Poisoning by toxic animals in China—18 autopsy case studies and a comprehensive literature review

Forensic Science International xxx (2013) xxx–xxx

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FSI-7324; No. of Pages 12

Case report

Poisoning by toxic animals in China—18 autopsy case studies and acomprehensive literature review

Long Chen a,*, Guang-zhao Huang b,**a Department of Forensic Medicine, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, Chinab Department of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hang Kong Road, Wuhan 430030, China

A R T I C L E I N F O

Article history:

Received 20 August 2012

Received in revised form 26 July 2013

Accepted 9 August 2013

Available online xxx

Keywords:

Poisoning

Toxic animals

Autopsy

China

A B S T R A C T

Although exposure to animal venom and poison, such as snakebites, bee stings, and contact, with toads,

is a common problem, reported deaths are rare. The present report discusses 18 fatal cases in China.

Causes of death were grouped into 6 categories, including 1 case of tetrodotoxin poisoning, 1 case of

gallbladder poisoning, 3 cases of snake venom toxicity, 4 cases of melittin toxicity, 4 cases of cantharidin

poisoning and 5 cases of venenum bufonis poisoning. The epidemiology of each venom-induced death,

the mechanism of exposure to venom, and the target organs and tissues affected by these toxic animals

were here systematically reviewed. Such details are important to even suspected cases of venom

damage. The associated problems related to forensic medicine, such as manner of death and possible

attribution to the toxic effects of various animals, are also discussed herein.

� 2013 Elsevier Ireland Ltd. All rights reserved.

Contents lists available at ScienceDirect

Forensic Science International

jou r nal h o mep age: w ww.els evier . co m/lo c ate / fo r sc i in t

Poisoning is a significant global public health problem.According to WHO data, an estimated 346,000 people diedworldwide in 2004 from unintentional poisoning. Of these deaths,91% occurred in low- and middle-income countries [1]. Poisoningincidents have a tremendous impact on families and society. TheMinistry of Health of the People’s Republic of China postspoisoning cases every year. In 2012, the network system recorded174 events of food poisoning; out of 6685 poisonings, 146 peopledied (a number of events involved more than one person). Thefatalities caused by food poisoning from the consumption ofpoisonous animals, plants, and mushrooms accounted for thelargest proportion (99/146, 67.8%). From 2000 to 2011, there werea total of 881 reported events of food poisoning from toxic plantsand animals, in which 19,605 people were poisoned, and 941people died. In comparison to chemical and microbial foodpoisoning, the proportion of events and number of poisoningcases from the consumption of toxic plants and animals is lower,but the number of deaths is higher [2,3]. In 1988, we reported 19Chinese autopsy cases of poisoning by toxic plants and describedthe forensic autopsies [4]. In fact, poisoning by toxic animals,including puffer fish, snakebites, bee stings, and fish gallbladdersare also common in China. In China, Japan, several Southeast Asiacountries, and Australia, incidents of tetrodotoxin (TTX) poisoningoccur frequently, mainly from the ingestion of poisonous puffer

* Corresponding author. Tel.: +86 21 54237566; fax: +86 21 64931016.** Corresponding author. Tel.: +86 27 83693653; fax: +86 27 83692644.

E-mail addresses: [email protected] (L. Chen), [email protected]

(G.-z. Huang).

Please cite this article in press as: L. Chen, G.-z. Huang, Poisonincomprehensive literature review, Forensic Sci. Int. (2013), http://dx

0379-0738/$ – see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.forsciint.2013.08.013

fish flesh, viscera, skin, or fish roe [5–8]. According to the availabledata, 116 incidents of puffer fish poisoning occurred in Japan from2002 to 2006, which involved 223 patients and 13 deaths [9]. InSingapore, a total of 53 patients with a history of puffer fishingestion were admitted to a regional hospital from 2001 to 2006;eight of the patients died [10]. In Taiwan, there were 30 TTXintoxication outbreaks from 1994 to 2003 with 124 patients and 8deaths [9,11]. A more recent outbreak of TTX poisoning occurred inBangladesh in 2008 involving 141 patients, and 17 died [12]. Thereare approximately 50 types of puffer fish in the coastal and YangtzeRiver areas of China. Statistics from China for Jiangyin city revealed29 puffer fish poisoning events from 1977 to 2007, which poisoned312 people and killed 12 [13].

Snakebites are a public health issue in many tropical andsubtropical countries. Approximately 5 million snake bites occureach year, resulting in up to 2.5 million poisonings and at least100,000 deaths [14,15]. Fish gallbladder has long been used as afolk remedy for various ailments in China. This type of poisoningoccurs primarily in China, occasionally in Japan, and rarely in othercountries [16–18]. The majority of these cases have been reportedin Chinese journals. Bee venom poisoning is accidental. Accordingto reports, an average of 79.5 animal-related fatalities occursannually, accounting for 713 deaths from 1999 to 2007 in the USA.The most common fatalities involved contact with hornets, wasps,and bees [19]. In China, there are approximately 200 types ofwasps, including the vespid, wasp, and hornet.

In this article, we will report 18 poisoning cases caused bypoisonous animals. We classified these poisonings into sixsections. The basic demographic data, clinical manifestations,

g by toxic animals in China—18 autopsy case studies and a.doi.org/10.1016/j.forsciint.2013.08.013


mailto:[email protected]

mailto:[email protected]


http://www.sciencedirect.com/science/journal/03790738


Table 1Eighteen cases of lethal animal venom toxicity in China.

Category Case Gender Age

(y)

Poisoning manner Clinical course Time of

death

PMI (h) Autopsy findings Manner of

death

Toxicology

Tetrodotoxin

poisoning

Case 1 F 3 Ingestion of dried

puffer fish

Dizziness, vomiting,

breathing difficulty,

coma

4.5 h 19 Internal organ

congestion and edema,

focal hemorrhage, and

inflammatory cell

infiltration

Accident Y

Gallbladder

poisoning

Cases 2 F 38 Ingestion of

gallbladder

Nausea, vomiting,

diarrhea

10 days 24 Toxic nephropathy,

liver toxicity

Accident N

Snake venom

toxicity

Case 3 M 43 Bitten on foot

back

Irritability, vomiting,

dizziness, vision loss

5 days 36 Toxic shock and

multiple organ

damage

Accident N

Case 4 M 4 Bitten on foot

malleolus medialis

Treated with

acupuncture, anti-

venin, irregular

pulse, tidal breathing

48 h 36 Foot diffuse

subcutaneous

hemorrhaging,

internal organ

congestion and edema

Accident N

Case 5 F 36 Injected with

snake venom

Numbness, blurred

vision, vomiting

5 days 14 Multiple organ

damage

Homicide N

Melittin

toxicity

Case 6 F 7 Stung by bees Skin reddening and

swelling, bee sting

terebras in the scalp

0.5 h 29 Multiple organ

congestion and edema,

anaphylactic shock

Accident Y

Case 7 M 4 Stung by bees Facial swelling,

cyanotic lips and

nails

0.5 h 23 Anaphylactic shock,

accidental suffocation

Accident Y

Case 8 M 13 Stung by wasps Coma with severe

cerebral edema, dark

brown urine

5 days 36 Stung skin necrosis,

acute cellulitis, acute

tubular necrosis

Accident N

Case 9 M 47 Stung by wasps Stung by a wasp in

head, rapid-onset

coma

0.5 h 40 Anaphylactic shock,

acute laryngeal edema

Accident Y (IgE)

