Incidence of subclinical mastitis and prevalence of major mastitis pathogens in organized farms and unorganized sectors.

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10(5)/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.

Sindbis virus replicase-based DNA vaccine construct encoding FMDV-specific multivalent epitope gene: studies on its immune responses in guinea pigs.

Foot-and-mouth disease (FMD) is still a perennial global menace affecting livestock health and production. It is imperative to figure out new ways to curb this disease. In this study, a sindbis virus replicase-based DNA vaccine, pSinCMV-Vac-MEG990, encoding a multivalent epitope gene (representing tandemly linked VP1 C-terminal halves of three foot-and-mouth disease virus (FMDV) serotypes) was constructed. In vitro transfection studies in BHK-21 cells revealed that the construct was able to express FMDV-specific antigen but does not overproduce the antigen. Immunization of guinea pigs with the construct at dose rate of 10, 5, 2 and 1 µg per animal through intramuscular route showed significant neutralizing antibody induction at all doses against all serotype tested as compared to non-immunized controls. On viral challenge of guinea pigs 4 week post-immunization with 1000 GPID(50) of FMDV serotype A, it was observed that the immunization not only delayed the appearance and reduced the severity of FMD lesions significantly (P < 0.05) but also provided complete protection in several guinea pigs. In fact, two of six and one of six guinea pigs were completely protected in 10 and 5 µg immunized groups, respectively. These results suggest that the development of the replicase-based DNA vaccine may provide a promising approach as an alternative vaccine strategy for controlling FMD.

Influence of copy number on the expression levels of pandemic influenza hemagglutinin recombinant protein in methylotrophic yeast Pichia pastoris.

The hemagglutinin (HA) gene of novel Swine Origin Influenza A/California/04/2009 (H1N1) was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA-synthetic gene having α secretory tag under the control of AOX1 promoter was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having single and multiple copy integrants of the expression cassettes were screened for the expression of full length HA protein in the culture supernatant. In order to completely exploit the expression potential of the P. pastoris expression system, a systematic investigation on the influence of gene copy number on the expression of the recombinant protein was made. A panel of Pichia clones carrying increasing copies of the heterologous gene was selected based on Geneticin resistance and SYBR green-based quantitative real-time PCR approach. Using these strategies, recombinant Pichia transformants carrying up to a maximum of four to six copies of the transgene were identified. After optimising the expression conditions for shaker flask culture, the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the expression level of H1N1HA recombinant protein. Our findings clearly suggest that the gene dosage effect play a vital role in high level expression of the pandemic Influenza HA protein in yeast system.

Genetic diversity and population genetic analysis of bovine MHC class II DRB3.2 locus in three Bos indicus cattle breeds of Southern India.

The present study was performed to evaluate the genetic polymorphism of BoLA-DRB3.2 locus in Malnad Gidda, Hallikar and Ongole South Indian Bos indicus cattle breeds, employing the PCR-RFLP technique. In Malnad Gidda population, 37 BoLA-DRB3.2 alleles were detected, including one novel allele DRB3*2503 (GenBank: HM031389) that was observed in the frequency of 1.87%. In Hallikar and Ongole populations, 29 and 21 BoLA-DRB3.2 alleles were identified, respectively. The frequencies of the most common BoLA-DRB3.2 alleles (with allele frequency > 5%), in Malnad Gidda population, were DRB3.2*15 (10.30%), DRB3*5702 (9.35%), DRB3.2*16 (8.41%), DRB3.2*23 (7.01%) and DRB3.2*09 (5.61%). In Hallikar population, the most common alleles were DRB3.2*11 (13.00%), DRB3.2*44 (11.60%), DRB3.2*31 (10.30%), DRB3.2*28 (5.48%) and DRB3.2*51 (5.48%). The most common alleles in Ongole population were DRB3.2*15 (22.50%), DRB3.2*06 (20.00%), DRB3.2*13 (13.30%), DRB3.2*12 (9.17%) and DRB3.2*23 (7.50%). A high degree of heterozygosity observed in Malnad Gidda (H(O) = 0.934, H(E) = 0.955), Hallikar (H(O) = 0.931, H(E) = 0.943) and Ongole (H(O) = 0.800, H(E) = 0.878) populations, along with F(IS) values close to F(IS) zero (Malnad Gidda: F(IS) = 0.0221, Hallikar: F(IS) = 0.0127 and Ongole: F(IS) = 0.0903), yielded nonsignificant P-values with respect to Hardy-Weinberg equilibrium probabilities revealing, no perceptible inbreeding, greater genetic diversity and characteristic population structure being preserved in the three studied cattle populations. The phylogenetic tree constructed based on the frequencies of BoLA-DRB3.2 alleles observed in 10 Bos indicus and Bos taurus cattle breeds revealed distinct clustering of specific Bos indicus cattle breeds, along with unique genetic differentiation observed among them. The results of this study demonstrated that the BoLA-DRB3.2 is a highly polymorphic locus, with significant breed-specific genetic diversities being present amongst the three studied cattle breeds. The population genetics and phylogenetic analysis have revealed pivotal information about the population structure and importance of the presently studied three Bos indicus cattle breeds as unique animal genetic resources, which have to be conserved for maintaining native cattle genetic diversity.

