Browsing by Author "Filia, Gursimran"
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ThesisItem Open Access Ante mortem diagnosis of co-infection of bovine tuberculosis and paratuberculosis(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2019) Singh, Inderjeet; Filia, GursimranThe aim of this study was to diagnose co-infection of bovine tuberculosis (bTB) and paratuberculosis (JD) in live animals using ante-mortem techniques such as comparative intradermal tuberculin test (CITT) and bovine gamma interferon assay (IFN-γ) for bTB and Single intradermal test (SID) for JD. Further molecular diagnosis of Mycobacterium tuberculosis complex (MTC) and M. avium subspecies paratuberculosis (MAP) by multiplex polymerase chain reaction (mPCR) was carried out using in house designed primers. Three hundred animals (94 cattle and 188 buffaloes) were selected from an organized dairy farm, while 18 cattle were from an unorganized dairy farm. The animals of age group between 2-12 years were included in the study. Blood and faecal samples were collected from the animals to be tested by CITT, IFN-γ, SID, conventional IS6110 PCR, IS900PCR and mPCR. Twenty one and nine animals were positive for bTB by CITT and IFN-γ assay respectively, out of which only nine animals were positive by both CITT and IFN-γ. Further by SID, nine animals were tested positive for JD. Four animals were positive reactors for both CITT and SID. All the blood samples were processed for conventional IS6110 PCR, IS900 and in-house mPCR. In IS6110 PCR 23 samples positive for MTC while four blood samples and five faecal samples were detected positive for MAP by IS900 PCR. A mPCR was developed to differentiate MAP and MTC in blood samples of cattle and buffaloes by observing a band size of 210 bp and 263 bp respectively. By in-house mPCR, one animal was positive result for both MTC and MAP. This animal was also positive for CITT/SID and IFN-γ. Thus, the in-house multiplex PCR may be useful to diagnose co-infection of bTB and paratuberculosis simultaneously.ThesisItem Open Access A comparison of some serological tests for diagnosis of brucellosis in vaccinated and unvaccinated bovines(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2018) Kaur, Ravneet; Filia, GursimranBovine brucellosis is an emerging zoonotic disease of livestock and is of major threat to dairy industry causing late term abortions and infertility. For the control and eradication of brucellosis, accurate serodiagnostic tests are required. The present study was carried out for serodiagnosis of brucellosis in vaccinated and unvaccinated bovines and to evaluate the Superagglutination test by comparing its efficacy with other serological tests like Rose Bengal Plate Test (RBPT), Microagglutination test (MAT), Indirect Haemagglutination Assay (IHA), Indirect Enzyme Linked Immunosorbent Assay (iELISA) based on sensitivity, specificity and predictive values. The seroprevalence of brucellosis from 486 animals by RBPT, MAT, IHA, i-ELISA and Superagglutination test was 224 (46.09%), 258 (53.09%), 216 (44.44%), 264 (54.32%) and 279(57.41%), respectively. The mean titre of MAT in unvaccinated animals was 1.84±0.91 and in vaccinated animals was 2.63±0.56. Superagglutination showed highest sensitivity (98.21%) when compared with RBPT and highest negative predictive value (NPV) of 98.07% when compared with RBPT and IHA. The specificity (85.53%) and positive predictive value (PPV) of 88.17% of Superagglutination was highest when compared with MAT. Superagglutination showed highest agreement with MAT. Agreement of Superagglutination with other test was found to be strong. Keeping i-ELISA as gold standard, the sensitivity of RBPT, MAT, IHA, c-ELISA and Superagglutination was 79.55%, 87.50%, 77.27%, 100.00% and 92.05%, while specificity was 93.69%, 87.84% and 87.84%, 66.67% and 83.78%, respectively. RBPT showed PPV of 93.75% and NPV of 79.39% while PPV of MAT and IHA was 89.53% and 88.31% while NPV 85.53% and 76.47%, respectively. C-ELISA showed PPV 97.37% and NPV 100.00%. Superagglutination showed PPV 87.10% and NPV 89.86%. All the tests showed strong degree of agreement while c-ELISA showed moderate degree of agreement when compared with i-ELISA. Superagglutination showed highest sensitivity and negative predictive value among all the tests with advantage of better visualization and ease of carrying of the test.ThesisItem Open Access Detection and differentiation of pathogens of Mycobacterium tuberculosis complex in bovines(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2017) Gill, Amandeep Kaur; Filia, GursimranBovine tuberculosis, chronic disease of animals, caused by species of Mycobacterium tuberculosis complex (MTC) remains a potential threat to animals as well as humans. Differentiation of the species of MTC is required for epidemiological and diagnostic purpose. The present study evaluated the presence of different species of MTC in bovines using gyrB-restriction fragment length polymorphism analysis. In this study, blood and milk samples from 50 milch animals which were positive reactors of comparative intradermal tuberculin test (CITT) were collected. Screening of MTC was done by IS6110-PCR using specific primers INS1/INS2 specific for MTC. Four (8%) animal were positive for MTC by IS6110-PCR. The positive samples were further confirmed by primers specific for gyrB