OUTBREAK OF TRICHOTHECENE MYCOTOXICOSIS ASSOCIATED WITH CONSUMPTION OF MOULD-DAMAGED WHEAT PRODUCTS IN KASHMIR VALLEY, INDIA

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Public Health OUTBREAK OF TRICHOTHECENE MYCOTOXICOSIS ASSOCIATED WITH CONSUMPTION OF MOULD-DAMAGED WHEAT PRODUCTS IN KASHMIR VALLEY, INDIA

SASHIDHAR R. BEEDU1 K. L. MUNSHI2

RAMESH V. BHAT1 Y. RAMAKRISHNA1

Food and Drug Toxicology Research Centre, National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania PO, Hyderabad-500 007, AP, India;1 and Department of Social and Preventive Medicine, Government Medical College, Srinagar, India2 to September, 1987, there that a considerable segment of the population of Kashmir Valley, India, were affected by a gastrointestinal disorder. Epidemiological investigations and laboratory based studies indicated that the outbreak was associated with the consumption of bread made from mould-damaged wheat. The disease was not age or sex specific. Evidence of mould damage of wheat consisted of the presence of moulds (such as Fusarium sp, Aspergillus sp), and varying quantities of trichothecene mycotoxins (such as deoxynivalenol, nivalenol, acetyldeoxynivalenol, T-2 toxin) in samples tested. The symptoms were reproduced in dogs fed extracts of contaminated samples. The finding that trichothecene mycotoxins, especially deoxynivalenol trichothecene, cause symptoms in man emphasises the need for a reappraisal of its safety limits in food.

Summary

During June were reports

INTRODUCTION

DURING July to September, 1987, an outbreak of a gastrointestinal disorder occurred in the Kashmir Valley, especially in the city of Srinagar and the surrounding areas. Since gastrointestinal symptoms are common among poor urban communities in certain seasons, the outbreak was initially not considered serious enough to need detailed investigation. However, as the numbers affected reached several thousands and seemed to come from all socioeconomic strata, a systematic investigation1 was begun. The objectives of the study were to find out whether there was a distinct disease entity and, if so, the most likely cause of the outbreak, so that control measures could be applied. In addition, it was considered that such an investigation would add to knowledge on causative factors of such outbreaks. First, a hypothesis had to be formulated. Since there was no established system for reporting an outbreak of a disease

of unknown aetiology, the hypothesis that the outbreak was a foodborne disease, due presumably to a chemical toxin or infectious agent, was based on information from press reports, especially in the local language newspapers, and from the preliminary analysis of the food samples sent by Government agencies for possible naturally occurring and man-made toxins. It was considered essential to differentiate between the man-made chemicals (such as pesticides), chemicals of plant and animal origin (such as pyrollizide alkaloids), and microbial toxins. EPIDEMIOLOGICAL INVESTIGATION

For field studies, the recommendations made by the International Programme on Chemical Safety (IPCS) for establishing chemical aetiology of specific diseases as a basis for their prevention were followed,2 but an open mind was kept about the nature of the outbreak, which was investigated according to WHO guidelines.The survey was conducted in the city of Srinagar and in the jurisdiction of primary health care centres of Mattan, Bijabehera Kulgam, and Quimoh of Anantnag District, in the state of Jammu and Kashmir during the first week of November, 1987. Information was collected according to a pre-tested questionnaire on whether any family member had been affected by gastrointestinal illness, its symptoms and clinical course, and the treatment received; the type of foods consumed; environmental factors; and unusual happenings in the areas. In the villages a house-to-house survey was conducted, while in the city of Srinagar, specific beats of certain municipal wards were selected at random for the survey. A cluster of a consecutive houses in each of the above mentioned areas in the city were visited. 150 families were interviewed. 39 of them reported at least one having been affected by a distinct gastrointestinal disease during June to September, 1987. The disease was characterised by mild to moderate abdominal pain or a feeling of fullness of abdomen within 15 min to 1 h of consuming the breakfast or evening snack, which consisted of local bread made of refined wheat flour or unleavened bread prepared at home from wheat flour purchased in the local market. Although the 97 affected members who were interviewed had had the above symptoms (for children, mothers were interviewed), 61 (63%) of them noticed irritation of the throat, and 38 (39%) had had diarrhoea (table 1). 5% had had blood in the stools. 7% had vomitted, and 2% had had an allergic reaction (eg, facial rash). Other minor symptoms reported included nausea and flatulence. All age-groups (table II) and all socioeconomic strata were affected. In children who had consumed considerable amounts of wheat preparations over periods of more than a week, secondary infections such as upper respiratory tract infections were reported. The higher TABLE II-AGE AND SEX DISTRIBUTION OF AFFECTED INDIVIDUALS

