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Kerala Veterinary and Animal Sciences University, Wayanad

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    ThesisItemOpen Access
    COMPARATIVE EFFICACY OF DIFFERENT TESTS FOR ANTE-MORTEM DIAGNOSIS OF BOVINE TUBERCULOSIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES, MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-11-07) SARIKA N; Dr. Binu K. Mani
    Blood and nasal swab samples, 50 each and 17 milk samples were collected from animals with symptoms suggestive of bTB, presented for slaughter, at Thrissur corporation slaughterhouse, Kuriachira. The samples were subjected to detection by ZN staining, IS6110 insertion element specific PCR to detect MTBC, 12.7 kb fragment specific mPCR to differentiate M. bovis and M. tuberculosis, real-time PCR specific for a region upstream of p34 gene to detect the presence of M. bovisand M. tuberculosis, bovine gamma interferon immuno assay of blood and isolation and further characterisation. Histopathology was also performed on post-mortem tissue samples. The presence of drug resistance genes (viz., rpoB for rifampicin, pncA for pyrazinamide and katG for isoniazid) were detected in the isolates obtained using mPCR. Further, comparison of the results of different ante-mortem tests to that of isolation from the tissue samples, which being the gold-standard for confirmative diagnosis of TB, was made and analysed statistically. Finally, in order to confirm efficacy of the various ante-mortem diagnostic methods assessed in the study, 118 apparently healthy dairy cattle, maintained in organised farms and households in and around Thrissur were screened for TB. In the present study, ZN staining on nasal swab/milk/tissue samples was found to be highly specific, but showed poor sensitivity. The sensitivity of the PCR (nasal swab), real-time PCR (nasal swab), isolation (nasal swab) and gamma interferon assay was 100, 100, 100 and 50 per cent, respectively. The specificity of PCR (nasal swab), PCR (blood), real-time PCR (nasal swab), isolation (nasal swab) and gamma interferon assay was 95.83, 85.42, 97.92, 100 and 91.67 per cent, respectively. The sensitivity and specificity of PCR and real-time PCR from tissue samples was 100. None of the molecular methods could detect mycobacteria in any of the milk samples. Visible lesions were absent in the tissue samples. The HP of PCR positive tissue samples revealed multiple areas of mononuclear infiltration, epitheloid cells and mildchanges in lung samples. Present study revealed that, nasal swabs were found to be a better choice for ante-mortem diagnosis of bTB, than milk. Isolation of bacteria from nasal swab and PCR of the nasal swab were found to have maximum sensitivity and specificity for diagnosing bTB. Screening of 118 cattle maintained in organised and unorganised farms in Thrissur district, revealed a positivity rate of 2.54, 7.63 and 2.54 respectively for PCR, gamma interferon immuno assay and isolation, respectively. Drug resistance genes were detected in five of the isolates obtained, of which, all the M. bovis isolates amplified all three genes namely rpoB, pncA and katG. However, the M. tuberculosis isolates showed discordant results and failed to amplify all genes. In conclusion, a single method alone did not specifically detect mycobacteria in the present study. Therefore, a combination of tests could be used for screening and accurate diagnosis of bTB, considering other factors like, availability of samples, cost of the test and diagnostic facilities available in the laboratory.
