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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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  • ThesisItemOpen Access
    Physicochemical properties of live attenuated duck plague vaccine and evaluation of stabilizer efficacy for lyophilization
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022) Barua, Jonmoni; Das, Sutopa
    Duck plague (DP) or Duck Viral Enteritis (DVE) is an acute contagious herpesvirus infection of ducks and waterfowl of the family Anatidae of the order Anseriformes. Anatid Herpesvirus-1 (AHV-1) or duck enteritis virus (DEV)of the family Herpesviridae is the responsible agent for DP or DVE which is a member. The disease is known to have a global distribution and is associated with significant economic losses worldwide. The only method for preventing and controlling the disease is vaccination. Also, an active decontamination process for an effective vaccination programme in field conditions is important. So, in the present study emphasis has been laid to understand the physicochemical properties of a DPV vaccine strain along with evaluation of thermostability of freeze-dried vaccine with different combinations of stabilizers. In the present study, a vaccine strain of DPV available in the DBT-ADMaCDepartment of Veterinary Microbiology, College of Veterinary Science, AAU, Khanapara was revived in CEF and selected for study on the basis of identity with DPV by observing CPE, PCR and molecular characterization. Characteristic CPE like vacuolation, rounding, syncytium formation and ultimately detachment of cells were observed, in case of PCR band was observed at 1510 bp which proves similar identity with DPV. Molecular characterization revealed homology with DPV isolates from India (Kerala and Assam) and China. Quantitation was done at each step to find out the titre by TCID50/ml after every evaluation right from initial titre, loss of titre during lyophilization, loss of titre during the evaluation of physicochemical treatment, stability evaluation of the freeze-dried vaccine vial, as well as reconstituted vials. The initial titre was found to be 6.9±0.17. The vaccine virus was found to be sensitive to temperatures exceeding 56ºC and above, pH 3 and below, and pH 11 and above. It was also found sensitive to ether and trypsin. On sterility test, no growth was found on the culture. Lyophilization was carried out with 3 combinations of stabilizers namely LS, PTI and LHT. On quality evaluation, PTI and LHT showed uniform cake formation along with minimal loss of titre due to lyophilization. To check the thermostability of freeze-dried vaccines and reconstituted vaccines, vials were exposed at different temperatures. Among the freeze-dried vaccine, LHT could keep the highest titre when exposed to different temperatures and sampled at different time intervals. Although, LS and PTI too could keep with the infectivity titre with minimal loss of titre. In case of the reconstituted vaccine, NSS showed better stability at different temperatures than PBS, though the differences were minimum between the two. Finally, it can be concluded that LHT is one of the better stabilizers for DPV freeze-dried vaccine production. Alternatively, LS and PTI can be used by utilizing a suitable freeze-drying protocol. PBS and NSS both can be used as a diluent for the lyophilized DPV vaccine although in this study NSS was found to be superior. Hence, stabilizer LHT with diluent NSS was found to be superior for the DPV vaccine strain under this study.
  • ThesisItemOpen Access
    Neutralization efficacy of classical swine fever c-strain specific antibody to different genotypes circulating in North Eastern states, India
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Sarma, Jayashree; Barman, N N
    Classical swine fever (CSF) or Hog cholera is a highly contagious viral diseases affecting domestic and wild pigs. It has been a big threat to the piggery sector globally, causing negative impact on the economic background. The disease is highly endemic in India including NER. Assam too records highest CSF outbreaks. The recent outbreaks recorded occurrence of genotype 2.2, 2.1 and 1.2 besides wide prevalence of the historical genotypes 1.1. Outbreaks in vaccinated herds and shift in genogroup 1 to 2 globally, has raised the concern over the antigenic variation, protective immune response and neutralizing capacity of C-strain vaccine antibodies. Thus the present study was undertaken to explore the cross- protection efficacy of C-strain vaccine antibodies to the different genotypes or the need for potential vaccine candidate. Tissue samples and lyophilized isolates were selected for the study from CSFV repository, Department of Veterinary Microbiology. Sandwich ELISA and nested RT-PCR was done to determine the presence of the virus. Out of total 49 samples, overall positivity in SELISA was 36.0% (18) and in nested RT-PCR was 30.0% (15). The recovery rate of tissue samples was lower (35.0%) in comparison to lyophilized isolate 40.0%. Molecular characterization of the samples found positive in screening test was done based on E2 full length gene. Five isolates representing each state from north-east was successfully amplified at 1119bp for full length amplification of E2 gene. Genogrouping and phylogentic analysis revealed, genogroups 1.1 and 2.2 circulating in NER showing 98% and 84% nucleotide identity, respectively with the reference Alfort/187 strain. Whereas 99% nucleotide identity within the genogroup. The five isolates with known genogroups were propagated in PK-15 cell line upto 5th passage level and confirmed by S-ELISA and nested RT-PCR. Four isolates were isolated successfully except the isolate from Assam. The OD value at different passage level ranged from 0.589 to 1.763, showing an increase in titre with each subsequent passage. CSFV_AAU_Mg01 showed highest OD value at 5th passage. In-situ demonstration of CSFV by IPT revealed reddish brown cytoplasm indicating replication of the virus in the cytoplasm.TCID50 of the passaged viruses were done by FAT showing comaparble titre (4.49-5.16) with that of vaccine strain at 5th passage level. CSFV_AAU_Mg01 showing highest log TCID50 10 5.16 log TCID50 per ml. Hyperimmune sera was raised using purified cell culture adapted lapinised vaccine showing titres of 1:800 and 1:1600 and used for immunological characterization of the isolates by cross neutralization assay. A 50% neutralization titre of the hyperimmune serum ranged from 1/133 to 1/158 when assayed against the different viruses by FAT. Neutralization and cross – neutralization assay with C-strain specific antibody showed 100% neutralization with genotype 1.1, whereas 84% in geno-type 2.2. The study revealed genotypes 1.1 and 2.2 widely circulating in NER with lower neutralization efficacy of vaccine antibodies to heterologous genotypes.
