A histological evaluation of pansteatitis-affected Mozambique tilapia, Oreochromis mossambicus (Peters 1852), from different geographical locations in South Africa.

Pansteatitis is the leading cause for the decline in Nile crocodile populations and the sporadic mortality of fish in the Olifants River System, South Africa. To determine the prevalence of this disease in lentic systems, Mozambique tilapia, Oreochromis mossambicus, were collected from Lake Loskop, Lake Flag Boshielo, Phalaborwa Barrage and Lake Luphephe-Nwanedi. The former three impoundments are located within the main stem of the Olifants River, while the latter, which is geographically isolated and situated in the Limpopo River System, served as a reference site. Mesenteric adipose, liver, serosa of the swim bladder, gill and the skeletal muscle of fish sampled were examined for gross and microscopic evidence of pansteatitis. Microscopically observed changes were used to statistically compare pansteatitis prevalence between samples and sites. Based on histopathological evaluation, the adipose tissue in the liver, swim bladder serosa and coelom from severely debilitated individuals showed the most significant pathological changes. Lesions indicative of steatitis were observed in fish collected from Lake Loskop (75%), Lake Flag Boshielo (22%) and Lake Luphephe-Nwanedi (15%). Further investigation is warranted to understand the pervasiveness and mechanisms driving pathological changes of pansteatitis at Lake Flag Boshielo, Phalaborwa Barrage and Lake Luphephe-Nwanedi.

Lipidomics for wildlife disease etiology and biomarker discovery: a case study of pansteatitis outbreak in South Africa.

INTRODUCTION: Lipidomics is an emerging field with great promise for biomarker and mechanistic studies due to lipids diverse biological roles. Clinical research applying lipidomics is drastically increasing, with research methods and tools developed for clinical applications equally promising for wildlife studies. OBJECTIVES: Limited research to date has applied lipidomics, especially of the intact lipidome, to wildlife studies. Therefore, we examine the application of lipidomics for in situ studies on Mozambique tilapia (Oreochromis mossambicus) in Loskop Dam, South Africa. Wide-scale mortality events of aquatic life associated with an environmentally-derived inflammatory disease, pansteatitis, have occurred in this area. METHODS: The lipidome of adipose tissue (n = 31) and plasma (n = 51) from tilapia collected from Loskop Dam were characterized using state of the art liquid chromatography coupled to high-resolution tandem mass spectrometry. RESULTS: Lipid profiles reflected pansteatitis severity and were significantly different between diseased and healthy individuals. Over 13 classes of lipids associated with inflammation, cell death, and/or oxidative damage were upregulated in pansteatitis-affected adipose tissue, including ether-lipids, short-chained triglyceride oxidation products, sphingolipids, and acylcarnitines. Ceramides showed a 1000-fold increase in the most affected adipose tissues and were sensitive to disease severity. In plasma, triglycerides were found to be downregulated in pansteatitis-affected tilapia. CONCLUSION: Intact lipidomics provided useful mechanistic data and possible biomarkers of pansteatitis. Lipids pointed to upregulated inflammatory pathways, and ceramides serve as promising biomarker candidates for pansteatitis. As comprehensive coverage of the lipidome aids in the elucidation of possible disease mechanisms, application of lipidomics could be applied to the understanding of other environmentally-derived inflammatory conditions, such as those caused by obesogens.

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.

Thyroid function of steatitis-affected Mozambique tilapia Oreochromis mossambicus from a sub-tropical African reservoir.

Thyroid function and nutritional indicators were measured in obese, steatitis-affected Mozambique tilapia Oreochromis mossambicus from Loskop Reservoir (LR), South Africa. Plasma thyroid hormones (especially T3) and thyroid follicle histomorphology revealed high levels of activity in every aspect of the thyroid cascade measured in fish from LR compared to a reference population of steatitis-free fish. Concurrent measurements of nutritional state including plasma lipids, liver lipid content and hepatocyte size showed that fish from LR had significant energy stores indicative of abundant nutritional intake. There were distinct sex and seasonal differences, with the highest plasma lipids and T3 levels observed in steatitis-affected females during spring and summer. Positive correlations were observed between plasma lipids (especially cholesterol) and T3 concentrations in fish from both populations, indicating a link between lipid metabolism and thyroid function. There was no direct evidence of thyroid disruption, but this cannot be ruled out until further research determines the factors that underlie the homeostatic shift leading to elevated plasma and liver lipids and T3 levels in steatitis-affected tilapia.

