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Birsa Agricultural University, Ranchi
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ThesisItem Open Access Comparative Study on Isolation and Identification of Methicillin Resistant and Non Methicillin resistant Staphylococcus Aureus from Mastitic Milk(Birsa Agricultural University, Ranchi, Jharkhand-6, 2019) Kumari, Sweeta; Prasad, ArunComparative Study on Isolation and Identification of Methicillin Resistant and Non Methicillin resistant Staphylococcus Aureus from Mastitic MilkThesisItem Open Access STUDIES ON IMMUNOMODULATOY EFFECT OF ETHANOLIC EXTRACT OF Trigonellafoenum-graecumLEAVES IN BROILER CHICKS(Birsa Agricultural University, Ranchi, Jharkhand-6, 2018) MURMU, SUNITA KUMARI; Prasad, ArunOn the basis of above finding it can be concluded that the dose 50mg/kg b.wt and 100mg/kg b.wt of ethanolic extract of fenugreek leaves has immunomodulatory effect in broiler chicks, but as the dose increased above from the range of 200 to 400 mg/kg b.wt the toxicity has been observed in liver and kidney which is evident by histopathlogy. Effect of ethanolic extract of fenugreek leaves of treatment on blood biochemical profile showed a mild increase in total serum protein and total serum globulin indicating better humoral immune response. The effect of ethanolic extract of fenugreek leaves showed mild increase in hemoglobin, PCV, TEC. Maternal haemagglutination inhibition titre against NDV vaccine was detectable only up to 21 days of age in non-vaccinated control group of chicks. Food Conversion Ratio(FCR) of higher weight gain per bird at the sale counter fetching more money to farmers.ThesisItem Open Access IMMUNOMODULATORY EFFECT OF COW URINE DISTILLATE ON HUMORAL AND CELL MEDIATED IMMUNE PARAMETERS IN BROILER CHICKS(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2010) Jojo, Rakesh; Prasad, ArunThe present study on the topic “Immunomodulatory effect of cow urine distillate on humoral and cell mediated immune parameters in broiler chicks” was conducted to asses the immunomodulatory effect of feeding CUD (@ 10ml/ltr. of drinking water) with comparison to that of a known positive immunomodulator, LevamisoleHCL(@ 10mg/kg b.wt.) feed supplement daily for 42 days on humoral and cell mediated immune parameters including effect on serum protein, av. body weight gain, cumulative feed intake and feed conversion ratio(FCR). For this purpose Eighty day old vencobb chicks were procured and maintained under standard farm condition in Avian Research and Development centre (ARDC), RVC, Kanke, Ranchi. Initially, the all chicks were kept under hover for first 6 days and on 7th day chicks were randomly allocated to different treatment groups comprising of 20 chicks in each were done as follows Gr.T1- received CUD and vaccinated with NDV vaccine (LaSota strain), Gr.T2- received Levamisole and vaccinated, Gr.T3- received normal feed but was vaccinated and Gr.T4 was kept as untreated and unvaccinated control. Birds of three groups (Gr.T1, Gr.T2& Gr.T3) received scheduled IBD and booster NDV vaccine (R2B strain) on 14th and 28thday but not Gr.T4. For monitoring humoral immune response against NDV vaccine, HI test was done using β procedure as per the standard protocol of Buxton and Frazer (1977). Prevaccination MHI antibody titre was measured on day 0 and 7 for knowing the status of maternally derived antibodies. Post vaccination MHI antibody titre was measured on weekly interval after RD vaccination to asses the effect of treatment to different groups. For monitoring cell mediated immune response status in all the four groups, contact sensitivity test using DNFB (2’ 4’ dinitrofluorobenzene) as contact sensitizer was used as per method of Tiwary and Goel (1985). Besides measuring mean skin thickness at test skin sites for comparison in all the four groups of birds, the skin section were also examined histopathologically to authenticate the nature of reaction i.e. whether they were a DTH reaction or not. 1. The chicks carried a detectable level of maternal antibody upto three weeks of their age. From 28th day of age, Gr.T4 did not show any level of MHI antibody titre against RD virus vaccine due to complete depletion of maternal antibody. On all weekly intervals post RD vaccination, higher MHI antibody titre was observed in levamisole treated group than any other groups. There was significantly(p<0.01) higher MHI titre observed in the levamisole treated group T2 than T1(CUD) treated group and two control groups at most ages post vaccination. Maternal antibody showed decreasing value with the advancement of age but MHI titre against RD vaccination showed increasing trends after vaccination on day 7 and 28. 