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ThesisItem Open Access Biofilm production, associated genes and antimicrobial resistance of escerichia coli isolated from bovine mastitis(2022-09) Das, Himasri; Saikia, G KLivestock 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.ThesisItem Open 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).ThesisItem Open 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 KumarNecrotic 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.ThesisItem Open 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.ThesisItem Open Access PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF Riemerella anatipestifer ISOLATES FROM DUCKS(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati) DOLEY, MONUJ KR.; Das, SutopaRiemerella 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.ThesisItem Open 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.ThesisItem Open Access MOLECULAR DETECTION AND CHARACTERIZATION OF NEWCASTLE DISEASE VIRUS STRAINS FROM POULTRY(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2021-10) NEOG, BHRIGU KUMAR; Sarma, D. K.Newcastle disease is a highly transmissible and acute fatal disease of poultry caused by virulent strains of Avian paramyxovirus type 1 (APMV-1) which is commonly known as the Newcastle disease virus (NDV). Avian paramyxovirus type 1 exhibit great variation in their pathogenicity and the severity of the disease produced varies with the host species and the strain of virus involved. Newcastle disease can have devastating effects on the poultry industry due to the high morbidity and mortality associated with virulent strains of the virus. A study was undertaken to detect and characterize different NDV strains circulating among the native poultry population. To investigate the presence of NDV in clinically suspected backyard chickens, a total of 289 tissue samples were collected from 74 birds at necropsy from nine districts of Assam and tested using haemagglutination inhibition (HI) and reverse transcriptase polymerase chain reaction (RT-PCR). Out of the 289 tissue samples, 24.57 % and 47.05 % samples were found to be positive for NDV in HI assay and RT-PCR respectively. Of the 74 clinically suspected chickens 52.70 % birds were found to be positive for NDV in HI assay while 91.89 % birds were found to be positive for NDV in RT-PCR. Among the different tissue samples tested for presence of NDV, a significantly higher number of tissue samples from spleen, trachea, lung, proventriculus and caecal tonsil tested positive for NDV irrespective of the test used. To detect NDV in apparently healthy chickens, 186 numbers of oropharyngeal swabs and 146 numbers of cloacal swabs were tested using HI assay and RT-PCR. Of the 186 oropharyngeal swabs tested, 24.57 % and 15.05 % swab samples were found to be positive for NDV in HI assay and RT-PCR respectively. Further, out of 146 cloacal swabs tested, 6.16 % and 21.23 % swab samples were found to be positive for NDV in HI assay and RT-PCR respectively. A total of 18 tissue samples, identified as NDV positive using HI assay and RT-PCR, were processed for isolation of NDV using SPF embryonated chicken eggs (ECEs) of 9-11 days of incubation. The presence of the virus in the allantoic fluid of the inoculated ECEs was confirmed by two RT-PCR techniques, one of which targeted a 767 bp sequence of the F gene while the other targeted a 426 bp sequence of the HN gene of NDV. NDV from all the 18 tissue samples (100 %) was detected in the allantoic fluid of ECEs using the RT-PCR techniques. Six representative NDV isolates were sequenced by outsourcing and subjected to phylogenetic study. The consensus sequences of the isolates were subjected to multiple sequence alignment with reference sequences from GenBank databases. A phylogenetic tree was then constructed where five of the isolates clustered with genotype XIII of Class II NDV while one clustered with genotype II of Class II NDV cluster. Evaluation of the amino acid composition of the F0 cleavage site revealed the presence of the consensus sequence 112R-R-Q-K-R-F117 in case of the five genotype XIII isolates whereas the genotype II NDV isolate possessed the sequence 112G-R-Q-G-R-L117 at the F0 cleavage site of the fusion gene. Thus, five isolates from the present study were identified as virulent NDV strains while one isolate was identified as an avirulent strain. A multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) was standardized for simultaneous detection and differentiation of different pathotypes of NDV. Three specific oligonucleotide primers were used in the mRT-PCR for amplification of the target sequences of the F gene of NDV. The pathotypes of NDV was differentiated based on the products generated by the mRT-PCR. A product in size of 364 bp was obtained in case of the lentogenic strains while for mesogenic strains two products in size of 364 bp and 204 bp