Cantharidin

poisoning

Case 10 F 21 Ingested poison for

private abortion

Vomiting, abdominal

pain, weakness,

coma, moist rales

24 h 36 Toxic nephropathy and

mild toxic

hepatopathy

Accident Y


private abortion

Abdominal pain,

diarrhea, vomiting

and severe chills

60 h 24 Toxic nephropathy and

mild toxic

hepatopathy

Accident Y


treatment

Throat and stomach

burning, vomit

blood, abdominal

pain, hematuresis,

oliguria

60 h 3 Multiple organ

congestion and edema

Accident Y

Case 13 28 Ingested poison for

treatment over the

course of 3 days

Abdominal pain,

vomiting with blood,

red urine

3 days 17 Toxic nephrosis and

acute toxic

nephropathy. multiple

organ congestion and

edema

Accident Y

Toad-venom

poisoning

Case 14 M 42 Eat for bet with

people

Drowsiness and

abdominal pain,

coma after 3 days,

arrhythmia with a

galloping rhythm

and rales in lungs

48 h 4 Toxic nephrosis and

acute toxic

nephropathy. multiple

organ congestion and

edema

Accident Y

Case 15 M 7 Drank clay pot

toad soup

Suffered vomiting,

tongue numbness,

abdominal

distension,

abdominal pain,

diarrhea within

30 min

4 h 5 Cyanotic lips and nails.

hydropic degeneration

of internal organ

(cardiac myocytes,

hepatocytes, and renal

proximal tubule

epithelial cells, as well

as lung, spleen)

Accident Y

Case 16 F 7

Case 17 F 9

Case 18 F 11

L. Chen, G.-z. Huang / Forensic Science International xxx (2013) xxx–xxxe2

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and forensic pathological diagnoses are summarized in Table 1. Ofthese cases, nine were autopsied by the authors, and the other ninecases were autopsied by other forensic medical examiners inChina. The discussion will focus on analyzing the mechanism ofdeath caused by poisoning from toxic animals. The target organs ortissues affected by toxic animals determines the basis of thesymptoms, the pathological changes and the toxicological analysis,and the associated problems related to forensic medicine, such asthe cause of poisoning, are also discussed in combination with acomprehensive review of the literature.


1. Case reports

1.1. Tetrodotoxin poisoning

1.1.1. Case 1

A 3.5-year-old girl shared one piece of dried puffer fish with her26-year-old mother. One hour later, she was dizzy, had difficultybreathing, and vomited, and her lips, tongue, and limbs werenumb. Gastric lavage treatment was performed after she was sentto the hospital. Three hrs later, she fell into a coma and was



Table 2Mouse experiment in which death was caused by exposure to dried puffer fishes.

Group 1 2 3 4 5

Intraperitoneal injection 1/30 3/50 0 3/2 2/30

Gastric lavage 1/180 1/120 0 3/60 1/60

Note: Number of dead mice/times (min).

L. Chen, G.-z. Huang / Forensic Science International xxx (2013) xxx–xxx e3

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declared dead only 4.5 h after having ingested the fish. Her motherhad similar symptoms and was fortunate to recover. The forensicpathologist found the following symptoms: cyanosis of the lips andnails, flatulence, stomach mucosa focal hemorrhage, acute enteri-tis, focal pulmonary hemorrhage, focal myocardial necrosis andinflammatory cell infiltration in the myocardial interstitial space,small focal melting necrosis of the myocardial muscle, brainedema, cerebellum tonsillar and hippocampal uncal herniation,multiple organ congestion, and edema. For the toxicology analysis,thin layer chromatography (TLC) was used for qualitative analysis,and high performance liquid chromatography (HPLC) was used forquantitative analysis. The results indicated that the tetrodotoxinconcentration in the leftover dried puffer fish was 7.1 mg/g. Notetrodotoxin was detected in the stomach contents, most likelybecause of the gastric lavage treatment at the hospital.

Upon receipt of approval from the animal ethics committee ofFudan University, we designed a mouse experiment as follows: 30healthy male mice weighing 20 � 5 g were randomly divided into 5groups; each group included six mice. Groups 1–4 were eachadministered 2 g samples from 4 isolated pieces of dried puffer fish,and the remaining dried puffer fish was administered to group 5. Thesamples were homogenized in an appropriate amount of double-distilled water. The supernatant was extracted for preparation. A totalof 1.2 ml of supernatant was acquired from each group. From thissupernatant, 0.6 ml was intraperitoneally injected into 3 mice, andthe remaining 0.6 ml of supernatant was placed in the stomachs ofthe other 3 mice by gavage. The results showed that all of the micethat died from poisoning had cyanotic lips and tails, shortness ofbreath, mouth breathing, an unsteady gait, ataxia, and paralysis priorto death. These results are shown in Table 2. Our results showed thatthe dried puffer fish in groups 1, 2, 4, and 5 were toxic; the toxicitywas highest in group 4. However, group 3 was not toxic.

1.2. Gallbladder poisoning

1.2.1. Case 2

A 38-year-old female treated her own ocular disease byingesting a whole gallbladder from a herring fish that weighed1.5 kg. She subsequently experienced nausea, vomiting, anddiarrhea. On the 3rd day, symptoms of jaundice, urine retention,swelling and other signs of liver and renal insufficiency appeared.

Fig. 1. Autopsy in Case 3 and 9. Case 3 was bitten by a snake in the back of the right foot an

of the right foot (black arrow); his right foot had signs of swelling and ecchymosis (A). Ren

that had formed in the distal tubules (black arrow) (B). Case 9 was stung by a wasp in the h

edema that caused a blocked glottis (C).


After treatment, her urine output had increased, but heart failureand pulmonary edema occurred by day 9. She died on the 10th day.The forensic pathology observations were as follows: the proximaltubule epithelial cells had significant hydropic degeneration andnecrosis, interstitial edema (toxic nephropathy), hepatic steatosisand scattered focal necrosis (liver toxicity), and mild acuteinterstitial myocarditis.

1.3. Snake venom poisoning

1.3.1. Case 3

A 43-year-old male was bitten by a snake on the back of hisright foot. On the following day, he experienced whole body achesand pains, irritability, vomiting, dizziness, and vision loss. On the5th day, the patient’s condition worsened, and he was sent to thehospital. He had sighing respiration, coarse breathing sounds in thelungs, percussion pain in the kidney region, and an undeterminableblood pressure. His heart rate was 55 beats/min; the heartbeatswere low and blunt, and the rhythm was irregular. He wasresuscitated in the emergency room, but declared dead at noon.The autopsy found a 0.3 cm-long laceration made of two pairs ofthin stripes on the back of the right foot. His right foot had swollenand was ecchymotic (Fig. 1a). The forensic pathology examinationnoted the snakebite on the back of the right foot as well assubcutaneous hemorrhaging and acute cellulitis. His internalorgans (brain, pulmonary, and myocardial tissue) presentedhemorrhaging and edema, toxic nephropathy, renal proximaltubular epithelial degeneration and necrosis, and a protein andpigment cast had formed in the distal tubules (Fig. 1b). There wasalso congestion in the heart, liver, spleen, pancreas, and thyroidorgans. With assistance from local snake specialists, the victim wasdetermined to have been poisoned by an Agkistrodon halys bite inaccordance with the bite mark characteristics and the poisoningsymptoms.

