WHOLE GENOME SEQUENCE CHARACTERIZATION OF PASTEURELLA MULTOCIDA ISOLATED FROM DIFFERENT ANIMAL SPECIES

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Date
2015
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AAU, Anand
Abstract
Pasteurella multocida is a commensal microorganism of the upper respiratory track of many animal and avian species and is responsible for wide range of diseases in domestic animals and poultry. Despite vaccination of the dairy animals particularly against Haemorrhagic Septicaemia (HS), several outbreaks occur regularly in Gujarat as well as in other parts of India. Whole genome sequencing is a recent advanced approach for understanding of genetic makeup of an organism as well for identification of virulence genes/factors responsible for the disease process in host. In order to sequence whole genome of P. multocida and to elucidate virulence associated genes, five isolates of P. multocida were sequenced using pyrosequencing based approach of 454 GS FLX Titanium. All the five isolates viz. P52 vaccine strain (P52VAC), poultry (Anand l_poultry), goat (Anand l_goat), buffalo (Anand l_buffalo) and cattle (Anand 1 cattle) were identified and characterized based on biochemical and cultural characters and subsequently confirmed by PM-PCR. For sequencing of the whole genome of organisms, dsDNA libraries were prepared for all the five isolates and quantity as well as quality checks were done using Agilent Bioanalyzer as well as TBS flurometer. dsDNA library of each of the five isolates was amplified using emPCR and positive clonal amplified DNA beads were used for sequencing after annealing of sequencing primers. After completion of the sequencing run, data generated in form of images were converted into reads using GS Run Browser. After signal processing, total 118843, 113997, 105729, 134886 and 31346 reads were generated which yielded 42,598,100 (42.59Mb), 29,000,497 (29.00Mb), 21,890,353 (21.89Mb), 39,756,349 (39.75Mb) and 7,429,658 (7.42Mb) of sequence bases for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandlcattle, respectively. Coverage obtained for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandlcattle was 18.87, 12.85, 9.70, 17.61 and 3.29 respectively. All the reads after signal processing were mapped with the reference genome available for a poultry isolate Pm70 at NCBI using GS Reference mapper. Mapping of the isolates P52VAC, Anandl poultry, Anandlgoat, Anandl buffalo and Anandl_cattle resulted in 38,079,806 (89.52%), 20,085,356 (87.38%), 19,867,143 (90.81%), 25,095,466 (63.22%) and 6,145,156 (82.87%) mapped bases with 105327, 97674, 95092, 86765 and 24967 mapped reads. Remaining reads which were not mapped by GS Reference Mapper, were used for de novo assembly using GS De Novo Assembler for finding sequences which code for plasmid of P. multocida. None of the de novo assembled sequences matched to plasmid. For sequence analysis and finding of virulence associated genes in P. multocida, two different annotation pipelines were used viz. Rapid Annotation using Subsystem Technology (RAST) and Prokaryotic Genome Automatic Annotation Pipeline (PGAAP). For RAST analysis, all the contigs generated after reference mapping with Pm70 uploaded at RAST server. RAST is a subsystem based annotation pipeline which generated 2,273,366bp (2.27Mb), 2,227,943bp (2.22Mb), 2,285,382bp (2.28Mb), 2,045,610bp (2.04Mb) and 1,438,517bp (1.43Mb)of genome with 209, 489, 349, 2188, and 3152 contigs for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandl cattle, respectively and 68, 54, 54, 40 and 0 RNA. Based on RAST analysis, highest abundance of subsystem were assigned to 'amino acids and derivatives', 'carbohydrates', 'protein metabolism' and 'cofactor and vitamins, prosthetic groups and pigments'. As expected, no subsystem was assigned to 'photosynthesis' and 'motility and chemotaxis' group as Pasteurella is a nonmotile organism and is not photosynthetic. Due to less coverage (3.29X) obtained for the Anandl_cattle isolate, it was omitted from the RAST based comparative analysis. Subsystem based genes/proteins assigned to the other four isolates under 'virulence, disease and defence' category ranged from 47 to 54 in number. There were presence of DedA, DedD and toxin under 'colicin and bacteriocin production' in P52 vaccine strain, poultry and goat isolates. Genes gyrA, gyrB, Pare and ParD under 'resistance to fluroquinolones' were present in all the four isolates. There was also presence of negative regulator of betalactamase expression, BLR gene leading to resistance expressed by this organism as well as multidrug resistance efflux pump cluster genes, MATE (Multidrug and toxin extrusion), MacA and MacB (Macrolide specific efflux protein) in P52 vaccine strain, poultry and goat isolates. For PGAAP analysis, all the reads generated after sequencing run were submitted to the PGAAP pipeline of