  • ThesisItemOpen Access
    Seroprevalence and molecular detection of bovine brucellosis and leptospirosis in Assam
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Devi, Bandana; Saikia, G K
    Brucellosis and leptospirosis are neglected zoonotic disease prevalent throughout the world. Bovine brucellosis is predominantly caused by Brucella abortus. Leptospirosis in bovine is mainly caused by Leptospira serovars under the serogroup Sejroe. Both the diseases share some common clinical signs and symptoms and cause severe economic losses. The present study was undertaken to estimate the seroprevalence of bovine brucellosis and leptospirosis and diagnose both the diseases by molecular detection of Brucella and Leptospira organisms in clinically suspected and seropositive cases. The study was carried out in Assam during August 2021 to July, 2022. In this study, a total of 1013 cattle serum samples were collected from 11 districts of Assam viz. Tinsukia, Lakhimpur, Dhemaji, Sonitpur, Nagaon, Kamrup-M, Barpeta, Udalguri, Kokrajhar, Dhubri and Cachar, and screened for Brucella antibodyby Rose Bengal Plate Test (RBPT) and Indirect-enzyme Linked Immunosorbent Assay (i-ELISA) to estimate the seroprevalence of the disease. To detect seroprevalence of leptospirosis, a total of 512 cattle serum samples were collected from 7 districts of Assam viz. Dhemaji, Bishwanath, Nagaon, Kamrup-M, Bongaigaon, Kokrajhar and Dhubri, and tested by i-ELISA for Leptospira antibody. A total of 41 serum samples were found to be positive for Brucella antibody by both the tests with a seroprevalence rate of 4.04% and 19 out of 512 serum samples were found to be positive for Leptospira antibody by i-ELISA with a seroprevalence rate of 3.71%. Higher seroprevalence of brucellosis was recorded in female (4.60%) than in male animals (2.16%). Similarly, higher seroprevalence of leptospirosis was recorded in female (4.53%) than in male animals (1.45%). Age wise seroprevalence of brucellosis was found to be highest in animals of 2.1 to 5 years (1st to 3rd lactation) of age(6.32%) followed by animals of 5.1 years and above (4th lactation onwards) age group (2.90%). In case of leptospirosis, animals of 5.1 years and above (4th lactation onwards) age group showed highest seroprevalence (7.47%) followed by animals of 2.1 to 5 years (1st to 3rd lactation) of age (4.14%). In both brucellosis and leptospirosis, higher seroprevalence rate i.e., 6.54% and 4.33%, respectively was recorded in crossbred than in local cattle (1.07% and 2.97%, respectively). In case of brucellosis, animals reared in organised farms showed higher seroprevalence (8.94%) than the animals reared in semi-organised (3.08%) and backyard farms (1.63%). On the other hand, in case of leptospirosis, animals reared in backyard farms showed higher seroprevalence (7.20%) than the animals reared in semi-organised (2.63%) and organised farms (2.47%). In relation to animal health status, the seroprevalence of both the diseases were found to be highest in clinically ill animals with 40.90% for brucellosis and 8.18% for leptospirosis than apparently healthy animals i.e., 3.22% for brucellosis and 3.06% for leptospirosis. Again, among clinically ill animals, seropositivity for brucellosis was highest in animals with history of abortion (66.66%) followed by animals with retention of placenta (50.0%) and repeat breeding (33.33%). Similarly, in case of leptospirosis, highest seroprevalence was found in animals with history of abortion (33.33%) followed by animals with retention of placenta (25.0%) and repeat breeding (13.33%). In this study, both Brucella and Leptospira antibodies could be detected in 5 out of 512 serum samples screened by i-ELISA specific for both the diseases with a seropositivity rate of 0.976%. For molecular detection of brucellosis, 41 seropositive (32 apparently healthy and 9 clinically ill) and 23 clinically suspected (seronegative) samples (whole blood, aborted foetus, placenta, vaginal swab) were tested by Brucella genus specific PCR. Out of these, 9 clinical samples (39.13%) from seronegative cases and 4 samples (9.75%) from seropositive cases were found to be positive for Brucella genomic DNA in Brucella genus specific bcsp31 PCR. Overall, out of 64 samples