C.J. Botha

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

The South African Hartbeespoort Dam is known for the occurrence of heavy Microcystis blooms. Although a few other cyanobacterial genera have been described, no detailed study on those cyanobacteria and their potential toxin production has been conducted. The diversity of cyanobacterial species and toxins is most probably underestimated. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Hartbeespoort Dam, water samples werecollectedinApril2011.Inapolyphasicapproach,27isolatedcyanobacterialstrainswereclassifiedmorphologically and phylogenetically and tested for microcystins (MCs), cylindrospermopsin (CYN), saxitoxins (STXs) and anatoxin-a (ATX) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and screened for toxin-encoding gene fragments. The isolated strains were identified as Sphaerospermopsis reniformis, Sphaerospermopsis aphanizomenoides, Cylindrospermopsis curvispora, Raphidiopsis curvata, Raphidiopsis mediterrranea and Microcystis aeruginosa. Only one of the Microcystis strains (AB2011/53) produced microcystins (35 variants). Forty-one microcystin variants were detected in the environmental sample from Hartbeespoort Dam, suggesting the existence of other microcystin producing strains in Hartbeespoort Dam. All investigated strains tested negative for CYN, STXs and ATX and their encoding genes. ThemcyEgeneofthemicrocystingeneclusterwasfoundinthemicrocystin-producingMicrocystisstrainAB2011/53and in eight non-microcystin-producing Microcystis strains, indicating that mcyE is not a good surrogate for microcystin production in environmental samples. Additional keywords: Cylindrospermopsis, Hartbeespoort Dam, microcystin, Microcystis, Raphidiopsis, Sphaerospermopsis.

A comparison of in vivo and in vitro assays to assess the toxicity of algal blooms

The toxicity of purified microcystin-LR (MC-LR) and algal material collected during the winter and summer seasons (2005/2006) from the Hartebeespoort dam, South Africa, was investigated using the enzyme-linked immunosorbent assay (ELISA), mouse bioassay, catfish primary hepatocytes (in vitro assay) and protein phosphatase inhibition (PPi) assays. Microcystis aeruginosa, known producer of microcystins, was the dominant cyanobacteria present in the water samples. Exceptionally high cell numbers per millilitre were observed, especially with the summer samples (approximately 1.442 x 10(8)cells/ml), indicating a severe algal bloom in the dam. The toxin concentration as detected by ELISA and PPi assay in the winter and summer extracts was at least 1000 times more than the provisional guideline value (1 microg/l) set by the World Health Organization (WHO) for MC-LR in drinking water. Hepatotoxic effects and death of mice were observed after dosing with the summer extracts, while no hepatotoxic effects were observed with winter extracts. The EC(50) values obtained after exposure of the catfish primary hepatocytes for 72h to MC-LR, winter and summer extracts was about 0.091, 0.053 and 0.014 mg/l, respectively. Similar toxicity results were obtained when the mouse bioassay and primary hepatocytes were used.

An outbreak of canine aflatoxicosis in Gauteng Province, South Africa

Sporadic outbreaks of aflatoxicosis occur in dogs when they consume contaminated dog food. During 2011, low-cost brands of pelleted dog food were contaminated with very high concentrations of aflatoxins. Approximately 100 dogs were presented to the Onderstepoort Veterinary Academic Hospital. Clinically, the dogs were depressed to collapsed and icteric, with haematemesis, melaena and haematochezia. The most common pathological findings were icterus, gastro-enterorrhagia and hepatosis. On histopathological examination, fatty hepatosis and bile duct proliferation were observed. A consistent, very characteristic finding was the presence of a blue-grey granular material within the bile ducts. A total of 124 samples of the dog food fed to the affected dogs was analysed to determine aflatoxin concentrations. Concentrations ranged from below the limit of quantification (< 5 μg/kg) to 4946 μg/kg and six samples were submitted to determine the ratio of aflatoxins in the feed. It is estimated that well over 220 dogs died in the Gauteng Province of South Africa as a result of this aflatoxin outbreak.

Biotransformation and Oxidative Stress Responses in Captive Nile Crocodile (Crocodylus niloticus) Exposed to Organic Contaminants from the Natural Environment in South Africa

In the present study, the biotransformation and oxidative stress responses in relation to chemical burden in the liver of male and female Nile crocodiles—Crocodylus niloticus—from a commercial crocodile farm passively exposed to various anthropogenic aquatic pollutants was investigated. In general, the data showed that male crocodiles consistently produced higher biotransformation and oxidative stress responses compared to females. Relationships between these responses and concentrations of aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) were also observed. Specifically, the catalytic assays for EROD and BROD (not PROD and MROD) showed sex-differences between male and female crocodiles and paralleled immunochemically determined CYP1A and CYP3A protein levels; the relatively similar levels of PAHs in both sexes suggest an estrogen-mediated reduction of this pathway in females. The antioxidant system exhibited higher levels in male crocodiles with slight or significant higher values for catalase (CAT), glutathione reductase (GR), glutathione peroxidases-H2O2 (GPx-H2O2), glutathione peroxidases-Cu (GPx-Cu), total antioxidant capacity towards peroxyl radicals (TOSC-ROO) and hydroxyl radicals (TOSC-HO), total glutathione (GSH) and malondialdehyde (MDA). On the other hand, the activities of acyl-CoA oxidase (AOX) and glutathione S-transferases (GST) were significantly higher in females. Principal component analysis (PCA) produced significant groupings that revealed correlative relationships (both positive and negative) between biotransformation/oxidative stress variables and liver PAHs and aliphatic hydrocarbon burden. The overall results suggest that these captive pre-slaughter crocodiles exhibited adverse exposure responses to anthropogenic aquatic contaminants with potentially relevant effects on key cellular pathways, and these responses may be established as relevant species biomarkers of exposure and effects in this endangered species.