1.3.2. Case 4

A male child of 4 years and 7 months old was bitten at 16:00 pmby a snake in the medial malleolus of his right foot. He was sent tothe snake specialist immediately, who provided an Agkistrodon

halys bite diagnosis. He was treated by bleeding detoxificationacupuncture around the bite marks, in addition to antiveninmedication. However, the symptoms became severe, and he wassent to hospital the next day, where he presented with delirium, anirregular pulse, tidal breathing, mydriasis, light reflex disappear-ance, and right foot and right lower extremity edema, and largeareas of bleeding were observed on the back of the right foot andright groin; he died that night. The autopsy found a1.2 cm � 0.5 cm snakebite wound in the right foot medialmalleolus and the back of the right foot was swollen to a high

d died 5 days later. A 0.3-cm-long laceration with two pairs of thin stripes in the back

al proximal tubular epithelial degeneration and necrosis, protein and pigment casts

ead, and he died 0.5 h later. The autopsy reported a high degree of throat and glottis




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degree and had extensive subcutaneous bleeding. The forensicpathology diagnosis was as follows: right foot medial malleoluswith diffuse subcutaneous hemorrhaging, acute cellulitis withpartial nerve and small artery degeneration and necrosis;myocardial cell and liver cell edema; brain, kidney, heart, liver,and thymus congestion; and an anemic-like spleen.

1.3.3. Case 5

The husband of the 36-year-old female case subject hadpurchased 13 kraits in order to acquire approximately 2 ml ofbungarotoxin. The woman was injected with 1 ml of venom using asyringe to the left hip. Ten minutes later, she developed a numbtongue, difficulty swallowing, and blurred vision. In the hospital,she started to vomit and had slurred speech and facial cyanosis.She died 5 days later. A needle eye was found in the left side of thedeceased’s buttocks and was accompanied by light swelling. Theforensic pathology diagnoses indicated a high degree of braincongestion and edema with focal hemorrhaging and softening fociformation. Her lung membranes had spot bleeding and lobularpneumonia. She also had extensive epicardial and endocardial spotbleeding, liver subcapsular punctate bleeding, diffuse fattyhepatocyte degeneration, liver sinusoidal congestion, acute spleeninflammation, and gastric mucosa spot bleeding.

1.4. Melittin poisoning

1.4.1. Case 6

The 7-year-old female victim was stung by bee swarms andconfirmed dead after being sent to the hospital. The autopsydetermined that the face and neck skin was notably red andswollen, and many bee sting terebras could still be found in thesurface of the scalp. Many pinpoint bleeding spots could be foundin the two palpebral conjunctivas. The lips and peripheral limbswere highly cyanotic. Her hands were red and swollen. An autopsyand microscopic investigation revealed that the larynx, glottis,epiglottis, tracheal mucosa, and lungs had congestion and edema.A punctate hemorrhage and eosinophilic leukocyte infiltration,spleen congestion, considerable eosinophilic leukocytes in thespleen sinus, a punctate hemorrhage in the liver and kidneymucosa, multiple organ (i.e., brain, kidney, liver, thymus, thyroid,and esophagus) congestion and edema were also observed. Thecause of death was anaphylactic shock caused by multiple beestings. We had planned to test the DNA extracted from the terebrasin the scalp of the deceased along with bee samples frombeekeepers to verify if the victim was stung by bees from thenearby beehives. However, we failed to do so because oftechnological limitations.

1.4.2. Case 7

The 4-year-old male victim was stung by bees after strikingthe beehive with a branch. He was taken to a hospital andconfirmed dead. The corpse arrived approximately half an hourafter the child was stung to death. The corpse surface showedfacial swelling and cyanotic lips and nails. In the right parietaloccipital scalp, a 1 cm � 1 cm area of edema was observed. Pin-like holes were scattered across the left cheek, left upper lip andchin; the surrounding skin was red and swollen. The autopsy andmicroscopic investigation results included pale throat mucosa, ahigh degree of edema in the throat, tracheal and bronchialcongestion, bronchospasms, and a mucus-filled cavity. Eosino-phil infiltration was found in the tracheal and bronchial tissuemucosa, spleen, liver and lung interstitial tissue, and kidney.Three bees were isolated from the scene for biologicalidentification; the bee specimens were Apis mellifera L. 1750,as classified Hymena FERA, Apida C and Api S. A part of the terebraremained in the bee specimen and was matched with the terebra


that was found in the scalp of the deceased. The cause of deathwas acute anaphylactic shock and accidental suffocation frombee stings.

1.4.3. Case 8

The 13-year-old male victim threw stones at a wasp honey-comb, and was stung in the head, face and chest. Upon clinicalexamination, the patient arrived in a light coma with severecerebral edema. He was unable to open his eyes and was short ofbreath. Dark brown spots caused by the bee stings extended allover his head, face, hands, chest and abdomen. He was unable tourinate, and experienced a severe shortness of breath, cyanotic lips,and moist rales in the lungs. He died 5 days after being stung.Laboratory examination results demonstrated the following: whiteblood cells, 3 � 109/L; neutrophils, 90%; lymphocytes, 8%; redblood cells, 3 � 1012/L; hemoglobin, 69 g/L; bleeding time, 10 min;clotting time, 12 min; platelet count, 92 � 109/L; CO2 combiningpower, 33.6%; and non-protein nitrogen, 66.6 mg/ml. The urineexamination results on day 16 were as follows: the urineappearance was red, turbid, and alkaline, protein +++, red bloodcells +, epithelial cells ++, pyocyte ++, and urinary casts ++. Theclinical diagnosis was wasp sting toxemia. The forensic pathologistdiagnosis indicated acute tubular necrosis; hemoglobin andgranular casts in the tubular lumen; stung skin necrosis andacute cellulitis in the head, face, and neck; acute spleeninflammation; mild fatty degeneration in the liver cells; lungcongestion and edema; petechiae of the epicardium; and mucousin the pleura and pelvis.

1.4.4. Case 9

The 47-year-old male victim was stung suddenly by wasps inthe occipital region of his head as he walked in the park; he rapidlyfell into a coma and died 0.5 h later. The corpse surface had a1.8 cm � 0.1 cm subcutaneous hemorrhage in the left occipitalregion, which was the area of the wasp sting. The autopsy andmicroscopic investigation results reported left occipital subcuta-neous edema, vascular dilation and congestion, a high degree ofthroat and glottis edema that caused a blocked glottis (Fig. 1C),lung congestion and edema, a high degree of myocardial interstitialcongestion, brain edema, and mild fatty hepatic degeneration. TheIgE level in the cardiac blood was 1384.32 ng/ml (normal value<800 ng/ml). The cause of death was hornet sting-inducedanaphylactic shock and acute laryngeal edema.

1.5. Cantharidin poisoning

1.5.1. Case 10

The 21-year-old female victim ingested approximately 25 g of acantharides mixture and attempted a private abortion. Shortlyafter ingestion, she started to vomit and felt abdominal pain andweakness. She became comatose 23 h later. Upon clinicalexamination, she had the following symptoms: undetectableblood pressure, low heart sound, a heart rate of 140 beats/min, andmoist rales at the bottom of both lungs. She died 24 h afteringestion. The forensic pathological diagnosis was skin blisterformation in the axilla, back, and neck. A gravid uterus (the fetuswas approximately 3 months old), a gastric mucosa punctatehemorrhage, acute gastroenteritis, small intestinal mucosalsurface necrosis, toxic nephropathy (significant hydropic degen-eration and mild necrosis of renal tubules, especially in theproximal tubule) and mild toxic hepatopathy (spotty necrosis ofthe liver cells and low neutrophilic infiltration) were observed. Thetoxicological analysis detected cantharidin in the gastric contentand cantharidin residue in the medicine she used with gaschromatography. The cause of death was toxic nephropathy andcirculatory failure caused by cantharidin.




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1.5.2. Case 11

Because of menolipsis and a suspected pregnancy, the 18-year-old female victim ingested cantharides and attempted an abortion.She suffered abdominal pain, diarrhea, and frequent vomiting onthe first day. On the second day, her abdominal pain had goneaway, but she experienced headache, precordial discomfort, severevomiting and severe chills. She died on the morning of the thirdday. The forensic pathology diagnosis was as follows: punctatehemorrhages under the epicardium, pulmonary pleura, renalcapsule and gastrointestinal mucous, proximal tubule epithelialnecrosis, a small amount of reddish fluid in the vagina, a smallblood clot in the uterine cavity, slight toxic hepatic disease (i.e.,slightly fatty degeneration of the hepatic cells with focalneutrophilic infiltration), spleen congestion, and bronchial pneu-monia. Cantharides debris was found in her pocket, and a GCanalysis indicated cantharidin in her gastric content. The cause ofdeath was toxic nephropathy from cantharidin.