Tissue culture independent transformation of the forage crop sunnhemp (Crotalaria juncea L.): an easy method towards generation of transgenics.

A transformation system which is free of in vitro plant regeneration following Agrobacterium infection is established for the forage legume, Sunnhemp (Crotalaria juncea L.) where in the entire embryo axis of the germinating seed was used as the target tissue for transformation. After standardization of transformation conditions, the cotyledonary node of the embryo axis was infected with Agrobacterium host LBA 4404 harboring the recombinant vector pCAMBIA 2301. The bivalent 1D gene of the two major foot and mouth disease virus (FMDV) serotypes 'O' and 'A22' and the neomycin phosphotransferase (nptII) gene were used as the markers for optimization of the protocol. The embryo axes were pricked randomly on the cotyledonary node and co-cultivated with Agrobacterium. The germlings were then allowed to grow under standard growth room conditions in to mature fertile plants. 60 T0 plants were established from 3 separate experiments. Three hundred seeds from the 60 T0 plants were sown to raise the T1 generation of which 180 were analyzed for integration of bivalent FMDV gene 1D "O" and "A22" and the nptII gene. Eighteen out of these 180 plants amplified both the marker genes. Two independent transgenic lines 24 and 37, showed elevated levels of expression of 12 µg and 8 µg (per gm of fresh leaf) of the bivalent ID antigen "O" and "A22" . The results showed that the transformation efficiency was 3 %. To the best of our knowledge, this is the first successful attempt of Agrobacterium tumefaciens mediated transformation of Sunnhemp. The protocol can generate whole plant transformants with relative ease and should be compatible to all genotypes of Sunnhemp.

Cloning, expression and immunoprotective efficacy of rHaa86, the homologue of the Bm86 tick vaccine antigen, from Hyalomma anatolicum anatolicum.

The Bm86 homologue of Hyalomma anatolicum anatolicum Izatnagar isolate was cloned and expressed in methylotropic yeast Pichia pastoris as intracellular, glycosylated and particulated form. It was named as rHaa86, the first recombinant protein of H. a. anatolicum. Seven epidermal growth factor-like domains predicted in Haa86 were structurally similar with that of its Bm86 counterpart. The identity between the corresponding EGF like domains of Bm86 and Haa86 were ranging from 51.3% to 78.3%. The molecular weight of the rHaa86 was 120-140 kDa, with possible 50-70 kDa glycosylation. The purified rHaa86 was characterized immunologically and evaluated for its immunoprotective potential against homologous challenge infestation in three groups of cross-bred calves. The immediate rejection percentage of females of H. a. anatolicum was 36 5%, 12.4% and 10.1% fed on immunized (group 1), adjuvant control (group 2) and untreated control (group 3) calves, respectively. The percent rejection of female ticks fed on immunized calves was 24.1% and 26.4% higher than for the ticks fed on control groups 2 and 3, respectively (P < 0.05). The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 58.0%, 9.0%, 5.0% and 61.6%, respectively. The mean reproductive index of females fed on group 1 calves was significantly lower (P < 0.05) than the females fed on the control groups and 44% reduction in the number of engorged larvae was recorded from the group 1 calves. The data demonstrated that rHaa86 antigen based vaccine could serve as one of the effective components in the integrated control of H. a. anatolicum.

