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ABSTRACT: Rinderpest causes a devastating disease, often fatal, in wild and domestic ruminants. It has been eradicated successfully using a live, attenuated vaccine from most part of the world leaving a few foci of disease in parts of Africa, the Middle East, and South Asia. We have developed transgenic peanut (Arachis hypogaea L.) plants expressing hemagglutinin (H) protein of rinderpest virus (RPV), which is antigenically authentic. In this work, we have evaluated the immunogenicity of peanut-expressed H protein using mouse model, administered parenterally as well as orally. Intraperitoneal immunization of mice with the transgenic peanut extract elicited antibody response specific to H. These antibodies neutralized virus infectivity in vitro. Oral immunization of mice with transgenic peanut induced H-specific serum IgG and IgA antibodies. The systemic and oral immunogenicity of plant-derived H in absence of any adjuvant indicates the potential of edible vaccine for rinderpest.
Virology 07/2004; 323(2):284-91. · 3.35 Impact Factor
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ABSTRACT: A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects antibodies unique to rinderpest virus (RPV) has been developed. This test can differentiate antibodies against RPV and those against peste des petits ruminants virus. The recombinant RPV hemagglutinin (H)-protein C-ELISA (recH C-ELISA) is based on the ability of a well-characterized monoclonal antibody (MAb) produced with the soluble, secreted form of the H protein (Sec H protein) of RPV made in a baculovirus expression system to compete with the binding of RPV antibodies in the serum of vaccinated or infected, recovered animals to the Sec H protein. The B-cell epitope recognized by the MAb corresponds to amino acids 575 to 583 on the H protein, which is not present on the antigenically closely related peste des petits ruminants virus hemagglutinin-neuraminidase protein. Initially, a positive-negative threshold cutoff value for percent inhibition of 34 was established with 500 known RPV-negative serum samples. The recH C-ELISA was developed with the enzyme immunoassay software of a commercial RPV C-ELISA kit. Comparative analysis of the test results for 700 serum samples obtained with the commercial kit gave a sensitivity of 112.4% and a specificity of 72.4%. Variations in percent inhibition values were observed for the two assay systems. These variations may have been due to the undefined amount of antigen present in the commercial kit as well as the use of a different MAb. The recH C-ELISA detected more positive serum samples compared to the number detected by the commercial kit, with the results confirmed by a virus neutralization test. Thus, recH C-ELISA is a sensitive tool for RPV serosurveillance in disease eradication programs.
Journal of Clinical Microbiology 04/2003; 41(3):943-7. · 4.15 Impact Factor
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ABSTRACT: In India, brucellosis was first recognised in 1942 and is now endemic throughout the country. The disease is reported in cattle, buffalo, sheep, goats, pigs, dogs and humans. B. abortus biotype-1 in cattle and buffaloes and B. melitensis biotype-1 in sheep, goats and man are the predominant infective biotypes. The long-term serological studies have indicated that 5% of cattle and 3% of buffaloes are infected with brucellosis. Economic losses due to brucellosis in livestock are considerable in an agrarian country like India. There is no organised and effective brucellosis control programme in the country. With the indigenous development of serum and milk based ELISA kits, the population survey of the disease has been undertaken on a large scale in several states and plans for the control of the disease through calf-hood vaccination are being worked out. An innovative approach--Bovine Brucellosis Progressive Control Programme (BBPCP) is targeted to overcome the basic problems of ban on cow slaughter, distress sale of animals following the positive serological diagnosis of brucellosis and absence of a disease control strategy. The work plan for the implementation of BBPCP is presented.
Veterinary Microbiology 01/2003; 90(1-4):183-95. · 3.33 Impact Factor
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ABSTRACT: A recombinant baculovirus expressing membrane bound form of hemagglutinin-neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein. Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells. The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263-368 and 538-609 and hence the epitopes identified are conformation-dependent. The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera. Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity. Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%). Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties. All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay.
Virus Research 01/2003; 90(1-2):171-85. · 2.94 Impact Factor
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ABSTRACT: Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus HN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination.
