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Article: Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples

Ashmi, Marcia / Kumar, Bablu / Agrawal, Ravi Kant / Prakash, Chandan / Abhishek / Singh, Karam Pal

Molecular and cellular probes. 2021 Oct., v. 59

2021

Abstract: Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of ... ...

Abstract	Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 10⁴ fold higher than conventional PCR and equivalent to real-time PCR (RT-PCR). The assay didn't exhibit cross-reaction with selected pathogenic non-Brucella bacteria and Brucella spp. other than B. abortus. Forty clinical samples were also tested using this novel assay and it was able to detect 25 samples as positive, however, conventional PCR could detect the targeted organism in 22 samples only.To the extent of our knowledge, this is the first report towards the development of a PSR assay for specific detection of B. abortus. The assay can be used as a quick, sensitive and accurate test for the diagnosis of bovine brucellosis in the field setting. Relatively one of the paradigm-shifting aspects of this assay would be it does not require any expensive equipment and the results can be easily visualized by the unaided eye, therefore making PSR a valuable diagnostic tool in field conditions.
Keywords	Brucella melitensis biovar Abortus ; bovine brucellosis ; cross reaction ; dairy industry ; detection limit ; diagnostic techniques ; disease transmission ; equipment ; genes ; quantitative polymerase chain reaction
Language	English
Dates of publication	2021-10
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	639082-1
ISSN	1096-1194 ; 0890-8508
ISSN (online)	1096-1194
ISSN	0890-8508
DOI	10.1016/j.mcp.2021.101761
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Article ; Online: Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples.

Ashmi, Marcia / Kumar, Bablu / Agrawal, Ravi Kant / Prakash, Chandan / Abhishek / Singh, Karam Pal

Molecular and cellular probes

2021 Volume 59, Page(s) 101761

Abstract	Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 10
MeSH term(s)	Animals ; Biological Assay ; Brucella abortus/genetics ; Brucellosis/diagnosis ; Brucellosis/veterinary ; Cattle ; Humans ; Nucleic Acid Amplification Techniques ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity
Language	English
Publishing date	2021-08-14
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	639082-1
ISSN	1096-1194 ; 0890-8508
ISSN (online)	1096-1194
ISSN	0890-8508
DOI	10.1016/j.mcp.2021.101761
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Article: Patho-epidemiological study of Streptococcus suis infections in slaughtered pigs from North and North-Eastern Region, India

Dinesh, Murali / Thakor, Jigarji Chaturji / Singh, Karam Pal / Singh, Rajendra / Anbazhagan, Subbaiyan / Chauhan, Renu / Qureshi, Salauddin / Sahoo, Nihar Ranjan / Sahoo, Monalisa

Indian journal of veterinary pathology. 2022 Mar. 28, v. 46, no. 1

2022

Abstract: Streptococcus suis, is widely distributed in all pig rearing countries causing huge economic loss to the pork industry. However, the information about the epidemiology of this pathogen with the associated pathologies in Indian pigs is very limited. The ... ...

Abstract	Streptococcus suis, is widely distributed in all pig rearing countries causing huge economic loss to the pork industry. However, the information about the epidemiology of this pathogen with the associated pathologies in Indian pigs is very limited. The study was conducted using pathological, immunohistochemistry, and molecular detection methods from four states of India viz. Uttar Pradesh (Lucknow), Uttar Pradesh (Bareilly), Delhi, Assam (Guwahati), and Punjab (Chandigarh). Out of 664 morbid tissues of slaughtered pigs, 41 cases (6.14%) were found to be positive for S. suis by PCR assay by amplifying gdh gene. The highest rate of prevalence was recorded in Bareilly (24%) while lowest in Guwahati (4.44%). Age wise significantly higher prevalence was recorded in growers followed by finishers. Lungs (58.5%) showed more tropism for S. suis showing predominant pathological lesions of bronchopneumonia. The microscopic lesions in lungs, palatine tonsils, spleen, lymph nodes and brain of naturally affected pigs corroborated well with the distribution of S. suis antigen in these organs on immunohistochemistry. The phylogenetic analysis showed close homology to the isolates of Japan, United Kingdom and China. The study shows that S. suis in slaughtered pigs could be a potential cause of serious public health concern.
Keywords	Japan ; Streptococcus suis ; animal pathology ; antigens ; brain ; bronchopneumonia ; epidemiology ; financial economics ; genes ; immunohistochemistry ; lymph ; pathogens ; phylogeny ; pork industry ; public health ; spleen ; swine ; China ; India ; United Kingdom
Language	English
Dates of publication	2022-0328
Size	p. 26-32.
Publishing place	Indian Association Of Veterinary Pathologists
Document type	Article
ISSN	0973-970X
DOI	10.5958/0973-970X.2022.00004.9
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Article: Effect of inhibition of Toll-like receptor 3 signaling on pathogenesis of rabies virus in mouse model