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    ThesisItemOpen Access
    DETECTION AND CHARACTERISATION OF BACTERIAL AND VIRAL AGENTS ASSOCIATED WITH NEONATAL DIARRHOEA IN CALVES
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD , LERALA VETRINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-25) SARITHA BABY; Dr. Chintu Ravishankar
    Neonatal calf diarrhea (NCD) is a major threat to cattle farmersworldwide. It causes huge financial losses mainly due to morbidity andmortality of the calves. The etiology of NCD is complex disease in which both infectious agents and non infectious factors are involved. A comprehensiveanalysis was carried out to detect the major bacterial and viral agentsresponsible for NCD in Kerala and also to assess non-infectious factors thatcontribute to the condition. A total of 120 diarrhoeic faecal samples collected from different cattle farms in Thrissur and Wayanad districts of Kerala duringthe period from October 2019 to September 2022 were used for the study.Each farm has its own management practices that were found to influence the onset of diarrhoea. In the study it was observed that unhygienic conditions, overcrowding and changes in nutrition were the major predisposing factors for NCD. The incidence of diarrhoea was higher in farms housing large number of animals. Calves below one month of age were affected the most and the maximum number of diarrhoeic calves (26 per cent) was found in the 22-30 days age group. Among the infectious agents, E. coli, Salmonella, rotavirus and coronavirus were focused in the current investigation. The isolated bacteria were characterized by biochemical testing and E. coli was found to be the main infectious agent associated with the condition with a prevalence of 84.17 per cent. From the 120 samples, a total of 101 isolates of E. coli could be isolated. None of the samples were found to be positive for Salmonella. Antibiotic sensitivity testing revealed that the all E. coli. isolates were resistant to Penicillin G, Cefotaxime/Clavulanic acid and Cefpodoxime. The percentage of isolates resistant to Amoxicillin/sulbactam and Enrofloxacin, Ciprofloxacin, Co-Trimoxazole and Tetracycline, Gentamicin, and Nitrofuratonin were 92.08 per cent, 85.15 per cent, 80.2 per cent, 77.23 per cent, and 72.25 per cent respectively. Only 53.47 and 51.49 per cent of the isolates were resistant to chloramphenicol and streptomycin, respectively. When the E. coli isolates were subjected to phylogrouping using a quadruplex PCR, it was observed that B1 was the most prevalent group in this study with 28 isolates. Group A, D, C, E, F and clade I were also detected but with a lower prevalence. The fimbrial genes are responsible for virulence of E. coli and played a role in causing diarrhoea. The presence of fimbrial genes F5, F41 and F17 was also tested employing PCR. Of the isolates tested ten were detected as positive for F17 with a positivity of 16.13 per cent. Fimbrial genes F5 and F41 were not detected in any of the isolates. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed for the detection of the viral agents. For rotavirus and coronavirus, VP6 gene and N gene were targeted respectively. Out of the 120 samples, 14 (11. 67 per cent) were found to be positive for rotavirus. The diarrhoeic faeces of calves infected with rotavirus were gray or creamy white and watery and all the affected calves were above one week of age. Coronavirus could not be detected in any of the samples. The results of the study indicate that diarrhoea in calves in Kerala was caused by both bacterial and viral agents. Varying degrees of antibiotic resistance were also detected against the common antibiotics. Good care of the new born calves and hygienic practices in farms will go a long way in control of the condition
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    ThesisItemOpen Access
    DETECTION AND MOLECULAR CHARACTERISATION OF VIRUSES ASSOCIATED WITH REPRODUCTIVE DYSFUNCTION IN PIGS WITH SPECIAL REFERENCE TO PORCINE TESCHOVIRUS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-22) JISHNU HARIDAS P.; Dr. Chintu Ravishankar
    The study was undertaken to detect viruses associated with reproductive dysfunction in pigs in Kerala with special reference to porcine teschovirus (PTV). Apart from PTV, the other viruses tested were porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV). A total of 45 tissue samples and faecal samples from animals with history of enteritis and reproductive dysfunction were collected. Faecal samples were also collected from 30 live pigs in farms with history of enteritis and reproductive dysfunction. Total RNA/DNA were extracted separately from the tissue samples using RNA/DNA extraction kit. Total RNA was also extracted from fecal samples. Complementary DNA (cDNA) was synthesized from extracted RNA using cDNA synthesis kit. The cDNA was used as template in the reverse transcription polymerase chain reaction (RT-PCR) for detection of 5’UTR region of PTV. The results of the test revealed that 18 out of 45 samples were positive for the virus with positivity rate of 40 per cent. The positive samples were further subjected to RT-PCR with primer sets for detecting the VP1 gene of the virus. However, sequence was obtained and the genotyping of the virus could not be carried out. In order to obtain the complete VP1 sequence, the forward primer binding to 5’UTR region and reverse binding to VP1 region was used to amplify a 3.3 kb region of the virus. In this test, a specific amplicon was obtained in one of the samples. The complete 3.3 kb amplicon was sequenced by Nanopore single strand sequencing technology using MinION equipment. The obtained sequences were mapped to PTV1 reference sequence using Geneious Prime software. On analysis of the VP1 sequence thus obtained, and subsequent phylogenetic analysis, it was observed that the sequence was similar to