2. A significantly (p<0.01) higher mean skin thickness was observed in Gr.T1 and Gr.T2 as compared to control groups (T3&T4) at 24 and 48 hrs. post challenge with DNFB. Further decreasing trend was observed at 72hrs. On histopathological examination, the stained tissue sections of test side showed mild to heavy infiltration with mononuclear cells and congestion suggesting a DTH reaction while right side challenged with vehicle only, stained section showed normal skin histology. 3. Higher values of serum protein, albumin and globulin were observed in treatment groups as compared to control groups. 4. At the end of experiment, the effect of treatments had significant influence on body weight at 42 days and observed higher weight in treatment groups which differed significantly(p<0.01) than control groups . Weight gain and feed conversion ratio was better in group T2 followed by T1, T3 and T4. By seeing the immunopotentiating effect of CUD on humoral immune response against NDV virus vaccine as direct test (HI test) and also in contact sensitivity a (DTH) test using DNFB as an indirect correlate of CMI, the use of CUD as immunomodulating agent may be advocated. It may be used as safety immunopotentiator to the birds @ 10ml/ltr. of drinking water to boost their immunity and to overcome immunosuppression and vaccine failure. Better CMI in treated groups advocate its better protective role in case of viral infections including other intracellular pathogens. These may also increase the body weight of broiler birds due to better FCR giving better gain to farmers and poultry industrialists.ThesisItem Open Access ROLE OF INACTIVATED VACCINE IN THE PRESENCE OF HIGH MATERNAL ANTIBODIES IN PUPS FOR PARVOVIRUS IMMUNIZATION(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2011) SINGH, SHREENIWAS; Prasad, ArunThe present research was carried out to study the prophylactic efficacy of inactivated vaccine and interference of maternal antibodies in eliciting immune response to inactivated vaccine against canine parvovirus. For this purpose, a five week’s vaccination trial was conducted in pups. This study was carried out in the duck unit of Ranchi Veterinary College, where the animals were housed throughout the period of study. The pups were grouped in 5 batches of 6 pups each and were monitored closely for two weeks before their inclusion in the study group. The pups were de-wormed at least seven days prior to vaccination. Regular cleaning and washing of rooms was followed with disinfectant to avoid any contamination and concurrent spread of parvovirus infection. Certain inclusion and exclusion criterion were followed while enrolling pups in this study group. Gr 1- received primary vaccination on 45-50 days of age with inactivated vaccine followed by boosters with live vaccine on 15th and 45th day from day of primary vaccination. Gr 2- received primary vaccination on 60 days of age with inactivated vaccine followed by boosters with live one on 30th and 60th day from day of primary vaccination. Gr 3 and Gr 4 received vaccination on 0th day, 30th day and 60th day with live vaccines whereas Gr 5 served as control group with no vaccination. For monitoring immune response against CPV vaccine in each group, estimation of maternal antibody haemagglutination inhibition antibody titer and serum neutralization antibody titer was observed on 0th day prior to administration of vaccine and the data was compared with titers obtained on 28th day and 35th day serum samples using 8HA unit of CPV(ka/BE) strain antigen. Mean HI antibody titer against Canine Parvovirus vaccine virus was found to be higher in Gr 2 (107±47.7, 153±47.7, 443±190 ) than Gr 1 (120±43.6, 133±16.8, 267±33.7 ) while Gr 3 (117±45.3, 143±44.0, 320±71.4 ) and Gr 4 (113±46.5, 140±40.8, 280±82.0 ) showed similar pattern in antibody titer. Similarly, mean SNT antibody titer against Canine Parvovirus vaccine virus was found to be higher in Gr 2 (57±22.7, 120±48.1, 240±94.2 ) than Gr 1 (60±8.9, 113±22.3, 227±124.6 ) while Gr 3 (53±12.2, 87±25.6, 187±44.6 ) and Gr 4 (67±21.6, 83±25.5, 146±33.7 ) showed similar pattern in antibody titer. Analysis of variance test showed no significant effect of groups on HI and SNT antibody titers against Canine Parvovirus. On day 28 , it was observed that there was marginal increase in antibody titers in all the vaccinated group, probably due to interference by maternal antibodies and less antigen stimulation by vaccine but it increased substantially on day 35 due to depletion of maternal antibody titer. Control group showed progressive decrease in HI and SNT antibody