gene. The amplified product of the gyrB-PCR was digested by RsaI and SacII enzymes and restriction fragment length polymorphism was carried out. The result of gyrB-RFLP revealed two animals positive for M. bovis and two for M. tuberculosis. Thus, gyrB-RFLP could be used as an additional tool in differential diagnosis of mycobacterial diseases caused by different species of MTC.ThesisItem Open Access Detection of drug resistance in mycobacterial isolates in cattle and buffaloes(2021) Deepa, Poloju; Filia, GursimranBovine tuberculosis is a chronic disease of animals caused by M. bovis. Culture is considered as gold standard for identification of a case of tuberculosis. Transmission of infection to humans is by consumption of unpasteurized milk. The emergence of drug resistant strains is a public health issue. A total of 200 animals (cattle and buffaloes) from organized dairy farm were screened by CITT. A total of 15 blood samples from the reactors tested positive by CITT, 57 tissue samples from PM Hall, GADVASU and from dead animals in the field, 6 trans-tracheal washes from animals with respiratory distress were collected. The samples were subjected to isolation by inoculating on Middlebrook 7H11 media and L-J media after proper decontamination. All the tissue specimens and transtracheal washes were subjected to ZN staining. The isolates obtained were subjected to acid fast staining and identified by biochemical testing. Extracted DNA from specimens and isolates were subjected to PCR for detection of MTC. The isolates were subjected to PCR for detection of drug resistance to rifampicin, isoniazid and streptomycin. Out of 57 tissue samples, 15 blood samples and six transtracheal washes, 24 tissue samples, two blood samples and one transtracheal wash, respectively were found to be positive for MTC. Out of all the samples subjected to isolation only 17 isolates were obtained (16 from tissue and one from transtracheal wash), six isolates were found to be positive for MTC by polymerase chain reaction, five from tissue samples and only one isolate from transtracheal wash. No isolate was obtained from blood samples. All the isolates showed clumps of acid-fast bacilli by ZN staining. Based on the biochemical tests six isolates were identified as Mycobacterium bovis. Out of six, four isolates which were positive for MTC and remaining five isolates other than MTC showed resistance to rifampicin. None of the isolates were resistant to streptomycin and isoniazid. This study emphasizes that antitubercular drug susceptibility testing of mycobacterial isolates from animals should be performed in order to estimate the magnitude of the risk of drug-resistant tuberculosis transmission to humans.ThesisItem Open Access Diagnosis of bovine tuberculosis in cattle and buffaloes using molecular and immunological approaches(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2021) Kaur, Harnoor; Filia, GursimranThe present study was carried out to diagnose bovine tuberculosis in live animals using techniques such as Comparative Intradermal Tuberculin Test (CITT), Gamma Interferon Assay and Enzyme Linked Immunosorbent Assay to detect the antibodies against the disease. Further molecular diagnosis of Mycobacterium tuberculosis complex (MTC) by conventional PCR was carried out on animals that were reactors in cell-mediated immune response. Two hundred thirty animals (74 cattle and 156 buffaloes) were selected from an organised dairy farm at Ludhiana and one each unorganised dairy farm from Jalandhar and Gurdaspur district. Blood samples were collected from animals to carry out É£-IFN assay, study the antibody response and conventional IS6110 PCR in reactor animals. Seventeen animals were positive for bTB by CITT and twenty one by É£-IFN assay. Seventeen animals that were tested positive by CITT, displayed positive result in É£-IFN assay. Four animals which were not tested positive for TB with CITT were positive with É£-IFN. Thirteen animals were positive for antibody response. Blood samples from 50 CITT reactor animals were processed for conventional IS6110 PCR, out of which 23 animals were positive for MTC. Six CITT inconclusive reactors were positive by PCR. Four É£-IFN assay negative animals tested positive by PCR. Thus multiple tests involving both the cell-mediated immune response and the humoral response should be carried out for the accurate diagnosis of bovine tuberculosis.ThesisItem Open Access Molecular detection of Mycobacterium bovis in milk of cattle and buffaloes suspected for bovine tuberculosis(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2016) Ahir, Paramjeet; Filia, GursimranThe aim of this study was to diagnose bovine tuberculosis (TB) in live animals using comparative intradermal tuberculin test (CITT) and bovine Gamma Interferon assay (?-IFN), detection of antibody response and molecular diagnosis of Mycobacterium bovis by multiplex polymerase chain reaction (mPCR) and real time PCR. One hundred forty animals (84 cattles and 56 buffaloes) were selected from an organized dairy farm, while 60 (52 cattle and 8 buffaloes) were from four unorganized dairy farms. The animals of age group between 2-12 years were included in the study. Blood and milk samples were collected from the animals to be tested by CITT, ?