Heavy metal mediated innate immune responses of the Indian green frog, Euphlyctis hexadactylus (Anura: Ranidae): Cellular profiles and associated Th1 skewed cytokine response.

Immune cell and cytokine profiles in relation to metal exposure though much studied in mammals has not been adequately investigated in amphibians, due mainly to lack of suitable reagents for cytokine profiling in non-model species. However, interspecies cross reactivity of cytokines permitted us to assay levels of IFNγ, TNFα, IL6 and IL10in a common anuran, the Indian green frog (Euphlyctis hexadactylus), exposed to heavy metals (Cd, Cr, Cu, Zn and Pb, at ~5ppm each) under field and laboratory settings in Sri Lanka. Enumeration of immune cells in blood and melanomacrophages in the liver, assay of serum and hepatic cytokines, and Th1/Th2 cytokine polarisation were investigated. Immune cell counts indicated overall immunosuppression with decreasing total WBC and splenocyte counts while neutrophil/lymphocyte ratio increased with metal exposure, indicating metal mediated stress. Serum IL6 levels of metal exposed frogs reported the highest (~9360pg/mL) of all cytokines tested. Significantly elevated IFNγ production (P<0.05) was evident in heavy metal exposed frogs. Th1/Th2 cytokine ratio in both serum and liver tissue homogenates was Th1 skewed due to significantly higher production of pro-inflammatory cytokines, IFNγ in serum and TNFα in the liver (P<0.01).Metal mediated aggregations of melanomacrophages in the liver were positively and significantly (P<0.05) correlated with the hepatic expression of TNFα, IL6 and IL10 activity. Overall, Th1 skewed response may well be due to oxidative stress mediated nuclear factor κ-light chain enhancer of activated B cells (NFκB) which enhances the transcription of pro-inflammatory cytokines. Xenobiotic stress has recently imposed an unprecedented level of threat to wildlife, particularly to sensitive species such as amphibians. Therefore, understanding the interactions between physiological stress and related immune responses is fundamental to conserve these environmental sentinels in the face of emerging eco-challenges.

Artemisia vulgaris L. ethanolic leaf extract reverses thrombocytopenia/thrombocytosis and averts end-stage disease of experimental severe Plasmodium berghei murine malaria.

BACKGROUND & OBJECTIVES: Artemisinin isolated from Artemisia annua is the most potent antimalarial against chloroquine resistant Plasmodium falciparum malaria. We previously reported that the ethanolic leaf extract of Artemisia vulgaris, an invasive weed and the only Artemisia species in Sri Lanka, possess both potent and safe antimalarial activity (in terms of antiparasitic properties) in a P. berghei murine malaria model. We report here a prototype study that investigated antidisease activities of A. vulgaris ethanolic leaf extract (AVELE) in a P. berghei ANKA murine malaria model that elicit pathogenesis similar to falciparum malaria. Profound thrombocytosis and thrombocytopenia in mice were detected in early-stage (Day 3), and at a later stage of infection (Day 6), respectively. Plasmodium berghei infected mice, 7 or 8 days post-infection reached end-stage disease with rapid drop in body temperature and usually die within 24 h, as a consequence of cerebral malaria. METHODS: Three doses of the AVELE (500, 750 and 1000 mg/kg) were used to assess antidisease activity of A. vulgaris in terms of survival, effects on thrombocyte related pathology and end-stage disease, antipyretic activity, and antinociception, using standard methodology. RESULTS: The 1000 mg/kg dose of AVELE significantly increased survival, reversed the profound thrombocytopenia/ thrombocytosis (p ≤0.01), altered the end-stage disease (p ≤0.05), and manifested significant antipyretic and antinociceptive (p ≤0.05) activities. INTERPRETATION & CONCLUSION: We conclude that a crude ethanolic leaf extract of A. vulgaris, showed potent antimalarial properties, in terms of antidisease activities; antipyretic activity, peripheral and central antinociception, increased survival, averted end-stage disease and reversed thrombocytopenia/thrombocytosis.

Antimalarial properties of Artemisia vulgaris L. ethanolic leaf extract in a Plasmodium berghei murine malaria model.

BACKGROUND & OBJECTIVES: Artemisinin isolated from Artemisia annua is the most potent antimalarial drug against chloroquine-resistant Plasmodium falciparum malaria. Artemisia vulgaris, an invasive weed, is the only Artemisia species available in Sri Lanka. A pilot study was undertaken to investigate the antiparasitic activity of an A. vulgaris ethanolic leaf extract (AVELE) in a P. berghei ANKA murine malaria model that elicits pathogenesis similar to falciparum malaria. METHODS: A 4-day suppressive and the curative assays determined the antiparasitic activity of AVELE using four doses (250, 500, 750 and 1000 mg/kg), Coartem® as the positive control and 5% ethanol as the negative control in male ICR mice infected with P. berghei. RESULTS: The 500, 750 and 1000 mg/kg doses of AVELE significantly (p ≤ 0.01) inhibited parasitaemia by 79.3, 79.6 and 87.3% respectively, in the 4-day suppressive assay, but not in the curative assay. Chronic administration of the high dose of AVELE ruled out overt signs of toxicity and stress as well as hepatotoxicity, renotoxicity and haematotoxicity. INTERPRETATION & CONCLUSION: The oral administration of a crude ethonolic leaf extract of A. vulgaris is non-toxic and possesses potent antimalarial properties in terms of antiparasitic activity.

Effects of diazinon on larvae of the Asian common toad (Bufo melanostictus, Schneider 1799).

Continuous subchronic exposure experiments were conducted to assess the effects of diazinon, an organophosphate pesticide, on the survival, growth and activity of larvae of the Asian common toad Bufo melanostictus. Two larval stages, the gill stage (Gosner stages 21 and 22) and gill-atrophy stage (Gosner stages 24 and 25), were continuously exposed to 4 microg/L, 400 mirog/L, and 10 mg/L of commercial-grade diazinon for 30 d. Treatments and untreated controls were maintained in triplicate with water changed and pesticide concentrations renewed every 3 d. Observations showed that subchronic exposure to 400 microg/L and 10 mg/L diazinon caused a significant dose-dependent increase in mortality compared to the control, regardless of the age at which larvae were exposed. One hundred percent mortality was observed in larvae exposed to 10 mg/L. No clear age-related sensitivity was evident in this study. The lethal concentrations at which 50% of the tadpoles (LC50) died during 30 d of continuous exposure were 6 and 7.5 mg/L for gill stage and gill-atrophy stage larvae, respectively. Diazinon impaired larval growth and activity. Tail abnormalities were apparent in larvae exposed to 400 microg/L and 10 mg/L of diazinon. This investigation provides the first empirical evidence of the negative effects of diazinon on the survival, growth and activity of B. melanostictus. The high degree of diazinon toxicity in this study highlights the need to consider important nontarget groups when recommending safe levels of pesticide application.