VP1 sequence of PTV 14. Out of the samples tested, 11 out of 45 were positive in RT-PCR for detection of ORF6 of PRRSV with positivity of 24.44 per cent. The BLAST analysis of the ORF6 sequences of PRRSV revealed that the isolates were similar to isolates from China (96.86 - 99.21 per cent similarity) and Switzerland (98.60 per cent similarity). The PPV was detected in 4 positives out of 45 samples (8.89 per cent similarity). The BLAST analysis of the sequences revealed that the isolates were similar to isolates from Italy (100 per cent similarity), China (100 per cent similarity), Germany (100 per cent similarity), South Korea (100 per cent similarity) and Brazil (100 per cent similarity) while the sequences were similar to isolates from Thrissur, Kerala only by 99.50 per cent. One of the animals had mixed infection with PTV and PRRSV and three animals had mixed infection with PPV and PTV. It is concluded that PTV is present at an alarming rate in pigs of Kerala. Also present are PRRSV and PPV, but to a lesser level compared to PTV. The presence of these viruses alone or in combination may be responsible for the reproductive dysfunction observed in pigs in Kerala. Key words: Reproductive dysfunction, PTV, PRRSV, PPV, RT-PCR, MinION
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    ThesisItemOpen Access
    DEVELOPMENT OF DIAGNOSTIC PROTOCOL FOR RIEMERELLOSIS IN DUCKS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-03-08) GREESHMA RAJU; Dr.Priya P. M.
    Riemerellosis or new duck disease is a bacterial disease caused by Riemerella anatipestifer affecting ducks and is accountable for massive mortality and low production rates. Effective prevention of the disease lies with prompt diagnosis and timely treatment, as vaccination against riemerellosis is not widely practised yet. The present study focussed on development of field-oriented diagnostic tests which are rapid, easy and cost-effective as opposed to the current diagnostic methods which are time consuming and expensive. Four diagnostic assays were standardised, viz., latex agglutination test (LAT) and dot-enzyme linked immunosorbent assay (Dot-ELISA), to detect antibodies, indirect immunoperoxidase test (IPT) and indirect immunofluorescence test (IFT) as antigen detection tests against riemerellosis. The local isolate of R. anatipestifer, RA1, preserved in the Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Mannuthy was utilised in the experimental study. The outer membrane protein (Omp) of the organism was used as antigen in the development of antibody detection tests and its efficacy was compared with indirect enzyme linked immunosorbent assay (ELISA). Whereas, the efficacy of antigen detection tests were compared to the gold standard method, isolation and identification of the organism. The study revealed that, though the antibody detection tests were capable of detecting antibodies against riemerellosis, indirect ELISA was more effective in that aspect and LAT detected antibodies better than Dot-ELISA. The IPT and IFT were standardised by experimental inoculation of RA1 to ducklings and was found to be equally efficient as that of isolation and identification of the bacteria. In conclusion, though LAT and Dot-ELISA were easy, rapid and cost effective, they cannot be used as sole diagnostic methods but could effectively support clinical and necropsy findings to arrive at a diagnosis. The IPT and IFT were time consuming like isolation and identification but the results were easily read and interpreted and IPT was found to be more advantageous, as it did not require expensive equipment and also provided a permanent record of results
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    ThesisItemOpen Access
    GENOTYPIC CHARACTERISATION OF INFECTIOUS BRONCHITIS VIRUS ISOLATES FROM KERALA
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARYA AND ANIMAL SCIENCES UNIVERSITY, 2022-03-08) SRUTHY CHANDRAN B.; Dr. Surya Sankar
    Infectious bronchitis virus (IBV) is the aetiological agent of infectious bronchitis (IB), an acute and contagious disease with high economic significance in the global poultry industry. Circulation of diverse variants of IBV make the control of the disease difficult, despite extensive vaccination programmes. In Kerala, vaccination against the disease is not strictly followed and the occurrence has been proved in earlier studies. In this scenario, the present study is contemplated on genotypic characterisation of IBV isolates across the state, focusing on the sequencing of whole S1 subunit of S gene, N and M genes followed by restriction fragment length polymorphism (RFLP) on representative amplicons of S1 subunit of S gene. Out of the 103 samples collected, 32 were found to be positive for the disease with detection primers that targeted the hypervariable region 3 of S1 subunit of S gene. The tissue homogenate from positive samples were inoculated in 9-11 day-old embryonated chicken eggs via., allantoic route. Allantoic fluid was harvested after 48 h and reverse transcriptase polymerase chain reaction (RT-PCR) with the same detection primers was done to confirm the presence of the virus. All the 32 samples were positive for RT-PCR targeting S1 subunit of S gene, N and M genes. The representative amplicons of the above mentioned genes were sequenced, analysed and compared with current vaccine strain H120 and sequences of other Indian IBV isolates available in the GenBank. On nucleotide sequence and amino acid profile analysis, it was found that variations exist among isolates as well as with vaccine strain and other Indian isolates. On phylogenetic analysis based on these genes, the isolates clustered with each other and also with isolates from different parts of India. The representative amplicons of S1 subunit of S gene when subjected to RFLP, generated restriction patterns similar to the vaccine strain, Massachusetts type.
