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Assam Agricultural University, Jorhat
Assam Agricultural University is the first institution of its kind in the whole of North-Eastern Region of India. The main goal of this institution is to produce globally competitive human resources in farm sectorand to carry out research in both conventional and frontier areas for production optimization as well as to disseminate the generated technologies as public good for benefitting the food growers/produces and traders involved in the sector while emphasizing on sustainability, equity and overall food security at household level.
Genesis of AAU -
The embryo of the agricultural research in the state of Assam was formed as early as 1897 with the establishment of the Upper Shillong Experimental Farm (now in Meghalaya) just after about a decade of creation of the agricultural department in 1882. However, the seeds of agricultural research in today’s Assam were sown in the dawn of the twentieth century with the establishment of two Rice Experimental Stations, one at Karimganj in Barak valley in 1913 and the other at Titabor in Brahmaputra valley in 1923. Subsequent to these research stations, a number of research stations were established to conduct research on important crops, more specifically, jute, pulses, oilseeds etc. The Assam Agricultural University was established on April 1, 1969 under The Assam Agricultural University Act, 1968’ with the mandate of imparting farm education, conduct research in agriculture and allied sciences and to effectively disseminate technologies so generated. Before establishment of the University, there were altogether 17 research schemes/projects in the state under the Department of Agriculture. By July 1973, all the research projects and 10 experimental farms were transferred by the Government of Assam to the AAU which already inherited the College of Agriculture and its farm at Barbheta, Jorhat and College of Veterinary Sciences at Khanapara, Guwahati.
Subsequently, College of Community Science at Jorhat (1969), College of Fisheries at Raha (1988), Biswanath College of Agriculture at Biswanath Chariali (1988) and Lakhimpur College of Veterinary Science at Joyhing, North Lakhimpur (1988) were established. Presently, the University has three more colleges under its jurisdiction, viz., Sarat Chandra Singha College of Agriculture, Chapar, College of Horticulture, Nalbari & College of Sericulture, Titabar. Similarly, few more regional research stations at Shillongani, Diphu, Gossaigaon, Lakhimpur; and commodity research stations at Kahikuchi, Buralikson, Tinsukia, Kharua, Burnihat and Mandira were added to generate location and crop specific agricultural production packages.
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ThesisItem Open Access Toxin gene profiling of Clostridium difficile in food and food products of animal origin(Assam Agricultural University, Khanapara,Guwahati, 2017-07) HAZARIKA, RITAM; SHARMA, R.K.The present work was undertaken with a view to investigate the presence of Clostridium difficile in food and food products of animal origin and profiling of toxin genes. A total of 235 samples were collected from different sources including raw meat (beef, pork, chicken and chevon), meat products (dry beef, dry pork, cooked chicken, chicken sausage and chicken salami), raw cow milk, milk product (Indian paneer) and fish products (dry fish and canned fish). Out of the total 235 samples, initially 56 (23.83%) samples revealed suspected colonies of C. difficile. All the suspected colonies exhibited the typical colony morphology with horse manure odour and typical Gram-positive rod shaped cell with sub-terminal spores. Among 56 tentative isolates, 17 (7.23%) could be confirmed as C. difficile, based on the detection of species specific gluD (GDH) and tpi (triose phosphate isomerase) genes. The 17 confirmed C. difficile isolates comprised of 15 (8.24%) from raw meat and meat product samples and remaining two (11.11%) from fish product samples. None of the samples from cooked chicken, raw chevon, chicken sausage, chicken salami, canned fish, cow milk and paneer yielded any C. difficile isolates. All the 17 C. difficile isolates were screened for detection of glutamate dehydrogenase (GDH) protein and toxin A and /or toxin B by enzyme immuno assay (EIA), out of which all isolates were