Aflatoxicosis (acute aflatoxin poisoning).

It was the recognition of a distinct and fatal disease in 1960 that led to the discovery of aflatoxin (Blount, 1961; Aslin, 1961; Goldblatt, 1969). Deaths occurred chiefly in turkey poults and ducklings, chickens being relatively unaffected (Austwick, 1978). The features of aflatoxicosis in poultry relate to their age, species, the amount of toxin consumed and the length of time over which it is consumed (Austwick, 1978). The most prominent features in the early outbreaks were sudden loss of appetite, nervous symptoms such as ataxia and opisthotonous and a high, rapid mortality (Goldblatt, 1969). These features represent very acute poisoning and were more marked in the young birds. More chronic effects include growth retardation, oedema and haemorrhages of the legs and feet (Austwick, 1978).

Pathologically many organs were involved in fatal cases (Austwick, 1978). Hepatic swelling and congestion was prominent with superficial hyperplastic nodules developing in the more chronic cases. The kidneys were congested and pericardial distension, sometimes with haemopericardium was noted. The viscera were usually oedematous and ascites was sometimes present. The histology of affected livers is quite distinctive and similar to the changes caused by ragwort poisoning with pyrrolizidine alkaloids (Schoental, 1963). The avian liver reacts differently to toxic substances and the histology is correspondingly different (Campbell, 1967). The most prominent features were bile dust proliferation, which could lead in advanced cases to the development of hyperplastic superficial nodules. There was a notable lack of fibrosis or inflammatory reaction (Austwick, 1978; Siller, 1961). Eosinophilic patches were seen by one early author along with diffuse necrosis (Campbell, 1967). The individual parenchymal cells were swollen with prominent vacuoles and enlargement of the nucleus. The nucleolus and cell membrane became more obviously basophilic. In ducklings the bile duct proliferation extended from the portal tracts as cords of basophilic tubules. This feature was developed as a bioassay for aflatoxins which was universally adopted until the later development of chemical assays.

Aflatoxicosis has been studied in many animals now, including monkeys (Keyl, 1968; Cysewski, 1968; Miller, 1984; Carlton, 1978). Schoental has graded different species susceptibility to the acute effects of aflatoxin into three groups (Schoental, 1967).

A) Very susceptibile - LD50 AFB1 approximately 1mg/kg body weight:
duckling, rainbow grout, guinea pig, rabbit, dog, newly-born rat and turkey
poult;
B) Susceptible - LD50 of AFB1 approximately 10mg/kg body weight: pig, rat,
monkey, calf, pheasant, chick, ferret, hamster, cow, mink, quail, coho salmon
and chicken.
C) Resistant - can tolerate large doses of AFB1 without ill effect: mice and sheep.

The species relevant to man is the monkey because of its phylogeny and similar metabolism. The quail also exhibits similar metabolic and excretory mechanisms. The clinical features of acute toxicity in monkeys include vomiting, anorexia, depression, jaundice, listlessness, drowsiness progressing to coma and death (Carlton, 1978). Liver function was markedly disturbed, as determined by enzyme and bilirubin blood estimations (Carlton, 1978). Hepatomegaly was common at autopsy and the other organs affected were the kidneys, heart and brain. Histologically the features were very similar to those described in humans. An oral dose of 13.5mg/kg was uniformly fatal in macaque monkeys and 4.5mg/kg killed one of four monkeys (Shank, 1971).

The first report of acute intoxication with aflatoxin in human came from Taiwan in 1967 (Ling, 1967). An intoxication of 26 people with 3 deaths occurred among three families. Mouldy rice was suspected as the culprit and culture of this grew A. flavus. Aflatoxin was also demonstrated in the 2 of 10 samples of the rice at a concentration of 23 and 18 ug/kg. The symptoms were non-specific and consisted of abdominal discomfort, malaise, oedema of the extremities, and vomiting without fever. Hepatomegaly was also noted.

The next case was reported from Uganda (Serck-Hanssen, 1970). This 15 year old boy developed abdominal pain and swelling over several days. He was afebrile with ankle oedema and hepatomegaly. An ECG revealed first degree and partial right bundle branch block. He died two days after admission and postmortem revealed pulmonary oedema, a dilated 'flabby' heart and diffuse centrilobular necrosis of the liver. Histology showed patchy liver cell necrosis with a polymorphonuclear cell infiltrate and fibrin in the sinusoids. The histology was similar to that described in aflatoxin poisoning of the African monkey (Alpert, 1970) and led to the author to look for aflatoxin in the child's home. Mouldy cassava was found there and contained aflatoxin 1.7mg/kg. Two siblings had also been taken ill as the same but recovered.