TABLE I-FAMILY SIZE, NUMBERS AFFECTED, AND SOURCE OF CONTAMINATION --

-

36 TABLE III-MYCOFLORA PROFILE IN SAMPLES COLLECTED FROM KASHMIR VALLEY

TABLE IV-ERGOSTEROL AND MYCOTOXIN PROFILE

I

I

I

I

J

*In addition ten samples each of refined wheat flour, wheat flour, and wheat were collected in the local Hyderabad market, were negative for toxins and ergosterol, and served as controls. DON deoxynivalenol; NIV nivalenol; AC-DON acetyldeoxy=

nivalenol;

*Number of samples tested. tOther fungi include Aspergillus niger; A candidus; A terreus; Penicillurra sp. % seed infect % number of grains infected. =

of males among those affected could be accounted for by the possibility that men were more likely than women to go out to work and hence to eat bread purchased from local bakers, whereas the rest of the family tended to eat food prepared at home. The interviews indicated that only individuals who consumed wheat products were affected. In most instances the disease lasted as long as the incriminated food (bread) was being consumed, generally 1-2 days. Treatment included antidiarrhoeal drugs such as diphenoxylate hydrochloride (’Lomotil’, Searle) and, in some cases, antibiotics; however, therapeutic agents had little or no effect and the disease subsided as soon as the consumption of incriminated food ceased. In Srinagar production of refined wheat flour in the city’s 8 roller flour mills fell by almost about 6000 quintals per month. 9 samples of refined wheat flour, 3 samples of wheat flour, and 3 samples of wheat were collected either from households that gave a history of the disease after consuming wheat preparations or from traders who reliably confirmed that the consumption of such products had resulted in the outbreak. In addition, 9 samples of wheat and wheat products seized from traders by Government authorities under the Prevention of Food Adulteration Act3 were also

proportion

analysed. The staple food of the people of the Kashmir Valley is rice, which is consumed at lunch and dinner. However, in urban and semi-urban areas, a considerable segment of the population consume local bread made from refined wheat flour; the bread is sold as rolls at street comers, a roll being made from about 10-30 g of refined wheat flour. Generally people in the Valley consume bread along with tea in the mornings and evenings. Some households also take unleavened bread (roti) prepared at home from ordinary wheat flour. The average intake of bread/unleavened bread varies from 1-6 pieces, depending on the size and age group. Events Preceding the Disease Outbreak

May, 1987, saw unseasonal rains at wheat harvest time in the Jammu Division of the State of Jammu and Kashmir and in the neighbouring State of Punjab. A large unspecified quantity of standing crop of wheat became rain-damaged and mouldy. Farmers sold such wheat on the open market at

=

ND

=

not

=

detected.

a nearly 50% discount, most to wheat flour mill owners from the Kashmir Valley, and some to mill owners from other parts of the country, such as Andhra Pradesh and Maharashtra. The millers, fearful that the mould-damaged wheat would deteriorate fast, mixed it with ordinary (atta) or refined (maida) wheat flour, or with good wheat. An interesting observation made by the general population in and around Srinagar was that the quality of ordinary wheat flour, refined wheat flour, and bread sold by the traditional bakers was poor in June-September, 1987. The inferior-quality wheat flours were popularly referred to as "chewing gum" wheat flour because of the stickiness or "chewiness", and the bread had poor crumb characteristics.