found positive for GDH protein and six were found to be phenotypically positive for toxin production. The C. difficile isolates were characterized by PCR for detection of toxin genes (tcdA, tcd B and binary toxin) and PaLoc region. Antimicrobial resistance pattern was also tested against nine different antimicrobial agents by E-test. Altogether, six C. difficile isolates were found to be toxigenic. Out of which two were from chicken samples and four from pork samples. All the toxigenic isolates from chicken (2) samples possessed both tcdA and tcdB (A+ B+), while all the pork isolates (4) carried variant toxin genes (A-B+). All the (A-B+) isolates from pork were found to harbour the binary toxin genes (cdtA and cdtB). Based on the detection of PaLoc region comprising regulatory genes tcdC, tcdR and tcdE revealed that the two toxigenic chicken isolates (A+ B+) possessed tcdC and tcdE ,while the remaining four toxigenic pork isolates (A-B+) carry tcdR and tcdE, respectively. All the 17 C. difficile isolates showed higher resistance pattern to ciprofloxacin (70.59%), followed by cefotaxime (58.82%), clindamycin (29.41%) and tetracycline (17.64%) but 100 per cent sensitive to chloramphenicol, moxifloxacin, tigecycline, metronidazole and vancomycin. All the beef isolates were clindamycin- resistant with an MIC of 96 - 128 mg/ml. While, all C. difficile isolates from pork and dry fish were sensitive to the antimicrobials tested except ciprofloxacin and cefotaxime, the four chicken isolates were sensitive to the antimicrobials except ciprofloxacin.ThesisItem Open Access Toxin gene profiling of Clostridium difficile in food and food products of animal origin(Assam Agricultural University, Khanapara,Guwahati, 2017-07) HAZARIKA, RITAM; SHARMA, R. K.The present work was undertaken with a view to investigate the presence of Clostridium difficile in food and food products of animal origin and profiling of toxin genes. A total of 235 samples were collected from different sources including raw meat (beef, pork, chicken and chevon), meat products (dry beef, dry pork, cooked chicken, chicken sausage and chicken salami), raw cow milk, milk product (Indian paneer) and fish products (dry fish and canned fish). Out of the total 235 samples, initially 56 (23.83%) samples revealed suspected colonies of C. difficile. All the suspected colonies exhibited the typical colony morphology with horse manure odour and typical Gram-positive rod shaped cell with sub-terminal spores. Among 56 tentative isolates, 17 (7.23%) could be confirmed as C. difficile, based on the detection of species specific gluD (GDH) and tpi (triose phosphate isomerase) genes. The 17 confirmed C. difficile isolates comprised of 15 (8.24%) from raw meat and meat product samples and remaining two (11.11%) from fish product samples. None of the samples from cooked chicken, raw chevon, chicken sausage, chicken salami, canned fish, cow milk and paneer yielded any C. difficile isolates. All the 17 C. difficile isolates were screened for detection of glutamate dehydrogenase (GDH) protein and toxin A and /or toxin B by enzyme immuno assay (EIA), out of which all isolates were found positive for GDH protein and six were found to be phenotypically positive for toxin production. The C. difficile isolates were characterized by PCR for detection of toxin genes (tcdA, tcd B and binary toxin) and PaLoc region. Antimicrobial resistance pattern was also tested against nine different antimicrobial agents by E-test. Altogether, six C. difficile isolates were found to be toxigenic. Out of which two were from chicken samples and four from pork samples. All the toxigenic isolates from chicken (2) samples possessed both tcdA and tcdB (A+ B+), while all the pork isolates (4) carried variant toxin genes (A-B+). All the (A-B+) isolates from pork were found to harbour the binary toxin genes (cdtA and cdtB). Based on the detection of PaLoc region comprising regulatory genes tcdC, tcdR and tcdE revealed that the two toxigenic chicken isolates (A+ B+) possessed tcdC and tcdE ,while the remaining four toxigenic pork isolates (A-B+) carry tcdR and tcdE, respectively. All the 17 C. difficile isolates showed higher resistance pattern to ciprofloxacin (70.59%), followed by cefotaxime (58.82%), clindamycin (29.41%) and tetracycline (17.64%) but 100 per cent sensitive to chloramphenicol, moxifloxacin, tigecycline, metronidazole