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    ThesisItemOpen Access
    DETECTION AND MOLECULAR CHARACTERISATION OF PATHOGENS CAUSING RESPIRATORY INFECTIONS IN GOATS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-01-25) ARUN P. M.; Dr. Rajasekhar R.
    Small ruminants contribute significantly to the Indian animal husbandry economy. Respiratory diseases of small ruminants are multifactorial and there are multiple etiological agents. The major infectious diseases which affect the respiratory tract of goats are Mycoplasmosis, Peste des petits ruminants, bluetongue, and goatpox. So, a comprehensive study was undertaken to detect and characterise the pathogens viz. Mycoplasma capricolum subsp. Capripneumoniae (Mccp), Small ruminant morbillivirus (SRMV), goatpox virus (GPV), and bluetongue virus (BTV) by molecular methods. Of the total 64 samples tested, 10 samples were positive in the RT-PCR targeting Fusion (F) gene of SRMV, two samples were positive in PCR targeting DNA polymerase gene of GPV and one sample was positive in the PCR targeting A32 gene of Orf virus (ORFV). None of the samples were positive for BTV and Mccp. The molecular characterisation of full N and F gene of the identified SRMV isolates were carried out. Phylogenetic analysis of the SRMV isolates based on the nucleocapsid (N) and F genes revealed that, all the isolates shared a common ancestor with Tamil Nadu isolate and were grouped under lineage IV. Phylogenetic analysis also revealed that, there are two genetic groups circulating in Kerala. Analysis of the F protein of SRMV showed two unique mutations (A18E and S430I) in Kerala isolates. Amino acid analysis of nucleoprotein revealed that most of the changes were in the C- terminal region. Four unique mutations were also observed in the Nucleoprotein (NP) of the present SRMV isolates (I153V, A431V, R458M and G461K). Among the 19 B cell epitopes of NP, at least one amino acid variation was detected in four epitopes. These changes may affect the monoclonal antibody based diagnostic assays. These changes in F and N gene indicates continuous emergence and circulation of new variants of virus within same geographical area. Phylogenetic analysis of GPV based on the P32 gene revealed that both the isolates of the present study clustered along with other Indian isolates with the highest similarity with the Puducherry isolate. The sequencing and phylogenetic analysis of ORFV showed similarity to the Indian isolates and isolates from China. A SYBR Green real time RT-PCR assay targeting F gene of SRMV was developed. The detection limit of the assay was estimated using cloned template and was found to be 13 copies. The assay was 1000 times more sensitive than the conventional RT-PCR and efficiency of the test was found to be 98.2 per cent. Thus, the developed SYBR Green real-time RT-PCR assay could be used as an alternative to diagnose SRMV.
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    ThesisItemOpen Access
    DETECTION AND MOLECULAR CHARACTERISATION OF BACTERIAL AND VIRAL PATHOGENS FROM RESPIRATORY INFECTIONS IN DOGS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-01-24) MANEESH K. M.; Dr. Sumod K.
