MARKUS ISA FRANCIS DVM (UNIMAID) 2007, M.Sc (ABU) 20142023-09-222023-09-222019-11-01https://teras.ng/api/asset/document/33edfc45-938d-491c-a1a6-cb968ef5800bhttps://teras.ng/catalog-item/b73539ff-1b66-414a-95ac-25280089b265http://dspace.teras-network.net:4000/handle/123456789/32101Mycoplasma bovis (M. bovis) and Mycoplasma mycoides subspecies mycoides (Mmm) are the two most important pathogenic species of mycoplasmas of cattle. This study was aimed to isolate, identify and confirm M. bovis and Mmm by culture and PCR as well as to characterize the genomes of identified Mmm isolates from cattle in Adamawa and Taraba States, Nigeria. A total of 480 samples of lung tissues (180), pleural fluid (180), nasal swab (60) and ear swab (60) were collected from 190 heads of cattle at slaughter in two abattoirs, namely; Yola Modern Abattoir and Jalingo Abbatoir in Adamawa and Taraba States respectively. Samples were processed based on standard laboratory protocols. Identification and confirmation were done using standard techniques. Genomic DNA was extracted using Maxwell 16 Tissue/cell DNA purification kit and sequenced on Illumina NextSeqTM 500 platform. Reads were assembled by a de novo strategy using the SPAdes, while strains comparison was performed by a gene-by-gene approach. Pangenome analysis was performed with USEARCH and virulence factors were identified by similarity searching against a specialized virulence factor database. Thirty nine (8.13%) Mycoplasma species were isolated from the samples, 25 (8.80%) and 14 (7.14%) from Adamawa and Taraba States respectively. Four (0.83%; 4/480) isolates were identified as M. bovis. Out of these, 1 (0.91%) was isolated from pleural fluid in Adamawa State, whereas 2 (2.86%) and 1 (3.57%) were respectively isolated from lungs and ear swab samples in Taraba State. Similarly, 33 (6.87%; 3/380) of the isolates were identified as Mmm. Out of these, 12 (10.91%) were isolated from both lung tissues and pleural fluid in Adamawa State, whereas, 5 (7.14%) and 4 (5.71%) were isolated respectively from lung tissues and pleural fluid in Taraba State. No Mmm was isolated from nasal and ear swab samples in the study area. Histopathological examination of the positive lung showed severe congestion and fibrin exudation into interalveolar spaces with the collapse of almost all the alveoli. Four M. bovis isolates were confirmed positive by PCR with the presence of one band of 734-bp; two isolates from lung tissues and one isolate from both pleural fluid and ear canal. All the 33 Mmm isolates were confirmed to be Mycoplasma mycoides subspecies based on amplification of CAP-21 genomic region yielding a band size of 574-bp. Following digestion of the amplicon with restriction endonuclease Vsp1, the production of two restricted fragments of 180-bp and 380-bp indicated typical fingerprinting pattern of Mmm. Whole genome sequencing was undertaken for the 20 field strains of Mmm isolates, comprising of thirteen isolates from Adamawa State and seven from Taraba State. The 20 genome assemblies were highly similar and resulted in 19 and 115 contigs with N50 value from 25,646 to 119,472. The total length of the genomes varied by approximately 22,000-bp and were between 1,180,728-bp and 1,202,919-bp per genome. Smallest genome size of 1,180,728-bp was observed in strain AL103 and largest genome size of 1,202,919-bp in strain AP68b. The G+C content of the Mmm strains varied between 23.92% and 24.03% and the genomes harboured about 2,053 to 2,127 predicted protein-coding genes per genome. The annotation of the genome revealed CDS between 2,016 and 2,087, and between 29 and 33 tRNA were identified per genome. Six (6) rRNA was identified per genome, 2 each of 5S rRNA, 16S rRNA and 23S rRNA. Genomic comparisons of the 20 field strains of Mmm revealed high orthologous average nucleotide identity (OrthoANI) values of between 99.59 and 99.92%. BLAST nucleotide comparison showed 90-100% relatedness of these strains to vaccine strain T1/44. The pan-genome of the strains of Mmm contains 3,081 protein-coding genes which comprised of 1,707 core genes (55.4% of the pangenome) and 1,374 accessory genes (44.6% of the pangenome). Functional annotation of the 20 Mmm gene products assigned a COG category for just 1,198 ortholog cluster of proteins occurring mainly in the core genome. Phylogenetic tree of the 20 strains showed 3 distinct phylogroups of 8, 4 and 8 strains MAT-1, MAT-2 and MAT-3 respectively and were confirmed by a Neighbor-Joining tree. The 20 field strains of Mmm had a virulence factor range between 9 and 25 and virulence genes varying between 11 and 68 with some strain (AP103, AP68b, AL90, AL107) harbouring high number of virulence factors. In conclusion, this work has established the presence of M. bovis and Mmm in Adamawa and Taraba States, Nigeria. We thus recommend large scale epidemiological studies and genomic analysis of Mmm strains circulating in other States of Nigeria.Post Graduate Theses