Virology 08/2002; 298(2):214-23. · 3.35 Impact Factor
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ABSTRACT: The avidin-biotin enzyme-linked immunosorbent assay (A-B ELISA), for use in surveillance for bovine brucellosis in India was developed and calibrated using the indirect brucellosis ELISA kit of the International Atomic Energy Agency (IAEA) as a reference. The reagents used in the A-B ELISA were as follows: the smooth lipopolysaccharide of Brucella abortus strain 99 (antigen); biotinylated anti-bovine immunoglobulin G (detection antibody); avidin-horseradish peroxidase (conjugate); and O-phenylenediamine dihydrochloride (chromogen). The test results were interpreted using the IAEA software EDI version 2.1.1, which was modified for use in the A-B ELISA. The cut-off percentage positivity value was established using 500 brucellosis-positive and 500 brucellosis-negative serum samples, confirmed with reference to the sample data using the indirect ELISA kit. The overall specificity of A-B ELISA was 98.8% and overall sensitivity was 98.2%. Field validation of the A-B ELISA kit was undertaken in six laboratories in India. Screening of 7,040 cattle and 678 buffalo serum samples from 12 states revealed serological evidence of brucellosis in 8.7% of cattle and 10.2% of buffalo. This kit proved to be robust and performed with a similar sensitivity and specificity to the indirect ELISA. The kit can be supplied at a lower cost than current commercial ELISA kits.
Revue scientifique et technique (International Office of Epizootics) 01/2002; 20(3):749-56. · 1.10 Impact Factor
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ABSTRACT: Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus within the family Paramyxoviridae, causes a fatal disease 'peste des petits ruminants' in goats and sheep. This enveloped virus is antigenically closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants. PPRV harbors two major surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. The surface glycoproteins of morbilliviruses are highly immunogenic and confer protective immunity. In this study, we investigated the immune responses generated in goats immunized with low doses of purified recombinant extracellular baculovirus carrying a membrane bound form of the HN protein of PPRV without any adjuvant. We report that the immunized goats develop both humoral and cell-mediated immune responses. Antibodies generated in the immunized animals could neutralize both PPRV and RPV in vitro. Further, using a combination of Escherichia coli expressed deletion mutants of PPRV-HN and RPV-H proteins, and synthetic peptides corresponding to the highly conserved N-terminal sequences of MV-H protein, we have mapped an N-terminal T cell determinant (amino acids 123-137) and a C-terminal domain (amino acids 242-609) harboring potential T cell determinant(s) in goats.
Vaccine 10/2001; 19(32):4816-23. · 3.77 Impact Factor
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ABSTRACT: Rinderpest virus causes a highly contagious and often fatal disease in domestic and wild ruminants. The surface glycoproteins, hemagglutinin (H) and fusion (F) proteins of this enveloped virus are known to confer protective immunity in cattle. We have reported the generation of a recombinant baculovirus expressing H protein and studied its protective properties in cattle. In this report, we demonstrate that the recombinant baculovirus encoded H protein expressed in insect cells gets incorporated into extracellular baculovirus. Single administration of low doses of purified recombinant extracellular virus with or without adjuvant induces virus neutralizing antibody responses and bovine leukocyte antigen (BoLA) class II restricted helper T cell responses in cattle.
Vaccine 08/2001; 19(28-29):3870-6. · 3.77 Impact Factor
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ABSTRACT: Rinderpest virus (RPV), a member of the genus Morbillivirus within the Paramyxoviridae family, causes a highly contagious and often fatal disease known as rinderpest in wild and domestic ruminants. The envelope of the virus contains two surface glycoproteins, namely the hemagglutinin (H) and the fusion (F) proteins, both of which have been shown to confer protective immunity in animals. In this paper, we demonstrate that single administration of low doses of recombinant H protein of RPV expressed in insect cells in the form of extracellular virus induces long lasting bovine leukocyte antigen class I restricted cytotoxic T-cell (CTL) responses in cattle in the absence of adjuvant. This is the first report of CTL responses in cattle against one of the protective antigens of RPV.
