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 NFkß 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.

Pansteatitis in polluted Olifants River impoundments: nutritional perspectives on fish in a eutrophic lake, Lake Loskop, South Africa.

This study compares the aetiology of pansteatitis in Lake Loskop, relative to two other impoundments along the Olifants River. Macroscopic and microscopic pathology, age determination and analysis of stomach content, fatty acids and stable isotopes explain the high prevalence of pansteatitis in Oreochromis mossambicus (Peters) and several other species in Lake Loskop. All the dietary indicator comparisons between pansteatitis-affected and healthy fish fail to support a systemic cause. Pansteatitis in Lake Loskop was linked to size and weight of O. mossambicus, but not to ontogenic age. Fish in Lake Loskop showed abnormally high omega-3 to omega-6 fatty acid ratios normally only found in marine fish with no significant difference in degree of assimilation of these fatty acids between pansteatitis-affected and healthy fish. This explains the vulnerability to, but not the occurrence of, pansteatitis. As a cause for the pansteatitis, these results point towards sporadic vitamin E-depleting trigger events, known sporadic fish die-off occurrences that provide surviving fish with a rich source of rancid fats on which to scavenge. The mechanism ties pansteatitis to eutrophication and trophic cascade effects, the intrinsic drivers of the disease and suggests an adaptive management strategy that might be applied by relevant conservation authorities.

Pan-steatitis in farmed northern bluefin tuna, Thunnus thynnus (L.), in the eastern Adriatic.

Clinical, gross and histopathological investigations were carried out into large-scale mortalities on eastern Mediterranean bluefin tuna, Thunnus thynnus (L.), farms. Fish showed only nervous signs and darkened colour. At post-mortem the liver was bronze coloured and the pyloric area waxy in consistency. There was no evidence of any other gross pathology. Histopathology showed severe hepatic necrosis and lipidosis. Peri-pancreatic lipoid tissue was heavily infiltrated with an inflammatory round cell infiltrate. Fish on all three farms had been fed on a North African pilchard diet rather than traditional local or Baltic species. Once the diet was modified, losses ceased. A diagnosis of pan-steatitis as seen in other farmed fish species, as well as in terrestrial animals, on particular fish-based diets was made, although the actual factor within the diet which induced the inflammatory effect is not known.

The ins and outs of mitochondrial dysfunction in NASH.

Rich diet and lack of exercise are causing a surge in obesity, insulin resistance and steatosis, which can evolve into steatohepatitis. Steatosis and nonalcoholic steatohepatitis (NASH) can also be induced by drugs such as amiodarone, tamoxifen and some antiretroviral drugs. There is growing evidence that mitochondrial dysfunction, and more specifically respiratory chain deficiency, plays a role in the pathophysiology of NASH whatever its initial cause. In contrast, the B-oxidation of fatty acids can be either increased (as in insulin resistance-associated NASH) or decreased (as in drug-induced NASH). However, in both circumstances, the generation of reactive oxygen species (ROS) by the damaged respiratory chain is augmented, as components of this chain are over-reduced by electrons, which then abnormally react with oxygen to form increased amounts of ROS. Concomitantly, ROS oxidize fat deposits to release lipid peroxidation products that have detrimental effects on hepatocytes and other hepatic cells. In hepatocytes, ROS and lipid peroxidation products further impair the respiratory chain, either directly or indirectly through oxidative damage to the mitochondrial genome. This, in turn, leads to the generation of more ROS and a vicious cycle ensues. Mitochondrial dysfunction can also lead to apoptosis or necrosis depending on the energy status of the cell. ROS and lipid peroxidation products also activate stellate cells, thus resulting in fibrosis. Finally, ROS and lipid peroxidation increase the generation of several cytokines (TNF-alpha, TGF-B, Fas ligand) that play sundry roles in the pathogenesis of NASH. Recent investigations have shown that some genetic polymorphisms can significantly increase the risk of steatohepatitis and that several drugs can prevent or even reverse NASH. For the next decade, reducing the incidence of NASH will be a major challenge for hepatologists.