examined, Brucella genomic DNA could be detected in 13 number of samples with a positivity rate of 20.31%. All 13 Brucella DNA were confirmed as Brucella abortus by multiplex PCR (AMOS). For molecular detection of leptospirosis, 19 seropositive (15 apparently healthy and 4 clinically ill) and 33 clinically suspected (seronegative) samples (whole blood, aborted foetus, placenta, vaginal swab and urine) were tested by Leptospira lipL32 gene PCR. Out of 33 clinically suspected (seronegative) samples, Leptospira DNA could be detected in 6 number of samples with positivity rate of 18.18%. Leptospira DNA could not be detected from seropositive samples. As a whole, out of 52 samples, Leptospira DNA could be detected in 6 sample with an overall positivity rate of 11.53%.
  • ThesisItemOpen Access
    Biofilm production, associated genes and antimicrobial resistance of escerichia coli isolated from bovine mastitis
    (2022-09) Das, Himasri; Saikia, G K
    Livestock production sector acts as one of the greatest contributors towards economic development of the country. Mastitis is considered to be one of the most common diseases of high yielding dairy cows which can cause decline in the milk production that ultimately leads to great economic loss in both developed and developing countries. Bovine mastitis can be divided into two types, clinical mastitis and subclinical mastitis. The present study was undertaken on phenotypic and genotypic detection of biofilm producing E. coli isolated from bovine mastitic milk and their antimicrobial resistance profile against commonly used selected groups of antibiotics. To carry out the study, a total of 560 quarters from 140 animals of both organized and unorganized dairy farms in and around Guwahati were screened for mastitis by California Mastitis Test (CMT) out of which 108 animals were found positive for mastitis. The overall prevalence of mastitis including clinical (15%) and subclinical form (62.14%) in both types of farms was 77.14%. In quarter wise distribution of mastitis, involvement of hind quarter was found to be more frequent. A total of 33 E. coli were isolated from 108 milk samples of mastitic dairy cows. All the isolates were screened for biofilm producing ability when tested by using on qualitative as well as quantitative detection methods viz., Congo red agar, Christensen tube and Tissue culture plate methods and all of them were found to be biofilm producers. All the E. coli isolates were tested for presence of biofilm associated genes, viz., csgA, fimH and luxS. The csgA gene was detected in 30 (90.90%) isolates, fimH in 31(93.93%) isolates and luxS was found in 30 isolates (90.90%). On relative quantification of mRNA expression of csgD gene revealed that the ΔCT value is significantly and negatively associated with biofilm production (P value<0.05). The E. coli isolates showed 100% sensitivity to Gentamicin, Neomycin and Amoxicillin+Sulbactam followed by Streptomycin (96.97%), Colistin (84.85%), ciprofloxacin and Ceftriaxone+Sulbactam (72.73% to each), Cefoperazone+ Sulbactam (69.70), Enrofloxacin and amoxycillin (63.64% to each) and Ceftriaxone (39.39%). However 100% resistance was observed for Cloxacillin followed by Ampicillin (96.97%) and Sulfadiazine (90.91%) on Disc diffusion test. In the present study, a total of 15 (45.45%) isolates were found to be multidrug resistant. Among all the MDR biofilm producing isolates, 6 were strong biofilm producers, 5 were moderate and 4 were weak biofilm producers and a significant correlation has been found between the strength of biofilm production and presence of MDR isolates (p<0.01). Our present finding has shown that the MIC values of Ceftriaxone, Amoxycillin, Gentamicin, Streptomycin were significantly correlated with strength of biofilm (P value<0.05). Out of 33 E. coli isolates tested, 18 (54.54%) were confirmed as ESBL producers based on double disk synergy test (DDST) and E-test. Further genotypic characterization of ESBL producing E. coli showed that ESBL encoding gene blaCTX-M was detected in 13 (39.39%) isolates with a product size of 585bp, blaSHV gene was detected in 3 (9.09%) isolates with a product size of 393bp and blaTEM gene was detected in 6 (18.18%) isolates with a product size 506bp.
