Indian Journal of Public Health Research & Development, 2014
Background : Rabies is an enzootic, fatal disease and early detection of this disease is necessar... more Background : Rabies is an enzootic, fatal disease and early detection of this disease is necessary. Till now a reliable diagnosis is possible only after death of animal. Objectives : So the present study was undertaken to use the molecular approaches like nested RTPCR and SYBR Green real time PCR for early diagnosis of rabies in suspected animals using less invasive non neural tissues like skin biopsy and hair follicles. Method : 12 cases of rabies suspected live animals were presented to the Veterinary Clinics, GADVASU, Ludhiana, India. Results : The sensitivity of nested RT-PCR for hair follicles and skin samples was 50% and 57.1% respectively. A sensitivity of 62.5% and 71.4% was observed with real time PCR in hair follicles and skin samples. Conclusion : We suggest that non-invasive molecular approaches may be used for the early and accurate diagnosis of rabies in animals.
Indian Journal of Public Health Research & Development, 2014
Background : Outward spread of rabies virus from the CNS leads to infection of almost all organs ... more Background : Outward spread of rabies virus from the CNS leads to infection of almost all organs and have been reported in various secretions and excretions viz. saliva, urine, CSF, tears (corneal smear). Although transmission of rabies virus from consuming unpasteurized milk from an infected animal is theoretically possible, no human or animal has ever been reported to develop rabies via this route. Neither there had been any practical proof of making claim about shedding of rabies virus in milk. This work aimed to identify the presence of rabies virus in unpasteurized raw milk from naturally-infected animals that were confirmed positive for rabies by FAT on brain tissue. Method : Out of total 25 animals showing clinical signs of rabies, milk sample was obtained from 17 lactating animals. Brief history was recorded and an attempt was made by employing advanced molecular approach TaqMan real time PCR on milk samples with an aim to detect the presence of rabies viral RNA. Confirmatory diagnosis of rabies was made by applying immunofluorescence techniques on brain tissue after death of an animal. Results : The present finding depicts the presence of rabies viral RNA in 4/17(23.52%) milk sample with a sensitivity of 60% when compared with FAT applied on brain tissue. Conclusion : A complete perusal of literature revealed that there had not been a single reported study to detect the presence of rabies viral RNA in milk samples. Although with this study it is now proved practically that rabies viral RNA can be found in milk samples from lactating animals however the infectivity of unpasteurized raw milk needs to be evaluated.
Porcine parvovirus (PPV) is a predominant infectious agent responsible for infertility in pigs. G... more Porcine parvovirus (PPV) is a predominant infectious agent responsible for infertility in pigs. Globally, it leads to colossal economic loss to the pig rearing communities, especially in the developing countries. The nonstructural gene 1 (NS1) of PPV is responsible for the virus replication, transcription regulation and cytotoxicity. Variations in the NS1 gene could cause increased virulence of the virus and the enhanced virulence raises concern about the effectiveness of the PPV vaccines against newly emerging strains. So, in the present study, we collected 84 samples from different regions of Punjab and Guwahati (Assam) during 2019-2022. Out of 84 samples, 12samples were positive for PPV-1. The NS1 gene was cloned and sequenced followed by analysis of Indian PPV-1 isolates to understand its evolutionary background, level of divergence and nucleotide/amino acid substitutions. The findings revealed that Indian PPV-1 isolates exhibit nucleotide substitutions with high percent sequenc...
The present study was undertaken to detect the presence of canine distemper virus (CDV) in suspec... more The present study was undertaken to detect the presence of canine distemper virus (CDV) in suspected dogs by immunochromatographic (IC) strip test and nested RT-PCR. A total of 100 samples (50 each of conjunctival and nasal swabs) were collected from dogs suspected of canine distemper. These samples were tested by IC strip test using Ubio quickVET canine distemper virus Ag detection kit for preliminary diagnosis and then subjected to molecular detection by nested RT-PCR. Twenty eight samples were found positive by the Ubio quickVet kit and out of these positive samples 17(34%) were conjunctival swabs and 11(22%) were nasal swabs. A total of 50 samples were subjected to nested RT-PCR for the detection of CD virus RNA by amplification of N gene and desired amplicon of 419bp was observed in nine samples, out of which 6 (24%) were conjunctival swabs and 3(12%) were nasal swabs. Nested RT-PCR of N gene is the reliable technique for the detection of CDV and conjunctival swab samples would...