1.5.3. Case 12

The 54-year-old female victim was suffering from irregularmenstruation. She took a packet of drugs at night. Two hours later,she felt burning in her throat and stomach, experienced heavyvomiting and observed blood in her vomit. Two days later, she tookanother bag of the same medicine, and suffered from abdominalpain, hematuresis, hematochezia, and oliguria, and died in themorning on the 3rd day. An autopsy found scattered bleeding inthe epicardium, pulmonary pleura, and pancreatic serosa; exten-sive hemorrhaging in the upper esophageal mucosa; two renalcapsule and pelvis mucosa that were covered with hemorrhagepoints; and a diffuse punctate hemorrhage in the bladder mucosa.The forensic pathology diagnosis indicated acute gastroenteritis,toxic nephrosis in which the proximal convoluted tubulesexhibited degeneration and necrosis, acute hemorrhagic cystitis,acute interstitial hepatitis, acute splenitis, pulmonary congestionand hemorrhage, and mild pancreatitis. Qualitative tests of thegastric content were performed by three different toxicologicalmethods, namely TLC, reaction to 4-(N,N-dimethyl amino) benzoicacid (DMABA), and sublimatography, all of which confirmed thepresence of cantharidin. A quantitative inspection indicated thateach package of medicine contained 1.1 g of cantharides. The causeof death was cantharidin poisoning, resulting in acute toxicnephropathy.

1.5.4. Case 13

The 28-year-old female victim used a folk prescription to treather enlarged neck lymph nodes, which had troubled her for 3 years.The folk prescription consisted of placing the powder from 5 to 7cantharides into the holes of drilled duck eggs. The duck eggs werethen steamed and one egg was ingested daily. The victim ingestedthe first egg, suffered general malaise, headache, abdominal pain,lumbago, nausea, and vomiting with blood. Her urine color was redthat night. The victim ingested another egg the next day, and theaforementioned symptoms were aggravated, in addition tohematuria and hematochezia. She ingested the third egg on thethird day, her condition was exacerbated, and she finally died thatday. The autopsy found the following: cyanotic lips and nails andseveral bilaterally swollen lymph nodes in the thyroid cartilage.The stomach content was a brown and turbid liquid ofapproximately 250 ml. The forensic pathology diagnosis was toxicnephrosis (i.e., mild renal tubular epithelial cell necrosis and renaltubular hyaline casts), esophageal mucosa shallow ulcers andhemorrhages, gastric mucosa punctate hemorrhages, gastric wallchronic inflammation, slight edema of the brain tissue, pulmonarycongestion and edema, bronchiolar spasm and liver, spleen, andbladder congestion. Toxicological analyses using gas chromatog-raphy-mass spectrometry (GC/MS) verified the presence of


cantharidin in the cantharides powders and in the blood, urine,liver, and stomach contents of the decedent. However, noquantitative analysis was reported. The cause of death wascantharidin poisoning, resulting in acute toxic nephropathy.

1.6. Toad venom poisoning

1.6.1. Case 14

The 42-year-old male victim placed a bet with other people thathe would swallow a live toad. He felt drowsiness and abdominalpain later that night. On the morning of the third day, he suddenlyfell into a coma. His limbs convulsed; white foam overflowed fromhis mouth, and he excreted a fetid black stool. At the clinicalexamination, he was in a deep coma, his pupils had contracted,pink foamy liquids overflowed from the nasal cavity, his heart ratewas 140 beats/min, he was experiencing arrhythmia with agalloping rhythm and he had rales in his lungs. The electrocardio-gram (ECG) data indicated multi-source frequent prematureventricular contractions (FPVC), bigeminy, ST-segment depression,and a flattened and inversed wave. He died 48 h after ingestion.The autopsy found his lips, fingers, and toes to be cyanotic, andpetechiae were found on the right bulbar conjunctiva. Uponmicroscopic examination, myocardial interstitial congestion, lungcongestion, edema and a focal hemorrhage, severe cerebral edema,liver edema, renal congestion, and a ballooning degeneration of therenal tubule were found. A toxicological analysis by TLC verifiedthe presence of venenum bufonis in the liver and stomach tissues.The cause of death was venenum bufonis poisoning.

1.6.2. Cases 15–18

Four 7- to 11-year-old case subjects (a 7-year-old male, 7-year-old female, 9-year-old female, and 11-year-old female) drank claypot soup that included a toad, and within 30 min, they sufferedfrom vomiting, tongue numbness, abdominal distension, abdomi-nal pain, diarrhea, and other symptoms. The four case subjects diedwithin 4 h. The four autopsy observations were the same, i.e.,bloody fluid in the nasal cavity and cyanotic lips and nails. Lightgreen stool was observed in the perianal areas of the 7-year-oldand 11-year-old girls. Scattered hemorrhage sites in the pulmo-nary pleura and epicardium, bloody and foamy liquid in thetrachea, lung congestion and edema, and a hemorrhage in thegastric mucosa were observed. Microscopic evidence showing thehydropic degeneration of cardiac myocytes, hepatocytes, and renalproximal tubule epithelial cells, as well as lung, spleen, and kidneycongestion were found. Additionally, necrosis in the intestinalmucous surface was found in the 7-year-old boy. A small numberof neutrophilic leucocytes infiltrated the hepatic portal area as wellas the myocardial interstitial and alveolar septa in the 9-year-oldgirl. Toxicological analysis by thin-layer chromatography verifiedthe presence of venenum bufonis in the soup residue. The cause ofdeath for the four children was venenum bufonis.

2. Discussion

The term ‘‘toxic animal’’ refers to a type of organism, theirmetabolites, or the organism itself that can affect the normalphysiological activities of humans or other organisms. In 2006, theInjury Surveillance System of China reported 345,897 injury cases.Among these cases, poisoning ranked seventh as a cause of injuryand included 8884 cases (2.57%). The poisoning category wasdivided into eight types as follows: drug poisoning, drug abuse,alcoholism, pesticide poisoning, toxic food poisoning, carbonmonoxide poisoning, animal bite poisoning, and other poisoning[20]. From 2004 to 2009, the People’s Hospital in Sichuan Provincetreated 232 acute poisoning or accidental injury cases, in which thepatient age ranged from 2 months to 14 years old. There were 26




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toxic animal poisoning cases, which included 11 cases of melittinpoisoning, seven cases of fish gallbladder poisoning, threesnakebite cases, one scorpion sting case, and one poisonous spidersting case, which accounted for 11.21% (26/232) of all acutepoisoning or accidental injury cases [21]. Therefore, the frequentoccurrence of toxic animal poisoning has become a prominentpublic health problem in China.

Studies have reported the presence of dozens of toxic animals inChina that can cause poisoning or death. Poisonous animals, suchas snakes, bees, poisonous spiders, centipedes, ticks, the pine mothInimicus japonicus, and octopus can cause injuries by biting orstinging. Animals or their organs, such as puffer fish, herring bile,Mylabris, venenum bufonis, Ruvettus pretiosus, Nassarius sp.,Huechys, Lytta, leeches, sea urchins, and starfish can causepoisoning when ingested [22].