    Numerous diseases commonly affect dogs for a number of reasons, many of those being brought on by inadequate management techniques. Respiratory diseases in dogs can result from a wide variety of infections, with the majority of cases being polymicrobial and multifactorial in origin. The canine infectious respiratory disease complex (CIRDC) is a significant subset of these diseases. The major pathogens that were classically associated with CIRDC are Bordetella bronchiseptica (Bb), canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canid alpha herpesvirus-1 (CHV-1), and canine parainfluenza virus (CPiV). Canine respiratory coronavirus (CRCoV), canine pneumovirus (CnPnV), Mycoplasma cynos, and Streptococcus equi subspecies zooepidemicus are some of the most recent infections to be associated with the development of CIRDC. Additionally, canine pneumovirus (CnPnV) and canine non-primate hepacivirus (CNPHV) have been occasionally linked to CIRDC. Despite the fact that CIRDC is typically thought to affect kennelled dogs, there is significant evidence of the disease in pet dogs, many of which have moderate to severe disease symptoms. A total of 59 samples were tested and 11 were positive for CDV nucleic acid targeting the N gene. Of a total of 59 tested samples 13 were found positive for Mycoplasma spp. by PCR targeting the 16S rRNA gene. After analysis of the amplified sequences using BLAST, one of the samples was confirmed as Mycoplasma cynos and one sample was confirmed as Mycoplasma maculosum. None of the samples tested were positive for Bb, CAV-2, CHV-1, CRCoV, Streptococcus equi subspecies zooepidemicus CnPnV, and CNPHV. Sequencing and Phylogenetic analysis revealed that all the CDV isolates are related but different strains. The isolates from Wayanad were related with the south Indian isolates. The isolates from Ernakulam, Malappuram, and Kannur showed close resemblance with the North Indian isolates.
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    ThesisItemOpen Access
    MOLECULAR CHARACTERISATION OF PORCINE CIRCOVIRUS 2 AND DEVELOPMENT OF A REAL-TIME POLYMERASE CHAIN REACTION FOR DETECTION OF THE VIRUS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-01-23) SHASHANK S; Dr. Chintu Ravishankar
    Porcine circovirus 2 (PCV2) is an emerging swine pathogen causing enormous economic losses to the global swine industry. The virus has been reported in many of the pig-producing states of India including Kerala. However, no systematic study has been performed to characterize the virus and elucidate the lineage of the virus prevalent in pigs in Kerala. Hence, the present study was undertaken to characterise PCV2 in pigs in Kerala by molecular methods, and to develop a rapid and specific assay for the detection of the virus. A total of 62 tissue samples taken for the study were screened for the presence of PCV2 by ORF2-based PCR, in which 36 samples (58.06 per cent) were positive. The complete genome of the virus was sequenced from 11 representative positive samples. The sequences obtained revealed similarity to PCV2 sequences reported from India previously, and to sequences from China and Vietnam. Phylogenetic analysis was carried out based on complete genome, and partial and complete ORF2 sequences. The analysis revealed that of the 11 PCV2 genomes analysed, eight, two and one belonged to genotypes PCV2d, PCV2h, and PCV2b respectively. It was also noticed that prior to 2021, only genotype 2d was prevalent in North Kerala. Since 2021, 2b and 2h were detected in the region which may be due to introduction of these genotypes from other areas where these genotypes are prevalent. In case of analysis of ORF2 sequences, close clustering of Kerala sequences with those from Tamil Nadu, Uttar Pradesh and Mizoram were observed. The sequences were also similar to PCV2 sequences reported from Kerala previously. On analysis of the complete amino acid sequence of ORF2, it was observed that sample 118/MIB/2021 had Asparagine (N) at the 234th position, which was not been recorded in any Kerala or Indian sequences so far. Based on the ORF2 nucleotide sequences obtained in the study, primers and TaqMan probes were designed for the detection of PCV2 genotypes prevalent in Kerala. A single TaqMan real-time PCR that could detect all the genotypes could not be standardised. However, the designed primers (but not the probe) were able to detect these genotypes. Hence, another TaqMan assay specific for detection of 2d was designed as that genotype was predominant in North Kerala. The detection limit estimated using the cloned template was found to be 310 copies of the viral genome. The designed TaqMan assay was used to test 40 samples (20 positive and 20 negative samples) and 23 samples (57.5 per cent) were positive indicating a higher sensitivity of the test.
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    ThesisItemOpen Access
    ISOLATION AND MOLECULAR CHARACTERISATION OF BACTERIAL PATHOGENS FROM RESPIRATORY INFECTIONS IN SWINE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD, 2023-06-17) ANJITHA G S; Sumod K.
