunoreactivity of 8-OHdG in the brain indicating high level of oxidative stress. Further, the higher immunosignaling of caspase-3 and caspase-9 in the brain points towards the association with intrinsic pathway of apoptosis. The toxicological analysis of feed samples detected high amounts of AFB1 (0.38ppm). These findings suggest that AFB1 in younger goat kids has more of neurotoxic effect mediated through caspase dependent intrinsic pathway.

Porcine respiratory disease complex (PRDC) in Indian pigs: a slaughterhouse survey.

Porcine Respiratory Disease Complex (PRDC) is an unequivocally leading cause of economic losses to the pig industry. To investigate the pathogens associated with PRDC, a total of 900 lungs with gross lesions and 125 lungs with no appreciable gross lesions were collected from the abattoirs and subjected to pathological investigation for distribution of lesions/and types of exudates, as well as to molecular confirmation of bacterial and viral pathogens by PCR. The pneumonic lungs showed the higher prevalence of Mycoplasma spp. (31.22%), with evidence of M. hyorhinis, P. multocida (21.33%), S. suis (18.66%), B. bronchiseptica (16.77%), and viral pathogens as porcine circovirus type 2 (PCV2) (28.11%), porcine reproductive and respiratory syndrome virus (PRRSV) (2.7%) and swine influenza virus (SIV) (1.2%). On histopathological examination, high prevalence of bronchopneumonia (37.88%) followed by enzootic pneumonia­like lung lesions (11.44%), and interstitial pneumonia (7.44%) was recorded in the majority of affected pigs. The winter season was found to be more conducive for highest prevalence of pneumonia as compared to other seasons. The present study reports the high prevalence of PRDC in slaughtered pigs of India. M. hyorhinis showing the EP­like lesions, PCV2 and their combination were likely to be the prime contributors of PRDC in Indian pigs.

Novel pathologic findings and viral antigen distribution in cattle and buffalo calves naturally infected with Foot-and-Mouth disease virus.

The Foot-and-Mouth disease is highly contagious acute viral disease of livestock inflicting huge economic loss to the farmers. The limited knowledge regarding the pathological lesions vis-a-vis distribution of the FMDV in lesser explored endocrine glands and important vital organs other than the target organs of infected calves prompted us to take the present investigation to have detailed insight into the pathogenesis. The systematic necropsy of 37 dead calves (cattle-28 and buffalo-9) was conducted, and thin representative tissue pieces from the affected organs were collected in 10% neutral buffered formalin (NBF) for pathological and immunohistochemical investigations. The genomic detection and its serotyping were done by RT-PCR and multiplex-PCR, respectively. Necropsy examination in all cases showed myocardial lesions resembling 'tigroid heart appearance'. Other organ specific lesions include vesiculo-ulcerative stomatitis, edema of the lungs, petechial hemorrhages, edema of the endocrines, and gastroenteritis. Histopathological examination showed varying sizes of vesicles and ulcerations in stratified squamous epithelium of the tongue, acute necrotizing myocarditis, lymphoid depletion in lymphoid tissues, hepatitis, pancreatitis, thymic hyperplasia, thyroiditis, adrenitis, and enteritis. Positive immunolabeling for viral antigens was observed in endocrine glands, lymphoid organs, lungs, liver, kidneys, and intestine, in addition to other typical locations. The thyroid, adrenal glands, and pancreas, in addition to the tongue and heart, are the tissue of choice for sampling in the field during epidemics. Further, the viral genome and serotype A was confirmed in the affected tissues. This study provides insights into novel tissue tropism and pathogenesis in young calves naturally infected with FMDV.

Pathomorphology, immunohistochemical, and molecular detection of an atypical porcine dermatitis and nephropathy syndrome (PDNS) due to PCV-2d-2 in naturally affected grower pigs of India.

Porcine circovirus type 2 (PCV-2) is of great economic significance to porcine industry worldwide. PCV-2 variants and genotypes, alternating world over, are the etiological agent of several clinical syndromes such as porcine dermatitis and nephropathy syndrome (PDNS), post-weaning multi-systemic wasting syndrome (PMWS) and others in pigs. This study is reporting an atypical manifestation of PDNS in twelve grower pigs, 3- to - 4.5 months age and either sex, died of the disease, with predominant lesions of nephropathy and no obvious clinical lesions in skin. Necropsy examination of pigs showed lesions of petechial -to- ecchymotic hemorrhages in the kidneys and in the right auricular musculature of the hearts. Microscopic lesions in H & E sections of the kidneys showed acute glomerulonephritis, interstitial nephritis, and vasculitis, but the skin morphology and architecture remained unaltered in contrast to the pathognomonic lesions of PDNS described in the literature. Other syndromic associations of PDNS in these cases included-perimyocarditis, interstitial pneumonia, depleted lymphoid tissues, tonsillitis, enteritis, and meningo-encephalitis. The lesional sites in duplicate paraffin tissue sections of kidneys, heart, lungs, spleen, lymph nodes, intestine, and brain demonstrated PCV-2 antigen in the cytoplasm of cells as highlighted by the intense immunolabeling on IHC staining. The PCV-2 positive organs reconfirmed by PCR, targeting ORF2 gene, which yielded 481bp size of products. The sequencing results of 481bp products on phylogenetic analysis showed 94% similarity with that of PCV-2 sequences in the database that grouped into PCV2d-2 genotype. The present report confirms, probably for the first time, the atypical PDNS cases due to PCV2d-2 genotype in naturally affected grower pigs of India.

Novel pathological findings and immunohistochemical detection of FMDV antigens in the brain of calves naturally infected with foot-and-mouth disease.