Accidental poisoning is the most common cause of toxic animalpoisoning. In China, if someone has witnessed the entire poisoningprocess, the cause of death is undisputed, and the cases would notbe sent for medico-legal investigation. When the sequence ofevents is unclear, a forensic autopsy will be performed whensuicide or homicide is suspected, or the relatives request it.Therefore, most of the poisoning death cases were not referred formedico-legal investigation. It took several years for us collectedthese 18 cases in China.

Accidental poisoning by toxic animals can happen in a variety ofsituations. First, the most common cause of poisoning is byingesting a poisonous animal or its organ or by ingesting food thatcontains toxic animal ingredients. Examples of this type ofpoisoning are found in case 1, in which a dried puffer fish wasingested, and in case 14, in which a venenum bufonis-containinganimal was swallowed. Second, another cause of accidentalpoisoning is a toxic animal bite, sting, or stab. In cases 3 and 4,the case subjects were bitten by a poisonous snake, and in cases 6–9, the subjects were stung by a bee, which caused melittinpoisoning and death. Third, many toxic animals can be used as apharmacological treatment according to the Chinese pharmaco-poeia [23]. However, poisoning can occur in cases of improper useor overdose. Drugs derived from toxic animals are an importantscientific component of traditional Chinese medicine. In traditionalChinese medicine, toxic animal-derived drug prescriptions areoften used unscientifically, or they are used in an informal attemptto treat a particular disease, which can involve either incorrectusage or an overdose and subsequent poisoning. For example,cantharidin has been used for illegal abortions anecdotally (e.g.,cases 10 and 11), to treat irregular menstruation (e.g., case 12), andto treat swollen lymph nodes (e.g., case 13) and has beenreportedly used in cancer treatment [24]. Additionally, becausecantharidin was reported to stimulate genital congestion, it may beused as an aphrodisiac, which could lead to poisoning and death[25,26]. The ingestion of a whole, raw fish gallbladder to cure eyediseases is mentioned in folklore; this practice often causesgallbladder poisoning, such as what happened in case 2 with theingestion of herring bile. Anecdotally, the consumption ofvenenum bufonis soup is believed to remove toxins and internalheat; this practice can lead to accidental poisoning [27], asobserved in cases 15– 18 in which four children who consumedvenenum bufonis soup died by poisoning. Fourth, suicide orhomicide cases caused by toxic animals are relatively rare.However, the consumption of cantharides or a cantharides tinctureor injection of a pure snake venom solution to commit suicide hasbeen reported [28]. Moreover, homicides employing a poisonousanimal to produce wine or mix with food, such as the addition ofcantharidin to soup, have been reported both in China and in otherAsian countries [29,30]. Case 5 is a rare case in which the criminalextracted coral snake venom and performed an intramuscularinjection to commit homicide.


2.1. Tetrodotoxin (TTX) poisoning

TTX is one of the most potent and oldest known neurotoxins. In1964, the formula for TTX (C11H17N3O8) and its chemical structurewere determined, and synthetic TTX was reported in 2003 [31,32].Chemically speaking, TTX is water-soluble, stable and difficult todestroy by general cooking methods [33,34]. Death results fromrapidly developing paralysis with hypotension and respiratoryfailure. There is no antidote and treatment is supportive [34,35].TTX exists not only in the puffer fish, but also in a variety of otherbiological organisms, such as chaetognaths, gastropods, mollusks,echinoderms, amphibians, nemerteans, and algae [36]. TTX canreportedly be produced by a variety of bacteria, is transmittedthrough the food chain and then accumulates in animals [37–39].Studies have found that some living organisms can produce TTX[40].

TTX binds to the sodium channel receptors, especially to theexcitable nerve and muscle cell membrane, and prevents sodiumions from entering the cells, which thereby blocks the nerves andmuscles from generating action potentials [41]. This loss of activitycauses neuromuscular paralysis and severe breathing difficultiesthat may cause death. TTX is highly selective for the sodiumchannel and only blocks voltage-dependent channels. Additionally,it is much more sensitive for the sodium channels of nerve cellsthan those of cardiac and skeletal muscles [42,43]. TTX can bedetected by several methods, such as GC–MS, UV spectrophotom-etry, HPLC, and TLC [44]. An immunochemical detection methodwas first reported by Water in 1989. At present, an enzyme-linkedimmunosorbent assay (ELISA) can be used for TTX detection, andthis method is in common use [45]. TTX is one of the deadliestpoisons; the lethal dose (LD) for TTX in humans is 6–8 mg/kg,which is much lower than the minimum lethal dose of sodiumcyanide (10 mg/kg). Therefore, TTX is about 10,000 times morelethal than cyanide by weight [9].

The latent period of TTX poisoning is short; the general onset ofsymptoms occurs within 3 h, and the process progresses rapidly,but a portion of cases have a delayed onset of up to 20 h [5]. Theprognosis for severe TTX poisoning is good with modern intensivecare [46]. Although the severity of the symptoms and the prognosisgenerally depends on the amount of toxin ingested, it can also beinfluenced by the victim’s medical condition, as in diabetic oruremic patients. A medical condition that alters sodium channelfunction can result in a worse outcome [5]. Gastrointestinalsymptoms, such as nausea, vomiting, and diarrhea, appear early inthe process. Nerve palsy occurs first in sensory nerves and then inthe motor nerve system [34]. Paralysis will occur in the vasomotorcenter and the respiratory center if the dose is large. Numbness ofthe lips, tongue tip, and extremities occurs first and is followed by awide range of muscle weakness and movement disorders (i.e.,drooping eyelids, slurred speech, aphonia, limb weakness, andataxia) [47].

Victims were reportedly poisoning by eating dried dressed fishfilets [48]. The levels of TTX in patient urine or plasma can beanalyzed by HPLC or LC–MS/MS. A urine analysis within the first24 h after consumption seems to be the most sensitive method fordetecting TTX poisoning [49,50]. Unfortunately, we did not save aurine sample from case1.

2.2. Gallbladder poisoning

Gallbladder poisoning refers to an acute chemical poisoningthat is caused by ingesting a gallbladder from a freshwater fish.Chinese folk medicine presents the idea that the ingestion of fishbile cures eye disease, cough, dermatoses, and relieves asthma. Thefish gallbladder is generally ingested raw; however, the gallbladdercan also be cooked or soaked in white wine before ingestion.




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Chinese publications from between 1965 and 1999 reported that451 patients were diagnosed with acute fish gallbladder poisoning,from which 83 people died, resulting in a mortality rate of 18.04%[4,51].

In China, there are 11 types of poisonous bile-containing fish,which include grass carp (Ctenopharyngodon idellus), herring(Mylopharyngodon piceus), silver carp (Hypophthalmichthys moli-

trix), bighead carp (Aristichthys nobilis), common carp (Cyprinus

carpio), crucian carp (Carassius auratus), group head bream(Megalobrama amblycephala), dace (Cirrhinus molitris), Alice mouth(Culter alburnus), golden fin bream (Paracanthobrama guichenoti),and squaliobarbus (Squaliobarbus curriculus). Their relative toxi-cities in order of strength are as follows: crucian carp > group headbream > herring > dace > silver carp > bighead carp > Alicemouth > common carp > grass carp > golden fin bream > squa-squaliobarbus. The bile of round mouth copper fish (Coreius

guichenoti) is also toxic. These 12 types of fish all belong to theorder Cypriniformes and the family Cyprinidae. To date, onlycyprinids have been found to be have poisonous bile. More than80% of the gallbladder poison victims were poisoned by ingestinggrass carp bile, which may be explained by the large scaleproduction of grass carp and the large size of its gallbladder.Sodium cyprinol sulfate has been isolated and purified from grasscarp bile and it is the major toxic component in cyprinid fish bile.The toxin is heat stable and it is not altered by either dissolving inether or precipitating with ethanol [52]. Therefore, the ingestion ofa cooked or wine-soaked gallbladder can also cause poisoning. Allof the 12 aforementioned types of fish contain sodium cyprinolsulfate. Aside from sodium cyprinol sulfate, cholic acid, cheno-deoxycholic acid, and goose taurocholic acid can react withpotassium ions to form bile salts that damage cell membranes. Biletoxins can act directly on the gastrointestinal system, liver, andkidney, resulting in liver and renal tubular epithelial (especially theproximal convoluted tubule) cell degeneration, necrosis, and acutegastroenteritis. Additionally, the consumption of bile toxin canlead to liver and renal failure in the short term [18,53–55].Exposure can also damage cardiac and brain cells, and causemultiple organ dysfunction syndrome (MODS); thus, the kidney,liver, and gastrointestinal system are the most commonly involvedorgans [56].