In India in 1974 an outbreak of hepatitis occurred affecting 397 patients in 200 villages with 106 deaths (Krishnamachari, 1975). The outbreak lasted two months and was confined to tribal population groups whose staple food was maize. Adverse rains drenched the standing maize crop and the outbreak began a few weeks after harvesting. The disease was associated with the consumption of maize contaminated heavily with Aspergillus flavus. The clinical features were jaundice preceded by a brief febrile period, vomiting and anorexia. Ascites and ankle oedema then rapidly developed. The liver was enlarged but not usually tender, as was the spleen. Death occurred because of gastro-intestinal haemorrhage. Hepatic coma was rare. No infant was affected. AFB1 was detected in two of seven serum samples and not in the one liver examined. Hepatic histology showed bile duct proliferation, periductal fibrosis and occasional multinucleated giant cells. Bile stasis was considerable. The maize samples from affected households contained 6-15 ug/kg aflatoxin, much lower levels than those seen in the Kenyan outbreak of 1982. Those affected could have consumed between 2 and 6 mg of aflatoxin daily over a period of a month.

Another outbreak of toxic hepatitis in India in 1974 affected both humans and dogs (Tandon, 1977). High fever at the onset was marked and although hepatic histology was not dissimilar to that described above in aflatoxicosis, the level of aflatoxin is contaminated samples was only 0.1 ug/kg. This level of aflatoxin contamination was the same in affected as in unaffected households. It was thought likely that other mycotoxins or the combination of aflatoxin and an infection might have been implicated.

A further outbreak of aflatoxicosis occurred in Kenya (Ngindu, 1982). Twenty patients developed hepatitis and twelve died in the Machakos area of Kenya. The clinical features were abdominal discomfort, anorexia, malaise and low grade fever. Jaundice appeared at 7 days with oedema of the legs, face and trunk. The liver was tender, sometimes enlarged and three patients had splenomegaly. Gastro-intestinal haemorrhage was a common terminal event but hepatic failure developed in all 12 fatal cases in some with massive ascites. Aspartate transaminase and alanine transferase levels were slightly elevated. The liver at autopsy showed centilobular necrosis with minimal inflammatory reaction with no bile duct proliferation. AFB1 was measured in two livers at autopsy and detected in both, the concentrations being 39 and 89 ug/kg. Levels of AFB1 in food samples from affected households were high, 3.2 -12mg/kg in maize. Unaffected homes had levels not exceeding 0.5mg/kg.

In October 1988, 13 Chinese children died of acute hepatic encephalopathy in the
northwestern state of Perak in peninsular Malaysia (Chao, 1991; Lye, 1995). The cases were geographically scattered in six towns in two districts. The clinical features included vomiting, pyrexia, diarrhoea, abdominal pain, anorexia, giddiness, seizures, and eventual coma. Initially, many presented with a Reye-like syndrome. Eleven post-mortem examinations were performed. The pathological findings included extensive coagulative necrosis of the liver with proliferative 'ductal/ductular metaplasia of the hepatocytes'. Giant cell formation, central vein sclerosis, bile stasis, and steatosis were also noted. There was presence of acute tubular necrosis, superficial upper gastrointestinal erosions, and ensuing encephalopathy. Aflatoxins were confirmed in postmortem samples from patients. Epidemiologic investigations determined that the children had eaten a Chinese noodle, “Loh See Fun”, hours before they died. The manufacturer of the noodle had used a banned food preservative containing boric acid was added to make the noodles. The attack rates among those who had eaten the noodles were significantly higher than those who had not (P < 0.0001). All the affected children lived in villages along the route of distribution of the noodle supplied by one factory in Kampar town. The acuteness of the illness differed from previously reported outbreaks described in Kenya, India, and
Thailand.

Acute aflatoxicosis in humans almost certainly occurs as evidenced by the above studies. The clinical features vary from report to report, as does the hepatic histology, from case to case. This may reflect nutritional status, race and concomitant mycotoxin consumption or infectious illness. The failure to detect AFB1, in body fluids after several days of illness does not exonerate aflatoxin. Also the failure to detect high levels in the home as in the 1974 outbreak in India only means that the aflatoxin contaminated food had probably been consumed.

Investigation of possible outbreaks of mycotoxicoses can be difficult for several reasons. However the timely determination of the cause could lead to useful prevention strategies, including destroying the incriminated food. Prevention of aflatoxicosis after ingestion may be possible with clay-based enterosorbents that bind aflatoxin in the gastrointestinal tract (Phillips et al, 1999 and 2002). Suggested guidelines for investigation and action have been outlined. The relevant phases include:
• a descriptive phase (case definition, descriptive pattern of disease),
• hypothesis generation (based on pathologic studies and indications from environmental and epidemiological investigations),
• hypothesis testing and confirmatory studies.
After establishing a causal relationship, steps need to be taken to transmit information to health authorities, to disseminate knowledge to health delivery personnel and the
general public, to identify borderline cases and plan management, to identify
mechanisms of exposure/contamination and relevant measures to remove or minimize
exposure to the causal agent.