Viral Immunology 02/2001; 14(4):349-58. · 1.97 Impact Factor
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S Sreevatsan,
J B Bookout,
F Ringpis,
V S Perumaalla,
T A Ficht,
L G Adams,
S D Hagius,
P H Elzer,
B J Bricker,
G K Kumar, M Rajasekhar,
S Isloor,
R R Barathur
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ABSTRACT: A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.
Journal of Clinical Microbiology 08/2000; 38(7):2602-10. · 4.15 Impact Factor
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ABSTRACT: A study was undertaken regarding the prevalence of bovine viral diarrhoea virus (BVDV) antibodies in bovine sera which tested negative for rinderpest and peste des petits ruminants virus antibodies. The samples were collected between January 1996 and December 1997. A total of 439 samples (327 cattle and 112 buffalo from 17 states of India) were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The mean prevalence of BVDV antibodies in cattle was 15.29% (50/327) in 16 states compared to 23.21% (26/112) in buffalo in 9 states, with an overall prevalence of 17.31% (76/439) in 17 states. The serological evidence that BVDV infection is widespread in India is of utmost practical importance because of the clinical resemblance to rinderpest. A differential diagnosis between these two diseases is critical in view of the declaration by India of provisional freedom from rinderpest disease; active sero-surveillance is to begin in 2000 to achieve certification of freedom from rinderpest infection by the Office International des Epizooties.
Revue scientifique et technique (International Office of Epizootics) 01/2000; 18(3):667-71. · 1.10 Impact Factor
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ABSTRACT: A serological survey of brucellosis in cattle and buffalo was performed in 23 States of India. A total of 30,437 bovine samples, comprising 23,284 cattle and 7,153 buffalo (Bubalus bubalis), were screened. The screening initially used the rose bengal plate test: doubtful and positive samples were then titrated in the serum tube agglutination test. The overall prevalence rate of antibodies was 1.9% in cattle and 1.8% in buffalo. In a detailed study of 47 organised farms in the southern State of Karnataka, 207 of 4,995 (4.1%) serum samples from cattle showed titres for brucellosis. This result was in contrast to the low rate of seropositive results reported in cattle owned by individual farmers in Karnataka (0.7% of 2,424 serum samples). In organised farms with a history of abortion, placenta retention and repeat breeding, the prevalence rate was 17%.
Revue scientifique et technique (International Office of Epizootics) 01/1999; 17(3):781-5. · 1.10 Impact Factor
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ABSTRACT: The authors describe a serological survey on the prevalence of infectious bovine rhinotracheitis (IBR) among cattle and buffalo (Bubalus bubalis) in three southern states of India. A local isolate of bovine herpesvirus 1 (BHV-1) from an outbreak of the respiratory form of IBR was used as a source of virus antigen in avidin-biotin enzyme-linked immunosorbent assay (ELISA). The overall prevalence of antibodies to BHV-1 in cattle was 50.9%, and in buffalo was 52.5%. Among breeding bulls, 114/120 samples from Tamil Nadu (95%) and 41/99 samples from Karnataka (41.4%) were seropositive. The possible association of IBR with bovine abortions was recorded in 31/56 samples (55.4%) from aborted crossbred cows. However, virus isolation was not performed on these animals. The authors also highlight the economic importance of IBR to the rapidly developing livestock industry in India.
Revue scientifique et technique (International Office of Epizootics) 10/1996; 15(3):1021-8. · 1.10 Impact Factor
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ABSTRACT: Rinderpest antibodies were demonstrated in 37 per cent of 2400 sheep and 29.5 per cent of 1000 goats in the southern states of India by the avidin-biotin enzyme linked immunosorbent assay technique. This technique, besides quantifying rinderpest antibodies, has provided a simple method for the differentiation of positive and negative serum samples on the basis of the development of a pink colour, a great advantage in the screening of large numbers of sera for field epidemiological studies. The presence of the antibodies in apparently healthy small ruminants is of great significance in the context of the control of rinderpest in India.