Using an enzyme histochemical test, we detected renal tubularepithelial alkaline phosphatase (AKP), acid phosphatase (ACP),succinate dehydrogenase (SDH), and cytochrome oxidase (CCO)activities. They were all reduced to varying degrees, whichillustrates that the toxic gallbladder components not only directlydamage cellular structure, but also increase damage to the cellsthrough the inhibition of enzyme activity (data reported inChinese). The bile components in fish can be detected by severalmethods, such as HPLC and gas chromatography–mass spectrom-etry (GC–MS) [57–59].

2.3. Snake venom poisoning

In China, there are approximately 226 types of snakes, 67 ofwhich are poisonous. The 10 most poisonous snakes to humans areas follows: cobra (naja), king cobra (Ophiophagus hannah),Bungarusmulticinctus, krait (Bungarus fasciatus), Vipera russelli

siamensis, Agkistrodon halys, Deinagkistrodon acutus, Trimeresurus

stejnegeri, Trimeresurus mucrosguamatus and Calliophis macclellan-

dii [60,61].The composition of venom is complex; one study found that the

composition of venom is more than 90% protein/peptide based, andthe remaining components are metal ions, lipids, and carbohy-drates. Venom peptides are classified into enzymatic and non-enzymatic categories. Venom poison can target many organs ortissues, such as the heart, kidney, brain, muscle, and blood. The


enzyme types and functions are primarily as follows: (1)phospholipase can catalyze lecithin hydrolysis, damage the cellmembrane structure, cause muscle toxicity, cytotoxicity, cardiactoxicity, and neurotoxicity and inhibit blood coagulation; (2)phosphodiesterase can cause an extreme drop in blood pressure;(3) hyaluronidase can hydrolyze hyaluronic acid and enable therapid penetration of other toxin components. These three enzymesare present in all types of venom. Additionally, the followingenzymes are common components in snake venom: (4) cholines-terase can hydrolyze acetylcholine to block nerve conduction andis present in at least 50 types of snake venom but not in the venomof the Viperidae family and rattlesnakes; (5) L-amino acid oxidasecan be found in more than 70 types of snake venom and it acts onthe human coagulation system and fibrinolytic system proteins,which can lead to bleeding or thromboembolism [62].

Non-enzymatic proteins or polypeptides include the following:(1) neurotoxins can block neuromuscular transmission [63]; (2)muscle toxins can lead to skeletal muscle focal necrosis, whichresults in myoglobinuria and acute renal failure; (3) cytotoxins cancause hemolysis and cytolysis, which results in hematuria andrenal tubular damage (there are 59 cytotoxins in the Elapidaefamily) [64]; (4) cardiotoxins (CTX) can cause myocardial damage[65]; (5) hemorrhagic toxins can cause tissue edema, hemorrhageand necrosis, which leads to multi-organ bleeding [66]; (6)cathelicidin exists in higher mammals, such as humans, and isalso found in the Elapidae family; (7) nerve growth factor (NGF)can elicit nerve tissue damage; and (8) protease inhibitors havebeen isolated from the venom of the Elapidae family and fromvipers [67].

Venom is divided into 3 categories, namely neuro-venom,blood-circulating venom, and mixed venom (including neuro-toxicity and blood-circulating toxicity), based on their clinicalmanifestations. The bungarotoxin of Bungarus multicinctus andBungarus fasciatusis is primarily a neurotoxin. The venoms of Vipera

russelli siamensis, Agkistrodon halys, Deinagkistrodon acutus, andTrimeresurus mucrosguamatus are blood-circulating venoms. Thesymptoms of mixed venom poisoning appear in the nervous, bloodand circulatory systems. Cobra, king cobra, and Trimeresurus

stejnegeri have mixed venoms [4,61,68]. The cause of death from avariety of snake bite poisonings is primarily related to the toxicsnake venom components and includes the following symptoms:the paralysis of respiratory muscles, which causes asphyxia andhypoxic-ischemic encephalopathy, hemorrhagic shock, toxiccardiomyopathy, acute tubular necrosis, infection, and death fromanaphylactic shock [67,69].

There are many ways to detect snake venom, such as bioassays,immunodiffusion, immunoelectrophoresis, radioimmunoassays(RIA), agglutination assays, and fluorescence immunoassays. Thefastest and most sensitive detection method is ELISA [70].Theakston et al. first detected snake venom and its antibody byELISA in 1977; the assay detection limit was 1–5 ng/ml, and theassay had good sensitivity and low cross-reactivity [71]. In 1988,Labrousse used a rapid ELISA for the direct detection of vipervenom in blood; the time to get results was 20 min, and only a 200-ml sample was required [72]. Wound extracts and urine, local bitetissue and neighboring lymph nodes, and even vitreous humorfrom the affected body, can be used to detect the venom [73].

Over the course of a snake bite poisoning identification, thelegal examiner should be aware of the details of the biteexperience, local and systemic symptoms, and they shouldcarefully check for poisonous snakebite teeth marks localized inthe skin, take samples from bitten localized tissue, blood, liver, andkidney. The snake venom antigen should be detected, and changesin organ pathology should be microscopically observed. The site ofthe snakebite is usually in the lower limbs. Agkistrodon halys

venom can be absorbed from the bite site later into the




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bloodstream by the blood and lymph, target blood, heart, kidney,brain, and other organ systems and can cause organ systemdysfunction [68]. Acute lymphadenitis occurs when venom flowsthrough the lymph nodes (case 3). Myocardial degeneration mayoccur, in addition to small focal necrosis, interstitial hemorrhageand inflammatory cell infiltration; myocardial edema wasobserved in cases 2 and 3, and case 3 showed a mild necroticribbon in the heart muscle. The prominent changes caused byAgkistrodon halys venom are hemolysis and bleeding, hemolysisand toxicity in the renal tubular epithelial cells, which causes toxicnephropathy, acute renal failure and death. Prominent lesions inthe urinary system, including anuria, proximal tubular epithelialcell degeneration and necrosis of the distal tubule and medullarytubule protein casts, and pigment casts are the main signs of blood-circulating venom poisoning [74]. We determined that the victimsin cases 3 and 4 were all bitten by Agkistrodon halys in the lowerlimbs, but their experiences differed greatly. Although case 4received medical attention and treatment, he displayed seriousdeterioration shortly after had been bitten. In fact, the sameamount of venom affects children more severely than adultsbecause of the reduced total dilution volume in children [75].

2.4. Melittin poisoning

Poisonous bees are a class of bees and wasps and have a venomgland that can secrete an aromatic transparent venom known asbee venom, which is stored in the poison sac and discharged uponstinging.