were generated. In case of velogenic strains only one product in size of 204 bp was generated. All the 18 NDV isolates from the present study was characterized using the mRT-PCR. Out of the eighteen isolates one isolate was identified as a lentogenic and two were identified as mesogenic. The other 15 isolates were identified as velogenic strains. A standard RFLP technique was also used to validate the results of the mRT-PCR. The RFLP involved digestion of a RT-PCR amplified 363 bp fusion gene product by HinfI restriction enzyme. The virulent and avirulent strains of NDV were differentiated based on the HinfI digestion patterns exhibited on 3 % agarose gels. The results of pathotyping obtained using RFLP analysis, corroborated the results of the mRT-PCR. A local isolate of NDV (AS/KM/CG 01) from non-vaccinated backyard chicken was subjected to complete genome sequencing by outsourcing. Evaluation of the genomic data revealed that the genome of the NDV isolate AS/KM/CG 01 consists of six genes arranged in tandem that encodes for six structural proteins namely, the nucleocapsid protein (NP), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin-neuraminidase protein (HN), and the polymerase protein (L). The isolate possessed a genome of approximately 15 kb. Evaluation of the F0 cleavage site within the fusion gene of the isolate revealed the presence of the consensus amino acid sequence 112G-R-Q-G-R-L117 which is typical of lentogenic strains of NDV. Phylogenetic study revealed that the NDV isolate belong to genotype II of class II NDV cluster. It was also found that the isolate has close relationship with previously reported genotype II NDV isolates from India and China. The isolate also showed more than 97 % homology with NDV vaccine strain LaSota. The study was summarized with the findings that genotype XIII NDV of class II cluster is predominant among the poultry flocks of Assam. Detection of genotype II NDV of class II closely related to NDV vaccine strain LaSota in the present study suggests a possible spillover of vaccine-type viruses from vaccinated poultry or feral avian reservoirs to non-vaccinated backyard chickens. However further studies are needed on this aspect.ThesisItem Open Access MOLECULAR AND ANTIGENIC CHARACTERIZATION OF CLASSICAL SWINE FEVER VIRUS ISOLATED FROM NORTH EASTERN REGION OF INDIA(Assam Agricultural University, Khanapara, Guwahati, 2017-07) Roychoudhury, Parimal; Sarma, Dilip KumarThe present study “Molecular and antigenic characterization of classical swine fever virus isolated from North Eastern region of India” was undertaken to explore the genogroups and subgroups of CSF virus isolated from different geographical location of North Eastern region of India, their genetic relatedness within and when compared to other isolates from different areas from the available data. Antigenic relatedness of the isolates to vaccine virus and to a local isolate(Lab. No.852) were studied by serological tests such as liquid phase blocking ELISA (LPB-ELISA) and neutralization peroxidase linked assay(NPLA).Antisera for the serological tests were raised against vaccine virus and a local isolate(Lab. No.852) in pigs and rabbits. Tissue samples collected during January 2011 to October 2012 were screened for Classical swine fever(CSF) virus antigen by sandwich ELISA(sELISA) and subsequently confirmed nested reverse transcriptase polymerase chain reaction (RT-nPCR). Out of 32 positive samples, virus could be isolated from 26 samples in PK-15 cell line. Seventeen isolates were selected for molecular characterization and 20 isolates for antigenic characterization. Virus Infectivity titres are expressed as log value of tissue culture infectious dose(logTCID50) per volume(0.1ml) of virus suspension, which varies in the range of 3.75 to 4.50 among the 20 isolates used for antigenic characterization. Cloning of three partial genomic region i.e.271nt fragment of 5′UTR, 271nt of E2 and 449nt of NS5B were carried out in pDrive vector and were subsequently sequenced by outsourcing. Phylogenetic analysis of the isolates using 150nt of UTR, 190nt of E2 and 409nt of NS5B revealed that 15 out of 17 isolates belonged to genogroup 1.1 and 2 isolates belong to genogroup 2.2. Nucleotide polymorphism at several locations were observed when compared to Alfort/187(GenBank Acc. No. X87939).Pair wise distance analysis of the E2 sequences of the 17 isolates showed 98.4% to 100% similarity within the same 1.1 genogroup While, two sequences of 2.2 genogroup showed 100% similarity with each other. Pair wise distance analysis of the 5′UTR sequences of the 17 isolates showed very close similarities, ranged between 99.3% to 100% While, two sequences of 2.2 genogroup showed 100% similarity with each other. Pair wise distance calculation of the NS5B sequence within the seventeen isolates from different parts of North Eastern India revealed overall similarity ranges from 