Canine distemper (CD), caused by canine distemper virus (CDV) is a highly contagious disease that... more Canine distemper (CD), caused by canine distemper virus (CDV) is a highly contagious disease that infects a variety of carnivores. Although only one CDV serotype has been reported worldwide, lot of genetic variation in the virus genome particularly in the haemagglutinin (H) gene has been observed in CDV strains of different regions which are thought to be one of the causes of vaccine failure. In this study, a total of 50 ocular and nasal swab samples from CDV suspected dogs when processed for viral RNA detection, desired amplicons of 268bp could be detected in three cases. The CDV positive samples were then subjected to virus isolation first in primary dog lymphocyte culture and then in MDCK cells which were again harvested, passaged and viral RNA was confirmed in cell culture harvest by diagnostic RT-PCR. The individual CDV genes of a local isolate viz. full length fusion protein (F), phosphoprotein (P) matrix protein (M) and partial coding sequence of haemagglutinin (H) and largep...
A total of 90 birds comprising two native breeds viz. Aseel and Kadaknath and one synthetic broil... more A total of 90 birds comprising two native breeds viz. Aseel and Kadaknath and one synthetic broiler stock i.e. IBL-80 were utilized to evaluate and compare antibody response to Sheep Red Blood Cells by haemagglutination test at 0, 5 and 10 days post primary inoculation, to study in vivo cell mediated immune response to mitogen Phytohaemagglutinin (PHA-P) and to evaluate immune responsiveness to IBDV vaccine. The presence of natural antibodies against SRBC was evident in all the genetic groups. All groups showed an increase in HA titre upto 10 days post immunization. The HA titre on 10 day was significantly higher in Aseel (1.88±0.10) followed by IBL-80 (1.13±0.05) and Kadaknath (1.09±0.06). However, the differences among Kadaknath and IBL-80 at day 10 PPI were failed to attain statistical significance. The in vivo cell mediated response to mitogen was highest in Aseel (0.68 mm) followed by IBL-80 (0.59 mm) and Kadaknath (0.43 mm).There was significant difference between the layer br...
Mastitis and metritis are the common inflammatory diseases of buffalo and result huge economic lo... more Mastitis and metritis are the common inflammatory diseases of buffalo and result huge economic losses to the small-scale farmers. Neutrophils are recruited during inflammation and cleared from site of inflammation by apoptosis to prevent further tissue damage. However, data are lacking on this aspect in buffalo. Hence, the study was aimed to determine the viability and apoptosis of neutrophils of buffalo during mastitis and metritis. Peripheral blood samples were collected from healthy, mastitis and metritis group (n=5, each group). Neutrophils were isolated and maintained up to 48 hr in RPMI-1640. The viability of neutrophils at different time intervals (6, 12, 24, 36 and 48 hr) was determined using trypan blue dye exclusion method. DNA Laddering, light microscopy, caspase-3 and caspase-9 colorimetric assays were used to observe the apoptosis. Freshly isolated neutrophils showed ≥ 97% purity and ≥ 92% viability. Healthy group showed decrease (p<0.05) in viable neutrophils 12...
Background: Swinepox is an economically important, classical pox disease of piglets. The present ... more Background: Swinepox is an economically important, classical pox disease of piglets. The present study was undertaken with a view to develop rapid serological tests to diagnose the disease. Methods: During the study period, 25 suspected swinepox outbreaks in Assam were confirmed by polymerase chain reaction with sequencing and phylogenetic analysis, further the outbreaks were confirmed by transmission electron microscopy (TEM) for identification of swinepox positive samples. The positive samples were used to isolate the virus in PK-15 cell line and develop indirect and sandwich ELISA. Result: The cell culture-based indirect ELISA was developed that demonstrated an accuracy of 88.8% compare to VNT and 100% sensitivity with 66.67% specificity, could identify 61.71% seroprevalence of swinepox in random pig serum samples. A sandwich ELISA was also developed with polyclonal sera raised in rabbits as coating antibody and swinepox positive pig serum as tracing antibody. The sandwich ELISA ...
Marek’s disease is a Lymphoprliferative disease of poultry caused by alphaherpesvirus. Despite th... more Marek’s disease is a Lymphoprliferative disease of poultry caused by alphaherpesvirus. Despite the ubiquitous use of vaccination to control losses, MD still affects a poultry industry with an estimated annual loss up to US $2 billion. Therefore, it is essential to better the ability of detection of MD virus during the early stages of an outbreak. The aim of the study was to develop a loop-mediated isothermal amplification (LAMP) method for the simple, rapid and inexpensive detection of MDV from the tissues of MD positive birds and to compare with diagnostic efficacy of conventional PCR. The LAMP was established using a set of three pair of primers specific to MEQ gene for detecting serotype 1 MDV. The amplification was able to finish in 60 min under isothermal condition at 65 o C by addition of Bst polymerase. The results were visualized directly under daylight or ultraviolet light by addition of SYBR Green I dye. The Assay demonstrate a significant advantage of loop-mediated isothe...
Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industr... more Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industry in developing countries like India. For evaluating the true extent of porcine infections, a total of 1308 representative sera samples were collected from 92 different pig farms covering 8 North-Eastern states and Punjab state of Northern India during a period of 2 years (2011–2013). Sera samples were tested for the presence of antibodies against porcine parvovirus (PPV), porcine circovirus-2 (PCV-2), and classical swine fever virus (CSFV) using commercial enzyme-linked immunosorbent assay (ELISA) kits. In the North-Eastern states, the seroprevalence of CSFV in non-vaccinated animals was 6.30% and that of PCV2 and PPV was 6.28% and 1.24%, respectively. In Punjab, the seroprevalence of CSFV in non-vaccinated animals was 44.44% and seroprevalence of PCV-2 and PPV was 34.07% and 39.10%, respectively. Detection of antibodies against more than one virus revealed that 4.66% animals had co-inf...
Background: This study aimed at the evaluation of the efficacy of TLR ligands (viz. Poly I:C, Lip... more Background: This study aimed at the evaluation of the efficacy of TLR ligands (viz. Poly I:C, Lipopolysaccharide and CpG:ODN) as vaccine adjuvants in combination with a suitable carrier (oil-in-water emulsion, normal saline solution, or alum) by studying the expression profile of cytokines belonging to Th1 and Th2 biased immune systems. Methods: Whole blood was collected from 108 birds that were divided into 9 different experimental groups: 3 TLR ligands each with 3 delivery vehicles. Th1 (IFN-gamma) and Th2 (IL4) biased cytokines were detected by qPCR followed by ELISA. Both the results showed the highest level of IL4 (Th1 biased) with LPS and the highest level of IFNg (Th1 biased) with CpG-ODN. Uneven TLR expression was detected from day 3 onwards till day 21 but ligands and delivery vehicles administered along with NDV vaccine did not show significant influence on the immune response. Result: It is evident that the TLR-3, 4 and 9 expressions increase following exposure to the res...
Indian Journal of Public Health Research & Development, 2014
Background : Rabies is an enzootic, fatal disease and early detection of this disease is necessar... more Background : Rabies is an enzootic, fatal disease and early detection of this disease is necessary. Till now a reliable diagnosis is possible only after death of animal. Objectives : So the present study was undertaken to use the molecular approaches like nested RTPCR and SYBR Green real time PCR for early diagnosis of rabies in suspected animals using less invasive non neural tissues like skin biopsy and hair follicles. Method : 12 cases of rabies suspected live animals were presented to the Veterinary Clinics, GADVASU, Ludhiana, India. Results : The sensitivity of nested RT-PCR for hair follicles and skin samples was 50% and 57.1% respectively. A sensitivity of 62.5% and 71.4% was observed with real time PCR in hair follicles and skin samples. Conclusion : We suggest that non-invasive molecular approaches may be used for the early and accurate diagnosis of rabies in animals.
Indian Journal of Public Health Research & Development, 2014
Background : Outward spread of rabies virus from the CNS leads to infection of almost all organs ... more Background : Outward spread of rabies virus from the CNS leads to infection of almost all organs and have been reported in various secretions and excretions viz. saliva, urine, CSF, tears (corneal smear). Although transmission of rabies virus from consuming unpasteurized milk from an infected animal is theoretically possible, no human or animal has ever been reported to develop rabies via this route. Neither there had been any practical proof of making claim about shedding of rabies virus in milk. This work aimed to identify the presence of rabies virus in unpasteurized raw milk from naturally-infected animals that were confirmed positive for rabies by FAT on brain tissue. Method : Out of total 25 animals showing clinical signs of rabies, milk sample was obtained from 17 lactating animals. Brief history was recorded and an attempt was made by employing advanced molecular approach TaqMan real time PCR on milk samples with an aim to detect the presence of rabies viral RNA. Confirmatory diagnosis of rabies was made by applying immunofluorescence techniques on brain tissue after death of an animal. Results : The present finding depicts the presence of rabies viral RNA in 4/17(23.52%) milk sample with a sensitivity of 60% when compared with FAT applied on brain tissue. Conclusion : A complete perusal of literature revealed that there had not been a single reported study to detect the presence of rabies viral RNA in milk samples. Although with this study it is now proved practically that rabies viral RNA can be found in milk samples from lactating animals however the infectivity of unpasteurized raw milk needs to be evaluated.