David W. Denning
May 2004

References
Campbell JG. An investigation of the hepatotoxic effects in the fowl of Ragwort (Senecio jacobaean) with special reference to the induction of liver tumours with seneciophylline. Proc Roy Soc Edin Sec B 1957; 66: 111-130.

Blount WP. T urkey "X" disease. J. Br. Turkey Fed 1961; 9: 52-77.

Siller WG, Oster DC. The histopathology of an enterohepatic syndrome of turkey poults. Vet Rec 1961; 73: 134-138.

Asplin FD, Carnaghan RBA. The toxicity of certain groundnut melas for poultry with special reference to their effect on ducklings and chickens. Vet Rec 1961; 73: 1215-1219.

Schoental R. Liver disease and "natural" hepatotoxins. Bull WHO 1963; 29: 823-833.

Schoental R. Aflatoxins. Ann Reve Pharmacol 1967; 7: 343-56.

Cysewski SJ, Pier AC, Engstrom GW, Richard JL, Dougherty RW, Thurston JR. Clinical pathologic features of acute aflatoxicoses of swine. Am J Vet Res 1968; 29: 1577-1590.

Goldblatt LA. Introduction. In: Goldblatt LA ed. Aflatoxin. New York: Academic Press, 1969: 1-11.

Ling KH, Wang JJ, Wu R, Tung RC, Lin CK, Lin SS, Lin TM. Intoxication possibly caused by aflatoxin B1 in the mouldy rice in Shaung-Chih township. J Formosan Med Assc 1967; 66: 517-525.

Carnaghan RB. Pathological and public health aspects of aflatoxicosis. Ann Ist Super Sanita 1967;3:307-15.

Serck-Hanssen A. Aflatoxin-induced fatal hepatitis? A case report from Uganda. Arch Environ Health 1970; 20: 729-31.

Alpert E, Serck-Hanssen A, Rajagopolon B. Aflatoxin-induced hepatic injury in the African monkey. Arch Environ Health 1970; 20: 723-28.

Shank RC, Johnsen DO, Tanticharoenyos P, Wooding WL, Bourgeois CH. Acute toxicity of aflatoxin B1 in the macaque monkey. Toxicol App Pharmacol 1971; 20: 227-31.

Edds GT. Acute aflatoxicosis: a review. J Am Vet Med Assoc 1973;162:304-9.

Krishnamachari KAVR, Bhat RV, Nagerajan V, Tilak TBG. Hepatitis due to aflatoxicosis. An outbreak in Western India. Lancet 1975; i: 1061-63.

Tandon BN, Krishnamuthy L, Kosby A et al. Study of an epidemic of jaundice, presumably due to toxic hepatitis, in Northwest India. Gastroenterology 1977; 72: 488-494.

Keyl AC. Mycotoxicosis in cattle. In Wyllie TD, Morehouse LG, eds. Mycotoxic fungi, mycotoxins, mycotoxicoses. An encyclopaedic handbook. New York, Marcel Dekkar, 1978; 3: 9-28.

Carlton WW, Szczech GM. Mycotoxicoses in laboratory animals. In: Wyllie TD, Morehouse LG, eds. Mycotoxic fungi, mycotoxins, mycotoxicoses. An encyclopaedic handbook. New York, Marcel Dekkar, 1978; 3: 407-414.

Austwick PKC. Aflatoxicosis in Poultry. In: Wyllie TD, Morehouse LG, eds. Mycotoxic Fungi, Mycotoxins, Mycotoxicoses. An encyclopaedic handbook. New York: Marcel Dekker, 1978; 2: 279-301.

Ngindu A, Jonson BK, Kenya PR et al. Outbreak of acute hepatitis caused by aflatoxin poisoning in Kenya. Lancet 1982; i: 1346-48.

Miller DM, Clark JD, Hatch RD, Jain AW. Caprine aflatoxicosis serum electrophoresis and pathologic changes. Am J Vet Res 1984; 45: 1136-41.

Chao TC, Maxwell SM, Wong SY. An outbreak of aflatoxicosis and boric acid poisoning in Malaysia: a
clinicopathological study. J Pathol. 1991;164:225-33.

Tandon HD. Handling toxicoses of unknown origin. Food Addit Contam 1993;10:105-13.

Lye MS, Ghazali AA, Mohan J, Alwin N, Nair RC. An outbreak of acute hepatic encephalopathy due to severe aflatoxicosis in Malaysia. Am J Trop Med Hyg. 1995 Jul;53(1):68-72.

Phillips TD. Dietary clay in the chemoprevention of aflatoxin-induced disease. Toxicol Sci. 1999;52(2 Suppl):118-26.

Phillips TD, Lemke SL, Grant PG. Characterization of clay-based enterosorbents for the prevention of aflatoxicosis. Adv Exp Med Biol 2002;504:157-71.