85.1% to 100%. Homologous 2.2 group isolates showed 100% similarity with each other. Six representative isolates (ML-1, ML-2, ML-3, ML-4, AS-1 and AS-3) recovered during August and September 2011 within the genogroup 1.1 were compared among themselves as well as with available published sequences during 2005-2007. The results clearly indicate the close relation of the present isolates with the previously isolated virus from same North Eastern Region. However, the sequences are slightly diverge from each others, compared during 2005 to 2012 clearly indicates the endemic status of the region. Antigenic characterization of 20 isolates were carried out by comparing 50% inhibition log titre in LPB-ELISA and neutralization inhibition log titre in NPLA using hyperimmune serum against vaccine virus and a local isolate(Lab. No.852) raised in rabbit and pig.The 50% inhibition log titre of CSF virus isolates in LPB-ELISA obtained by using rabbit hyperimmune serum against the vaccine virus as well as against local isolate (Lab. No. 852) ranged from 1.505 to 2.107. The overall mean titre of CSF virus isolates in LPB-ELISA with the vaccine virus antiserum was 1.820±0.032 and with the local isolate (Lab. No.852) antiserum was 1.806±0.029 . The results of statistical analysis revealed no significant difference (’t’ value=0.373; P<0.05) between the mean of the 50% inhibition titre of CSF virus isolates with the vaccine and local isolate (Lab. No.852) antiserum in LPB-ELISA. The 50% inhibition log titre of the present isolates in LPB-ELISA obtained by using pig hyperimmune serum against the vaccine virus as well as against local isolate (Lab. No. 852) ranged from 1.505 to 2.107. The overall mean titre of CSF virus isolates in LPB-ELISA with the vaccine virus antiserum was 1.806±0.095 and with the local isolate antiserum was 1.818±0.025 . The results of statistical analysis revealed no significant difference (’t’ value=0.331; P<0.05) between the mean of the 50% inhibition titre of CSF virus isolates with the vaccine and local isolate antiserum in LPB-ELISA. Neutralization log titre of CSF virus isolates in NPLA using the rabbit hyperimmune serum raised against the vaccine virus as well as serum raised against a local isolate(Lab. No.852) ranged from 1.505 to 1.982. The overall mean titre of CSF virus in NPLA using rabbit hyperimmune serum, the vaccine virus antiserum was 1.754±0.028 and with the local isolate (Lab. No.852) antiserum was 1.789±0.026. The results of statistical analysis revealed no significant differences (‘t’ value,0.882; P<0.05) between the mean of the neutralization titre of CSF virus isolates with the vaccine virus and reference field virus antiserum in NPLA. The neutralization log titre of CSF virus isolates in NPLA using pig hyperimmune serum raised against the vaccine virus, ranged from 1.505 to 1.982 and against the local isolate(Lab. No.852), ranged from 1.505 to 1.806. The overall mean titre of CSF virus in NPLA using pig hyperimmune serum, the vaccine virus antiserum was 1.758±0.03 and with the local isolate (Lab. No.852) antiserum was 1.746±0.021 . The results of statistical analysis revealed no significant differences (‘t’ value,0.319; P<0.05) between the mean of the neutralization titre of CSF virus isolates with the vaccine virus and local isolate(Lab. No.852) antiserum in NPLA. Comparison of neutralization pattern of the isolates compared to vaccine virus antiserum and local isolate(Lab. No.852) antiserum by LPB-ELISA and NPLA using rabbit and pig hyperimmune serum revealed that the 50% inhibition mean log titre obtained in LPB-ELISA was slightly higher than the mean neutralization log titre obtained in NPLA in all the cases, but the mean titre obtained in the tests did not differ significantly. Antigenic characterization of six selected isolates (MZ-1,AS-1,MN-1,TR-1,AR-1,ML-1) representing genogroup 1.1 and 2.2 were selected for LPBE and NPLA using monoclonal antibody specific for CSF virus E2 glycoprotein. In LPBE, 50% inhibition log titre of the five isolates (MZ-1,AS-1,MN-1,AR-1 and ML-1) along with the vaccine virus and field isolate was 0.903.The neutralization log titre of CSF virus isolates in NPLA using monoclonal antibody showed similar log titre of 0.779 in four isolates(MZ-1,AS-1,AR-1,ML-1) along with the vaccine virus and field isolate, while two isolates (MN-1 and TR-1) showed a titre of 0.602.The overall mean titre of six CSF virus isolates with the reference vaccine and field virus isolate using monoclonal antibody in LPB-ELISA was 0.865±0.037 and in NPLA was 0.734±0.028. Statistically, there was no significant difference (‘t’ value=0.015; P<0.05) between the mean of the 50% inhibition log titre and neutralization log titre of CSF virus isolates in LPB-ELISA and NPLA using monoclonal antibody. Comparison of 50% inhibition log titre and neutralization log titre of the isolates among the two geno groups and their subgroup 