titers on day 28 and day 35. Pups with haemagglutination inhibition titers greater than or equal to 1:80 showed more interference to active immunization. Within group variation in antibody titer was noticed in all the groups, though the pups belonged to the same litter. Haemagglutination test, unlike Serum Neutralization test, does not always detect differences in amount of antibody titers when the amount of antibody titer is low, indicating Serum Neutralization test is more specific in detecting serum antibody titer. The age of the pups belonging to Group 1 falls in the “Window of Vulnerability” period. Hence the within group variation in antibody titer is less pronounced in this group suggesting more interference to both inactivated and live vaccines. Killed vaccines can also be blocked by maternally derived antibodies. Killed vaccine only prime the immune response, requiring further doses to provide protective immunity. From the findings of the present study, the role of inactivated vaccine in the presence of high maternal antibodies in pups has been discussed and its potential inclusion in vaccination protocol for puppies has been advocated.ThesisItem Open Access STUDIES ON COMPARATIVE EFFICACY AND SAFETY OF MEGAVAC-6 WITH VANGUARD 5L AND NOBIVAC DHPPI FOR PARVOVIRUS IMMUNIZATION(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2012) Kumari, Meera; Prasad, ArunThe present study on the topic “Studies on comparative efficacy and safety of Megavac 6 with Vanguard 5L and Nobivac DHPPI for parvovirus immunization” was carried out to eliciting immune response in pups against parvovirus infection. For this purpose 24 unvaccinated healthy puppies (Non descript), 8-18 wks were enrolled and divided into four groups. Each contained 6 pups. GrpI received Megavac 6, grpII received Vanguard 5 L, grpIII received Nobivac DHPPI and grp IV was taken as control. To determine efficacy of vaccines estimation of maternal antibody was observed by HI and SNT as per Buxton and Frazer 1997 on 0th day prior to administration of vaccine and data was compared with titres obtained on 28th day and 35th day. The pups carried a detectable level of maternal antibody on 0th day not showed any significant difference between different groups. After vaccination on day 28th and 35th Megavac 6 treated groups showed highly significant (p<0.01) MHI and SNT antibody titre than Vanguard 5L treated group and Nobivac DHPPI treated groups. Control group i.e. grp IV showed significant decrease in HI and SNT antibody titre. No unusual symptoms were noted. Non of the group showed any adverse reaction during and after vaccination. Based on the findings of the trial, following conclusion can be drawn. Megavac 6, Vanguard 5L & Nobivac DHPPI can be used to protect dogs against parvovirus infection. Efficacy of Megavac 6 was better than vanguard5L & Nobivac DHPPI & was more active during maternal antibody interference. Megavac 6, Vanguard 5L &Nobivac DHPPI were safe & did not show any adverse reaction during &after vaccination. The health status of the pups vaccinated with Megavac 6 Vanguard 5L & Nobivac DHHPI were sound and better in comparison to unvaccinated animals.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 STUDIES ON IMMUNOMODULATORY EFFECT OF AQUEOUS EXTRACT AND DRIED POWDER OF Moringa oleifera LEAF IN BROILER CHICKS(Birsa Agricultural University, Kanke, Ranchi, Jharkhand, 2012) Akhouri, Swechha; Prasad, ArunIn our present study on the topic “Studies on immunomodulatory effect of aqueous extract and dried powder of Moringa oleifera leaf in Broiler Chicks, the immunomodulatory properties of a reputed Ayurvedic herb, Moringa oleifera was assessed using various experimental parameters. During the study, fifty broiler chicks(day old) were procured and grouped into 5 experimental groups viz. T1, T2, T3, T4 and T5 comprising of 10 chicks each, on day 7. The chicks were vaccinated against Ranikhet disease (RD) virus on day 7(F1 strain) and day 28 (R2B strain) except the fifth group which served as unvaccinated control group. All the groups of chicks were vaccinated against Infectious bursal disease (IBD) on day 14 (Georgia strain). The birds were maintained under similar managemental conditions and exposure to any sort of injury or stress was avoided. First two groups of chicks, T1 and T2 were fed with aqueous extract and dried powder of Moringa oleifera respectively each @ 250 mg/kg b. wt. For comparison, a Levamisole fed group T3 was included in the experimental design to compare the immunomodulatory effect of the preparations of M.oleifera with that of a known positive immunomodulator Levamisole fed @10 mg/kg b.wt. The other