Porcine parvovirus (PPV) is a predominant infectious agent responsible for infertility in pigs. G... more Porcine parvovirus (PPV) is a predominant infectious agent responsible for infertility in pigs. Globally, it leads to colossal economic loss to the pig rearing communities, especially in the developing countries. The nonstructural gene 1 (NS1) of PPV is responsible for the virus replication, transcription regulation and cytotoxicity. Variations in the NS1 gene could cause increased virulence of the virus and the enhanced virulence raises concern about the effectiveness of the PPV vaccines against newly emerging strains. So, in the present study, we collected 84 samples from different regions of Punjab and Guwahati (Assam) during 2019-2022. Out of 84 samples, 12samples were positive for PPV-1. The NS1 gene was cloned and sequenced followed by analysis of Indian PPV-1 isolates to understand its evolutionary background, level of divergence and nucleotide/amino acid substitutions. The findings revealed that Indian PPV-1 isolates exhibit nucleotide substitutions with high percent sequenc...
The present study was undertaken to detect the presence of canine distemper virus (CDV) in suspec... more The present study was undertaken to detect the presence of canine distemper virus (CDV) in suspected dogs by immunochromatographic (IC) strip test and nested RT-PCR. A total of 100 samples (50 each of conjunctival and nasal swabs) were collected from dogs suspected of canine distemper. These samples were tested by IC strip test using Ubio quickVET canine distemper virus Ag detection kit for preliminary diagnosis and then subjected to molecular detection by nested RT-PCR. Twenty eight samples were found positive by the Ubio quickVet kit and out of these positive samples 17(34%) were conjunctival swabs and 11(22%) were nasal swabs. A total of 50 samples were subjected to nested RT-PCR for the detection of CD virus RNA by amplification of N gene and desired amplicon of 419bp was observed in nine samples, out of which 6 (24%) were conjunctival swabs and 3(12%) were nasal swabs. Nested RT-PCR of N gene is the reliable technique for the detection of CDV and conjunctival swab samples would...
Canine distemper (CD), caused by canine distemper virus (CDV) is a highly contagious disease that... more Canine distemper (CD), caused by canine distemper virus (CDV) is a highly contagious disease that infects a variety of carnivores. Although only one CDV serotype has been reported worldwide, lot of genetic variation in the virus genome particularly in the haemagglutinin (H) gene has been observed in CDV strains of different regions which are thought to be one of the causes of vaccine failure. In this study, a total of 50 ocular and nasal swab samples from CDV suspected dogs when processed for viral RNA detection, desired amplicons of 268bp could be detected in three cases. The CDV positive samples were then subjected to virus isolation first in primary dog lymphocyte culture and then in MDCK cells which were again harvested, passaged and viral RNA was confirmed in cell culture harvest by diagnostic RT-PCR. The individual CDV genes of a local isolate viz. full length fusion protein (F), phosphoprotein (P) matrix protein (M) and partial coding sequence of haemagglutinin (H) and largep...
A total of 90 birds comprising two native breeds viz. Aseel and Kadaknath and one synthetic broil... more A total of 90 birds comprising two native breeds viz. Aseel and Kadaknath and one synthetic broiler stock i.e. IBL-80 were utilized to evaluate and compare antibody response to Sheep Red Blood Cells by haemagglutination test at 0, 5 and 10 days post primary inoculation, to study in vivo cell mediated immune response to mitogen Phytohaemagglutinin (PHA-P) and to evaluate immune responsiveness to IBDV vaccine. The presence of natural antibodies against SRBC was evident in all the genetic groups. All groups showed an increase in HA titre upto 10 days post immunization. The HA titre on 10 day was significantly higher in Aseel (1.88±0.10) followed by IBL-80 (1.13±0.05) and Kadaknath (1.09±0.06). However, the differences among Kadaknath and IBL-80 at day 10 PPI were failed to attain statistical significance. The in vivo cell mediated response to mitogen was highest in Aseel (0.68 mm) followed by IBL-80 (0.59 mm) and Kadaknath (0.43 mm).There was significant difference between the layer br...
