Next Generation Sequencing [FEED METAGENOMICS] and virulent genes detection of Aspergillus Niger from chicken blood in the Federal Capital Territory, Nigeria

Aspergillus niger is pathogenic and causes aspergillosis, which is a fungal infection caused by spores of indoor and outdoor Aspergillus mold species. Due to the ubiquitous nature of Aspergillus niger , its spores are commonly inhaled by humans from the environment which posses as a severe health threat. Aspergillus niger has not been isolated and reported from chicken blood in Nigeria. This study aims to molecularly characterize Aspergillus niger isolated from chicken blood using real time polymerase chain reaction [RT – PCR] and to carry out multiplex-PCR for detection of virulent genes from the isolate. Eleven isolates of Aspergillus niger from the three hundred and sixty chicken blood samples collected using convenient sampling method from six Area Councils of the Federal Capital Territory [FCT], Nigeria was analysed. DNA extraction and purification were carried out to enable amplification of Internal Transcribed Spacer [ITS] genes using Real Time Polymerase Chain Reaction [PCR]. These amplicons were further subjected to Multiplex PCR for virulent genes evaluation. Fungal ITS1 and ITS4 sequencing was carried out and the similarity search using BLAST on NCBI was used for species identification and MEGA 5.0 was used for phylogenetic analysis. Multiplex PCR amplified four virulent genes namely Azole antifungal resistance gene [cyp51A], Ochratoxin A [ota1] production gene, Fumonisin B [Fum 6] and Fumonisin B2 [Fum 8] production gene. Analyses of sequenced ITS genes of eleven Aspergillus isolates were identified by the GenBank database which confirmed the isolates as A. niger as the BLAST results correspond to the similarity between the sequence queried and the biological sequences within the NCBI database. Phylogenetic analysis of sequences yielded genetic relatedness of 99% between the local species that have been previously isolated from soil and cereal samples in Nigeria, and also had 97% relatedness to isolates obtained from waste water effulent and leguminous plants from European countries like Pakistan, China, India and Egypt. The isolates were detected to have evolutionary origin from the A. niger subgenus circumdati. This study has shown the presence of A. niger from poultry feed in the FCT and also its isolation from chicken blood which has never been established in Nigeria. • This article explores the use of metagenomics Next Generation Sequences (mNGS) to analyse poultry feed. 18 fungal species including Aspergillus niger (0.22%) were identified and 4 virulent genes of A. niger (ota1, fum 6 and fum 8 and cyp51A gene) were detected. • The analysis of sequenced ITS genes of eleven Aspergillus isolates were investigated and deposited in the GenBank. • Phylogenetic analysis of sequences was carried out in order to establish the genetic relatedness between the local species in Nigeria and foreign species from European countries like Pakistan, China, India and Egypt. • This study detected A. niger from poultry feed in the FCT and also its isolation from chicken blood in Nigeria is a novel finding. • The long duration of fungal culture (3 days to 2 weeks) limits the diagnostic utility of microbial cultures hence the use of molecular diagnostic techniques, such as Metagenomics Next Generation Sequencing and Polymerase Chain Reaction, which are faster, more sensitive, and more specific as observed in this study.