two groups T4 and T5 included in the experimental design served as the vaccinated and unvaccinated control groups respectively. The effect of the herb on humoral immune response was evaluated using beta procedure of haemagglutination inhibition test against RD virus, as per the standard protocol of Buxton and Frazer (1977). The effect of treatment on cell mediated immune response of the chicks was monitored through contact sensitivity test as per Tiwary and Goel (1985) with slight modification. The effect of treatment on non specific immune response was measured through nitroblue tetrazolium reduction test as per Culling et al., Certain blood biochemical profiles viz. total serum protein, serum albumin, serum globulin; albumin-globulin ratio and haemoglobin concentration were also estimated at the end of the experiment. Effect of feeding of M.oleifera preparations on average body weight and feed conversion ratio (FCR) of birds was also estimated at weekly intervals. The HI test conducted on serum samples of 6 randomly selected birds of each group at weekly intervals showed higher MHI Ab titre in the Levamisole treated group than any other experimental group, after 21 days. M.oleifera treated groups T1, T2 showed a stimulatory effect on humoral immune response as judged by the MHI Ab titre of the broilers. The contact sensitivity test was conducted with DNFB as contact sensitizer to assess the effect of M.oleifera on Cell mediated immune response which was observed in the form of an increase in the mean skin thickness (MST) of the DNFB challenged area of skin of the birds. The peak delayed type hypersensitivity reaction, represented by maximum MST, was observed 24 hrs. post challenge. The MST of Levamisole treated group was significantly higher than the control at 6, 24, 48 and 72 hrs post challenge. M.oleifera showed higher MST than the control groups at up to 48 hrs. Post challenge. Feeding of M.oleifera showed positive effect on the MST of the DNFB challenged birds. All the chicks of all the groups showed significantly higher MST on the challenged site when compared to MST of control site of the chicks. The non specific immune response was measured by counting formazan positive cell. Levamisole treated group showed significantly highest formazan positive cells from day 21.The Formazan counting of group T1 and T2 was higher than groups T4 on day 21 onwards and the difference was significant. No significant difference was observed in the MHI Ab titre of M.oleifera leaves aqueous extract treated group T1 and M.oleifera leaves powder treated group T2 up to day 42 but group T1showed a higher formazan positive cell Blood biochemical profile viz. total serum protein and serum albumin using Coral diagnostic kit Percentage Serum samples collected on 42 day were used for the estimation of the albumin; serum globulin and albumin-globulin ratio were further evaluated. The treatment groups showed significant difference in total protein, serum albumin level, but no significant difference was observed in serum globumin and albumin globulin ratio. At the end of the experiment, uncoagulated blood was collected from representative samples of each group for haemoglobin estimation. The treatments showed significant effect on the Hb% of the broilers. The highest Hb % was recorded in group T2 treated with dried powder of M.oleifera followed by T1 treated with aqueous extract of M. oleifera, followed by levamisole treated group, positive control, and Negative control group. M.oleifera treated group showed higher average body weight than any other experimental group at most of the time and the average body weight of the group was observed to be significantly higher than the control groups on day 42. The average body weight of the three test groups however did not differ significantly from each other at any point of time. M. oleifera fed group showed better FCR than other groups followed by levamisole treated groups followed by the vaccinated and unvaccinated control groups of chicks. Based on the above findings, it may be said that M. oleifera treated, groups when fed @ 250 mg/kg b. wt., has a stimulatory effect on immune response and feed conversion efficiency of the broilers. However the immunomodulatory effect of Levamisole is higher than the herbal preparations. Thus, we can conclude that aqueous extract and dried powder of M. oleifera may be recommended as a safe and commercially beneficial immunomodulator for better immune response on mass vaccination and side by side to get better body weight gain and haemoglobin % in the flock for commercial benefit.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.