Sardana, Sumit / Singh, Karam Pal / Saminathan, Mani / Vineetha, Sobharani / Panda, Shibani / Dinesh, Murali / Maity, Madhulina / Varshney, Rajat / Sulabh, Sourabh / Sahoo, Monalisa / Dutt, Triveni

Acta tropica. 2022 Oct., v. 234

2022

Abstract: Rabies is a zoonotic viral disease with inevitably fatal outcome. Toll-like receptor 3 (TLR3) could sense dsRNA viral infections, and implicated in pathogenesis of rabies and Negri bodies (NBs) formation. Present study was undertaken to elucidate the ... ...

Abstract	Rabies is a zoonotic viral disease with inevitably fatal outcome. Toll-like receptor 3 (TLR3) could sense dsRNA viral infections, and implicated in pathogenesis of rabies and Negri bodies (NBs) formation. Present study was undertaken to elucidate the role of TLR3 in pathogenesis, NBs formation, and therapeutic potential of blocking TLR3/dsRNA interaction in rabies infection. Young Swiss albino mice were infected with 100 LD₅₀ of street rabies virus (SRABV) intracerebrally (i/c) on day 0 and treated with 30 μg of CU CPT 4a (selective TLR3 inhibitor) i/c on 0, 3 and 5 days post-infection (DPI). Three mice each were sacrificed at 1, 3, 5, 7, 9, 11, and 13 DPI to study sequential pathological consequences through histopathology, Seller's staining, immunofluorescence, immunohistochemistry, TUNEL assay, flow cytometry, and viral and cytokine genes quantification by real-time PCR. CU CPT 4a inhibited TLR3 expression resulted in delayed development and decreased intensity of clinical signs and pathological lesions, low viral load, significantly reduced NBs formation, and increased survival time in SRABV-infected mice. These parameters suggested that TLR3 did influence the SRABV replication and NBs formation. Inhibition of TLR3 led to decreased expression of pro-inflammatory cytokines and interferons indicated an anti-inflammatory effect of CU CPT 4a during SRABV infection. Further, TLR3-inhibited group revealed normal CD4⁺/CD8⁺ T-cells ratio with less TUNEL-positive apoptotic cells indicated that immune cell kinetics are not affected during TLR3-inhibition. SRABV-infected and mock-treated mice were developed severe clinical signs and histopathological lesions, more NBs formation, high viral load, increased pro-inflammatory cytokines expression in brain, which were correlated with higher expression levels of TLR3. In conclusion, these data suggested that TLR3/dsRNA signaling pathway could play critical role in pathogenesis of SRABV infection in vivo and opens up new avenues of therapeutics.
Keywords	Rabies lyssavirus ; Toll-like receptor 3 ; albino ; anti-inflammatory activity ; apoptosis ; brain ; cytokines ; flow cytometry ; fluorescent antibody technique ; histopathology ; immunohistochemistry ; mice ; pathogenesis ; quantitative polymerase chain reaction ; rabies ; therapeutics ; viral load
Language	English
Dates of publication	2022-10
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	210415-5
ISSN	1873-6254 ; 0001-706X
ISSN (online)	1873-6254
ISSN	0001-706X
DOI	10.1016/j.actatropica.2022.106589
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Article ; Online: Pathomolecular epidemiology, antimicrobial resistance, and virulence genes of Streptococcus dysgalactiae subsp. equisimilis isolates from slaughtered pigs in India.