Characterization of VP2 gene of an Indian Bluetongue virus serotype 2 and its close phylogenetic relationship to the Taiwan isolate.

In this study we present the first report on partial amplification, sequencing and phylogenetic relationship of VP2 of the Indian isolate BTV-2. A PCR product of 1135 bp was amplified, cloned and sequenced. About 1063 bp of partial VP2 gene (1792-2854 bp region) of the Indian isolate was subjected to sequence analysis with already published sequences available in the genome database. The percent similarity of 85.2 was observed with Taiwan isolate and 59% with other isolates of BTV-2. However, 46.2% similarity with Australian BTV-1 and no significant similarity were noted with other serotypes. In-silico analysis and restriction enzyme digestion confirmed the presence of conserved SalI site at 2380 bp position in both Indian and Taiwan isolates. Phylogenetic analysis showed that all BTV-2 isolates formed one distinct group in which BTV-2 Indian and Taiwan isolate is more closely related and further demonstrated that BTV's of the same serotype from different geographical regions were closely related at nucleotide and amino acid level, respectively.

Enhanced mucosal immune response in cattle persistently infected with foot-and-mouth disease virus.

The mucosal immune response to foot-and-mouth disease virus (FMDV) type Asia 1 was examined in experimentally infected cattle by assaying antibodies by the virus-neutralizing test (VNT) and IgA ELISA in two secretory fluids, oesophageal pharyngeal fluid (OPF) and oro-nasal fluid (ONF). Out of 17 animals infected by the intradermo-lingual route, 12 became persistently infected (carriers), as defined by positive antigen capture RT-PCR reactions for FMDV RNA in OPF samples collected at 28 days or later after exposure. This proportion of carriers (71%) with FMDV Asia 1 is comparable to other serotypes of the virus. When the two groups were examined, the carriers and non-carriers showed no difference in the serum antibody titre until the end of the experiment at 182 days post-infection (DPI). However, despite an initial similarity significantly higher neutralizing antibody titres and FMDV-specific IgA response were detected among the carriers than the non-carriers in both of the secretory fluids. The response was higher and more stable in ONF compared to OPF. Thus, mucosal antibody assays have the potential to be used as a means of differentiating carrier from non-carrier cattle. Furthermore, the findings are consistent with the higher mucosal antibody response in carriers being an effect of persistent infection rather than the cause.

Sequencing, characterization and genetic variation of the Bos indicus glucose-6-phosphate-dehydrogenase gene.

The coding sequence of the bovine (Bos indicus) Glucose-6-phosphate-dehydrogenase (G6PD) gene was amplified by Reverse Transcriptase-PCR (RT-PCR), cloned, sequenced and characterized. The deduced amino acid sequence clustered the bovine G6PD sequence with the other mammalian G6PD proteins into a monophyletic group. The bovids (B. indicus and B. taurus) clustered clearly from the rodent (rat, mouse and hamster) subcluster and from humans. The multiple sequence alignment of the bovine G6PD with the mammalian species clearly revealed conservation of the substrate, coenzyme, catalytic and the dimer binding sites with the solved X-ray crystallographic structure of Homo sapiens. Also, four fragments of bovine (Bos indicus) G6PD gene viz. 118, 319, 683 and 408 bp were amplified and sequenced for the first time. A G/A and G/C single nucleotide polymorphisms in intron-9 and exon-10 were detected on PCR-RFLP of the 319 bp amplicon with Hae III and Pst I, respectively. This work is the first study on Bos indicus G6PD gene at the nucleotide level.

Cost effectiveness of colony lysis and colony PCR methods for screening of recombinant Escherichia coli colonies--a comparative study.

Economizing the research protocols by using low cost technologies is the need of laboratories of developing world. Screening of recombinant E. coli colonies is the crucial step in gene cloning and expression studies. In the present study, the cost effectiveness of colony lysis method and colony PCR method in the screening of recombinant E. coli colonies was compared. The colony lysis method was 20 two times more cost effective and less time consuming and can be used to screen the recombinant E. coli colonies in large scale instead of colony PCR method.

Processing of multimer FMD virus VP1-2A protein expressed in E. coli into monomers.