  • ThesisItemOpen Access
    PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF METHICILLIN SENSITIVE AND RESISTANT Staphylococcus aureus (MSSA & MRSA) ISOLATED FROM BOVINE MASTITIS
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2020-12) ALI, ARFAN; SAIKIA, G. K.
    The present study was undertaken on characterization of Staphylococcus aureus isolated from bovine mastitic milk in respect of their phenotypic and genotypic characteristics more particularly resistance to methicillin (MSSA & MRSA) and other groups of antimicrobial agents, presence of methicillin resistance and other virulence genes. To carry out the study, a total of 1328 quarter milk samples from 812 animals of organized and unorganized dairy farms of Kamrup (M), Kamrup (R) and Nalbari districts of Assam were screened by California Mastitis Test (CMT) out of which 630 animals (1328 quarter) were found positive for mastitis. The 630 mastitic animals comprised 117 clinically and 513 subclinically affected dairy cows. The overall prevalence of mastitis including clinical (14.41%) and subclinical form (63.18%) mastitis in these three districts was 77.59%. Maximum number of animals had infection involving two quarters in both clinical (47.86%) and subclinical (52.44%) mastitis. Involvement of right hind quarters was higher (28.91%) than the left hind quarters (28.13%) in clinical mastitis, while it was higher in left hind quarters (29.10%) than right hind quarters (26.21%) in subclinical mastitis. Higher prevalence rate of clinical (15.36%) and subclinical (68.76%) mastitis was recorded in organized farms in comparison to clinical (12.13%) and subclinical (49.79%) mastitis in unorganized farms. A total of 194 isolates of staphylococci were obtained from 630 bovine mastitic milk, out of which 151 (77.84%) coagulase-positive isolates identified as Staphylococcus aureus by phenotypic tests were confirmed genotypically by detection of S. aureus specific aroA gene by PCR. Of the 151 isolates, 54 (35.76%) were from clinical and 97 (64.24%) from subclinical mastitis and all of them produced coagulase and fermented mannitol. The prevalence of S. aureus associated mastitis was found to be 46.15% and 18.91% for clinical and subclinical forms, respectively. The prevalence of MRSA was 9.27% (14) as determined by cefoxitin resistance in phenotypic tests and confirmed by detection of mecA gene by PCR. The MRSA isolates were completely resistant (100%) to methicillin, cloxacillin, cefoxitin, tetracycline, streptomycin, colistin and mupirocin followed by higher degree of resistance to gentamicin and oxytetracycline (85.71% each) and moderate resistance to neomycin (50%). The MSSA isolates exhibited higher degree of sensitivity (73.72 – 100%) to tetracycline, amoxyclav, cefotaxime, ciprofloxacin, colistin, neomycin, streptomycin, mupirocin, ceftriaxone, gentamicin, cloxacillin, oxytetracycline, teicoplanin except cefepime to which they were least sensitive (54.01%). Out of 151 S. aureus isolates, 55 (36.42%) were multidrug resistant (MDR) which exhibited resistance against 4-12 antimicrobial agents. Among the MDR isolates, 14 (25.45%) were MRSA which showed resistance to 9-12 antimicrobial agents. A comparative study on antimicrobial resistance spectrum of MRSA and MSSA strains was conducted by disc diffusion and E-test using 10 antimicrobial agents which included penicillin, ampicillin, oxacillin, amoxyclav, cefoxitin, cefotaxime, ceftriaxone, gentamicin, ciprofloxacin and teicoplanin. All the MRSA isolates (14) exhibited similar pattern of resistance to all the agents except cefotaxime to which three isolates showed variation. All of the 38 representative MSSA isolates were sensitive to cefoxitin, oxacillin and teicoplanin in both the tests. One to three isolates showed variation in resistance pattern to rest of the antimicrobial agents. The E-test was found to be more effective than disc diffusion method for determining sensitivity of clinical isolates to antimicrobial drugs. In phenotypic characterization, all the coagulase positive isolates (MSSA and MRSA) caused alpha or beta haemolysis on sheep blood agar and showed susceptibility to novobiocin and resistance to polymyxin B which are typical characteristics of S. aureus. All the 151 S. aureus isolates harboured the virulence associated nuc (thermonuclease) and spa (staphylococcal protein A) genes and lukF-PV by six (6) and bap by two (2) isolates as revealed by PCR assay. The isolates which showed presence of lukF-PV and bap genes were methicillin resistant strains of S. aureus (MRSA).