Patel, Sagar M / Sahoo, Monalisa / Thakor, Jigarji Chaturji / Murali, Dinesh / Kumar, Pradeep / Singh, Rajendra / Singh, Karam Pal / Saikumar, G / Jana, Chandrakanta / Patel, Shailesh Kumar / Mote, Akash B / Karthikeyan, Ravichandran / Vandre, Rajesh Kumar / Biswal, Jitendra Kumar / Sahoo, Nihar Ranjan

Journal of applied microbiology

2024 Volume 135, Issue 1

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Swine ; Mice ; Virulence/genetics ; Anti-Bacterial Agents/pharmacology ; Streptococcal Infections/epidemiology ; Streptococcal Infections/veterinary ; Streptococcal Infections/microbiology ; Drug Resistance, Bacterial/genetics ; Streptococcus
Chemical Substances	Anti-Bacterial Agents
Language	English
Publishing date	2024-01-02
Publishing country	England
Document type	Journal Article
ZDB-ID	1358023-1
ISSN	1365-2672 ; 1364-5072
ISSN (online)	1365-2672
ISSN	1364-5072
DOI	10.1093/jambio/lxae002
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Article ; Online: A retrospective study showing a high rate of seropositivity against SARS-CoV-2 in wild felines in India

Borkakoti, Richa / Karikalan, M / Nehul, Sanket Kumar / Jogi, Harsh Rajeshbhai / Sharma, Kirtika / Nautiyal, Sushmita / Mishra, Ragini / Mahajan, Sonalika / Biswas, Sanchay Kumar / Nandi, S. / Chander, Vishal / Pawde, Abhijit / Saikumar, G / Singh, Karam Pal / Sharma, Gaurav Kumar

Arch Virol. 2023 Apr., v. 168, no. 4 p.109-109

2023

Abstract: We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of ... ...

Abstract	We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of seropositivity was positively correlated with that of the incidence in humans, suggesting the occurrence of large spillover events.
Keywords	Severe acute respiratory syndrome coronavirus 2 ; neutralization ; retrospective studies ; seroprevalence ; India
Language	English
Dates of publication	2023-04
Size	p. 109.
Publishing place	Springer Vienna
Document type	Article ; Online
ZDB-ID	7491-3
ISSN	1432-8798 ; 0304-8608
ISSN (online)	1432-8798
ISSN	0304-8608
DOI	10.1007/s00705-023-05735-4
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Article ; Online: Exploring the possible use of saponin adjuvants in COVID-19 vaccine.

Sharma, Rinku / Palanisamy, Arivukarasu / Dhama, Kuldeep / Mal, Gorakh / Singh, Birbal / Singh, Karam Pal

Human vaccines & immunotherapeutics

2020 Volume 16, Issue 12, Page(s) 2944–2953

Abstract: There is an urgent need for a safe, efficacious, and cost-effective vaccine for the coronavirus disease 2019 (COVID-19) pandemic caused by novel coronavirus strain, severe acute respiratory syndrome-2 (SARS-CoV-2). The protective immunity of certain ... ...

Abstract	There is an urgent need for a safe, efficacious, and cost-effective vaccine for the coronavirus disease 2019 (COVID-19) pandemic caused by novel coronavirus strain, severe acute respiratory syndrome-2 (SARS-CoV-2). The protective immunity of certain types of vaccines can be enhanced by the addition of adjuvants. Many diverse classes of compounds have been identified as adjuvants, including mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticles, and liposomes. Several saponins have been shown to stimulate both the Th1-type immune response and the production of cytotoxic T lymphocytes against endogenous antigens, making them very useful for subunit vaccines, especially those for intracellular pathogens. In this review, we discuss the structural characteristics, mechanisms of action, structure-activity relationship of saponins, biological activities, and use of saponins in various viral vaccines and their applicability to a SARS-CoV-2 vaccine.
MeSH term(s)	Adjuvants, Immunologic/administration & dosage ; Adjuvants, Immunologic/chemistry ; Animals ; COVID-19/immunology ; COVID-19/prevention & control ; COVID-19 Vaccines/administration & dosage ; COVID-19 Vaccines/chemistry ; COVID-19 Vaccines/immunology ; Humans ; Saponins/administration & dosage ; Saponins/chemistry ; Saponins/immunology ; Structure-Activity Relationship ; Vaccines, Subunit/administration & dosage ; Vaccines, Subunit/chemistry ; Vaccines, Subunit/immunology
Chemical Substances	Adjuvants, Immunologic ; COVID-19 Vaccines ; Saponins ; Vaccines, Subunit
Language	English
Publishing date	2020-12-09
Publishing country	United States
Document type	Journal Article ; Review
ZDB-ID	2664176-8
ISSN	2164-554X ; 2164-5515
ISSN (online)	2164-554X
ISSN	2164-5515
DOI	10.1080/21645515.2020.1833579
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Article ; Online: Evaluation of Stem Cell Laden Collagen + Polycaprolactone + Multi-Walled Carbon Nano-Tubes Nano-Neural Scaffold with and Without Insulin Like Growth Factor-I For Sciatic Nerve Regeneration Post Crush Injury in Wistar Rats.