The composition of bee venom is complicated and includespeptides (e.g., melittin, neurotoxins, and mast cell degranulationpeptides), enzymes (e.g., phospholipase A2, protease, and hya-luronic acid enzymes), and non-peptide substances (e.g., hista-mine, phospholipids, and 5-HT) [76–79]. The primary activecomponent is melittin, which is a known hemolytic toxic peptide[77]. Melittin has a strong cytotoxic effect, with hemolytic andantibacterial activities, and it can act directly on the heart andblood vessels. Neurotoxin (apamin) can pass through the blood-brain barrier, it is a central nervous system toxin, and it targetspotassium ion channels. Phospholipase A2 and hyaluronidase cancause allergic reactions. Phospholipase A2 can cause hemolysis,destroy the phospholipids on the cell membrane, and coordinatewith MCDP, causing mast cell degranulation [80]. Hyaluronidasecan cause bee venom to infiltrate and proliferate in the localizedarea, which allows other bee venom components to infiltrate,resulting in rapid skin cellulitis [28,62].

Studies have shown that melittin and phospholipase can lead torhabdomyolysis [81]. Melittin, phospholipase A and lecithin canenhance red cell membrane permeability, causing intracellularcolloid exudation and red blood cell lysis by osmotic pressure,which results in colloid exudation hemolysis [82,83]. Hemolysisand rhabdomyolysis cause secondary acute tubular necrosis 2–3 days later, and the cytotoxic effect of melittin can directlydamage tubular epithelial cells, both of which can lead to acuterenal failure. Bee venom contains a non-phospholipid protein thatcan induce encephalitis [84,85]. Phospholipase A in the phospho-lipid membranes of the nervous system can promote the release ofphospholipid base protein and other antigens and induce animmune response, which causes brain tissue damage [86]. Melittininhibits myocardial mitochondrial and synaptic Na-K+ATPaseactivity and causes myocardial ischemia, hypoxia and cardiacarrhythmias. Therefore, the target organs or target tissues of beetoxin poisoning are the brain, heart, kidney, muscle, and blood [87].

The quantity and toxicity of melittin is greater in wasps than inbees. When compared with the bee, wasp venom contains a higherconcentration of biogenic amines and it contains kinin venom (e.g.,wasp kinin, golden wasp kinin, and yellow winged wasp kinin),


which can cause intense pain and vasodilation, increasing vascularpermeability. The antigen 5 peptide is the most importantcomponent of allergic reactions; other allergic components includea variety of phospholipases, hyaluronidases, and cholinesterases[88]. Wasp melittin-containing anti-coagulant serine protease hasa strong fibrinolytic activity, and it contributes to the dissolution offibrin and fibrinogen [62].

There are four classes of fundamental change after a bee sting.Class one is localized tissue redness, swelling, blister formation andtissue necrosis, which is caused by vasoactive substances in themelittin. Class two is a systemic allergic reaction, which ischaracterized by urticaria, angioedema, nausea, vomiting, difficul-ty breathing, hypotension and shock; these changes often occurimmediately after the sting. Death from bee stings is caused byallergens, which cause body hypersensitivity or extreme edema ofthe local respiratory tract, leading to airway obstruction. Classthree is mainly composed of rashes, subcutaneous hemorrhage,lymph node swelling and other serum sickness-like reactions;these reactions are stimulated by the immune complexes thatcirculate in the blood or by a late-onset allergic reaction. Class fourincludes other changes, such as a sting-induced accident or injuryfrom falling [89].

Deaths by bee sting can be divided into three causal categoriesas follows: (1) an accident or high fall after the bee sting; (2)systemic allergic reactions or a localized respiratory tractobstruction; and (3) sting venom poisoning. The third type isrelatively rare because more than 500 bee stings are required toprovide sufficient bee venom to cause a direct death. A large doseof venom can cause respiratory center paralysis [90]. We believethat the third type should include a death that occurs several dayslater with acute tubular necrosis, acute renal failure-induced orneurotoxin-induced toxic encephalopathy [91,92]. Anaphylacticshock, laryngeal edema, and pulmonary edema are the leadingcause of early deaths in patients, such as in case 8. Currently, thereis no specific melittin detection method. It is difficult to determinebee sting-induced anaphylactic shock and death in the forensicidentification [81,84,89]. The subjects in cases 6, 7, and 9 diedfrom anaphylactic shock, which manifested as larynx and glottisedema. In cases 6 and 7, the subjects experienced larynx, lung, andspleen eosinophilic leukocyte infiltration. In case 9, there was noapparent eosinophil infiltration, which may be explained by theshort time between the sting and the death (only half an hour fromthe bee sting to death). The most useful tests for diagnosing lethalanaphylaxis are measurements of allergen-specific IgE and serumtryptase [36,93]. The normal blood IgE value of a healthyindividual is less than 800 ng/mL, and the normal serum tryptaseconcentration is less than 13.5 mg/L. In case 9, the cardiac bloodIgE value was 1384.32 ng/mL, which was higher than normal, andthis data provided diagnostic evidence for bee sting-induceddeath.

At autopsy, the findings may be non-specific and notparticularly helpful in establishing the diagnosis. There were nospecific macroscopic or microscopic postmortem features, usuallybecause of the rapidity with which anaphylaxis-associated deathoccurs [94–97]. Death by bee sting allergy can be confused withother allergy-induced types of death in which there is laryngealedema and no significant lesion, which may indicate an unknowncause of death. Terebra detection is important in the diagnosis ofbee sting-induced death. The bee stingers are bee specific, whichcan facilitate the identification of the bee populations. In anautopsy, the head, face, limbs and other exposed parts of the bodiesshould be carefully examined. Using a scanning electron micro-scope, Huang et al. characterized the sting remaining on the skin ofdeceased people who had died from a bee sting allergy, and a clearbarb or a punctate, hollow and ditch ridge were used as evidence ofa bee venom allergy-induced death [98].




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3. Cantharidin poisoning

Cantharide insects belong to the phylum Arthropoda, classInsecta, order Coleoptera, and family Meloidae. The main toxicsubstance in cantharides is cantharidin. Most cantharides containapproximately 1–1.2% (0.2–0.7 mg) cantharidin [99]. When takenorally, 0.6–1 g of cantharides are generally poisonous; 1.5–3 g(from approximately 5 cantharide bodies) can be lethal [22,100].

Cantharidin is widely present in the body of over 1500 types ofcantharides and it is a natural defense toxin [101]. In 1810, theFrench pharmacologist Robiquet first extracted crude cantharidinfrom Lytta vesicatoria. In 1877, Piccard proposed the cantharidinmolecule structure as C10H12O4; the molecular weight is 196.2 g/mol. In 1953, Gilbert first synthesized cantharidin in thelaboratory.

Cantharide is a highly toxic Chinese herbal medicine with avariety of activities, such as anticancer, antiviral, and aphrodisiacactivities, and it can elevate white blood cell activity. Over the pastdecade, Chinese scientists have synthesized more than 100 speciesof cantharidin derivatives, including sodium cantharidate, methyl-cantharidimide, allylcantharidimide, and hydroxycantharidimide.In vitro test results demonstrate that cantharidin can inhibit themetabolism of HeLa cells and tumor cells in the human esophagus,cardiac, gastric cancer, liver cancer, lung cancer, breast cancer, andothers. Cantharidin and cantharidin derivatives have similar anti-tumor effects. Sodium cantharidate has a wide-spectrum anti-tumor effect, especially in anti-hepatocellular carcinoma. In China,cantharides and their preparations were widely used for thetreatment of acute and chronic hepatitis B, liver fibrosis, advancedhepatocellular carcinoma, ovarian cancer, urinary tract cancer,neurodermatitis, psoriasis, urticaria, purpura, eczema, flat warts,genital warts, and other diseases; cantharides had a positive effect,and no poisoning in relation to the treatment of these diseases wasreported [102–104].