Expressions of several genes in bacteria were carried out by independent promoter. However, in case of eukaryotes ribosome skipping and introduction of IRES are employed as alternative to multiple translation initiation. Foot and mouth disease virus (FMDV) 2A peptide has been widely used for co-expression of multiple genes in eukaryotic, plant and mammalian systems. The 18 amino acid 2A peptide of FMDV facilitates efficient co-translational dissociation of the polyprotein into discrete protein products. To study the role of 2A in multimeric protein production a construct consisting of tandem repeat of 4 units of C- terminal VP1 linked through 2A sequence was made and expressed in E. coli. Along with tetramer protein, trimer, dimer and monomer proteins were produced. Stability studies showed that the tetramer protein was cleaved to smaller monomer on storage. The results provide scope for using FMDV 2A for expressing multiple genes under a single promoter in prokaryotes.

Sero-diagnosis of Echinococcus granulosus infection in dogs using faecal supernatant antigen.

In the present study, sero-diagnosis of Echinococcus granulosus infection in dogs was studied using 250 field serum samples, collected from stray dogs. The three serological techniques viz., latex agglutination test (LAT), Dot-ELISA and enzyme immunotransfer blot (EITB) were used to detect E. granulosus specific anti-bodies using fecal supernatant antigen. The LAT detected positivity in 53 (21.2 %) serum samples with the sensitivity and specificity of 100 and 78.80 % respectively. Whereas, the Dot-ELISA, showed positivity in 45 (18.0 %) samples and the sensitivity and specificity were 100 and 82.0 % respectively. The specific antibodies were detected in 47 (18.80 %) serum samples by EITB and the polypeptides of 45 and 34 kDa were identified in all the positive serum samples. The sensitivity and specificity of EITB were found to be 100 and 86.80 %, respectively.

Protective immune response of the capsid precursor polypeptide (P1) of foot and mouth disease virus type 'O' produced in Pichia pastoris.

Foot and mouth disease virus (FMDV) is the aetiological agent of a highly contagious vesicular disease of cloven-hooved animals. The gene coding for the capsid polyprotein (P1) of FMDV from serotype 'O' vaccine strain (O75Madras) was cloned and expressed in yeast Pichia pastoris. The expressed P1 protein was characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analysis. Immunisation of Guinea pigs with recombinant P1 induced FMDV type O specific immune response. The humoral response to vaccine was measured by indirect ELISA and a serum neutralisation test (SNT). The Guinea pig sera showed high titres both in ELISA and SNT. Upon challenge with virulent Guinea pig adapted homologous type 'O' virus, the animals showed a protective index of 2.52. This study shows that the yeast expressed FMDV P1 could be a safe vaccine in non-endemic countries and a cost-effective vaccine in endemic countries. This is the first report on the production of FMDV structural proteins in yeast and their application as a vaccine.

Past and present vaccine development strategies for the control of foot-and-mouth disease.

Foot-and-mouth disease (FMD) virus (FMDV) was the first animal virus to be identified. Since then, it has become a model system in animal virology and more information has been obtained about FMDV. The disease causes heavy economic crises in enzootic countries both due to loss of animal health and productivity. The only way of its control in an enzootic area is strict vaccination and restricted animal movement. The first experimental vaccine against FMD was made in 1925 using formaldehyde inactivation of cattle tongue infected with the virus and this approach remained the basic one until late 1940s. Antigenic plurality and continuous co-circulation of different serotypes in a given geographical region and persistence of virus in infected or vaccinated animals make the disease very difficult to control. The latter is solely based upon the application of isolation, slaughter or aphtisation, and vaccination. With the advent of recombinant DNA technology, recombinant protein and/or DNA-based vaccines are being tested in various heterologous systems for development of FMD vaccines. The subunit vaccines, synthetic peptide vaccines, DNA vaccines, cytokine-enhanced DNA vaccines, recombinant empty capsid vaccines, chimeric viral vaccines, genetically engineered attenuated vaccines, recombinant viral vector vaccines, self-replicating genetic vaccines and transgenic plants with expressed FMDV proteins represent the present vaccine development strategies for control of FMD.

Sequence analysis of segment a of a field virus isolate from an outbreak of Infectious bursal disease in India.