Confirmed organophosphorus and carbamate pesticide poisonings in South African wildlife (2009–2014)

During a six-year period (from January 2009 to December 2014), specimens collected from 344 cases of suspected organophosphorus and carbamate pesticide poisonings in wildlife, including birds, were submitted to the Toxicology Laboratory (ARC-OVI) for analysis. A positive diagnosis was made in 135 (39%) of these cases. The majority of cases were from birds, which included Cape vultures (Gyps coprotheres) and African white-backed vultures (Gyps africanus) and bateleur eagles (Terathopius ecaudatus). In one incident 49 vultures were killed when a farmer intentionally laced carcasses with carbofuran in an attempt to control jackal predation. There were 22 incidents of poisoning in helmeted guineafowl (Numida meleagris). On nine different occasions blue cranes (Anthropoides paradiseus) were poisoned, in one incident 14 birds were reported to have been killed. Over the period of investigation, there were 20 cases of poisoning involving mammalian species, the majority being vervet monkeys (Chlorocebus pygerythrus). The carbamate pesticides were responsible for 57 incidents of poisoning. Aldicarb, carbofuran and methomyl were detected in 26, 18 and 12 cases respectively. The majority of organophosphorus pesticide poisonings were caused by diazinon (n = 19), monocrotophos (n = 13) and methamidophos (n = 10).

Vitellogenin in African sharptooth catfish (Clarias gariepinus): purification, characterization, and ELISA development.

Vitellogenin (Vtg) induction in African sharptooth catfish (Clarias gariepinus) was assessed in order to develop a method for monitoring estrogenic pollution in African freshwater systems. Clarias gariepinus Vtg (Cg-Vtg) was purified from serum obtained from 17α-ethynylestradiol (EE2)-exposed fish and polyclonal antibodies against Cg-Vtg were raised. An enzyme-linked immunosorbent assay (ELISA) was developed and the induction and kinetics of Vtg were assessed in male fish in three different exposure trials using both natural estrogen (17α-estradiol [E2]) and synthetic EE2. Concentrations of EE2 in water and levels of EE2 conjugates in bile were quantified by liquid chromatography–mass spectrometry (LC-MS). In addition, co-administration of E2 and benzo[a]pyrene (BaP) were studied. Vtg was induced in all exposure trials and the maximum induction was observed 1 wk after exposure. Exposure of male C. gariepinus to 1.4, 2.7, and 13.9 μg/ml EE2 induced Vtg synthesis at all concentrations. BaP did not influence the Vtg kinetics. However, an increased rate of biliary excretion of EE2 was observed when BaP was additionally administered. In conclusion, Vtg is induced in male C. gariepinus after exposure to both E2 and EE2, rendering it a suitable biomarker for endocrine-disrupting chemicals in African freshwater systems.

Stenocarpella maydis and its toxic metabolites: a South African perspective on diplodiosis

Stenocarpella maydis is one of the most prevalent ear and stalk rot pathogens of maize globally, causing reductions of grain quality and yield. Various molecular methods, including polymerase chain reaction (PCR)-based techniques and nucleotide microarrays, have been developed for the identification of S. maydis infestation in maize grain. In addition to diplodiatoxin, new metabolites, namely dipmatol, diplonine and chaetoglobosins K and L, have been isolated from S. maydis infected cultures. S. maydis infected maize is also associated with intoxication in ruminants. Diplodiosis, a nervous disorder of cattle and sheep, results from ingestion of mouldy ears, kernels and maize stubble infected by S. maydis. Although this disease is most common in southern Africa, it has also been reported in Australia, Argentina and Brazil. Diplodiosis is characterised by reluctance of the animals to move, a wide-based stance, incoordination, paralysis and death. Myelin degeneration (status spongiosis) is the only histopath...