  • ThesisItemOpen Access
    DEVELOPMENT OF A SUITABLE VACCINE FORMULATION AGAINST TYPE A Clostridium perfringens ASSOCIATED NECROTIC ENTERITIS IN BROILER CHICKEN
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2019-07) SARMAH, HIRAMONI; Sharma, Rajeev Kumar
    Necrotic enteritis (NE) is one of the most clinically dramatic and important bacterial disease of poultry industry. It has a great negative impact on broiler industry due to production losses, increased mortality, increased feed conversion ratio. The cost of NE worldwide was estimated to 2 billion dollars per year with 1% daily mortality. Most common age of outbreaks of NE in broiler flocks raised on litter are between the second and fifth week of age. NE in broiler chicken is commonly associated with Clostridium perfringens toxin type A, while involvement of type C is very rare. The study was undertaken to develop a suitable vaccine preparation against C. perfringens type A associated NE for broiler chicken. During the study clinical samples. viz., intestinal content, intestinal scrapings from broilers died of suspected form of NE and faecal swabs from live affected birds with clinical symptoms suggestive of NE were screened for C. perfringens. A total of 26 repository isolates of C.perfringens maintained in Department of Microbiology, College of Veterinary Science, Khanapara were also considered for the present study. All the isolated C. perfringens recovered from NE affected broiler birds along with the repository were characterized with respect to the toxin types, detection of gene(s) associated with virulence and secretory protein, pathogenicity for mice, release of toxins and secretory proteins in cell free supernatant and resistance patterns towards antimicrobial agents. The detailed characterization was carried out with an idea to identify a suitable vaccine candidate for the development of vaccine preparations against NE in broiler chicken. Clinical samples, comprising of intestinal scrapings (42), intestinal contents (30) were collected from 72 dead broiler chickens with suspected form of necrotic enteritis. Another 23 faecal samples were collected from an equal no. of clinically affected live broiler birds by swabbing. A total of 41 isolates were identified as toxin type A, only 10 isolates isolates isolates isolates isolates isolates isolates isolates isolates exhibited additional virulent genes viz netB, tpeL and gapC genes either alone or in combination. All the eluted amplified PCR products of target genes with respective band sizes were confirmed by DNA sequencing. All total of 10 isolates of C. perfringens type A positive for netB alone (5), and netB with tpeL and gapC (5), were subjected to mouse pathogenicity trial. The mouse pathogenicity trial revealed variable pathogenicity, producing clinical symptoms in 21 inoculated mice within 72 hrs of observation, while 17 of the clinically affected mice were succumbed to death. The highest mortality was observed in group of mice inoculated with S8. On SDS-PAGE analysis cell free supernatant of S8 could exhibit highest 16 different visible bands with MW, ranging from 12 to 250 kDa. The four additional virulence associated proteins, NetB (33 kDa), GPD (40 kDa), α- toxin (43 kDa) and tpeL(180 kDa) were also distinctly visible. On immunoblotting clear immune dominant antigenic proteins identified as netB (33 kDa), GPD (40 kDa), alpha (43 kDa) and tpeL (180 kDa). were observed in cell free supernatant of S8 and other few strain. On antimicrobial resistance profiling highest resistance pattern was observed against ciprofloxacin (80.0%), followed by norfloxacin and tetracycline (60.0% each), gentamicin (30.0%) and levofloxacin (20.0%). Gatifloxacin, cefmetazole,clindamycin, metronidazole, and tigecycline were found to be effective against all the isolates. After selection of a suitable strain of C. perfringens type A, six different vaccine formulations, i.e., non-adjuvanted crude toxoid (I), non-adjuvanted crude toxoid with bacterin (II), non-adjuvanted crude toxoid with sonicated supernatant (SS) and bacterin (III), adjuvanted crude toxoid (IV), adjuvanted crude toxoid with bacterin (V) and adjuvanted crude toxoid with SS and bacterin (VI) were prepared. Comparative evaluation of the six vaccine formulations was carried out in respective groups of broiler birds, with respect to their serum antibody titer. Among the vaccine formulations, combination of crude toxoid, bacterin and SS was found to be superior in respect to the mean serum antibody titer in vaccinated bird (group VI), throughout the study period throughout study period. The passive mouse protection study could reveal that the pooled immunized serum samples of 21st, and 28th day could protect the mice with the challenge with homologous strain of C. perfringens.
  • ThesisItemOpen Access
    CHARACTERIZATION OF OUTER MEMBRANE VESICLES (OMVs) OF Pasteurella multocida OF AVIAN ORIGIN
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2020-01) Gogoi, Anamika; Sharma, R. K.