and vancomycin. All the beef isolates were clindamycin- resistant with an MIC of 96 - 128 mg/ml. While, all C. difficile isolates from pork and dry fish were sensitive to the antimicrobials tested except ciprofloxacin and cefotaxime, the four chicken isolates were sensitive to the antimicrobials except ciprofloxacin.ThesisItem Open Access DEVELOPMENT AND EVALUATION OF IMMUNOPOTENCY OF OUTER MEMBRANE VESICLES VACCINE OF Salmonella Typhimurium ADJUVANTED WITH CHITOSAN NANOPARTICLES(Assam Agricultural University, Khanapara,Guwahati, 2017-07) SARMAH, RAKESH KUMAR; Tamuly, ShantanuDuring the past two decades, Salmonella Typhimurium has emerged as a leading cause of human infections in many countries and the Salmonella spp has a broad host range and can infect broad array of animals causing diseases ranging from gastroenteritis to life threatening systemic infections. Salmonella enterica serovar Typhimurium is most frequently isolated serovar causing global food born outbreaks. The control of Salmonellosis can be accomplished either by vaccination or medication. Antibiotic resistance and antibiotic residue is a major hurdle in medication. The present study was conducted to study the efficacy of the chitosan nanoparticle adjuvanted outer-membrane vesicle based vaccine candidate against Salmonella Typhimurium. The OMV was extracted from Salmonella Typhimurium (MTCC-98) strain and confirmed by SDS-PAGE. The (ChNP-OMV) vaccine was synthesized by standard method and nanometer size was confirmed by TEM studies. In the immunization study three vaccine formulation were compared and injected in the 7th day and followed by booster dose on the 14th day. The humoral immune response of the target vaccine was compared with OMV based vaccine with or without booster dose and oil adjuvanted bacterin vaccine with or without booster dose by indirect ELISA. Collection of serum was done on 0th day just before vaccination, and thereafter 7, 14, 28, 45 and 60 days post primary vaccination. Humoral immune response in mice of all the experimental groups determined by using indirect ELISA. The (ChNP-OMV) vaccine was able to elicit quick and higher antibody response in the 14th day followed by steady decline up to 60th day. All the vaccine formulation elicited similar IgG response on 7th day PPI, however chitosan nanoparticle adjuvanted outer-membrane vesicle based vaccine elicited highest IgG response on 14th days PPI (with or without booster dose) with statistical significance of 95 % confidence level. Serum enzymatic test revealed that the significantly lower serum creatine kinase level in 7th and 14th day in the group of mice that was administered chitosan nanoparticle adjuvanted outer-membrane vesicle based vaccine as compared to the group of mice that was injected oil adjuvanted bacterin vaccine on 14th days PPI. However a significant decrease in antibody response was observed for the groups containing (ChNP-OMV) complex. From this study, it can be concluded that the ChNP-OMV (S. Typhimurium) vaccine was indicative of quick immune response for immediate vaccination in outbreaks against salmonellosis.ThesisItem Open Access CHARACTERIZATION AND QUALITY EVALUATION OF PIG FIBROBLAST CELLS AT DIFFERENT SUBCULTURES(Assam Agricultural University, Khanapara,Guwahati, 2017-07) DHAR, PRIYA; Nath, N. Ch.Fibroblast cells are the most common cells of connective tissue. In the present study, porcine fibroblast cells were isolated, cultured and subcultured upto six passages from adult and fetal skin samples. The time required to attain 70% confluence in primary culture of fetal fibroblast cells was found to be 26.40±2.40 hours, which was significantly lower (p≤0.01) than 67±4.80 hours of adult fibroblasts culture. The cells were maintained upto six passages which was 38.8 days for adult and 26 days for fetal fibroblast cells. The fetal fibroblast cells (days) required significantly lower (p≤0.01) time to subculture than adult fibroblast cells. Morphologically the isolated cells had fibroblastic character like typical fusiform shape, turgor vitalis cytoplasm, centrally located nuclei and flame like migration pattern upto sixth passage. The subculture time and cell viability (%) was significantly (p≤0.01) better from third subculture onwards upto sixth subculture as compare to primary and secondary cultures. TUNEL assay revealed a decreasing trend of