Histomorphology, ultrastructure and fatty acid composition of the adipose tissue in pansteatitis, the potentials in understanding the underlying mechanism and diagnosis of pansteatitis in the Nile crocodile

BACKGROUND : In an effort to characterize the fat body and other adipose tissue in the Nile crocodile and the effects of pansteatitis on the structure and composition of the adipose tissue, we evaluated the regional variation in structure and fatty acid composition of healthy farmed crocodiles and those affected by pansteatitis. METHODS : Adipose tissue samples were collected from the subcutaneous, visceral and intramuscular fat and the abdominal fat body of ten 4-year old juvenile crocodiles from Izinthaba Crocodile Farm, Pretoria, South Africa while pansteatitis samples were collected from visceral and intramuscular fat of crocodiles that had died of pansteatitis at the Olifant River, Mpumalanga, also in South Africa. Histomorphology, ultrastrustucture and fatty acid composition by fatty acid methyl ester (FAME) analysis were conducted. RESULTS : Histological examination showed regional variations in the adipose tissue especially in the collagen content of the ECM, tissue perfusion and division into lobes and lobules by fibrous capsule. Considerable fibrosis, mononuclear cell infiltration especially by macrophages and lymphocytes and toxic changes in the nucleus were observed in the pansteatitis samples. Regional variation in lipid composition especially in Myristoleic (C14:1), Erucic acid (C22:1n9), and Docosadienoic acid (C22:2n6) was observed. Most of the saturated and trans fatty acids were found in significant quantities in the pansteatitis samples, but had very low levels of the cis fatty acid and the essential fatty acids with C18 backbone. CONCLUSION : This study demonstrates that there exists some regional variation in histomorphology and fatty acid composition in the healthy adipose tissue of the Nile crocodile. It also showed that pansteatitis in the Nile crocodile might have been triggered by sudden change in energy balance from consumption of dead fish; and probable exposure to toxic environmental conditions with the evidence of up scaled monounsaturated long chain fatty acids composition and toxic changes in the leucocytes observed in pansteatitis in the present study.http://www.sherpa.ac.uk/romeo/issn/1476-511X/am2017Anatomy and PhysiologyParaclinical SciencesVeterinary Tropical Disease

Next generation sequencing and RNA-seq characterization of adipose tissue in the Nile crocodile (Crocodylus niloticus) in South Africa: possible mechanism(s) of pathogenesis and pathophysiology of pansteatitis

BACKGROUND: Concerted efforts to identify the pathogenesis and mechanism(s) involved in pansteatitis, (a generalized inflammation of the adipose tissue), that was attributed to the recent crocodile die off in the Olifants River and Loskop Dam in Kruger National Park, Mpumalanga, South Africa have been in the forefront of research in recent time. As part of the efforts, molecular characterization of healthy and pansteatitis adipose tissue was carried out by RNA sequencing (RNA-Seq) using Next Generation Sequencing (NGS) and de novo assembly of the adipose transcriptome, followed by differential gene expression analysis. METHODOLOGY: Healthy adipose tissue consisting of fifty samples was collected from the subcutaneous, visceral, intermuscular adipose tissues and the abdominal fat body of ten 4 years old juvenile crocodiles from a local crocodile farm in Pretoria, South Africa. Ten pansteatitis samples were collected from visceral and intermuscular adipose tissues of five crocodiles that were dying of pansteatitis. RESULTS: Forty-two thousand, two hundred and one (42,201) transcripts were assembled, out of which 37, 835 had previously been characterized. The de novo assembled transcriptome had an N50 (average sequence) of 436 bp, percentage GC content of 43.92, which compared well with previously assembled transcripts in the saltwater crocodile. Seventy genes were differentially expressed and upregulated in pansteatitis. These included genes coding for extracellular matrix (ECM) signaling ligands, inflammatory cytokines and tumour necrosis factor alpha (TNFα) receptors, fatty acid synthase and fatty acid binding proteins, peroxisome proliferator-activated receptor gamma (PPARγ), nuclear factor and apoptosis signaling ligands, and mitogen activated protein kinase enzymes among others. Majority (88.6%) of the upregulated genes were found to be involved in hypoxia inducible pathways for activation of NFkβ and inflammation, apoptosis, Toll-like receptor pathway and PPARγ. Bicaudal homologous 2 Drosophila gene (BICD2) associated with spinal and lower extremity muscle atrophy was also upregulated in pansteatitis while Sphingosine -1-phosphate phosphatase 2 (SGPP2) involved in Sphingosine -1- phosphate metabolism was downregulated. Futhermore, Doublesex–mab-related transcription factor 1 (DMRT1) responsible for sex gonad development and germ cell differentiation was also downregulated. CONCLUSION: Thus, from the present study, based on differentially expressed genes in pansteatitis, affected Nile crocodiles might have died partly due to their inability to utilize stored triglycerides as a result of inflammation induced insulin resistance, leading to starvation in the midst of plenty. Affected animals may have also suffered muscular atrophy of the lower extremities and poor fertility.Tertiary Education Fund (TETFund), University of Ibadan, Nigeria and University of Pretoria Post Graduate Support Bursaryhttp://www.plosone.orgpm2020Anatomy and PhysiologyParaclinical SciencesVeterinary Tropical Disease