Sequence analysis of genome segment A of an Indian Infectious bursal disease virus (IBDV) field isolate (KT1/99) revealed total 95 nucleotide substitutions, resulting in 17 amino acid changes. Of these, five amino acid changes, namely F60S, T137I, I374V, V519I and E682D were unique to the KT1/99 isolate. The amino acid change P222A and the proposed hot mutation spot 680Y, reported to be present in very virulent IBDV isolates were also found in KT1/99. This isolate had nucleotide divergence of 1.1% to 4.95% from the other reported serotype 1 IBDV isolates and 19.6% from serotype 2 strain OH in polyprotein gene sequence, while divergence at amino acid level was 0.6% to 2.9% and 11.4%, respectively. Based on both nucleotide and amino acid sequence analysis, KT1/99 was grouped phylogenetically with the reported Bangladesh isolate BD3/99 in one cluster along with other reported very virulent isolates in same lineage.

Nucleotide sequence analysis of variable region of VP2 gene of two infectious bursal disease virus isolates from commercial poultry farms.

Two infectious bursal disease virus (IBDV) isolates were obtained from commercial poultry farms with a history of severe outbreaks. A 474-bp product encompassing hypervariable region of IBDV VP2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide sequences of two isolates, VMB1 and VMB2, were determined and compared with those of twenty IBDV strains, including seven very virulent, four classical virulent, four classical attenuated, three antigenic variants and two avirulent serotype 2 strains. The two isolates showed a similarity of 96.5-98.4% with very virulent strains, 84.6-94.6% with classical virulent strains, 90.0-91.4% with classical attenuated strains, 83.0-91.9% with antigenic variants and 65.8-68.7% with avirulent strains. The deduced amino acid sequences of the two isolates showed amino acid substitutions of V256I, N279D, L294I and N299S, specific for very virulent strains. Phylogenetic analysis showed that the two isolates, along with a reported very virulent Indian strain, were closely related to European, Japanese and Chinese very virulent strains indicating their evolutionary origin.

Isolates of infectious bursal disease virus from India are of very virulent phenotype.

Four Indian field isolates, a classical virulent and an attenuated vaccine strains of Infectious bursal disease virus (IBDV) have been characterized by sequence analysis of part of the VP1 gene (from nucleotide 1538-1979) comprising one of viral RNA dependent RNA polymerase motifs. Sequence alignment of these viruses with reported viruses of other countries revealed Indian IBDV field isolates to be 100% similar to very virulent Japanese (OKYM), European (UK661) and Bangladesh (BD3/99) IBD viruses at amino acid level, whereas they had 0.2-0.9% divergence at nucleotide level. Out of the total 24 nucleotide changes found in the Indian field isolates, as well as reported very virulent viruses, only one resulted in amino acid change S-P at 562 position. The Indian field isolates displayed nucleotide divergence of 10.6-11.6% and amino acid divergence of 2.8-3.5% from the classical virulent and attenuated vaccine strains. The RNA dependent RNA polymerase motif from amino acid 528-541, present in the sequence analyzed, was conserved among all the viruses, irrespective of pathotype and serotype. In the phylogenetic tree, based on nucleotide sequence, Indian field viruses were grouped with reported very virulent viruses in one lineage whereas, classical virulent, attenuated vaccine and serotype 2 strains formed part of the second lineage. But in the phylogenetic tree based on amino acid sequence alignment, the serotype 2 strain OH grouped with Indian field isolates and reported very virulent viruses in one lineage and classical virulent and attenuated vaccine strains formed the second lineage.

Detection of fowl adenovirus associated with hydropericardium hepatitis syndrome by a polymerase chain reaction.

Hydropericardium hepatitis syndrome (HHS), previously unknown in the broiler industry, is an emerging disease that causes severe hydropericardium. A polymerase chain reaction (PCR) was developed to detect the fowl adenovirus (FAV) associated with HHS. The virus from infected livers was purified, with confirmation by electron microscopy and experimental infection. Methods were developed to isolate the viral DNA from purified virus and infected tissues. Available sequence data on the hexon gene of fowl adenoviruses and other adenoviruses were aligned to determine the conserved and variable regions. Primers were constructed from the alignment data. The amplified fragment consisted of the variable region of the hexon gene flanked by conserved primer sites. Optimum conditions were standardized to achieve the amplification of the desired fragment. As expected, the amplified product was found to be of 0.7 kg size. The nucleotide sequence analysis confirmed the specific nature of the product. Amplification of the specific product could be obtained not only from the DNA isolated from the purified virus but also from the total DNA extracted from infected tissues. The PCR was useful for the detection of FAV associated with HHS.