1.1 and 2.2 by LPB-ELISA and NPLA revealed no significant differences in the neutralization pattern using antisera against vaccine virus and a local isolate (Lab. No.852).ThesisItem Open Access CHARACTERIZATION OF OUTER MEMBRANE PROTEINS OF Pasteurella multocida OF PORCINE ORIGIN(Assam Agricultural University, Khanapara, Guwahati, 2016-07) Borah, Bornali; Saikia, G. K.The present study was undertaken with a view to isolate and identify Pasteurella multocida from apparently healthy, diseased and dead pigs by both conventional and molecular methods, to study the pathogenicity of the isolates in mice, to prepare partially purified outer membrane proteins (OMPs) from most local virulent porcine strains (capsular types A and D), to study the protein profile of OMPs extract by SDS-PAGE, to identify the immunogenic proteins of OMPs by western blotting, to purify these proteins by size exclusion chromatography and to study the immunogenic potential of oil-adjuvanted vaccine prepared from the purified immunodominant OMPs in mice challenged with virulent homologous and heterologous capsular types of P. multocida. In the present investigation, a total of 357 samples including nasal swabs (187), tracheal swabs (18), lung (125) and heart blood (27) from apparently healthy, diseased and dead pigs were examined for isolation of P. multocida. Of these, 17 (4.76%) samples yielded P. multocida. More isolates were obtained from nasal swabs (9) from apparently healthy and diseased pigs than that of tracheal swabs (4) and lung tissues (4) from apparently healthy and dead pigs. All the 17 isolates showed cultural, morphological, staining and biochemical characteristics typical of P. multocida. The isolates were further confirmed as P. multocida on the basis of detection of species-specific gene (KMT1) by P. multocida species-specific PCR (PM-PCR). Among the 17 isolates, 6 (35.29%) were identified as capsular type A and 11 (64.71%) were identified as capsular type D based on multiplex capsular PCR results targeting hyaD-hyaC and dcbF genes, respectively. Mouse pathogenicity trial of 19 isolates of P. multocida revealed that the isolates induced 33.33 to 100.00 per cent mortality within 72 hours of inoculation. Two isolates (LS-3 and NS-4) were found to be comparatively more pathogenic causing 100.00 per cent mortality in the inoculated mice within 24-48 hours post inoculation and were selected for extraction of OMPs. Two most pathogenic strains of P. multocida, one each of types A and D (LS-3 and NS-4) were selected for extraction of OMPs. Analysis of OMPs of P. multocida type A by SDS-PAGE revealed presence of 21 protein bands with MWs ranging from 192.1 to 20.0 kDa. Among these protein, 36.8 and 25.0 kDa proteins appeared to be the major OMPs followed by 20.0, 56.5, 83.4, 47.4, 76.1, 51.2, 35.0, 99.2, 67.5 and 105.5 kDa protein bands based on band intensity in SDS-PAGE. While, OMPs of P. multocida type D showed presence of 22 protein bands with MWs ranging from 134.0 to 15.0 kDa. Among these protein, 35.7 and 25.0 kDa proteins appeared to be the major OMPs followed by 15.0, 56.2, 99.2, 47.1, 44.4, 66.5, 50.8, 78.5, 40.8 and 73.7 kDa proteins. The comparative evaluation of protein profiles of OMPs of serotypes A and D of P. multocida of porcine origin revealed that both the types shared three proteins with MWs 134.0, 99.2 and 25.0 kDa, of which the 25.0 kDa protein was found to be a major OMPs based on band intensity. The western blot analysis of the partially purified OMPs of P. multocida type A showed five major immunogenic proteins of MWs 83.4, 56.5, 36.8, 25.0 and 20.0 kDa giving strong immunostaining reaction with hyperimmune serum raised in rabbits. On the other hand, the partially purified OMPs of P. multocida type D showed five major immunogenic proteins of MWs 99.2, 56.2, 35.7, 25.0 and 15.0 kDa. Both the types shared a major immunogenic protein with MW 25.0 kDa. Size exclusion chromatography showed 10 peaks in OMPs extract of P. multocida type A and 13 peaks in OMPs extract of type D. In P. multocida type A, the five peaks of OMPs contained the protein fractions with molecular masses 83.4, 56.5, 36.8, 25.0 and 20.0 kDa, while in type D, the five peaks contained the protein fractions with MWs 99.2, 56.2, 35.7, 25.0 and 15.0 kDa. Mice immunized with oil-adjuvanted purified OMPs vaccines of P. multocida types A and D were challenged with 1x102 cfu of live P. multocida types A and D through subcutaneous (s/c) route and were found to be fully protective (100%). The organisms could not be re-isolated from the inoculated mice sacrificed after 7 days. The control group of mice showed 100 per cent mortality and died of septicaemia within 48 to 72 hours after challenge with 1x102 cfu of live P. multocida types A and D. Re-isolation of the organisms used for challenge infection was possible from the heart blood and the internal organs of dead mice of the control group.