The Veterinary record 01/1989; 123(23):595-7. · 1.25 Impact Factor
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ABSTRACT: The hemagglutinin (H) protein of Rinderpest virus expressed by a recombinant baculo-virus used as a vaccine produced high titres of neutralizing antibody to Rinderpest virus in the vaccinated cattle, comparable to the levels produced by live attenuated vaccine. The immunized cattle were protected against a vaccine-virus challenge, as demonstrated by the failure of development of antibodies to N protein of the vaccine virus. The lack of replication of vaccine virus in the immunized cattle indicated that they are capable of showing a protective response if challenged with a virulent virus.
Vaccine.
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ABSTRACT: Rinderpest antibodies in 166 lambs of different ages, born to vaccinated ewes were studied with avidin-biotin enzyme-linked immunosorbent assay. The mean serum background optical density (OD) value in 20 colostrum deprived lambs and 50 unvaccinated adult sheep was 0.12. The OD values of 166 one to six month-old colostrum-fed lambs ranged from 0.08 to 0.64 and the pooled rinderpest positive serum was 1.44. A peak value of 0.64 was obtained in the first week which gradually declined to become negative for the presence of rinderpest maternal antibodies at around six months of age (OD=< 0.24). Primary vaccination of lambs of vaccinated ewes is therefore considered appropriate at six months of age for effective immunisation with least interference of maternally-derived antibodies.
Small Ruminant Research.
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ABSTRACT: Rinderpest causes a devastating disease, often fatal, in wild and domestic ruminants. It has been eradicated successfully using a live, attenuated vaccine from most part of the world leaving a few foci of disease in parts of Africa, the Middle East, and South Asia. We have developed transgenic peanut (Arachis hypogaea L.) plants expressing hemagglutinin (H) protein of rinderpest virus (RPV), which is antigenically authentic. In this work, we have evaluated the immunogenicity of peanut-expressed H protein using mouse model, administered parenterally as well as orally. Intraperitoneal immunization of mice with the transgenic peanut extract elicited antibody response specific to H. These antibodies neutralized virus infectivity in vitro. Oral immunization of mice with transgenic peanut induced H-specific serum IgG and IgA antibodies. The systemic and oral immunogenicity of plant-derived H in absence of any adjuvant indicates the potential of edible vaccine for rinderpest.
Virology.
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[show abstract]
[hide abstract]
ABSTRACT: A recombinant baculovirus expressing membrane bound form of hemagglutinin–neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein. Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells. The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263–368 and 538–609 and hence the epitopes identified are conformation-dependent. The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera. Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity. Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%). Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties. All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay.
Virus Research.
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[show abstract]
[hide abstract]
ABSTRACT: Rinderpest virus causes a highly contagious and often fatal disease in domestic and wild ruminants. The surface glycoproteins, hemagglutinin (H) and fusion (F) proteins of this enveloped virus are known to confer protective immunity in cattle. We have reported the generation of a recombinant baculovirus expressing H protein and studied its protective properties in cattle. In this report, we demonstrate that the recombinant baculovirus encoded H protein expressed in insect cells gets incorporated into extracellular baculovirus. Single administration of low doses of purified recombinant extracellular virus with or without adjuvant induces virus neutralizing antibody responses and bovine leukocyte antigen (BoLA) class II restricted helper T cell responses in cattle.
Vaccine.
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[show abstract]
[hide abstract]
ABSTRACT: The hemagglutinin (H) protein of Rinderpest virus expressed by a recombinant baculovirus used as a vaccine produced high titres of neutralizing antibody to Rinderpest virus in the vaccinated cattle, comparable to the levels produced by live attenuated vaccine. The immunized cattle were protected against a vaccine-virus challenge, as demonstrated by the failure of development of antibodies to N protein of the vaccine virus. The lack of replication of vaccine virus in the immunized cattle indicated that they are capable of showing a protective response if challenged with a virulent virus.
Vaccine 15(6-7):603-7. · 3.77 Impact Factor
