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Birsa Agricultural University, Ranchi
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ThesisItem Open Access MOLECULAR CHARACTERIZATION OF THE VIRUS AND HOST FACTORS IN RESPECT TO FOOT AND MOUTH DISEASE INFECTION IN CATTLE(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2011) Das, Sutopa; Prasad, ArunIn India Foot and mouth disease (FMD) is endemic since many centuries. It is present almost in all parts of the country and occurs round the year. India is losing Rs 18,000 crore annually due to the dreaded FMD in cattle and livestock (Economic Times, 2011). Out of the possible seven serotypes of the foot and mouth disease virus (FMDV) , only four serotypes, viz, ‘O’, ‘A’, ‘C’ and Asia 1 were ever recorded in India. The molecular epidemiological studies have established that the Pan-Asian strain is the major cause of outbreak of FMD involving serotype ‘O’ in India . Since 1996, type C outbreaks have not been recorded in India. The FMDV has a single stranded positive sense RNA genome. The viral genome is translated as a single polyprotein, which is post-translationally cleaved by viral proteases into four structural proteins (VP1, VP2, VP3 and VP4). Among the 4 structural polypeptides, VP1 is the most immunogenic protein of FMDV. During the course of outbreaks, the high rate of mutation in the replicating virus population can lead to the accumulation of genomic changes and eventually to the emergence of immunogenic variants. This poses serious threat to the FMD control campaigns in endemic countries. The innate immune system comprises the cells and mechanisms that defend the host from infection by other organisms, in a non-specific manner. Induction of the antiviral innate immune response depends on recognition of viral components by host pattern- recognition receptors; one of them is the Toll-like receptors (TLRs). To date, 10 TLRs have been identified in cattle. Pathogen-associated molecular patterns (PAMPs) specific to viruses are recognized by four TLR family members (TLR 3, 7,8 and 9). TLRs play essential roles in the production of type I interferons (IFNs) and proinflammatory cytokines. The interferon(IFN) are a family of proteins that have antiviral properties and role in immunoregulatory actions. One of the IFNs, interferongamma (IFN-γ) is a dimerized soluble cytokine that is the only member of the type II class of interferons. The importance of IFN-γ in the immune system stems in part from its ability to inhibit viral replication directly, but, most important, derives from its immunostimulatory and immunomodulatory effects. On the other hand, interleukin-10 (IL10) is an anti-inflammatory cytokine and has pleiotropic effects in immunoregulation and inflammation. It down-regulates the expression of Th1 cytokines (IFN-γ). Most of the differences in the genome of the individuals of the same species are due to single base substitution polymorphisms, popularly known as single nucleotide polymorphisms (SNPs). SNP has been correlated to disease severity in human subjects. The TLR-3 mRNA expression was not found to be affected by FMDV infection. With the above facts, it was hypothesized that viral isolates causing FMDV infection among the cattle from Assam and Ranchi may have serotypic and genotypic diversity which may correlate with the disease severity. We also hypothesized that certain host factor molecules involved in innate immunity of mammals may also be associated with the severity of the disease. To that direction an effort was made to examine the expression or detection of certain host factors such as TLR3 (with its SNP analysis), status of IFN-γ and IL10 and finally to correlate the observations with severity of the disease. Therefore, the present research programme was undertaken to analyze the serotype and genotype of the FMD viral isolates from affected animals; to analyze the expression of TLR3 in healthy and infected cattle; to detect important mutation(s) (if any) in the TLR3 gene in affected cases and to correlate TLR3 expression and polymorphism with immunomodulation in FMD infected cases. APPROACH: In the present study, a total of 52 clinical materials were collected out of which 40 were from the FMD affected animals from Assam and Ranchi and12 numbers of tongue epithelial samples were collected as control samples from cattle slaughter houses. The clinical materials were in the form of tongue/feet epithelium and sera samples.The tissue and the sera samples were stored in -200C. The degree of severity of the infected sample was based on the visual observation of the severity of the manifestation of the symptoms. They were graded as ‘no disease’, ‘less severe’ and ‘more severe’ and were depicted as ‘-’, ‘++’ and ‘+++’ respectively. In order to confirm the serotype of the isolates collected, samples were tested by sandwich ELISA as per the bench protocol of Project Directorate on Foot -and-Mouth disease, IVRI, campus, Mukteswar, Uttarakhand. For genotyping, total RNA was isolated from the tissue sample by following standard protocol. cDNA was prepared using standard PCR (Eppendorf Germany) protocol. The cDNA thus prepared was stored at -200C and was used for PCR amplification of 3D gene. The 3D gene was amplified by using the cDNA. 3D specific