    The Fowl Cholera, an infectious disease of poultry, waterfowl and many other birds is caused by Pasteurella multocida. To overcome those hurdles in poultry industry, focus has been given to identify immunogenic subcomponent of the causative agent and their use in development of modern vaccines. The present study was undertaken with a view to evaluate immunogenic potential of Outer Membrane Vesicles (OMVs) of Pasteurella multocida as well as their release under the influence of various environmental and physico-chemical factors. The extraction of OMV fraction was made from a highly pathogenic strain of P. multocida capsular type A associated with Fowl Cholera. The release of OMVs by the selected isolates was found to be significantly (p˂0.001) highest under the influence of iron deficient condition (2, 2 bipyridyl), exhibiting a protein concentration of 18.3 mg/ml. Similarly, the influence of pH in iron restricted environment was also have an impact on OMV release, which was found to be significant (p˂0.05) in reverse direction. A positive correlation could also be made in respect to the oxidative and antibiotic stress with release of OMVs. The comparative protein profiling of OMVs, OMPs and whole cell lysate of the selected pathogenic P. multocida type A isolate could exhibit more distinct and prominent protein bands in OMV fraction. The OMV fraction could also reveal the ompA (37.7-38.1 kDa), which was not prominently observed in other two fractions. The immunogenic potential of the extracted OMV fraction revealed an increasing trend of the mean antibody titre in both the immunized groups, with (Group I) or without (Group II) booster. The immunized birds of group I exhibited a significantly rising trend (p<0.05) of the mean serum antibody titre from the day of the vaccination, until it reached its peak (5947.41±62.6). The peak titre was observed on 28th day of post primary immunization, following booster on 21st day post immunization. Similarly, the immunized birds of group II the mean serum antibody titre of 7th dpi was continued to increase significantly at every weeks of observation till it reached peak on 21st (4576.27±42.9). The declining trend of the mean serum antibody titre was observed in the birds of group II from the day 28th of post immunization (4219.12±64.5) and continued till end of the study, i.e. the 60th dpi (3813.83±148.5). No significant difference could be observed between the two preparations, with and without booster in respect to the mean serum antibody titre till 21st dpi. Challenge trial could establish 100 per cent protection of vaccinated birds against homologous challenge, while development of clinical signs in the immunized birds was observed, following heterologous challenge. There was no significant difference between OMVs administered group and control group was observed in terms of blood SOD and GPx activity.
  • ThesisItemOpen Access
    PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF Riemerella anatipestifer ISOLATES FROM DUCKS
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati) DOLEY, MONUJ KR.; Das, Sutopa
    Riemerella anatipestifer cause one of the most economically important infectious diseases among the domesticated duck population. The present study was conducted to isolate R. anatipestifer and also to phenotypic and molecular characterization of the isolates. During the study period, 27 suspected field outbreaks were attended in five district of Assam. A total of 624 samples were collected and processed for isolation followed by phenotypic and molecular characterization. All confirmed isolates (n=95) were screened for two important virulence genes ompA and cam gene. Further, the confirmed field isolates were also subjected for antimicrobial resistant pattern against 28 most commonly used antimicrobial agents to determined suitable antimicrobial regime. Pathogenicity test was also conducted from isolates recovered from dead ducks in suitable host system. On bacteriological examination, 121 isolate (19.39%) could be recovered based on phenotypic characteristics (cultural, morphological and biochemical). Phenotypically, highest bacteria could be isolated from brain and heart tissue (28.57%) followed by spleen and liver (26.19%) and least from ocular swab (12.50%). All the isolates produced small, smooth, circular, mucoid, glistening and dew drop like colonies on blood agar under micro-aerophilic condition for 18-24hours. The colonies were found to be non haemolytic on blood agar except 4 isolate (11.12%), watery, discrete, translucent