apoptotic index from primary culture onwards to sixth subculture for both adult and fetal fibroblast cells. Fetal fibroblast cells maintained a lower apoptotic index than adult fibroblast in each of the subcultures. Therefore, fibroblast cells were found to be better from third subculture onwards to sixth subculture in regards to subculture time, viability (%), apoptotic index and in maintaining its normal morphological characteristics. ALP is considered as important marker for identification of pluripotent embryonic stem cells as well as multipotent mesenchymal stem cells. Isolated fibroblast cells of all the subcultures showed activity for ALP test indicating their progressive and undifferentiated quality and may be of multipotent in nature.ThesisItem Open Access MOLECULAR CHARACTERIZATION AND GENOTYPING OF STAPHYLOCOCCI ASSOCIATED WITH BOVINE MASTITIS(Assam Agricultural University, Khanapara,Guwahati, 2017-07) DUTTA, MADHUSMITA; Borah, P.Mastitis is an important disease of dairy cows and buffaloes causing huge economic losses in the form of reduced milk production. It is an inflammation of the mammary gland (udder) that causes physical and chemical changes in milk, and leads to pathological condition of the glandular tissue. It is generally associated with poor hygienic and husbandry practices. The present study was undertaken with a view to isolate and identify Staphylococcus aureus from both mastitic and apparently normal bovine milk samples. The study also included molecular typing of representative isolates and detection of important virulence-associated genes by PCR. A total of 204 milk samples comprising both clinically affected (14) and apparently normal (190) milk were used for this study. The apparently normal milk samples were subjected to California mastitis test, of which 85.79 % tested positive for sub-clinical mastitis. Bacteriological and biochemical examinations were performed to isolate and identify staphylococci associated with mastitis. A total of 60 (33.8%) out of 177 milk samples yielded Staphylococcus aureus, which were confirmed by polymerase chain reaction (PCR) amplification of conserved sequences of aroA gene. All the isolates (100 %) were found to possess three virulence-associated genes, namely surface protein A (spa), thermonuclease (nuc) and coagulase (coa) genes, while 58 (96.6%) of the isolates showed the presence of clumping factor A (clfA) gene. Antimicrobial susceptibility testing revealed that all the 60 isolates were resistant to Ampicillin and Cotrimoxazole, while the highest susceptibility (100%) was shown to Gentamicin, Kanamycin and Chloramphenicol followed by Streptomycin (80%). On the other hand, significantly lower susceptibility was shown to Ceftriaxone (13.33%), Tetracycline (8.33%) and Cefapime (1.67%). Out of the total 60 isolates, seven were subjected to PCR-RFLP of the coagulase (coa) gene. Polymorphism was shown by all the isolates (100%) with four different restriction patterns. Ten isolates were subjected to staphylococcal protein A (Spa) typing and PFGE. Spa typing revealed two different types, t165 and t1611. On the basis of phylogenetic analyses based on spa typing and PFGE, it was concluded that isolate number 9 of Spa type t165 is the ancestral strain, the clonal descendents of which are endemic in the study area causing subclinical bovine mastitis.ThesisItem Open Access POLYMORPHISM OF PROLACTIN (PRL) AND PROLACTIN RECEPTOR (PRLR) GENES IN INDIGENOUS DUCKS OF ASSAM(Assam Agricultural University, Khanapara,Guwahati, 2017-07) BASUMATARY, KELESON; Das, BulaThe present study was conducted to investigate the polymorphism of Prolactin (PRL) and Prolactin receptor (PRLR) genes in 101 indigenous ducks of Assam. PCR-RFLP analysis of PRL gene using restriction enzyme DraI revealed three genotypes, arbitrarily designated as AA, AB and BB. Following digestion, the AA genotype yielded two fragments (141 and 316 bp), the AB genotype yielded three fragments (141, 316 and 457 bp), and the BB genotype yielded one fragment (457 bp). The frequencies of A and B alleles were found to be 0.847 and 0.154 respectively and the genotype frequencies for AA, AB and BB genotypes were found to be 0.812, 0.069 and 0.119, respectively. The PCR-RFLP analysis of PRLR gene using restriction enzyme PciI revealed two genotypes, arbitrarily designated as AA and AB. The AA genotype yielded two