primer were used in the process. Both positive and negative controls were run parallely along with the test samples. The 3D gene amplicons were sent to Macrogen® (Seoμl, Korea) for sequencing adhering to the guidelines of the vendors with respect to minimum concentration of the amplicons and the primers. The sequences received were subjected for Phylogenetic analysis of FMDV isolates using Molecular Evolutionary genetic Analysis (MEGA 4.1) software. The expression of TLR3 gene was studied by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) as well as validated by Real Time PCR. Total RNA was isolated from the tissues using the standard TRIZOL method. cDNA was prepared as stated above by employing standard protocol. Semiquantitaive rtPCR was performed for TLR3 using β-actin as internal controls. Expression of the RNA transcripts of TLR3 and β-actin, isolated from the epithelial tissue samples from FMDV infected samples was determined by Real Time RT-PCR by using SYBR Green Fluorescent Dye. The mRNA levels of the target genes were normalized to the transcript level of the housekeeping gene β -actin. For relative quantification, the expression of mRNA transcripts of the target genes from normal tissue was also determined. Primers were validated on an Applied Biosystems machine by using serial dilutions of total RNA with endogenous control and target primers, whose values were plotted as the log input amount versus ΔCT values (target CT − endogenous CT) for relative efficiency. Primers with a slope of less than 0.1 were used, due to similar amplification efficiencies as the endogenous control. For TLR3 SNP polymorphism, the genomic DNA from the freshly collected tissue samples was isolated by standard Proteinase-K digestion and phenol/ chloroform extraction procedure. The quantity and quality of DNA was measured by using Nanodrop spectrophotometer (GE NanoVue plus). Exon 3 of the TLR3 gene was amplified through PCR in an gradient thermocycler (Epphendroff) by using specific primers. The amplicon product was sent to Macrogen, Korea for sequencing. The variations in the nucleotide sequences were verified by comparing with the sequences reported in the National Centre for Biotechnology Information (NCBI) database and were analyzed using the Clustal X software output (Rosalind Franklin Centre for genomics Research; http://www. hgmp. mcc. ac.uk). The IFN-γ level was detected in the serum samples from infected and control samples using the Bovine IFN–γ ELISA kit (BioSource Bovine IFN–γ EASIA, Belgium) following the manufacturers instructions. Samples yielding mean OD below the Assay Cut off was considered as negative and samples yielding mean OD above the Assay Cut off was considered as positive for IFN- γ. Expression of the RNA transcripts of IL10 and β-actin, isolated from the epithelial tissue samples from FMDV infected samples was determined by Real Time RT-PCR using SYBR Green Fluorescent Dye. The mRNA levels of the target genes were normalized to the transcript level of the housekeeping gene β-actin. For relative quantification, the expression of mRNA transcripts of the target genes from normal tissue was also determined. Primers were validated on an Applied Biosystems machine by using serial dilutions of total RNA with endogenous control and target primers, whose values were plotted as the log input amount versus ΔCT values (target CT − endogenous CT) for relative efficiency. Primers with a slope of less than 0.1 were used, due to similar amplification efficiencies as the endogenous control. From the real time PCR plots ΔCt, ΔΔCt and 2 -ΔΔCt values were calculated and tabulated. Expression of the target gene normalized to the reference gene and relative to the calibrator = 2 -ΔΔCt indicates the fold change in expression of the target gene compared to that in the normal (reference). 2 -ΔΔCt values indicated fold changes in IL10 mRNA expression. All analysis was performed as per the method described by Snedecor and Cochran (1994) followed by the statistical package for social science, version 13.0 (SPSS, Chicago, IL) software.ThesisItem Open Access In Vitro Expression of Recombinaat Buffalo Lactoferrin Gene and its Antibacterial Activity(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2013) Kumari, Namita; Prasad, ArunLactoferrin (Lf) is an iron binding glycoprotein having many biological activities, such as facilitating iron absorption and having antimicrobial and anti-inflammatory effects. Therefore, the present study was undertaken to characterize the buffalo Lf at the genic level and express N-terminal of buffalo Lf in E. coli system to see its antibacterial activity. For that, total RNA was isolated from the mammary gland of lactating buffalo and reverse transcription-polymerase chain reaction (RT-PCR) was carried out for first strand cDNA synthesis. Buffalo lactoferrin gene of 2.2 kb was amplified using gene specific primers and cloned into pTZ57R/T cloning vector using DH5α strain of E. coli. The positive-negative selection of colony was done based on differential colour development by LacZ selection. Positive clones of buffalo Lf gene cDNA were sequenced and were analyzed with the help of laser gene software and submitted to NCBI GenBank (Acc. No. JF825526). Sequencing of the selected representative