with characteristics odour of culture. Biochemically, all the isolates showed positive for catalase and oxidase test (100%), 97 isolates for gelatin liquefaction test (80.16%) whereas found negative for indole, methyl red, Vokes-Proskauer, H2S, ornithine decarboxylase etc. On sugar fermentation tests, 10 isolates revealed positive for trehalsoe and xylose (8.26%), 7 isolates for lactose (5.78%) test. Among the phenotypically identified suspected isolates only 95 isolates (78%.51) could be recovered through PCR assay targeting 16S rRNA, ERIC sequence and gyrB gene with equal positivity. Phylogenetic analysis of the representative isolates based on species-specific 16S rRNA gene revealed formation of single clade with two reference strains HXb2 (CP011859.1) and D-743 (AY871831.2) of China. Moreover, within the clade four isolates (ASC/AAU/RA5, ASDi/AAU/RA4, ASS/AAU/RA3 and ASK/AAU/RA1) formed one sub-clade, whereas ASD/AAU/RA2 formed another sub-clade with HXb2 and D-743 strains. The analysis revealed that R. anatipestifer circulating in Assam is closely related to the Chinese strains of the organism and at least two different strains are prevalent in the study area. The pairwise sequence identity analysis of 16s rRNA gene sequences among the isolates were between 96.5-100 % with divergence ranged from 0 to 3.5 % among the strains. Based on pairwise sequence identity, all isolates ASC/AAU/RA5, ASDi/AAU/RA4, ASS/AAU/RA3 and ASK/AAU/RA1 formed one molecular sub-group whereas isolate ASD/AAU/RA2 was far from this molecular group. Among all the stains of R. anatipestifer, the largest divergence (3.5 %) was exhibited by the isolate ASD/AAU/RA2. There were no significant divergence among ASC/AAU/RA5, ASDi/AAU/RA4, ASS/AAU/RA3 and ASK/AAU/RA1, whereas ASD/AAU/RA2 showed 1.7% divergence when compared with other four isolates from Assam. These findings additionally support the phylogenetic analysis which is suggestive of circulation of at least two different strains in Assam. Similarly, phylogenetic analysis targeting tree gyrB gene elicited that all the five R. anatipestifer isolates of Assam forms one clade in the cluster 1 with five reference Chinese strains RA9913 (JN969056), WJ4 (CP041029), XG19 (CP076675), RA-CH-1 (CP003787), and HXb2 (CP011859). This analysis revealed that R. anatipestifer circulating in Assam are closely related to Chinese strains. The Pairwise sequence identity analysis of gyrB gene sequences of R. anatipestifer were between 92-100% and revealed that all the isolates of Assam formed one molecular sub-group with Chinese strains. The pairwise identity among the isolates of Assam is between 99-100%. These findings additionally support that the R. anatipestifer strains circulating in Assam has close resemblance with R. anatipestifer strains of China. Pathogenicity trial with pathogenic isolates in duckling revealed highest mortality within 48-72 hours (53.34%) followed by 24-48 hrs (33.34%) post inoculation (pi) and bacterium could be reisolated from the death duck. The antimicrobial (28) resistant pattern of field isolates (n=95) revealed 100 per cent sensitive to enrofloxacin, ciprofloxacin, cefotaxime, sulphadiazine and sulphafurazole whereas piperacillin+tazobactum, methicilin, rifampicin, colistin were found to be 100 per cent resistant. All the isolates of R. anatipestifer displayed an expanding resistance pattern to number of antibiotics such as 88.89% to clindamycin, 81.48% to oxytetracycline, 85.18% to ofloxacin, 70.37% to streptomycin, 51.85% to chloramphenicol, 37.03% towards cefixime, 29.62% towards gentamicin etc. The group wise antibiotic resistant patterns of R. anatipestifer isolates revealed that most of the isolates were resistant to tetracycline (81.46%) group followed by penicillin (74.69%), Phenicols (51.84%), aminoglysides (40.74%) and flouroquinolones (28.39%) etc while highest susceptibility were recorded towards carbapenems (100%) followed by Sulphonamides (95.07%), cephalosporins (90.13%),, quinolones (81.49%), and macrolids (77.77%), The molecular screening of the field isolates towards virulent gene through PCR assay revealed that all the isolates were found positive for conserved ompA gene (100%) whereas only 4 isolates (4.25%) were found to be positive for cam gene.
  • ThesisItemOpen Access
    MOLECULAR DETECTION AND CHARACTERIZATION OF FOOT AND MOUTH DISEASE VIRUS (FMDV) AND STUDY OF CYTOKINE EXPRESSION IN NATURALLY INFECTED LOCAL/CROSSBRED CATTLE FROM ASSAM
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2021-09) BRAHMA, DERHASAR; Sharma, K.