-IFN, detection of antibody response against M. bovis and molecular detection of M. bovis. A total of 29 (14.5%) animals were positive by CITT, 42 (21.0%) animals gave inconclusive results and 129 (64.5%) were negative reactors. A total of 23 animals were tested positive by ?-IFN assay (11 cattle, and 12 buffaloes). The mPCR assay targeting simultaneously M. bovis and Mycobacterium tuberculosis complex (MTC) was standardized which detected amplicons of 245bp for MTC and 500bp for M. bovis from the milk samples. One milk sample was positive for M. bovis and two samples for MTC by conventional PCR. Forty four DNA samples of milk were selected from the animals which were positive by either of the test i.e. CITT, ?-IFN assay, antibody kit test, mPCR or positive in more than one test and subjected to Taqman real-time PCR. Two samples which were positive for MTC by mPCR were diagnosis to be positive for M. bovis by this technique. Thus it is evident that to control bovine tuberculosis in a herd, diagnosis should be made by using two or more tests to detect the infected animals and segregate themThesisItem Open Access Molecular detection of Mycobacterium tuberculosis complex and analysis of cytokine profiles associated with bovine tuberculosis(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2019) Kaur, Gurleen; Filia, GursimranThe present investigation was carried out to diagnose bovine tuberculosis and to characterizse the tuberculous lesions. Tissue samples from 20 cases suspected for bTB along with five healthy tissue samples were used for the study. Ziehl- Neelsen staining was done and AFB were detected in 13 samples. Extraction of DNA from tissue samples was carried out and subjected to IS6110 PCR using INS1/INS2 primers specific for MTC with band size at 245 bp. Eleven samples were found positive by IS6110 PCR. For the differentiation of species of MTC, gyrB PCR-RFLP was performed. All the PCR positive samples showed presence of M.bovis subsp. bovis as the major causative agent of bTB. Histopathologically, eleven samples showed the presence of characteristic granulomas and majority of the granulomas observed were encapsulated with areas of central necrosis and calcification. IHC was performed on samples for detection of MTC antigen. Thirteen of the total samples were positive by IHC. Cytokines (IFN-γ, TNF-α, IL-10, IL-4, IL-13) were estimated in tissue homogenates by ELISA. A significant increase in the pro-inflammatory cytokine levels was observed in TB positive samples as compared to that of anti-inflammatory cytokine levels. However, both pro-inflammatory and anti-inflammatory cytokines levels along with IL-10 (immune-modulator) were increased in TB positive animals as compared to that of healthy animals. Thus, the present study showed that M. bovis is the main cause of bTB and cytokines play a major role in immunopathogenesis of bTB.ThesisItem Open Access Molecular diagnosis of caprine tuberculosis and paratuberculosis in goat herds(Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, 2018) Singh, Maninder; Filia, GursimranThe aim of the study was to diagnose caprine tuberculosis and paratuberculosis in goats with objectives to detect CMI response against Mycobacterium tuberculosis complex in goats and to detect Mycobacterium tuberculosis complex and Mycobacterium avium subspecies paratuberculosis in goats by molecular techniques. A total of 200 goats above 2 years of age, one hundred goats from one organized and one hundred from four unorganized farms were randomly selected for the study. Blood and fecal samples (n=200 each) were collected from the goats. For diagnosis of caprine tuberculosis, comparative intradermal tuberculin test (CITT) in animals for CMI response, while IS6110 PCR and IS6110 real time PCR on blood samples were performed for detection of Mycobacterium tuberculosis complex (MTC). While for diagnosis of paratuberculosis, Ziehl-Neelsen’s staining of acid fast bacilli in fecal samples, IS900 PCR and IS900 real time PCR for detection of Mycobacterium avium subspecies paratuberculosis (MAP) were carried out. Out of 200 animals, a total of three (1.5%) animals were positive by CITT, 38 (19%) animals showed inconclusive results and 159 (79.5%) were negative reactors. Out of 200 animals, four animals (2.0%) were positive for MTC in IS6110 PCR. MTC was further confirmed as M. bovis by IS6110 real time PCR. Four samples positive by conventional PCR were also positive for IS6110 real time PCR. Further, by ZiehlNeelsen’s staining, fifteen (7.5%) out of 200 fecal samples showed acid fast bacilli, while nine animals (4.5%) were positive for MAP in IS900 PCR. By IS900-TaqMan real time PCR, thirteen (6.5%) animals were detected positive for MAP including nine animals, which were positive in conventional PCR.The animals which were positive for M. bovis are different from the animals, which were positive for MAP. Thus the present study reported the prevalence of caprine tuberculosis and paratuberculosis in Punjab. Multiple diagnostic approaches were helpful for confirmatory diagnosis of these mycobacterial diseases in live animals. Further, the present study provides additional knowledge in the diagnosis of mycobacterial disease in goats from Punjab, which had not been carried out earlier.ThesisItem Open Access Studies On Immunogenicity Of Pasteurella Multocida B-2 Grown Under Iron Regulated Conditions(Punjab Agricultural University; Ludhiana, 2004) Filia, Gursimran; Jand, S.K.