LABORATORY INVESTIGATION OF WHEAT SAMPLES

Wheat samples collected during the survey were examined microscopically and for moisture content, total ash, ash insoluble in dilute HC1, alcoholic acidity, white worms, black beetles, webs, rodent hair, excreta, and gluten, as specified by the Prevention of Food Adulteration Act (PFA).3 The samples analysed conformed to the existing Indian PFA standards. Tests for pesticide residues4 were negative. However, fungi, some identified as Fusarium sp, or Aspergillus f Zavus, were isolated from the samples. Fusarium sp were isolated from all refined wheat flour, ordinary wheat flour, and wheat samples, whereas Aspergillusflavus could be isolated from only 2 of 12 refined wheat flour samples, 2 of 5 ordinary wheat flour samples, and 4 of 7 wheat samples (table III). The total seed infections in wheat samples ranged from 31 to 36% while the number of fungal propagules in wheat flours ranged from 44 000 to 378 000 propagules per gram. Mould damage was confirmed by detecting the presence of ergosterolaa chemical component of the cell walls of moulds, in all the samples of wheat and wheat products in quantities ranging from 1-8-5-4 g/g (table IV). In none of the 30 samples of wheat and wheat products obtained from Hyderabad markets could ergosterol be detected, although fungi such as A flavus and Fusarium sp could be isolated from these samples. Among the mycotoxins, aflatoxins7 and ergot alkaloids8 could not be detected in any of the samples. However, trichothecene toxins9-11 could be detected in 12 of the 24 samples (table IV). Several of the trichothecenes detected in refined and ordinary wheat flour samples by thin layer chromatography12 could not be identified. The Fusarium isolates from the above samples were toxigenic under laboratory conditions. 10,13

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The toxins were extracted12 from contaminated samples put into bread, and fed to seven (2-month-old) pups (mean weight 2-2 kg) at a dose of 200 pg/kg body weight. All the animals vomited within 1 to 3 h of ingestion of the toxin. The four control pups (mean weight 2-2 kg) did not show any toxic signs. It was estimated that the consumption of wheat flour in quantities ranging from 30 g to 150 g containing a minimum amount of 0-34 ug/g of deoxynivalenol caused mild symptoms. Blood counts were available for only 1 patient whose leucocyte counts were within the normal limits (total leucocyte count 11 000/ul, neutrophils 62%, lymphocytes 36%, and eosinophils 2%). DISCUSSION

The outbreak was identified as being due to trichothecene mycotoxins present in bread made from rain-damaged mouldy wheat. The symptoms were very similar to the red mould poisoning in Japanl4 caused by the consumption of mouldy wheat and rice. The feeling of fullness soon after consumption of toxin-containing food reduced food consumption, so the disease was self-limiting. However, the leucopenia reported during the outbreak of alimentary toxic aleukia in USSR,15 which, almost two decades later, has been thought to be due to trichothecene poisoning, could not be ascertained in the present case. The trichothecene groups of mycotoxins, mostly produced by moulds belonging to the genus Fusarium, were discovered in the 1960s and 1970s.14 Among these toxins, T-2 deoxynivalenol, and diacetoxyscripenol have been implicated in diseases affecting farm animals such as swine, dairy cows, and poultry. There is considerable controversy as to whether some of these mould metabolites are used in biological warfare. 11,16 Laboratory experiments on the storage of high-moisture wheat and their spoilage by moulds have indicated that mould counts increase from about 1000 to 3 000 000/g, with a predominance of species of Aspergillus sp and there is accompanying breakdown of lipids.1’ Gluten in such wheat is difficult to wash and the breadmaking properties are

impaired.18 Confirmed foodbome outbreaks of mycotoxicoses have taken the form of outbreaks of ergotism or alimentary toxic aleukia (from contaminated foodgrains),14,19 of aflatoxicoses (from contaminated maize) ,20,21 of enteroergotism (from contaminated pearl millet),19 and of red-mould poisoning (from contaminated grain).14 Mycotoxicosis in man and animals due to the ingestion of mould-damaged cereals have also been suspected from time to time. In 1934, Mundkur2 reported that in India wheat grains affected by "scab" caused by Fusarium contained an emetic principle rendering it unsuitable for feeding to animals. Several species of Fusarium isolated from such scabby wheat have produced metabolites that produced an emetic response in pigeons.23,24 Our report supports an earlier observation that trichothecene toxins occur naturally in subtropical Asian countries.25 It also provides evidence for the natural occurrence of disease in man due to them. Doubts have been raised about the natural occurrence of trichothecene in India, mainly because it was assumed that the temperature was 35 °C and that at this temperature the Fusarium are to grow and produce mycotoxins.16 However, in winter the temperature range in different parts of India may be anywhere between 0°C and 25°C depending on location, and such climatic conditions are indeed ideal for the production of trichothecene.