Mishra, Mamta / Maiti, Swapan Kumar / Elangovan, Kalaiselvan / Shivaramu, Shivaraju / Singh, Karam Pal / S, Amitha Banu / Mamachan, Merlin / Arya, Manish / Mishra, Divya / Hescheler, Jurgen

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

2023 Volume 57, Issue 6, Page(s) 452–477

Abstract: Background/aims: All body functions are activated, synchronized and controlled by a substantial, complex network, the nervous system. Upon injury, pathophysiology of the nerve injury proceeds through different paths. The axon may undergo a degenerative ... ...

Abstract	Background/aims: All body functions are activated, synchronized and controlled by a substantial, complex network, the nervous system. Upon injury, pathophysiology of the nerve injury proceeds through different paths. The axon may undergo a degenerative retraction from the site of injury for a short distance unless the injury is near to the cell body, in which case it continues to the soma and undergoes retrograde neuronal degeneration. Otherwise, the distal section suffers from Wallerian degeneration, which is marked by axonal swelling, spheroids, and cytoskeleton degeneration. The objective of the study was to evaluate the potential of mesenchymal stem cell laden neural scaffold and insulin-like growth factor I (IGF-I) in nerve regeneration following sciatic nerve injury in a rat model. Methods: The animals were anaesthetized and a cranio-lateral incision over left thigh was made. Sciatic nerve was exposed and crush injury was introduced for 90 seconds using haemostat at second locking position. The muscle and skin were sutured in routine fashion and thus the rat model of sciatic crush injury was prepared. The animal models were equally distributed into 5 different groups namely A, B, C, D and E and treated with phosphate buffer saline (PBS), carbon nanotubes based neural scaffold only, scaffold with IGF-I, stem cell laden scaffold and stem cell laden scaffold with IGF-I respectively. In vitro scaffold testing was performed. The nerve regeneration was assessed based on physico-neuronal, biochemical, histopathological examination, and relative expression of NRP-1, NRP-2 and GAP-43 and scanning electron microscopy. Results: Sciatic nerve injury model with crush injury produced for 90 seconds was standardized and successfully used in this study. All the biochemical parameters were in normal range in all the groups indicating no scaffold related changes. Physico-neuronal, histopathological, relative gene expression and scanning electron microscopy observations revealed appreciable nerve regeneration in groups E and D, followed by C and B. Restricted to no regeneration was observed in group A. Conclusion: Carbon nanotubes based scaffold provided electro-conductivity for proper neuronal regeneration while rat bone marrow-derived mesenchymal stem cells were found to induce axonal sprouting, cellular transformation; whereas IGF-I induced stem cell differentiation, myelin synthesis, angiogenesis and muscle differentiation.
MeSH term(s)	Rats ; Animals ; Rats, Wistar ; Insulin-Like Growth Factor I/pharmacology ; Insulin-Like Growth Factor I/therapeutic use ; Nanotubes, Carbon ; Sciatic Neuropathy/drug therapy ; Sciatic Neuropathy/pathology ; Sciatic Nerve/injuries ; Nerve Regeneration/physiology ; Crush Injuries/drug therapy ; Crush Injuries/pathology ; Mesenchymal Stem Cells/pathology ; Collagen
Chemical Substances	Insulin-Like Growth Factor I (67763-96-6) ; polycaprolactone (24980-41-4) ; Nanotubes, Carbon ; Collagen (9007-34-5)
Language	English
Publishing date	2023-11-18
Publishing country	Germany
Document type	Journal Article
ZDB-ID	1067572-3
ISSN	1421-9778 ; 1015-8987
ISSN (online)	1421-9778
ISSN	1015-8987
DOI	10.33594/000000670
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Article ; Online: Preliminary investigation reveals novel pathological consequences of bluetongue virus-1 infection in the endocrine glands of pregnant Indian sheep.