Buffalo alpha S1-casein gene 5'-flanking region and its interspecies comparison.

The expression of milk protein genes is tightly regulated in a spatio-temporal manner through the combinatorial interaction of lactogenic hormones and a set of transcription factors mediating developmental and tissue-specific gene expression. The recruitment of a unique set of transcription factors is determined by the cis-regulatory motifs present in the gene promoter region. Here, we report the isolation, sequencing, structural analysis and interspecies comparison of the 5'cis-regulatory region of the buffalo alpha S1 (αS1)-casein gene. The proximal promoter region of the buffalo αS1-casein gene harbored the insertion of a 72-bp fragment of long interspersed nuclear element of the L1_BT retrotransposon family. Among the core and vertebrate-specific promoter elements, the motifs for the binding of Brn POU domain factors (BRNF), Lim homeodomain factors (LHXF), NK6 homeobox transcription factors (NKX6), nuclear factor kappa B/c-rel (NFKB), AT-rich interactive domain factor (ARID), Brn POU domain factor 5 (BRN5), pancreatic and intestinal homeodomain transcription factor (PDX1), Distal-less homeodomain transcription factors (DLXF), T-cell factor/lymphoid enhancer-binding factor-1 (LEFF) and GHF-1 pituitary-specific POU domain transcription factor (PIT1) were over-represented in the αS1-casein gene regulatory region (Z score >4.0). The Multiple EM for Motif elicitation predicted three motifs which consisted of the sequences known to bind mammary gland factor/signal transducer and activator of transcription 5 (MGF/STAT5), estrogen receptor-related alpha (ERα), steroidogenic factor 1 (SF1) and glucocorticoid receptor (GR), indicating their potential role in the mammary gland-specific gene expression. The interspecies comparison of the proximal promoter region revealed conserved sequences for TATA boxes and MGF/STAT5 in all species, whereas activator protein 1 (AP1), pregnancy-specific mammary nuclear factor (PMF), CCAAT/enhancer binding protein (C/EBP), double-stranded and single-stranded DNA-binding protein 1 (DS1 and SS), ying and yang factor 1 (YY1), and GR half-sites were among ruminants. The functional significance of the L1_BT retrotransposon insertion on the buffalo αS1-casein gene expression needs to be experimentally validated.

Montanide ISA™ 201 adjuvanted FMD vaccine induces improved immune responses and protection in cattle.

Despite significant advancements in modern vaccinology, inactivated whole virus vaccines for foot-and-mouth disease (FMD) remain the mainstay for prophylactic and emergency uses. Many efforts are currently devoted to improve the immune responses and protective efficacy of these vaccines. Adjuvants, which are often used to potentiate immune responses, provide an excellent mean to improve the efficacy of FMD vaccines. This study aimed to evaluate three oil adjuvants namely: Montanide ISA-201, ISA-206 (SEPPIC, France) and GAHOL (an in-house developed oil-adjuvant) for adjuvant potential in inactivated FMD vaccine. Groups of cattle (n=6) were immunized once intramuscularly with monovalent FMDV 'O' vaccine formulated in these adjuvants, and humoral (serum neutralizing antibody, IgG1 and IgG2) and cellular (lymphoproliferation) responses were measured. Montanide ISA-201 adjuvanted vaccine induced earlier and higher neutralizing antibody responses as compared to the two other adjuvants. All the adjuvants induced mainly serum IgG1 isotype antibody responses against FMDV. However, Montanide ISA-201 induced relatively higher IgG2 responses than the other two adjuvants. Lymphoproliferative responses to recall FMDV antigen were relatively higher with Montanide ISA-201, although not always statistically significant. On homologous FMDV challenge at 30 days post-vaccination, 100% (6/6) of the cattle immunized with Montanide-201 adjuvanted vaccine were protected, which was superior to those immunized with ISA-206 (66.6%, 4/6) or GAHOL adjuvanted vaccine (50%, 3/6). Virus replication following challenge infection, as determined by presence of the viral genome in oropharynx and non-structural protein serology, was lowest with Montanide ISA-201 adjuvant. Collectively, these results indicate that the Montanide ISA-201 adjuvanted FMD vaccine induces enhanced immune responses and protective efficacy in cattle.