fragments (108 and 259 bp) and the AB genotype yielded three fragments (108, 259 and 367 bp). The frequencies of A and B alleles were found to be 0.956 and 0.045, respectively and the genotype frequencies for AA and AB genotypes were found to be 0.911 and 0.089, respectively. On the basis of the present study, it was found that the A variant of PRL gene was predominant in the indigenous ducks of Assam with the highest frequency of AA genotype followed by BB genotype (AA>BB>AB). For PRLR gene, the frequency of allele A was higher than that of allele B with a higher frequency of AA genotype (AA>AB). Chi-square (χ2) test revealed that the population under study was not in Hardy-Weinberg Equilibrium for PRL gene, while for PRLR gene, the population was in Hardy-Weinberg equilibrium.ThesisItem Open Access EFFECTS OF CRYOPROTECTANTS IN VITRIFICATION OF GOAT OOCYTES(Assam Agricultural University, Khanapara,Guwahati, 2017-07) Majumdar, Kabita; Biswas, R.K.A total of 1243 ovaries were obtained from local abattoirs and processed for recovery of oocytes by aspiration and slicing techniques from follicles within 2-3 hours of slaughter. A total of 621 and 1124 oocytes were recovered from 545 and 698 ovaries by aspiration and slicing techniques respectively and graded on the basis of cumulus cell layer adhered to the zona pellucida . The mean recovery rate differed significantly between grades of oocytes in both the techniques and interaction between technique and grades of oocytes differed significantly. A total of 1745 immature good quality goat oocytes were vitrified by using cryoprotectants viz., Ethylene Glycol (EG), Propylene Glycol (PG) and DMSO and their combinations i.e., EG+PG, EG+DMSO and PG+DMSO @ 6M, 8M, 10M and 12M concentrations in the vitrification solution with the addition of 0.5M sucrose in basic solution that contained DPBS and FBS (4:1) and Gentamicin (50µg/ml). The equilibration solution was prepared by adding the cryoprotectant at the rate of half of the concentration used for vitrification and 0.25M sucrose in basic solution. The vitrification solution contained 0.5M sucrose and half of the total concentration for each cryoprotectant when vitrification of oocytes was done by combining two cryoprotectants. The mean rate of recovery of morphologically normal oocytes after vitrification differed significantly between concentrations in PG, EG and EG+PG. The highest mean recovery rate of morphologically normal vitrified oocytes was 74.16±4.44 per cent which was obtained in 10M EG+PG (1:1). The mean rate of recovery of morphologically normal vitrified oocytes was the highest (71.82±5.21%) in 0.5M sucrose contained in 5M EG+5M PG, although it did not differ significantly from concentrations of 0.25M, 0.75M and 1M sucrose. The mean percentages of in vitro matured oocytes following vitrification revealing expanded cumulus cells (61.67±2.54) and polar body extrusion (47.04±2.70) were significantly lower for vitrified oocytes as compared to the corresponding values (70.91±1.98% and 63.51±2.83 %) in non-vitrified oocytes .ThesisItem Open Access DEVELOPMENT OF Pasteurella multocida BIVALENT OUTER MEMBRANE PROTEIN BASED VACCINE ENTRAPPED IN ALUMINIUM HYDROXIDE NANOPARTICLES AND EVALUATION OF ITS IMMUNE RESPONSE IN MICE(Assam Agricultural University, Khanapara,Guwahati, 2017-07) Pegu, Haladhar; Tamuly, ShantanuSwine pasteurellosis is one of the most economically important diseases of pig caused by Pasteurella multocida(P. multocida) capsular types A and D and these organisms are commensals and opportunistic pathogens in the upper respiratory tract in pig. The vaccine prepared from P52 stain of serotype B:2 of P. multocida is effective only in controlling bovine pasteurellosis but not swine pasteurellosis due to antigenic variation among capsular types A and D. In the present study, an attempt was made to develop a bivalent vaccine that could elicit immunity against both the capsular types of P.multocida. Whole outer-membrane proteins (OMP) were extracted from P.multocida capsular types A and D, and were mixed together in the ratio of 1:1 forming bivalent outer-membrane proteins. The bivalent OMP was adsorbed onto aluminum hydroxide nanoparticles. The size of aluminum hydroxide nanoparticles adsorbed outer-membrane protein was found to be in the range of 120 to 130 nm. The aluminum hydroxide nanoparticles adjuvanted bivalent OMP