    Respiratory illness in pigs are undoubtedly the most serious operational risk for swine farmers worldwide. Porcine respiratory disease complex occurs due to interplay of several factors such as viral agents, bacterial pathogens and adverse managemental conditions. The commonly recognized bacterial pathogens causing respiratory infections in pigs include Mycoplasma hyopneumoniae, M. hyorhinis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Haemophilus parasuis, Streptococcus suis, and Trueperella pyogenes, to name a few. A comprehensive study was undertaken to detect the presence of bacterial respiratory pathogens in swine with major interest directed towards Mycoplasma spp., Actinobacillus pleuropneumoniae, Bordetella bronchiseptica and Pasteurella multocida in northern Kerala, India. A total of 43 samples, including 27 tissue samples and 16 nasal swabs collected during 2021 were used for this study. Conventional bacteriology followed by molecular diagnostics and sequencing was the methodology adopted for detection of predominant bacterial pathogens other than Mycoplasma spp., in which case, classical bacteriology was excluded. The detection of Mycoplasma spp. was attempted initially with genus specific 16S rRNA primer set and 25 out of 43 samples were found positive. These twenty five positive samples were further subjected to amplification with another set of 16S rRNA primers specific for M. hyopneumoniae and M. hyorhinis. No samples were positive for M. hyopneumoniae whereas 12 out of the 43 samples were positive for M. hyorhinis with positivity rate of 27.91. Sequencing and subsequent phylogenetic analysis of the isolates revealed that the majority of the strains were similar to strains from USA and China, with the exception of one isolate which showed more resemblance to a strain from Brazil. The positivity rate of Pasteurella multocida isolates identified by means of culture and biochemical tests, and confirmed by PCR was 30.23 per cent. On phylogenetic analysis with kmt gene, these isolates showed more similarity to strains of serogroups D belonging to China and Iran. The isolation and identification of Bordetella bronchiseptica by conventional bacteriology was attempted and confirmation was done by PCR amplification of flagellin and fimbrial genes. The phylogenetic studies showed that Respiratory illness in pigs are undoubtedly the most serious operational risk for swine farmers worldwide. Porcine respiratory disease complex occurs due to interplay of several factors such as viral agents, bacterial pathogens and adverse managemental conditions. The commonly recognized bacterial pathogens causing respiratory infections in pigs include Mycoplasma hyopneumoniae, M. hyorhinis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Haemophilus parasuis, Streptococcus suis, and Trueperella pyogenes, to name a few. A comprehensive study was undertaken to detect the presence of bacterial respiratory pathogens in swine with major interest directed towards Mycoplasma spp., Actinobacillus pleuropneumoniae, Bordetella bronchiseptica and Pasteurella multocida in northern Kerala, India. A total of 43 samples, including 27 tissue samples and 16 nasal swabs collected during 2021 were used for this study. Conventional bacteriology followed by molecular diagnostics and sequencing was the methodology adopted for detection of predominant bacterial pathogens other than Mycoplasma spp., in which case, classical bacteriology was excluded. The detection of Mycoplasma spp. was attempted initially with genus specific 16S rRNA primer set and 25 out of 43 samples were found positive. These twenty five positive samples were further subjected to amplification with another set of 16S rRNA primers specific for M. hyopneumoniae and M. hyorhinis. No samples were positive for M. hyopneumoniae whereas 12 out of the 43 samples were positive for M. hyorhinis with positivity rate of 27.91. Sequencing and subsequent phylogenetic analysis of the isolates revealed that the majority of the strains were similar to strains from USA and China, with the exception of one isolate which showed more resemblance to a strain from Brazil. The positivity rate of Pasteurella multocida isolates identified by means of culture and biochemical tests, and confirmed by PCR was 30.23 per cent. On phylogenetic analysis with kmt gene, these isolates showed more similarity to strains of serogroups D belonging to China and Iran. The isolation and identification of Bordetella bronchiseptica by conventional bacteriology was attempted and confirmation was done by PCR amplification of flagellin and fimbrial genes. The phylogenetic studies showed that Respiratory illness in pigs are undoubtedly the most serious operational risk for swine farmers worldwide. Porcine respiratory disease complex occurs due to interplay of several factors such as viral agents, bacterial