Cantharidin itself has a strong stimulating effect on the skin,mucous and gastrointestinal tract and can cause skin erythema,blisters, or necrosis. After absorption by the skin or mucousmembranes, the urinary system and liver are the major targetorgans; cantharidin causes renal necrosis and urinary tractinflammation. Oral administration causes throat irritation andoral mucosal blisters and ulcers, and it can also cause acutegastroenteritis, abdominal pain, vomiting, and shock, and deathresults from peripheral circulatory failure. For delayed poisoningcases, the poison that is excreted by the kidneys causes kidneydamage, lower back pain, oliguria, urinary frequency, a urinaryburning sensation, dysuria, and hematuria; in severe cases, toxicnephropathy will occur along with renal tubular epithelialextensive degeneration and necrosis and, often, death by acuterenal failure [105,106]. Autopsy findings demonstrate that themain lesions are located in the gastrointestinal tract and kidneys.Other symptoms include esophageal mucosal congestion, swelling,ulcers, gastric mucosal hyperemia, hemorrhage, focal superficialerosion that extends to the upper segment of the small intestine,degeneration and necrosis of renal tubular epithelial cells in water,renal hemorrhagic cystitis, focal liver cell necrosis, acute splenicinflammation and brain edema.

In cases 10–13, the poisoned persons had significant gastroin-testinal stimulus damage and toxic nephropathy. Additionally,case 10 had skin irritation and mild liver toxicity; case 11 had mildliver toxicity; case 12 had hemorrhagic bladder inflammation,acute splenic inflammation and acute interstitial hepatitis; andcase 13 had superficial ulcerations in the esophageal mucosa withhemorrhage and cerebral edema. All of these cases had toxicnephropathy that was caused by cantharides poisoning. Thesepathological changes are consistent with other records. Thecantharidin content is mainly determined by gas chromatography;


this method is simple and has high sensitivity, high recovery, andgood reproducibility. There are other methods for determining thecantharidin content, such as HPLC, GC–MS, ultraviolet spectro-photometry, and the acid–base titration method [107,108]. Thevomit, gastrointestinal contents, excrement, liver, and kidney areusually sampled for toxicological analysis. In this paper, all 4 caseshad positive cantharidin test results. If a foot or broken wingresidue from a cantharide is found in the pockets of the deceased orin the gastrointestinal contents, these are not only good samplesfor toxicology analysis, but also direct evidence of cantharidinpoisoning.

3.1. Venenum bufonis poisoning

The toad belongs to the class Amphibia and family Bufonidae.There are more than 250 types of toad. Three types of toad arewidely found in China, including Bufo gargarizans Cantor, Bufo

melanosticus Schneider, and Bufo raddei Strauch [102]. Venenumbufonis (chansu) is secreted from the glands and skin of theBufonidae, and it is commonly used in traditional Chinesemedicine after appropriate processing. Pharmacological studiesindicate that venenum bufonis has many effects, such ascardiotonic (i.e., similar to digitalis) [109], anti-myocardialischemia (i.e., similar to urokinase), anti-endotoxin shock, anti-tumor, analgesic, and anti-inflammatory effects, and it is widelyused in China, Japan, Korea, and other Asian countries[103,110,111]. In Chinese folklore, toad ingestion leads to aclearing of inner heat, detoxification, and removal of prickly heat,thus supporting its use in food therapy.

Venenum bufonis can be detected by GC/MS [112]. Thechemical composition is as follows: (1) bufadienolide, whichincludes bufotoxins and bufogeinin compounds in approximately70 species; bufotoxins are derivatives of bufogeinins, whichcontain a variety of cardiac steroids that can increase cardiaccontractility; (2) bufotenines, which include indole alkaloids andhave been isolated from nearly 10 types of toads; (3) sterols, suchas cholesterol, 7a-hydroxycholesterol, 7b-hydroxycholesterol, b-sitosterol, ergosterol, and campesterol; and (4) other components,such as amino acids, organic acids, epinephrine, morphine,peptides, and polysaccharides [103,113].

Bufotoxin and bufogeinin poisoning can cause arrhythmia andcardiac arrest and stimulate gastrointestinal contractions andcause vomiting. Indole alkyl amines can cause hallucinations. Afteringestion, severe nausea, vomiting, abdominal pain, and diarrheaappear 0.5–2.0 h later followed by headaches, dizziness, drowsi-ness, numbness of the lips and limbs and a variety of cardiacarrhythmias, cardiogenic cerebral ischemic syndrome, and shocksymptoms. Severe poisoning can cause death within 1–4 h[106,114]. The pathological changes are non-specific, such asmyocardial hydropic degeneration and pulmonary edema; theheart, brain, liver, spleen and kidney show significant congestion[115].

In cases 15–18, the four poisoned subjects died within 4 h afteringestion; symptoms were present in the digestive system andincluded abdominal pain, vomiting, diarrhea, and tongue numb-ness. In case 14, the poisoning symptoms appeared later after livetoad ingestion, and because digestion and absorption took longer,the poisoning was prolonged for three days. The symptomsoccurred primarily in the nervous system but included abnormalheart rhythms and gastrointestinal symptoms. The target organs ortarget tissues of toad venom poisoning were the heart, gastroin-testinal tract, and brain.

The lethal dose of venenum bufonis is not specified herein. Incase 14, ingesting one toad caused severe poisoning and death. Incases 15–18, only three toads (i.e., an average of 45 g per person)caused death, which demonstrates the extreme toxicity of these




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animals. The methods for detecting venenum bufonis arespectrophotometry, TLC, and HPLC. Qualitative and quantitativetesting in the Chinese pharmacopoeia employed TLC and HPLC todetect resibufogenin and cinobufagin. Although the methodincluded a solid phase extraction method, high performanceliquid chromatography was successfully applied to qualitativelyand quantitatively detect 4 types of toad bufadienolides, whichincluded cinobufotalin, bufalin, cinobufagin, and resibufogeninfrom the liver tissue. The majority of laboratories in China cannotyet quantitatively detect venenum bufonis. Cases 14–18 werequalitatively analyzed for venenum bufonis with positive results.

A thorough case investigation of all details is important insuspected poisonous animal forensic cases. Most toxic animalpoisoning cases can be recognized by understanding the exposureprocess of the deceased, which includes ingestion, being stung orbitten, and the symptoms before death. Occasionally, the cause ofdeath was hard to determine, and a careful inspection of the corpsesurface and complete autopsy was performed. Extracting thenecessary samples for a toxicology analysis is also required.

In some cases, internal organs, such as the kidney, liver, cardiac,gastrointestinal system and other areas with toxic lesions, canprovide direction for the toxicology analysis. In case 10, theautopsy results yielded blisters on the skin of the armpit and neck,toxic nephropathy, and evidence of pregnancy, so the possibility ofcantharidin poisoning from the use of cantharides to cause anabortion should have been considered and was later confirmed bya laboratory test using the stomach contents. The broken bodiesand wings of cantharides mixed in the stomach contents are strongevidence of poisoning and a good source of samples for poisonanalysis. In case 11, fragments of cantharidin were found in thepockets of the deceased, which were positively identified bylaboratory tests using the stomach contents to quickly clarify thenature of the case.

Toxic components of poisonous animals are complex, and anumber of toxic ingredients cannot be identified; certain poison-ous animals have no special toxicology analysis method. Therefore,it may be necessary to ask zoologists for animal speciesidentification on occasion, to make an accurate forensic identifi-cation, and to perform an experiment using an animal model todetermine the specific poisoning symptoms and calculate thetoxicity.

Ethical standards

We declare that the present study complies with currentChinese law. The mouse experiment was approved by the animalethics committee of Fudan University.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgment

We gratefully thank those colleagues who took part in theautopsy work related to the cases mentioned in this article.

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Poisoning by toxic animals in China—18 autopsy case studies and a comprehensive literature review