Singh, Rohit / Singh, Karam Pal / Singh, Rajendra / Singh, Vidya / Kumar, Pawan / Varshney, Rajat / Yadav, Akanksha / Mote, Akash / Gangwar, Mukesh / Prasath, N Babu

Animal biotechnology

2023 Volume 35, Issue 1, Page(s) 2269428

Abstract: Bluetongue virus (BTV), a major peril to the sheep industry, infects a wide range of the cells in the infected animals including mononuclear, dendritic and epithelial cells. However, little is known about its tropism for the secretory epithelial cells of ...

Abstract	Bluetongue virus (BTV), a major peril to the sheep industry, infects a wide range of the cells in the infected animals including mononuclear, dendritic and epithelial cells. However, little is known about its tropism for the secretory epithelial cells of endocrine glands and the pathogenesis it induces. The aim of the study was to assess the BTV load, antigen distribution in the tissue of the pituitary, thyroid as well as adrenal glands and associated histopathological consequences. BTV antigens were localized using immunohistochemistry in the thyroid's epithelial cells, zona fasciculata and zona reticularis cells and the anterior pituitary epithelial cells. The real-time PCR portrayed the high viral load in adrenals at 7
MeSH term(s)	Sheep ; Animals ; Pregnancy ; Female ; Bluetongue/diagnosis ; Bluetongue virus ; Immunohistochemistry ; Endocrine Glands/pathology ; Sheep Diseases
Language	English
Publishing date	2023-10-18
Publishing country	England
Document type	Journal Article
ZDB-ID	2043243-4
ISSN	1532-2378 ; 1049-5398
ISSN (online)	1532-2378
ISSN	1049-5398
DOI	10.1080/10495398.2023.2269428
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Article ; Online: Development and evaluation of recombinant gD protein based ELISA for sero-surveillance of BoHV-1 in India.

Nautiyal, Sushmita / Nandi, Sukdeb / Sharma, Kirtika / Gairola, Vivek / Sai Balaji, K G / Biswas, Sanchay Kumar / Agrawal, Ravikant / Mahajan, Sonalika / Singh, Karam Pal / Sharma, Gaurav Kumar

Biologicals : journal of the International Association of Biological Standardization

2023 Volume 84, Page(s) 101720

Abstract: Bovine herpes virus-1 (BoHV-1) is responsible for production losses through decreased milk yields, abortions, infertility, and trade restrictions in the bovine population. The disease is endemic in many countries including India. As the virus harbors a ... ...

Abstract	Bovine herpes virus-1 (BoHV-1) is responsible for production losses through decreased milk yields, abortions, infertility, and trade restrictions in the bovine population. The disease is endemic in many countries including India. As the virus harbors a unique feature of latency animals once infected with the virus remain sero-positive for lifetime and can re-excrete the virus when exposed to stressful conditions. Hence, identification and culling of infected animals is only the means to minimize infection-associated losses. In this study, an economical indigenous assay for the detection of BoHV-1 specific antibodies was developed to cater to the huge bovine population of the country. The viral structural gD protein, expressed in the prokaryotic system was used for optimization of an indirect ELISA for bovines followed by statistical validation of the assay. The diagnostic sensitivity and specificity of the indirect ELISA were 82.9% and 91.3% respectively. Systematically collected serum samples representing organized, unorganized and breeding farms of India were tested with the indigenously developed assay for further validation.
MeSH term(s)	Animals ; Cattle ; Herpesvirus 1, Bovine ; Viral Proteins ; Enzyme-Linked Immunosorbent Assay ; Antibodies, Viral ; India ; Cattle Diseases/diagnosis
Chemical Substances	Viral Proteins ; Antibodies, Viral
Language	English
Publishing date	2023-11-07
Publishing country	England
Document type	Journal Article
ZDB-ID	1017370-5
ISSN	1095-8320 ; 1045-1056
ISSN (online)	1095-8320
ISSN	1045-1056
DOI	10.1016/j.biologicals.2023.101720
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