Mastitis and metritis are the common inflammatory diseases of buffalo and result huge economic lo... more Mastitis and metritis are the common inflammatory diseases of buffalo and result huge economic losses to the small-scale farmers. Neutrophils are recruited during inflammation and cleared from site of inflammation by apoptosis to prevent further tissue damage. However, data are lacking on this aspect in buffalo. Hence, the study was aimed to determine the viability and apoptosis of neutrophils of buffalo during mastitis and metritis. Peripheral blood samples were collected from healthy, mastitis and metritis group (n=5, each group). Neutrophils were isolated and maintained up to 48 hr in RPMI-1640. The viability of neutrophils at different time intervals (6, 12, 24, 36 and 48 hr) was determined using trypan blue dye exclusion method. DNA Laddering, light microscopy, caspase-3 and caspase-9 colorimetric assays were used to observe the apoptosis. Freshly isolated neutrophils showed ≥ 97% purity and ≥ 92% viability. Healthy group showed decrease (p<0.05) in viable neutrophils 12...
Background: Swinepox is an economically important, classical pox disease of piglets. The present ... more Background: Swinepox is an economically important, classical pox disease of piglets. The present study was undertaken with a view to develop rapid serological tests to diagnose the disease. Methods: During the study period, 25 suspected swinepox outbreaks in Assam were confirmed by polymerase chain reaction with sequencing and phylogenetic analysis, further the outbreaks were confirmed by transmission electron microscopy (TEM) for identification of swinepox positive samples. The positive samples were used to isolate the virus in PK-15 cell line and develop indirect and sandwich ELISA. Result: The cell culture-based indirect ELISA was developed that demonstrated an accuracy of 88.8% compare to VNT and 100% sensitivity with 66.67% specificity, could identify 61.71% seroprevalence of swinepox in random pig serum samples. A sandwich ELISA was also developed with polyclonal sera raised in rabbits as coating antibody and swinepox positive pig serum as tracing antibody. The sandwich ELISA ...
Marek’s disease is a Lymphoprliferative disease of poultry caused by alphaherpesvirus. Despite th... more Marek’s disease is a Lymphoprliferative disease of poultry caused by alphaherpesvirus. Despite the ubiquitous use of vaccination to control losses, MD still affects a poultry industry with an estimated annual loss up to US $2 billion. Therefore, it is essential to better the ability of detection of MD virus during the early stages of an outbreak. The aim of the study was to develop a loop-mediated isothermal amplification (LAMP) method for the simple, rapid and inexpensive detection of MDV from the tissues of MD positive birds and to compare with diagnostic efficacy of conventional PCR. The LAMP was established using a set of three pair of primers specific to MEQ gene for detecting serotype 1 MDV. The amplification was able to finish in 60 min under isothermal condition at 65 o C by addition of Bst polymerase. The results were visualized directly under daylight or ultraviolet light by addition of SYBR Green I dye. The Assay demonstrate a significant advantage of loop-mediated isothe...
Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industr... more Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industry in developing countries like India. For evaluating the true extent of porcine infections, a total of 1308 representative sera samples were collected from 92 different pig farms covering 8 North-Eastern states and Punjab state of Northern India during a period of 2 years (2011–2013). Sera samples were tested for the presence of antibodies against porcine parvovirus (PPV), porcine circovirus-2 (PCV-2), and classical swine fever virus (CSFV) using commercial enzyme-linked immunosorbent assay (ELISA) kits. In the North-Eastern states, the seroprevalence of CSFV in non-vaccinated animals was 6.30% and that of PCV2 and PPV was 6.28% and 1.24%, respectively. In Punjab, the seroprevalence of CSFV in non-vaccinated animals was 44.44% and seroprevalence of PCV-2 and PPV was 34.07% and 39.10%, respectively. Detection of antibodies against more than one virus revealed that 4.66% animals had co-inf...
Background: This study aimed at the evaluation of the efficacy of TLR ligands (viz. Poly I:C, Lip... more Background: This study aimed at the evaluation of the efficacy of TLR ligands (viz. Poly I:C, Lipopolysaccharide and CpG:ODN) as vaccine adjuvants in combination with a suitable carrier (oil-in-water emulsion, normal saline solution, or alum) by studying the expression profile of cytokines belonging to Th1 and Th2 biased immune systems. Methods: Whole blood was collected from 108 birds that were divided into 9 different experimental groups: 3 TLR ligands each with 3 delivery vehicles. Th1 (IFN-gamma) and Th2 (IL4) biased cytokines were detected by qPCR followed by ELISA. Both the results showed the highest level of IL4 (Th1 biased) with LPS and the highest level of IFNg (Th1 biased) with CpG-ODN. Uneven TLR expression was detected from day 3 onwards till day 21 but ligands and delivery vehicles administered along with NDV vaccine did not show significant influence on the immune response. Result: It is evident that the TLR-3, 4 and 9 expressions increase following exposure to the res...
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