Lead ingestion as a potential contributing factor to the decline in vulture populations in southern Africa

Abstract Vulture populations in southern Africa have been on the decline for years, which unlike the Asian vulture crisis, has no one specific cause. Reasons attributable are deliberate and secondary poisonings, drowning, power line injuries, electrocutions, traditional medicine (“muti” trade) and calcium deficiencies. However, lead toxicity as a potential causal factor is hardly mentioned. The potential for lead toxicity needs to be considered as substantial game hunting occurs in the region with little regulatory control on bullet types. In this study, we determined the whole blood lead concentrations of captive and wild vulture populations in South Africa and Namibia (n=185). Results were compared to previous published ranges indicative of background exposure (<10 &mgr;g/dL), non‐toxic point exposure based upon the range established from captive birds and subclinical exposure. In general, whole blood lead concentrations were higher for wild African White‐backed vultures (Gyps africanus)(AWBV) than Cape vultures (G. coprotheres)(CGV) at 15.54±12.63 &mgr;g/dL vs 12.53±8.88 &mgr;g/dL (non‐significantly different), while in the Bearded vultures (Gypaetus barbatus) no indication of exposure was evident. Very similar exposures resulted irrespective of the birds being in captivity or under wild, free‐roaming conditions. A proportion of wild birds did, however, appear to be exposed to another source of lead than purely environmental (±12% and 30.6% for AWBV and CGV respectively). One bird, which had a whole blood concentration of 100 &mgr;g/dL, died soon after capture. To find the relationship between whole blood lead concentration and likely exposure factors, birds were compared by their rural/urban location, vicinity to mines and surrounding soil lead concentrations. With no relationship being present for the latter factors, we believe that this is evidence that the portion of southern African vultures being exposed to unknown source of lead, which we suggest arises from leaded ammunition remaining from hunting Graphical abstract Symbol. No caption available. HighlightsWhole blood concentrations were measured in various captive and wild colonies of White‐back and Cape griffon vultures.Whole blood lead concentrations had a range of <3–84 &mgr;g/dL.12% of birds had concentrations had higher than expected whole blood lead concentration, when compared to the captive birds.Based on the high whole blood lead concentrations, it is likely that leaded bullets could be the source of exposure.

Cytotoxicity of diplodiatoxin, dipmatol and diplonine, metabolites synthesized by Stenocarpella maydis.

The cytotoxicity of three Stenocarpella maydis metabolites (diplodiatoxin, dipmatol and diplonine) was investigated on Neuro-2a, CHO-K1 and MDBK cell lines. Diplodiatoxin was the most cytotoxic followed by dipmatol. Conversely, diplonine was not cytotoxic. Diplodiatoxin and dipmatol affected mitochondrial succinate dehydrogenase (MTT assay) and the overall viability of cells as assessed in real-time (xCELLigence assay). The results obtained so far indicate that diplodiatoxin and dipmatol exert their toxicity possibly via the necrotic cell death pathway.

Multitoxin analysis of Aspergillus clavatus -infected feed samples implicated in two outbreaks of neuromycotoxicosis in cattle in South Africa

Aspergillus clavatus intoxication is a highly fatal neuromycotoxicosis of ruminants, especially cattle. It is caused by the ingestion of infected sprouting grain and sorghum beer residue. Locomotor disturbances, tremors and paralysis are observed. Histologically, degeneration and necrosis of larger neurons in the medulla oblongata, the midbrain, the thalamus and the ventral horns of the spinal cord are observed. Although a range of mycotoxins such as patulin, cytochalasin E and pseurotin A have been isolated, there is limited information on which specific mycotoxin or group of mycotoxins are involved during outbreaks of intoxication in livestock. In the present study, two outbreaks of A. clavatus poisoning in cattle are briefly described. Feed samples were collected for fungal identification, and culture and multitoxin analysis. A range of fungal metabolites were detected, and the estimated concentrations (μg/kg) are provided. Both the sprouting barley and brewers grain were predominantly infected with A. clavatus and, to a lesser extent, Rhizopus arrhizus. The only common Aspergillus secondary metabolite present in all the samples was pseurotin A. Patulin and cytochalasin E were present in the sprouting barley samples, as well as the A. clavatus isolates cultured on malt extract agar for 2 weeks; however, neither of these mycotoxins could be detected in the brewers grain sample.Aspergillus clavatus intoxication is a highly fatal neuromycotoxicosis of ruminants, especially cattle. It is caused by the ingestion of infected sprouting grain and sorghum beer residue. Locomotor disturbances, tremors and paralysis are observed. Histologically, degeneration and necrosis of larger neurons in the medulla oblongata, the midbrain, the thalamus and the ventral horns of the spinal cord are observed. Although a range of mycotoxins such as patulin, cytochalasin E and pseurotin A have been isolated, there is limited information on which specific mycotoxin or group of mycotoxins are involved during outbreaks of intoxication in livestock. In the present study, two outbreaks of A. clavatus poisoning in cattle are briefly described. Feed samples were collected for fungal identification, and culture and multitoxin analysis. A range of fungal metabolites were detected, and the estimated concentrations (μg/kg) are provided. Both the sprouting barley and brewers grain were predominantly infected with A. clavatus and, to a lesser extent, Rhizopus arrhizus. The only common Aspergillus secondary metabolite present in all the samples was pseurotin A. Patulin and cytochalasin E were present in the sprouting barley samples, as well as the A. clavatus isolates cultured on malt extract agar for 2 weeks; however, neither of these mycotoxins could be detected in the brewers grain sample.