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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open 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 RavishankarThe 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, MinIONThesisItem Open 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 resultsThesisItem Open 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 SankarInfectious 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.ThesisItem Open 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.ThesisItem Open 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.ThesisItem Open 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 RavishankarPorcine 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.ThesisItem Open 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.ThesisItem Open Access MULTI-LOCUS SEQUENCE TYPING FOR SPECIES/SEROVAR IDENTIFICATION OF CLINICAL ISOLATES OF LEPTOSPIRA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-12-30) DIVYA D.; Siju JosephLeptospirosis is an emerging and re-emerging zoonotic disease endemic in Kerala and close monitoring of emerging serovars is essential, for adopting appropriate control strategies. Multi-Locus Sequence Typing (MLST) is reported to be a less expensive serovar level identification tool as compared to much costly and cumbersome methods like whole genome sequencing. A study was undertaken to isolate Leptospira from infected animals and rats and to perform MLST for the serovar level identification. The seroprevalence recorded using MAT was 20.97 per cent. Among the 205 samples, 15 were confirmed to have the presence of pathogenic Leptospira using G1 and G2 genus specific PCR. However, when these were inoculated to EMJH semi-solid medium, only ten isolates were obtained. The ten isolates were further subjected to secY, icdA and GyraseB PCR and sequenced. The obtained sequences were analysed using BLAST and the isolates demonstrated more than 98.5 per cent homology. The sequences obtained from the isolates were fed into specified MLST database and using the Leptospira MLST scheme-3, the allelic profile and sequence type were generated. Among the ten isolates, nine could be identified at the serovar level. The serovar of the isolates identified by MLST were perfectly matching with that of MAT. The MLST results obtained in the present study indicated that the isolates S24 and S 33 belonged to the same serovar Canicola, S40 and S47 were Sejroe and S55, S71, S27, S69 and S19 were Pomona, Australis, Autumnalis, Icterohaemorraghiae and Bataviae, respectively. It was concluded that MLST could be used as an alternative to the whole genome sequencing and other cumbersome procedures for the serovar level identification of Leptospira. Further, it is recommended that for getting more promising results in MLST, the sequence data from at least seven genes need to be employed.ThesisItem Open Access CHARACTERISATION OF NEWCASTLE DISEASE VIRUS ISOLATES PREVALENT IN KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-12-30) RASHI U.; M. MiniNewcastle disease caused by Newcastle disease virus (NDV) is one of the highly pathogenic viral diseases of poultry and is considered to cripple poultry industry globally. The diagnosis of the disease is based on virus isolation and characterisation and detection of the genome of the virus by molecular techniques such as reverse transcriptase polymerase chain reaction (RT-PCR). Samples collected from dead and ailing birds showing lesions and signs suggestive of ND formed the materials for the study. Out of the 63 samples collected, eight revealed the virus in RT-PCR targeting the F gene, directly from the tissue samples. The NDV could be isolated in embryonated chicken eggs from the samples which yielded positive amplicons by RT-PCR. All the viral isolates showed haemagglutination activity (4 to 10 log₂) and was inhibited by the NDV specific antiserum. The HI titre for all the isolates was found to be 4 log₂. The ICPI and MDT indices were determined. The ICPI values were in between 0.75 to 1.53 and MDT values varied from 54 h. to 79.2 h. Based on the intracerebral pathogenicity index and mean death time, three of the isolates were classified under velogenic group and the remaining five under mesogenic group. The RT-PCR assay targeting the fusion protein cleavage site of the F gene was carried out and the specific amplified products were sequenced and phylogenetically analysed. All the isolates were of similar sequences and contained ¹¹²GRQGRL¹¹⁷ monobasic amino acid motifs at FPCS region, which is peculiar to lentogenic viruses. On phylogenetic analysis, all the isolates were grouped in genotype II of class II viruses. In this study, NDV isolates with lentogenic FPCS region belonging to velogenic and mesogenic pathotypes were obtained.