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.

Tissue distribution of perfluoroalkyl acids and health status in wild Mozambique tilapia (Oreochromis mossambicus) from Loskop Dam, Mpumalanga, South Africa.

This study examined concentrations of 15 perfluoroalkyl acids (PFAAs) in tissues from male Mozambique tilapia (Oreochromis mossambicus) collected at Loskop Dam, Mpumalanga, South Africa in 2014 and 2016. Nine of the 15 PFAAs were detected frequently and were included in statistical analysis and included two of the most commonly known PFAAs, perfluorooctanesulfonic acid (PFOS) (median, 41.6ng/g) and perfluorooctanoic acid (PFOA) (median, 0.0825ng/g). Of the tissues measured, plasma (2016 and 2014 median, 22.2ng/g) contained the highest PFAA burden followed by (in descending order): liver (median, 11.6ng/g), kidney (median, 9.04ng/g), spleen (median, 5.92ng/g), adipose (median, 2.54ng/g), and muscle (median, 1.11ng/g). Loskop Dam tilapia have been affected by an inflammatory disease of the adipose tissue known as pansteatitis, so this study also aimed to investigate relationships between PFAA tissue concentrations and incidence of pansteatitis or fish health status. Results revealed that healthy tilapia exhibited an overall higher (p-value<0.05) PFAA burden than pansteatitis-affected tilapia across all tissues. Further analysis showed that organs previously noted in the literature to contain the highest PFAA concentrations, such as kidney, liver, and plasma, were the organs driving the difference in PFAA burden between the two tilapia groups. Care must be taken in the interpretations we draw from not only the results of our study, but also other PFAA measurements made on populations (human and wildlife alike) under differing health status.

Comparison of the lipid properties of healthy and pansteatitis-affected African sharptooth catfish, Clarias gariepinus (Burchell), and the role of diet in pansteatitis outbreaks in the Olifants River in the Kruger National Park, South Africa.

Pansteatitis has been identified in wild populations of sharptooth catfish, Clarias gariepinus (Burchell), and Nile crocodiles, Crocodylus niloticus Laurenti, inhabiting the same waters in the Olifants River Gorge in the Kruger National Park, South Africa. Mesenteric and pectoral fat tissue was investigated microscopically and by fatty acid analysis in healthy and pansteatitis-affected catfish from both captive and wild populations. Variation in fatty acid composition between pectoral and mesenteric fat was noted. Composition of mesenteric fat differed between fish from various localities as a result of differences in diet. Pansteatitis in the captive population, resulting from ingestion of high amounts of dietary oxidized fat, reflected higher levels of unsaturated fatty acids within the mesenteric fat. Mesenteric fat of pansteatitis-affected wild catfish was characterized by an increase in moisture content, a decrease in fat content and a decrease in stearic and linoleic acids. The n-3 to n-6 fatty acid ratio of mesenteric fat was higher in pansteatitis-affected wild catfish than in healthy catfish from the same locality, reflecting higher polyunsaturated fat intake by pansteatitis-affected fish. The possible role of alien, invasive, phytoplankton-feeding silver carp, Hypophthalmichthys molitrix (Valenciennes), in the aetiology of pansteatitis in both catfish and crocodiles in the Olifants Gorge is discussed.

On-Site Classification of Pansteatitis in Mozambique Tilapia (Oreochromis mossambicus) using a Portable Lipid-Based Analyzer.

While no pansteatitis-related large-scale mortality events have occurred since 2008, the current status of pansteatitis (presence and pervasiveness) in the Olifants River system and other regions of South Africa remain largely unknown. In part, this is due to both a lack of known biological markers of pansteatitis and a lack of suitable non-invasive assays capable of rapidly classifying the disease. Here, we propose the application of a point-of-care (POC) device using lipid-based test strips (total cholesterol (TC) and total triglyceride (TG)), for classifying pansteatitis status in the whole blood of pre-spawning Mozambique tilapia (Oreochromis mossambicus). Using the TC strips, the POC device was able to non-lethally classify the tilapia as either healthy or pansteatitis-affected; the sexes were examined independently because sexual dimorphism was observed for TC (males p = 0.0364, females χ2 = 0.0007). No significant difference between diseased and pansteatitis-affected tilapia was observed using the TG strips. This is one of the first described applications of using POC devices for on-site environmental disease state testing. A discussion on the merits of using portable lipid-based analyzers as an in-field disease-state diagnostic tool is provided.