Foot-and-mouth disease (FMD) is an economically important contagious disease of livestock mainly cattle, buffalo, sheep, goats, and pig. There is limited data available on pathogenesis of foot and ...mouth disease in goats. In the study, the sheep and goats were infected experimentally with a serotype O foot-and-mouth disease virus by different challenge routes. The sheep and goats challenged by coronary band route and coronary band and intra-dermo-lingual route exhibited FMD clinical signs at 2–5 days post challenge. Whereas intra-dermo-lingual challenged sheep and goats did not exhibit FMD clinical signs. Live virus could be isolated from blood of infected sheep and goats at 2–5 days post challenge. Viral RNA could be detected from blood of infected sheep and goats at 1–10 days post challenge. The neutralizing antibody titre was detected at 10 days post challenge and maintained up to 35 days post challenge in all infected sheep and goats. Non structural protein (NSP) antibodies were detected as early as 5–10 days post challenge and remain positive up to 35 days post challenge in the infected sheep and goats. In conclusion, the pathogenesis of sheep and goats with serotype O foot and mouth disease virus by different challenge routes could be demonstrated.
Abstract Serology is used to predict vaccine induced protection against challenge with a heterologous strain of the same serotype of foot-and-mouth disease virus (FMDV). To evaluate the accuracy of ...such predictions, we compared the protection afforded to cattle vaccinated with the O1 Manisa strain of FMDV against challenge with either a homologous (O1 Manisa) or a heterologous strain (O1 Campos). Serology by virus neutralization test (VNT) using O1 Manisa antiserum predicted an acceptable protection against such a challenge. Two experiments were carried out to compare the results for consistency. A total of 78 naïve cattle were vaccinated with different antigen payloads (60–0.94 μg) of O1 Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O1 Manisa or O1 Campos. Unvaccinated naïve control cattle ( n = 20) were also challenged with either the O1 Manisa or O1 Campos viruses and all developed generalized FMD. The protection results for the vaccinated cattle revealed that higher payloads of O1 Manisa vaccine were needed to protect against heterologous challenge compared to that for homologous challenge. The 50% protective dose (PD50 ) values for the vaccine in experiments 1 and 2 were found to be 28.78 and 9.44 for the homologous challenge and 3.98 and 5.01 for heterologous challenge. Furthermore, protection against O1 Campos required a higher level of vaccine-induced antibody against this virus compared to the level of O1 Manisa neutralizing antibody associated with protection against homologous challenge. The 50% protective level of in vitro neutralizing antibody was found to be log10 1.827 for O1 Campos and log10 0.954 for O1 Manisa based on O1 Manisa based virus neutralization test.
Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using ...the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay.
► Mycobacterium (TB) protein ESAT-6 and CFP-10 is cloned as a fusion ESAT-6-CFP-10 protein and purified from E. coli. ► Fusion protein has been evaluated in IFN-gamma ELISPOT assay. ► Fusion protein can replace ESAT-6, CFP-10 or a combination of both in ELISPOT assay for BTB diagnosis.
Abstract Expression of Physalis mottle tymovirus (PhMV) coat protein (CP) in Escherichia coli ( E. coli ) was earlier shown to self-assemble into empty capsids that are nearly identical to the ...capsids formed in vivo . Aminoacid substitutions were made at the N-terminus of wild-type PhMV CP with single or tandem repeats of infection related B-cell epitopes of foot-and-mouth disease virus (FMDV) non-structural proteins (NSPs) 3B1, 3B2, 3AB, 3D and 3ABD of lengths 48, 66, 49, 51 and 55, respectively to produce chimeras pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3D and pR-Ph-3ABD. Expression of these constructs in E. coli resulted in chimeric proteins which self-assembled into chimeric tymovirus-like particles (TVLPs), Ph-3B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD as determined by ultracentrifugation and electron microscopy. Ph-3B1, Ph-3B2, Ph-3AB and Ph-3ABD reacted with polyclonal anti-3AB antibodies in ELISA and electroblot immunoassay, while wild-type PhMV TVLP and Ph-3D antigens did not react. An indirect ELISA (I-ELISA) was developed using Ph-3AB to detect FMDV-NSP antibodies in sera of animals that showed clinical signs of FMD. Field serum samples from cattle, buffalos, sheep, goats and pigs were examined by using these chimeric TVLPs for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The assay was demonstrated to be highly specific (100%) and reproducible with sensitivity levels (94%) comparable to the Ceditest kit ( P > 0.05).
► The haemagglutinin of highly pathogenic avian influenza was cloned into Pichia sp. ► The antigen was expressed, purified and the haemagglutination property was analysed. ► The recombinant ...haemagglutinin was found to elicit neutralizing antibodies in mice. ► This method of vaccine manufacturing in Pichia pastoris may be cheaper and quicker. ► This method will help to produce vaccines against an avian influenza pandemic.
Recombinant avian influenza vaccines offer several advantages over the conventional vaccines. In this study, the haemagglutinin (HA) gene of highly pathogenic avian influenza H5N1 was cloned and expressed as His tagged protein in methylotropic yeast Pichia pastoris. The expression of recombinant HA (rHA) protein was confirmed by SDS-PAGE and western blot analysis. The rHA protein was purified using Ni-NTA affinity chromatography under denaturing conditions and the functions of the protein was assessed by the haemagglutinin assay after refolding. The immunogenicity of the rHA was evaluated by immunizing four groups of mice with different payloads (2.5, 5.0, 10 and 25μg) of purified rHA and the production of rHA specific antibodies were analysed by haemagglutinin inhibition assay (HI) and enzyme-linked immunosorbent assay (ELISA). An antigen specific immune response was observed against rHA indicating that the rHA antigen could be used as a vaccine candidate against avian influenza. These results suggest that this strategy would pave the way for the development of rapid and cost effective method for the production of an avian influenza vaccine.
In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of
Rhipicephalus microplus strain A in controlling the tick ...infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography–mass spectrometry analysis of the purified protein confirmed its identity as Bm95. Vaccine was prepared by blending various concentrations of purified Bm95 with aluminium hydroxide as an adjuvant. Immunogenicity studies of the vaccine in rabbits and cattle indicated that the vaccine was highly immunogenic. The efficacy studies of the vaccine was done in cattle. Naïve
Bos indicus cattle were vaccinated with the recombinant vaccine and were challenged with the larval, nymphal and adult forms of
Rhiphicephalus haemaphysaloides. The vaccine protected the animals from larval, nymph and adult tick challenges with an efficacy of 98.7%, 84.6% and 78.9% respectively. The results obtained from the above studies clearly demonstrated the advantage and possibilities of the use of Bm95 in controlling
R. haemaphysaloides infestations in the field.
Bovine herpesvirus 1 (BoHV-1) infection in cattle and buffalo makes these animals life-long carriers of the virus which is intermittently excreted in semen. In the present study, a real-time ...polymerase chain reaction (PCR) was validated to screen frozen semen from cattle and buffalo for BoHV-1 by amplification of the gB gene of the virus. Analysing the intra- and inter-test variability, the assay was found to be highly reproducible. High sensitivity (100%) and specificity (90.04%) of this real-time PCR assay was recorded in comparison to virus isolation. Extended frozen semen samples from 574 cattle and buffalo bulls that were seropositive to infectious bovine rhinotracheitis (IBR) tested by real-time PCR indicated that 1.97% semen batches from cattle and 3.36% batches of buffalo semen were positive for BoHV-1. The real-time PCR protocol will be useful for screening large numbers of semen samples from IBR-seropositive cattle and buffalo bulls as the test is less time consuming and several batches of semen can be tested with ease compared to virus isolation in cell culture.
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