pathogens and adverse managemental conditions. The commonly recognized bacterial pathogens causing respiratory infections in pigs include Mycoplasma hyopneumoniae, M. hyorhinis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Haemophilus parasuis, Streptococcus suis, and Trueperella pyogenes, to name a few. A comprehensive study was undertaken to detect the presence of bacterial respiratory pathogens in swine with major interest directed towards Mycoplasma spp., Actinobacillus pleuropneumoniae, Bordetella bronchiseptica and Pasteurella multocida in northern Kerala, India. A total of 43 samples, including 27 tissue samples and 16 nasal swabs collected during 2021 were used for this study. Conventional bacteriology followed by molecular diagnostics and sequencing was the methodology adopted for detection of predominant bacterial pathogens other than Mycoplasma spp., in which case, classical bacteriology was excluded. The detection of Mycoplasma spp. was attempted initially with genus specific 16S rRNA primer set and 25 out of 43 samples were found positive. These twenty five positive samples were further subjected to amplification with another set of 16S rRNA primers specific for M. hyopneumoniae and M. hyorhinis. No samples were positive for M. hyopneumoniae whereas 12 out of the 43 samples were positive for M. hyorhinis with positivity rate of 27.91. Sequencing and subsequent phylogenetic analysis of the isolates revealed that the majority of the strains were similar to strains from USA and China, with the exception of one isolate which showed more resemblance to a strain from Brazil. The positivity rate of Pasteurella multocida isolates identified by means of culture and biochemical tests, and confirmed by PCR was 30.23 per cent. On phylogenetic analysis with kmt gene, these isolates showed more similarity to strains of serogroups D belonging to China and Iran. The isolation and identification of Bordetella bronchiseptica by conventional bacteriology was attempted and confirmation was done by PCR amplification of flagellin and fimbrial genes. The phylogenetic studies showed that Respiratory illness in pigs are undoubtedly the most serious operational risk for swine farmers worldwide. Porcine respiratory disease complex occurs due to interplay of several factors such as viral agents, bacterial pathogens and adverse managemental conditions. The commonly recognized bacterial pathogens causing respiratory infections in pigs include Mycoplasma hyopneumoniae, M. hyorhinis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Haemophilus parasuis, Streptococcus suis, and Trueperella pyogenes, to name a few. A comprehensive study was undertaken to detect the presence of bacterial respiratory pathogens in swine with major interest directed towards Mycoplasma spp., Actinobacillus pleuropneumoniae, Bordetella bronchiseptica and Pasteurella multocida in northern Kerala, India. A total of 43 samples, including 27 tissue samples and 16 nasal swabs collected during 2021 were used for this study. Conventional bacteriology followed by molecular diagnostics and sequencing was the methodology adopted for detection of predominant bacterial pathogens other than Mycoplasma spp., in which case, classical bacteriology was excluded. The detection of Mycoplasma spp. was attempted initially with genus specific 16S rRNA primer set and 25 out of 43 samples were found positive. These twenty five positive samples were further subjected to amplification with another set of 16S rRNA primers specific for M. hyopneumoniae and M. hyorhinis. No samples were positive for M. hyopneumoniae whereas 12 out of the 43 samples were positive for M. hyorhinis with positivity rate of 27.91. Sequencing and subsequent phylogenetic analysis of the isolates revealed that the majority of the strains were similar to strains from USA and China, with the exception of one isolate which showed more resemblance to a strain from Brazil. The positivity rate of Pasteurella multocida isolates identified by means of culture and biochemical tests, and confirmed by PCR was 30.23 per cent. On phylogenetic analysis with kmt gene, these isolates showed more similarity to strains of serogroups D belonging to China and Iran. The isolation and identification of Bordetella bronchiseptica by conventional bacteriology was attempted and confirmation was done by PCR amplification of flagellin and fimbrial genes. The phylogenetic studies showed that these isolates bore more resemblance to those found in Japan and Hungary. Attempts were also carried out to isolate A. pleuropneumoniae by culturing of the samples. The isolation of bacteria could not be done in culture, but molecular detection was employed with omlA gene and subsequent confirmation was done by sequencing. Phylogenetic analysis grouped the isolates in the study with serotypes 3 and 7 of Japan, Switzerland and Germany.