    Foot and mouth disease (FMD) is a transboundary and the most contagious disease of cloven-hoofed animals including domestic and wild ruminants and pig, and has a great potential for causing severe economic loss due to loss of production and deprivation from international trade of animal products to FMD free countries. FMDV may occur in all the secretions and excretions of acutely infected animals, including expired air. Following recovery from the acute stage of infection, infectious virus may persist in the oropharynx of some ruminants (carriers), where live virus or viral RNA may continue to be recovered from oropharyngeal fluids and cells for upto 6 months or more. In this study, besides Sandwich ELISA, molecular detection and typing of FMDV was done using multiplex Reverse Transcription Polymerase Chain Reaction (mRTPCR), Reverse Transcription Loopmediated Isothermal Amplification (RT-LAMP) and SYBR Green real-time PCR targeting 3D gene. Isolation and molecular characterization of FMDV by sequencing was done. Also, study of expression of cytokines like interferon (IFN-α, IFN-β, IFN-) as well as certain interleukins (IL-1α, IL-1β, IL-2, IL-6, IL-10 and IL-12) and tumour necrosis factor (TNF-α) was estimated at mRNA level by SYBR Green real-time PCR from whole blood (White Blood cells) samples during the natural infection and during the period of persistence. This study was carried out in a total of 129 animals, comprising of 93 crossbred (vaccinated) and 36 local (non-vaccinated) cattle and additionally 12 healthy in-contact animals were taken as control animals. For carrying out this study, Tissue (n=29), whole blood (n=36) and oropharyngeal fluid (n=190) samples were collected as per standard procedure in 50% glycerol, EDTA and 0.8 M PBS/transport media, respectively. OP fluid was collected from recovered animals until complete recovery (i.e. 1st, 3rd, 6thand 9thmonth) from FMD infection. All the RNA extractions were done using Qiagen RNA extraction kit. We found that, out of 29 tissue samples, 20 samples were positive for serotype O, 9 were positive for serotype A and none of the samples was positive for Asia-1 by the multiplex RT-PCR as well as RT-LAMP. FMDV could be detected in 86.21%, 100%, 100% and 100% of tissue samples by sandwich-ELISA, mRT-PCR, RT-LAMP and SYBR Green real-time PCR respectively. Sensitivity test was run using 10 fold serial dilution of RNA extracted from FMDV antigen and found that, the real-time PCR was more rapid and highly sensitive technique of all, secondly the RT-LAMP, followed by the mRT-PCR. From the follow-up cases of the FMD recovered cattle, 38 (23.75%), 47 (29.38%) and 49 (30.63%) OPF samples (n=178) were found to be positive for FMDV by the multiplex RT-PCR, RT-LAMP and SYBER Green real-time PCR respectively, indicating persistence (carriers).The SYBR green real-time PCR was very much useful for detection of persistence from the OPF samples.However, OPF (n=12) and blood (n=12) samples from all the healthy controls and blood (n=12) from persistent animals were negative for FMDV. All blood samples (100%, n=12) from the clinically FMD infected cattle were positive for FMDV. The persistence of FMDV in oropharyngeal region of cattle lasted for upto 3 to 4 months in most of the FMD infected cattle. Persistence in crossbred (vaccinated) cattle didn’t last for more than 4 months. Only 2 Local non-vaccinated cattle (1.6%) was found to have persistence upto 6-7 months after infection. The overall number of persistent animals and the rate of persistence in cattle (n=129) at 1st month, 3rd month and 6th month were 32 (24.81%), 15 (11.26%) and 2 (1.6%) respectively, and was slightly higher in the local non-vaccinated compared to the crossbred vaccinated cattle. No statistical significance was observed between the two groups as the P value was found to be 0.23 (>0.05) and the Chi-square value was 5.57. The sequencing results showed that the Serotype O sequence (MZ501211-G-02- 19, MZ501212-G-03-19 and MZ501213 Op) shared 98.81% identity with Pakistan isolate MN953620, 96.43% identity with India isolate KY579948.1 (Nagaland, submitted by RRC Assam) and 94.05% identity with India complete genome isolate MN983158.1; and theSerotype A sequence (MZ501214-Mg/01/19) shared 95.29% identity with Indian isolate HQ832583.1 and 94.24% identity with Bangladesh isolate KT982204. The identity range was 98.81%-96.43% and 95.29%-92.22% for type O and A respectively, based on the nucleotide sequence Blast search in NCBI. The multiple sequence alignment showed that there are some minor changes in the nucleotide sequences with the consensus sequences. There were nucleotide insertions in the 3953 and 3954 positions in two of the query sequences of FMDV type O. Whereas, in FMDV type A, there were nucleotide insertions at 3807, 3813-3815 and 3841 positions and deletions at 3771 and 3874 positions of the nucleotide sequences. The result from this study shows that cytokine genes had general trend of upregulation during acute infection and decreased level of expression or down regulation during persistence. Cytokines in blood were generally upregulated in both acute infection and persistence, but compared to acute, there was decreased mRNA level of expression of cytokines during persistence except the down regulation of IFN-β, IL-2 and IL-6, whereas, all but IFN-α and IL-1α were down regulated in OPF during persistence. These cytokines may have certain role in persistence of FMDV by suppression of immune response and also by having anti-inflammatory or immunomodulatory response in carrier cattle. Thus, from this study, we can conclude that, molecular detection techniques are the most sensitive and specific techniques for detection of FMDV and particularly for diagnosis of persistence from OPF samples. Persistence occurred in 32 cattle (25%) after 1st month of the FMDV infection, out of which the proportion of local non-vaccinated cattle was slightly higher. And that cytokines may have a role in persistence of FMDV in cattle.