Foot-and-mouth disease (FMD) is an extremely contagious and economically devastating viral disease of cloven-hoofed domestic and wildlife animals. The disease is endemic in India and other developing countries of the world. The disease is mainly characterized by the presence of vesicular lesions and "tigroid heart" in calves. The current report describes the novel pathologic findings along with the distribution of FMDV antigens in brain of young calves naturally infected with FMDV. The carcasses of 37 calves suspected to have died from FMD were presented for postmortem investigation. Out of 37 dead calves, 10 calves showed the clinical signs of neurological abnormalities like opisthotonos, muscle twitching and tremor in hind limbs, stiffening of the neck followed by death. Microscopically, the meninges were congested, hemorrhagic, and infiltrated with mononuclear cells. The various sub anatomical sites of the brain showed the varying degrees of vascular changes, perivascular cuffing, focal to diffuse gliosis as well as degeneration and neuronal necrosis, indicating the nonsuppurative encephalitis. The immunolabeling of FMDV antigen was demonstrated in the neurons, inflammatory cells, and microglial cells besides its typical locations. The neurons of the brain also showed strong immunopositivity for caspase-3, caspase-9 and p53 and negative for Bcl-2 and apoptosis-inducing factor (AIF) by both immunohistochemistry and western blotting indicating the role of caspase mediated intrinsic, and p53 dependent apoptotic pathway. Further, the TUNEL assay also confirmed the apoptosis in the neurons and glial cells of the brain of naturally infected calves. This study in calves establishes a basis for resemblance to other members of Picornaviruses, such as Enterovirus 71 and Coxsackievirus of humans and showing the neuropathological alterations along with the distribution of FMDV antigens associated with apoptosis in younger calves.

Localization of Pasteurella multocida antigens in the brains of pigs naturally infected with Pasteurellosis revealing a newer aspect of pathogenesis.

Pasteurella multocida is an economically important respiratory pathogen of pigs confronting swine industry worldwide. Despite extensive research over the decades, its pathogenesis is still poorly understood. Recent reports have demonstrated the nervous system affection as a newer aspect of pathogenesis by Pasteurella multocida type B:2 in Haemorrhagic Septicemia, but there are no reports of the involvement of nervous system by P. multocida in pigs. Therefore, the study was aimed to explore the neurovirulence of Pasteurella multocida in naturally infected pigs. A total of 15 brains were collected from the natural cases of pig mortality suggestive of Pasteurellosis. Grossly, the leptomeninges were markedly congested and brains were oedematously swollen. Histologically, there was moderate to severe fibrinohaemorrhagic and mononuclear cells exudates present in the leptomeningeal tissue and cerebrospinal spaces. Similar vascular inflammatory lesions (perivascular and perineuronal) along with gliosis, neuronal degeneration and necrosis were noted in various subanatomical sites of the brain (cerebrum, cerebellum, brainstem and spinal cord). The culture and biochemical tests showed the presence of P. multocida within the brain tissue. P. multocida type specific antibody staining in the brain tissues revealed intense distribution of antigens in the inflammatory exudates of meningeal vessels, neurons, glial cells and endothelial cells of the blood vessels contributing its association with neuropathological lesions. Pasteurella multocida specific PCR amplification of capsular polysaccharide gene yielded 460 bp and multiplex PCR showed the involvement of capsular serogroups A &D. All the isolates showed the presence of 10 genes for virulence factors. The disease confirmation of both serotypes was proven by Koch's postulates using Swiss albino mice. Further, histopathological brain lesions along with the immunohistochemical detection of bacterial antigens were corroborated with natural cases of P. multocida as described above. To the best of our knowledge, we first time report the neuroinvasion of P. multocida in naturally infected pigs.

Neuropathology mediated through caspase dependent extrinsic pathway in goat kids naturally infected with PPRV.

Peste des petits ruminant (PPR), a highly contagious viral disease of small ruminants, is characterized by erosive stomatitis and pneumo-enteritis. However, its neurovirulence potential as observed with other morbilliviruses has not been fully investigated. The present study describes the neuropathological alterations induced by PPR virus through apoptotic pathway. A total number of 12 carcasses of local breed goat kids of either sex were received for postmortem examination. The clinical history was described as symptoms of mucopurulent nasal discharge, high to low grade fever, erosive stomatitis, dyspnoea and profuse watery diarrhoea followed by mortality of 35 goat kids within a week. The pathoanatomical lesions and immunohistochemical demonstration of PPRV antigen in lungs, intestine, spleen and lymph nodes confirmed PPR disease in goats. Grossly, five brain specimens showed moderate to severe leptomeningeal congestion during necropsy. Microscopically, brain sections showed leptomeningitis and nonsuppurative encephalitis characterized by vascular congestion, haemorrhages in the parenchyma, perivascular cuffing with mild to moderate mononuclear cells (mainly lymphocytes and few macrophages), focal to diffuse microgliosis, neuronal degeneration, satellitosis and neuronophagia. Immunolabelling of viral antigen was observed in the cytoplasm of neurons and glial cells. The RT-PCR amplification of N gene fragment also confirmed the presence of PPRV in the brain. The strong immunoreactivity of Caspase-3, Caspase-8 and comparatively lower expression of caspase-9 along with the absence of any reactivity for Apaf-1 antigen in the brain sections indicated the role of caspase dependent extrinsic pathway in inducing neuropathological changes. The presence of apoptotic neurons in the brain by TUNEL assay further confirmed the apoptosis and strong immunoreactivity of iNOS in neurons which suggested the generation of oxidative stress, that might have induced the apoptosis. The overall findings confirm the neurovirulence potential of PPR virus, via the extrinsic pathway of apoptosis, in natural cases of PPR disease in goat kids.