based vaccine has shown quickest immune kinetics in terms of IgG response as compared to aluminum hydroxide microparticlesadjuvanted bivalent bacterin vaccine against Pasteurella multocida capsular type A. However, the IgG response against P. multocida capsular type A stimulated by aluminum hydroxide nanoparticles adjuvanted bivalent OMP based vaccine could not last longer compared to aluminum hydroxide microparticle adjuvanted bivalent bacterin. The immune kinetics in terms of IgG response against P. multocida capsular type D stimulated by aluminum hydroxide nanoparticles adjuvanted OMP was found to be similar to aluminum hydroxide microparticlesadjuvanted bivalentbacterin vaccine. The aluminum hydroxide nanoparticles adjuvanted bivalent OMP was efficient in stimulating IgG response in initial level against P.multocida capsular type A that may be helpful in use of vaccine formulation during the outbreak of the disease. Finally, the local inflammation induced by aluminum hydroxide nanoparticles in the injection sites was found to be milder than that induced by aluminum hydroxide microparticles.ThesisItem Open Access MOLECULR CHARACTERIZATION OF BIFIDOBACTERIA FROM ANIMAL SOURCES AND IN-VITRO ASSESSMENT OF THEIR PROBIOTIC POTENTIAL(Assam Agricultural University, Khanapara,Guwahati, 2017-07) PATHAK, BEDANTA; Borah, ProbodhThe present study was undertaken with a view to isolate bifidobacteria from various sources, viz. fresh bovine milk, faeces of calves and piglets, ruminal content of slaughtered bovines and intestinal/caecal content of chickenand to characterize the isolates by random amplified polymorphic DNA (RAPD) analysis and sequencing of amplified 16S rRNA gene fragments. A total of 12 (2.52%)out of477 samples from various sources examined were found positive forBifidobacterium spp. yielding the same number of isolates. All of these isolates were recovered from faecal samples of young piglets (6.12%), while samples from other sources examined did not reveal the presence of bifidobacteria. The isolates were characterized and identified by biochemical tests and phosphoketolase (F6PPK) enzyme assay, which showed characteristic features of Bifidobacterium spp. All the 12 isolates were also confirmed by genus-specific PCR based on amplification of 16S rRNA gene. Sequencing of the amplified products and subsequent sequence analysis by NCBI-BLAST revealed that out of the 12 isolates, 3 (25%) belonged to B. breve, 1 (8.33%) to B. pseudolongum, 6 (50%) to B. animalis subsp. lactis and 1 (8.33%) to B. thermacidophilum subsp. porcinum. However in respect of one isolate, the species could not be ascertained by 16S rRNA sequencing due to poor sequence data, although it was confirmed as belonging to Bifidobacterium spp. by genus-specific PCR. RAPD analysis could not clearly differentiate the strains belonging to different species. Phylogenetic analysis based on the bandings patterns also failed to differentiate the isolates belonging to different species of Bifidobacterium. In-vitro assessment of their antibacterial effect of the isolates revealed that two isolates ofB. animalis subsp.lactishadhigher inhibitory activity compared to the other isolates. Based on higher inhibitory effect on selected pathogens, five isolates were selected for further characterization in respect of their probiotic potential in vitro.The inhibitory effect of all these five isolateson the selected pathogens persisted even after heat-treatment at 100°C for 5 min. However, the inhibitory effect didn’t persist after treatment with pronase-E and proteinase-K. All the five isolates tested were able to grow at a minimal pH at 4.0. Two of the tested isolates belonging toB. animalis subsp.lactisspecies exhibited comparatively higher acid tolerance even after 180 min of exposure and showed the lowest percentage of mortality of 16.21 and 21.79 per cent, and 13.40 and 17.32 per cent, respectively on exposure to lysozyme (0.5 mg/ml) and ox bile salts @ 0.3% (w/v). In antibiotic resistant test, all the five isolates showed almost uniform susceptibility to different antimicrobial agents tested, except tetracycline. The present studysuggestedthat the two isolates from faeces of piglets obtained in the present studyof B. animalis subsp.lactispossess promising probiotic potentialand further in vivo study is required to be donefor validation of their probiotic activity.