NCBl after removing sequences less than 200bp. PGAAP analysis revealed genome size of 2,273,366bp (2.27Mb), 2,227,943bp (2.22Mb), 2,285,382bp (2.28Mb), 2,045,610bp (2.04Mb) and l,438,517bp (1.43Mb) with 40.40%, 40.20%, 40.50%, 40.90% and 41.00% of G+C contents for P52VAC, Anandl _poultry, Anandlgoat, Anandl _buffalo and Anandl _cattle, respectively. Total number of coding sequences (CDS) were 2066, 2337, 2319, 3258 and 3623; total number of protein encoding genes (PEG) were 2194, 2284, 2266, 3218 and 3590, and total number of RNA assigned were 64, 53, 53, 41 and 33 for P52VAC, Anandl_poultry, Anandlgoat, Anandl buffalo and Anandl cattle, respectively. Deciphering virulence mechanism is one of the most useflil application of bacterial genomics to understand the molecular intricacies involved in disease mechanism as well as'for understanding host-pathogen interactions. For this purpose, genes associated with virulence were downloaded from annotation files available at (http://www.ncbi.nlm.nih.gov/genome/genomes/912?) in 'Protien' column/section to find out gene locus/id. After manually searching for the virulence associated genes, 55 important genes were selected based on the available literature. These 55 genes grouped under seven broad categories viz. capsule, fimbriae and adhesion, iron metabolism, outer membrane protein, superoxide dismutase, sialic acid metabolism and transcription regulation. Out of these seven categories, all the five genes falling under three categories i.e. SodA and SodC under superoxide dismutase, NanH and NanB under sialic acid metabolism and Fis under transcription regulation category were present in all the five isolates. Nine genes involved in capsule production were found, out of which, PglA and Kmtl were present in all the five isolates, while HyaE was present only in the goat isolate. HexA and HexC genes were absent in buffalo and cattle isolates, while HexB and HexD were absent in goat and cattle isolates. KpsF gene was absent in poultry and cattle isolates. Gene LctP was present only in goat and cattle isolates. Sixteen genes were found under the category of fimbriae and adhesion, of which, Hsf, PfliBl, PfliR, PflB, PlpB, and Plp4 genes were found in all the five isolates of P. multocida studied. HofC gene was absent may in vaccine strain, wliereas PlpE gene was absent in cattle as well as buffalo isolates. ComE gene was absent in P52 vaccine strain, while TadE was absent in buffalo isolate. PfhBl and PlpP genes were absent in cattle isolate, while RcpA and RcpB were absent in buffalo isolate. ClpB gene was absent in P52 vaccine strain and cattle isolates, whereas TadF was absent in buffalo and cattle isolates. For iron metabolism, 16 genes were found, of which, ExbB, FbpB, HbpA, HgbA, HemU, OmpW, Rjh and RffG genes were present in all the five isolates studied. Genes FbpA and TonB were absent in cattle isolate, TbpA and TonB dependent lactoferrin and transferrin receptor were absent in goat isolate. Gene FbpC was absent in buffalo isolate. TonB-dependent receptor was present in poultry and goat isolates only. P52 vaccine strain was having a presence of translocation protein TolB, whereas HemR gene was absent. For outer membrane proteins, out of nine genes found, HasR, LppB, LspB, OmpH and PtfA genes were present in all the five isolates of P. multocida. Outer membrane protein LolB was found in P52 vaccine strain as well as in cattle isolate. VacJ gene was absent in goat and cattle isolates. Oma87 gene was absent only in poultry isolate. Gene PfhA was found present only in P52 vaccine strain. This study is apparently the first attempt in India involving local P. multocida isolates from four different species and a vaccine strain for the purpose of identifying virulence genes/virulence associated genes using modem biotechnological tools like pyrosequencing based whole genome sequencing. The study aids in data of whole genome sequencing of bacterial pathogens particularly for P. multocida and also provides new insight into their genomic characters and possible molecular mechanisms involved in disease process. The present findings would provide a much needed base for fijrther screening of virulence associated genes and identification of certain markers for early diagnosis as well as characterization of P. multocida, which continues to pose challenges as a menace against the health management of animals. Genes which have been found in all the P. multocida isolates under the study can be explored as specific probes for the early diagnosis of the disease. Further, future scientific endeavors targeting the vaccine design for P. multocida may get a scientific support from this data, so as to formulate modern and more effective vaccines, for better animal health.
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ANIMAL BIOTECHNOLOGY, CHARACTERIZATION
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