Evaluation of mouse bioassay results in an inter-laboratory comparison for paralytic shellfish poisoning toxins

An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing in China, and to analyze the main factors affecting the performance of this method. The toxic scallop Patinopecten yessoensis collected from coast of Bohai Sea, China, was used as a test sample in the comparison study. The results were reported and evaluated using robust statistical methods. The z scores showed that 80%, 8%, and 12% of laboratories reported satisfactory results, unsatisfactory results, and questionable results, respectively. This evaluation demonstrates that the PSP mouse bioassay is an appropriate method for screening and testing PSP toxicity in shellfish. However, it was found that the experience and skill of technicians, as well as the body weight and health status of mice being used significantly affected the accuracy of the method.

Development of An ICR Mouse Bioassay for Toxicity Evaluation in Neurotoxic Poisoning Toxins-Contaminated Shellfish

Objective To develop an ICR （female） mouse bioassay （MBA） for toxicity confirmation and evaluation of neurotoxins （brevetoxins）-contaminated shellfish. Methods Brevetoxins （BTX-B） as a causative agent of neurotoxic shellfish poisoning （NSP） under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. Results BTX-B could be detected in shellfish tissues at concentration of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit （80 μg/100 g shellfish tissues）. The LD 50 identified was 455 g/kg for BTX-B under general shellfish matrices （excluding oyster matrices） dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. Conclusion The two ELISA analyses agree favorably （correlation coefficient, r 0.96; Student＇s t-tests, P〉0.05） with the developed bioassay.

Toxicity assessment and screening of tetrodotoxin in the oblong blowfish(Takifugu oblongus)from the Tamil Nadu Coast of Bay of Bengal,India

Objective:To assess the toxicity of the pufferfish Takifugu oblongus,from Chennai coast,Tamil Nadu,India and to detect the presence of Tetrodotoxin(TTX).Methods:The toxicity was evaluated by mouse bioassay using Swiss Albino mice which were expressed in mouse units(MU).Gross anatomical features were observed which is followed by histopathology of the dead mice tissues to establish the toxicity.Instrumental analysis for the presence of tetrodotoxin was also performed through GC-MS and HPLC.Results:The toxicity of ovary was the maximum with 163 MU/g and lowest toxicity was observed in skin with 75.88 MU/g.Histopathological analyses of the dead mice showed various cellular degenerations and inflammations.The amount of Tetrodotoxin detected through GC-MS and HPLC was more reliable and sensitive than the customary mouse bioassay as instrumental analyses were able to detect even nanograms of the toxin.Conclusions:The present study evidently proved that Takifugu oblongus is highly toxic and consumption of the same can pose serious threat for health and possible lethality to humans.

Morphological and toxicity characteristics of Pseudo-nitzschia pungens strain PP0201-01 isolated from the East China Sea

The morphology and toxicity of Pseudo-nitzschia strain PP0201-01, isolated near the estuary of Changjiang (Yangtze) River in Year 2002 and tentatively identified as Pseudo-nitzschia pungens, were studied. The strain was identified morphologically as P. pungens using scanning electronic microscope (SEM). The ana- lytical results of LC-PDA (liquid chromatography with photodiode array detector), LC-MS (liquid chromatogra- phy with mass detector), and the toxicity data from mouse bioassay, showed that no domoic acid was produced by the strain. However, the mechanism of domoic acid production by Pseudo-nitzschia species is complicated. More works are needed to assess the toxic species and to evaluate the potential risk of amnesic shellfish poison- ing (ASP) in China.

Features of Ciguatera Fish Poisoning Cases in Hong Kong 2004-2007

Ohjective To review the clinical features and laboratory investigations of ciguatera patients in Hong Kong between 2004 and 2007 in order to show the timely sampling of implicated fish from ciguatera victims and application of validated mouse bioassay for confirming suspected clinical cases of ciguatera. Methods Diagnosis of the ciguatera victims was based on history of coral fish consumption and clinical presentations stated in official guidelines for clinical diagnosis of ciguatera fish poisoning in Hong Kong. Food remnants of coral fish samples were collected swiftly from ciguatera victims between 2004 and 2007 for ciguatoxins （CTXs） analysis. Results Major clinical symptoms in ciguatera patients included gastrointestinal and neurological effects including limb numbness and diarrhoea, which developed at 0.5 to 15 hours after consumption of fish. In most cases, neurological symptoms were more common than gastrointestinal symptoms. A broad range of attack rate （10%-100%） was observed in each ciguatera outbreak. Validated mouse bioassay on ether extracts of the food remnant samples confirmed that all were CTXs-positive （〈0.5 - 4.3 MU/20 mg ether extract） and directly linked to the corresponding ciguatera cases. Conclusion Consistency between clinical and laboratory analysis for ciguatera poisoning illustrates the application of laboratory mouse bioassay in a timely fashion for confirming ciguatera poisoning cases and implementing effective public health measures. With further improvement in laboratory techniques, features of ciguatera fish poisoning cases can be better defined, Further studies are needed to determine the risk of each class of CTXs （Pacific-, Indian- and Caribbean-CTXs） in Hong Kong.

A new simple screening method for the detection of paralytic shellfish poisoning toxins

The current testing for paralytic shellfish poisoning(PSP) in shellfish is based on the mouse bioassay(MBA).To alleviate animal welfare concerns,we evaluated the utility of using sublethal indicators of toxicity as an alternative to measuring time to death.Live mice were injected with a PSP congener and the changes in neurotransmitter levels were measured 60,90,and 120 min after injection.Acetylcholine(ACh) was the most sensitive marker for PSP toxicity.The changes in neurotransmitter levels were most pronounced in the blood.Thus,measurement of Ach levels in the blood may serve as a sensitive predictor for PSP that would not require sacrifice of the mice.This method was relatively simple,sensitive(less than 1 μg/kg weight,equivalent to 20 ng/mL),low maintenance,and rapid(less than 60 min).