clones revealed that nucleotide sequence of buffalo Lf contained an ORF of 2127 bp encoding a precursor peptide of 708 amino acids with a molecular weight of 77.65 kDa and isoelectric point of 7.979. Comparative study of buffalo Lf with other reported livestock available in GenBank showed that buffalo Lf had higher homology both at nucleotide and amino acid level with cattle. The phylogenetic analysis also indicated that buffalo Lf had a close relationship with that of cattle. To see the antibacterial activity of in vitro expressed buffalo Lf, 150 bp of Nterminal of buffalo Lf was amplified and cloned in pTZ57R/T cloning vector. Then it was further sub-cloned into the expression vector pQE30 and expressed in E. coli strain (SG13009). After induction with IPTG, the target fusion protein was successfully expressed and identified by SDS-PAGE and Western blotting. The protein purified using His-tag had a molecular weight of 7.0 kDa. Further the expressed buffalo N-Lf protein displayed bactericidal activity after activation by enzymatic proteolysis using porcine pepsin against S. aureus and E. coli comparable to commercially available bovine Lf. The successful expression of functionally active and intact buffalo N-Lf in this study can be used to study biochemical antimicrobial interaction and has the potential to be used as an immunomodulator in future, particularly against infectious diseases like mastitis, thus having significant impact on buffalo productivity.ThesisItem Open Access DEVELOPMENT AND STANDARDIZATION OF DOT-ELISA FOR RAPID AND RELIABLE DIAGNOSIS OF NEWCASTLE DISEASE, INFECTIOUS BRONCHITIS AND INFECTIOUS BURSAL DISEASE IN POULTRY(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2016) Kumari, Anuradha; Prasad, ArunThe present work was taken upon with an objective to develop a cheap, sensitive and ready to use technique even in field against ND, IBD and IB. For this development several stages of dot-ELISA has been standardized and the developed dot-ELISA may be helpful for recording the seroprevalence of ND, IBD and IB infections. For dot-ELISA under field conditions following quantification were found to be effective in achieving best results, (A) Antigen standardization ND antigen: 11.25μg Lasota/F1 strain of virus in 0.9 μl of diluent, IBD antigen: 15μg BursaB2K/Gumboro strain of virus in 1.2 μl of diluent, IB antigen: 6.25μg Georgia strain of virus in 0.5 μl of diluents. (B) Blocking solution standardization ND: skimmed milk (1%) + gelatin (0.5%) +BSA (0.5%) IBD: skimmed milk (1.5%) + gelatin (0.5%) +BSA (1%) IB: skimmed milk (2%) + gelatin (1%) (C) Sera standardization ND: 1:40 IBD: 1:10 IB: 1:20 (D) Conjugate standardization ND: 1:800 IBD: 1:500 IB: 1:1100 Standard dot-ELISA developed for ND, IBD and IB was compared with ELISA and AGID. For ND and IB, The result of dot-ELISA when compared with the results of ELISA has slightly lower value whereas dot-ELISA when compared with AGID has showed higher value. Sensitivity and specificity are very important parameter to judge accuracy of the test. The sensitivity and specificity of dot-ELISA calculated in present study was compared with result of ELISA was 68.97% and 61.76% and with AGID which was 95.74% and 82.22% respectively for ND. Similarly for IBD, the sensitivity and specificity of dot-ELISA calculated in the scenario was compared with result of ELISA as 67.57% and 81.82% and with AGID as 88.89% and 94.64% respectively. The sensitivity and specificity of dot- ELISA for IB was compared with result of ELISA as 67.44% and 66.67% and with AGID 93.88% and 67.44% respectively for IB. As far as literature is concerned, ELISA based seoprevalence of ND, IBD and IB has not been reported from Ranchi or from the Jharkhand, therefore it seems to be the first report from this area. In the present study, the overall seroprevalence of ND, IBD and IB on the basis of ELISA, AGID and dot-ELISA was 63.04%, 51.09% and 57.61%; 40.22%, 39.13% and 38.04%; 93.48%, 53.26% and 65.22% respectively. Seroprevalence is very important aspect to determine the threat intensity. Until now we don’t have any economical kit at farm level which helps in regular monitoring. ELISA kit in market as confirmatory diagnosis is quite expensive and it had to be incorporated. Another drawback is that a well equipped lab with trained personnel is required to carry out experiment. Due to these impairments diagnosis at farm level becomes difficult. Diagnosis by dot-ELISA may overcome these short comings and detect the disease at much cheaper rate with appreciable sensitivity and specificity. In comparison to plate ELISA, the developed dot-ELISA can be performed at farm level without sophisticated equipment like ELISA reader, expensive reagents and other specialized instruments. It will prove to be highly cost effective and also suitable for small sample size. The farm personnel can perform the test as per the protocol and read the results easily. Moreover, antibodies to three viruses can be screened at a time which further reduces the cost testing. The present study clearly indicates prevalence of ND, IBD and IB as high, moderate and extremely high in Ranchi respectively. Mere presence of prevailing antibody without the gross pathological lesion indicates mild or subclinical form of viral infection.