unlikely

Safe limits26 in wheat for human consumption have been prescribed for deoxynivalenol (but not for the other trichothecenes) in Canada (2000 p.g/kg in unclean soft wheat, 1000 pg/kg for unclean soft wheat for infant foods, and 1200 ug/kg for imported non-staple food), USSR (1000 flg/kg for durum wheat and 500 )J.g/kg for other wheat), and Romania (5 ug/kg in all feeds). The Food and Drug Administration of USA has issued only an advisory letter with no legal status to manufacturers and users suggesting a limit of 2000 ug/kg in wheat intended for milling and 1000 )J.g/kg for finished products meant for human consumption. The evidence provided in the present study that deoxynivalenol can cause an outbreak of disease in man emphasises the need for governments to formulate and implement laws on trichothecene mycotoxins. We thank the authorities of State Government of Jammu and Kashmir for inviting us to conduct the field investigation and for the facilities for the work; Mr Maria Desalphine, for coordinating the field visits; Prof G. Q. Allaqaband, Prof M. S. Kuru, and Mr R. N. Kaul for useful discussions; and Dr Teerath Singh, for arranging the field visits.

Correspondence should be addressed to R. V. B. REFERENCES 1. WHO guidelines, environmental health criteria 27. Guidelines on studies on environmental epidemiology. Geneva. WHO, 1983. 2. International Programme on Chemical Safety. Principles of studies on disease of suspected chemical etiology and their prevention. Environmental Health Criteria 72. Geneva WHO, 1987; 79 3. Trivedi HD, ed. AP Mathur’s commentaries on Prevention of Food Adulteration Act 1954, India. Lucknow. Eastern Book Company, 1986. 4. Health aspects of chemical safety Interim document 14. Pesticide residue analysis. Geneva: WHO, 1984 5. Booth C, ed. Methods in microbiology. London: Academic Press, 1971: 2: 2-47. 6. Sashidhar RB, Sudershan RV, Ramakrishna Y, Nahdi S, Bhat RV. Enhanced fluorescence of ergosterol by iodination and determination of ergosterol by fluorodensitometry. Analyst 1988; 133: 809-12. 7. Williams S, ed Association of Officials Analytical Chemists. Virginia. AOAC, 1984. 478 8. Bhat RV, Roy DN, Tulpule PG. The nature of alkaloids of ergoty pearl millet or bajra and its comparison with alkaloids of ergoty Rye and ergoty wheat. Toxicol Appl Pharmacol 1976, 36: 11-17 9. Kamimura H, Nishizima N, Naoi Y. Simultaneous detection of several Fusarium mycotoxins in cereal grains and food stuffs. J Assoc Off Analyt Chem 1981; 64: 1067-73. 10. Bennet GA, Peterson RE, Planner RD, Shotwell OL. Isolation and purification of DON and a new trichothecene by HPLC J Am Oil Chem Soc 1981; 58: 1002A-05A. 11. Anonymous. Changes in methods, DON in wheat TLC methods, final action. J Assoc Off Analyt Chem 1986; 69: 362-64. 12. Ramakrishna Y, Bhat RV. Comparison of different spray reagents for detection of trichothecenes Curr Sci 1987, 56: 524-26 13. Ueno Y, Sawano M, Ishii K. Production of trichothecene mycotoxins by Fusarium sp in shake culture Appl Microbiol 1975, 31: 4-9. 14. Ueno Y, ed. Developments in food science—trichothecenes—Chemical, biological and toxicological aspects. Tokyo: Kodansha, 1983: 4. 15. Mirocha CJ, Pawlosky RA, Chatterjee K, Watson S, Hayes W. Analysis for Fusarium toxin in various sample implicated in biological warfare in Southeast Asia. J Assoc Off Analyt Chem 1983; 66: 1489-99. 16. Marshall E. Bugs in the yellow rain theory. Science 1983; 220: 1356-58. 17 Rasiklal DD, Pomeranz Y, Sauer DB. Changes in wheat flour damaged by mould during storage. Effect on lipid, lipoprotein and protein. J Agr Food Chem 1970; 18: 613-16 18. Rasiklal DD, Pomeranz Y, Hoseney RC, Shogren MD, Finney KF. Changes in wheat flours damaged by mould during storage. Effect in breadmaking. J Agr Food Chem

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